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WO2025008499A1 - Methods and compositions for purifying particles and macromolecules - Google Patents

Methods and compositions for purifying particles and macromolecules Download PDF

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Publication number
WO2025008499A1
WO2025008499A1 PCT/EP2024/068977 EP2024068977W WO2025008499A1 WO 2025008499 A1 WO2025008499 A1 WO 2025008499A1 EP 2024068977 W EP2024068977 W EP 2024068977W WO 2025008499 A1 WO2025008499 A1 WO 2025008499A1
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WIPO (PCT)
Prior art keywords
solid support
resin
buffer
cells
vesicles
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French (fr)
Inventor
Joel DE BEER
Nicolas Meier
Patrick Weber
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Anjarium Biosciences AG
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Anjarium Biosciences AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the methods described herein can produce purified macromolecules, vesicles, cells, or viral particles that are suitable for various uses (e.g., gene therapies).
  • gene therapies e.g., gene therapies.
  • Many types of macromolecules, vesicles, cells and viral particles have been developed for therapeutic purposes. However, reliable production and purification of these therapies have been challenging as compared to conventional small molecular therapies. For example, while extracellular vesicle offers many advantages for targeted gene delivery (e.g. low immune response), current purification methods do not offer sufficient selectivity to efficiently capture the EVs and remove significant amount of impurities, including undesirable nucleic acids, host cell proteins, carbohydrates and lipids.
  • anion exchange resins have been used in the purification of vesicles, cells, or viral particles.
  • current purification methods of macromolecules, vesicles, cells, or viral particle are limited in terms of recover yield and efficiency.
  • yields of hybridosome purification on various types of commercially available resin, including anion exchange resin is low ( ⁇ 35 %).
  • Those methods also have low reproducibility, and are unable to isolate different types of vesicles.
  • Significant loss of vesicles, such as exosomes has been observed using currently available methods, which is at least partially due to the high retention of the vesicles on the resin.
  • compositions of solid support see Section 5.3.
  • kits for purifying vesicles, cells or viral particles see Section 5.4).
  • methods of purifying macromolecules are provided herein. [0006] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b.
  • the method further comprises a washing step at the first pH.
  • the first pH is lower than the pKa of the ionizable moiety.
  • the first pH is below 8.
  • the first pH is between 4.5 to 7.4
  • the second pH is above 7.
  • the second pH is between 7.4 to 9.
  • the first pH is between 6.5 to 7.4 and the second pH is between 7.4 to 9.
  • the second pH is 7.4.
  • the eluting step is performed at a conductivity from about 55 mS/cm to about 140 mS/cm.
  • the eluting step is performed at a salt concentration from about 0.01 M to about 2 M.
  • the solid support has a binding affinity to the vesicles, cells, or viral particles at the first pH that is at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times higher NAI-1540508512v1 -2- Attorney Docket No.14497-018-228 than the binding affinity to the vesicles, cells or viral particles at the second pH.
  • the solid support has an average pore size from about 10 nm to about 1000 nm. In one embodiment, the average pore size is from about 10 nm to about 200 nm.
  • the method further comprises a step of preparing the solid support.
  • the solid support is prepared by providing a resin comprising at least one reactive group on the surface.
  • the reactive group is covalently connected to the resin via a linker.
  • the resin is a cation exchange (CEX) resin, an anion exchange (AEX) resin, a mixed mode resin, an affinity resin, a pseudo affinity resin, a hydrophobic interaction resin, a hydrophobic charge induction resin, or any combination thereof.
  • the resin is an anion exchange (AEX) resin.
  • the reactive group can react with a ligand, wherein the ligand is an amine, an alcohol, a thiol, or a carboxylic acid.
  • the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and lysine.
  • NHS N-hydroxysuccinimide
  • Sulfo-NHS ester an imidoester
  • fluorophenyl ester an aldehyde
  • a carbonate an anhydride
  • the reactive group is an epoxy group.
  • the epoxy group is capable of reacting with the ligand via a ring- opening reaction to form an ionizable moiety and a hydroxyl group.
  • the reactive group has a density of from about 100 ⁇ mol to about 2000 ⁇ mol per gram of the resin.
  • the reactive group has a density of from about 500 ⁇ mol to about 1000 ⁇ mol per gram of the resin.
  • the ligand is an amine. In one embodiment, the amine is a secondary amine.
  • the secondary amine is R 1 -NH-R 2 , wherein R 1 , R 2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R 1 and R 2 together with the atoms they are attached to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is optionally substituted.
  • the amine has a pKa of from about 8 to about 11 as determined by titration experiment using hydrochloric acid.
  • the ligand is selected from a group consisting of diethylamine, NAI-1540508512v1 -3- Attorney Docket No.14497-018-228 diethanolamine, dimethylethanolamine, diisopropylamine, isopropylethylamine, diisopropanolamine, 2-methylethanolamine, 2-ethylethanolamine, 2-(isopropylamino)ethanol, 2- (butylamino)ethanol, 2-benzylaminoethanol, piperidine, morpholine, 2,6-dimethyl-morpholine, 4- (2-chloroethyl)morpholine, 4-morpholinecarbonyl chloride, piperazine, 4-methylpiperazine and 4- hydroxyethylpiperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide; wherein each piperidine, morpholine, piperazine, pyrrole, pyrrolidine, imi
  • the preparation of the solid support further comprises a step of contacting the reactive group with the ligand to form an ionizable moiety.
  • the ionizable moiety comprises a tertiary amino group.
  • the ionizable moiety has a pKa from about 7.6 to 8.8.
  • the ionizable moiety has a density of from about 50 ⁇ mol to about 1000 ⁇ mol per gram of the solid support.
  • the ionizable moiety is capable of binding and releasing the vesicles, cells or viral particles in a pH-dependent manner.
  • the solid support is capable of binding the vesicles, cells or viral particles at an efficiency of at least 80% at the first pH and releasing the vesicles, cells or viral particles at an efficiency of at least 50% at the second pH.
  • the vesicle, cell or viral particle is negatively charged at the first pH.
  • the vesicle, cell or viral particle is negatively charged at the second pH.
  • provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a.
  • the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C 1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer.
  • the polymer is agarose, polystyrene, polymethacrylate, NAI-1540508512v1 -4- Attorney Docket No.14497-018-228 polyacrylamide, or hydroxylated methacrylic polymer.
  • the ionizable In one embodiment, the ionizable . [0044] In one embodiment, the ionizable of from about 50 ⁇ mol to about 1000 ⁇ mol per gram of the solid support, or from about 250 ⁇ mol to about 800 ⁇ mol per gram of the solid support. [0045] In one embodiment, the loading buffer and the one or more elution buffers have the same pH.
  • the one or more elution buffers each independently has a salt concentration or conductivity that is at least 1.2 times, at least 1.4 times, at least 1.6 times, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, or at least 20 times higher than the salt concentration or conductivity of the loading buffer.
  • the method further comprises one or more washing steps.
  • the vesicle is a nanovesicle.
  • the viral particle is lentiviral particle.
  • the nanovesicle has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm.
  • the vesicle is an extracellular vesicle. In one embodiment, the extracellular vesicle is exosome.
  • the nanovesicle is an artificial nanovesicle.
  • the artificial nanovesicle is a liposome.
  • the nanovesicle is a hybrid nanovesicle. In one embodiment, hybrid nanovesicle is hybridosome.
  • the method further comprises a step of preparing the hybridosome by fusing one or more lipid nanoparticle with one or more exosome.
  • the fusing is performed at a pH below 6.5.
  • the sample comprises at least one impurity that is a small molecule, a macromolecule, a different vesicle, a different cell, or a different viral particle.
  • the sample comprises at least one impurity that is a different vesicle, wherein the different vesicle is a lipid nanoparticle, an exosome or a liposome.
  • the impurity has a binding efficiency with the solid support of less than 20% at the first pH.
  • the sample has a volume from about 0.1 mL to about 2000 L, from about 10 L to about 2000 L, or from about 100 L to about 1000 L.
  • the sample is pretreated before contacting with the solid support.
  • the sample is pretreated by chromatography step, centrifugation, ultra-centrifugation, filtration, ultra-filtration, diafiltration, clarification, lyophilization, lysing, digestion, drying, dilution, concentration, heating, cooling, or any combination thereof.
  • the sample is produced from a cell culture container or bioreactor.
  • the vesicle, cell or viral particle is eluted at a yield of at least 50%.
  • the vesicle, cell or viral particle is eluted at a yield of at least 80%.
  • the method further comprises a step of detecting the elution of the vesicle, cell or viral particle.
  • the detecting involves the detection of optical signal from the vesicle, cell or viral particle, wherein the optical signal is UV-Vis signal, fluorescence signal, luminescence signal, enzyme-linked immunosorbent assay (ELISA) or light scattering signal.
  • the optical signal is UV-Vis signal, fluorescence signal, luminescence signal, enzyme-linked immunosorbent assay (ELISA) or light scattering signal.
  • the resin has an average pore size of from about 10 nm to about 1000 nm; and [0070] In one embodiment, the density of the tertiary amino group on the resin is from about 50 ⁇ mol to about 1000 ⁇ mol per gram of the resin, preferably from about 250 ⁇ mol to about 800 ⁇ mol per gram of the resin. [0071] In one embodiment, provided herein is a solid support comprising the resin as described herein. In one embodiment, the solid support is in the form of membrane, bead, or chromatography column.
  • the solid support comprises the resin in an amount from about 0.05 gram to about 5000 gram.
  • a kit comprising the solid support as described herein, a loading buffer and an elution buffer, wherein the loading buffer has a pH between 4.5 to 7.4, and the elution buffer has a pH between 7.4 to 9.
  • a kit comprising the resin as described herein, a loading buffer, and an elution buffer, and wherein the loading buffer has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer.
  • the kit further comprises a washing buffer, wherein the washing buffer has a pH between 4.5 to 7.4.
  • the loading buffer is a MOPSO buffer, Tris buffer, phosphate buffer, MOPS buffer, Bis-Tris buffer, ADA buffer, MES buffer, HEPES buffer, citric buffer or acetate buffer.
  • the elution buffer is a phosphate buffer, HEPES buffer, Tris buffer, Bicine buffer or Tricine buffer.
  • the elution buffer has a conductivity from about 55 mS/cm to about 140 mS/cm.
  • the elution buffer has a salt concentration from about 0.01 M to about 2 M. In one embodiment, the elution buffer has a salt concentration of greater than 0.65 M. 4.
  • Figure 1 depicts a representative workflow of preparing hybridosomes, followed by loading and eluting from solid support in a pH-dependent manner.
  • Figure 2 depicts the measurements of pKa values of ionizable moieties prepared from diethanolamine, DiIsoPropylAmine (DIPA), 2, 6-dimethylmopholine, or mopholine.
  • DIPA DiIsoPropylAmine
  • Figure 3 depicts the measurements of pKa values of ionizable moieties prepared from 2- NAI-1540508512v1 -7- Attorney Docket No.14497-018-228 methylethanolamine, 2-ethylethanolamine, 4-methylpiperazine, or 4-hydroxyethylpiperazine.
  • Figure 4 depicts a representative purification of hybridosomes on commercially available AEX resins (POROS D), which gives an elution yield of vesicles of 6.6%. Elution of vesicles was detected by UV-Vis and fluorescence.
  • Figure 5 depicts a representative purification of hybridosomes to remove excess LNPs using different resins under various loading and eluting conditions.
  • FIG. 6 depicts a representative purification of hybridosomes comprising pxDNA cargo using TOYOPEARL-DIPA resin, which was detected by UV-Vis, DLS, conductivity, fluorescence and luminescence.
  • Loading condition 50 mM HEPES, pH 7.0, 0.2 M NaCl.
  • Elution condition 50 mM Tris, pH 8.0, 0.01-1 M NaCl gradient.
  • Figure 7 depicts a scheme of using pH gradient for the purification of exosomes.
  • Figure 8 depicts the recovery yields of exosomes using different commercial resins under various elution conditions. The recovery yields are generally below 25%.
  • Figure 9 depicts the recovery yields of exosomes using different resins prepared. Higher recovery yields are observed compared with the use of commercial resins.
  • Figure 10 depicts a correlation between the elution pH of exosomes and the pKa of the ionizable moieties.
  • Figure 11 depicts a representative purification process of exosomes using Agarose- Morpholine resin, which is detected by UV-Vis, DLS and conductivity. Loading condition: 50 mM NaOAc, 50 mM NaCl, pH 5.5. Elution condition: 50 mM Glycine, 50 mM NaCl, pH 10.
  • Figure 12 depicts a representative purification process of exosomes using TOYOPEARL-DIPA resin packed in a 6 mL XK16/20 column, which was detected by UV-Vis, DLS, conductivity, and luminescence.
  • Loading condition 50 mM HEPES, pH 7.0, 200 mM NaCl.
  • Wash condition 50 mM Tris pH 8.0, 10 mM NaCl.
  • Elution condition 50 mM TRIS, pH 8.0, 0.01- 1 M NaCl gradient.
  • Figure 13 depicts a representative purification process of exosomes using TOYOPEARL-Morpholine resin, which is detected by UV-Vis, DLS, conductivity, and luminescence.
  • FIG. 14 depicts a representative purification process of exosomes using NAI-1540508512v1 -8- Attorney Docket No.14497-018-228 TOYOPEARL-DIPA resin in 1 mL column, which was detected by UV-Vis, DLS, conductivity, and luminescence.
  • Loading condition 20 mM Tris, pH 6.8, 50 mM NaCl.
  • FIG. 15 depicts a representative purification process of exosomes using TOYOPEARL-Morpholine resin in 1 mL column, which was detected by UV-Vis, DLS, conductivity, and luminescence.
  • Loading condition 20 mM Tris, pH 6.8, 50 mM NaCl.
  • Elution condition 20 mM Tris, pH 7.9, 0.05-1 M NaCl gradient. Fractions collected are further analyzed by SDS-PAGE.
  • Figure 16 depicts a representative purification process of exosomes using TOYOPEARL-DIPA resin in 1 mL column, which was detected by UV-Vis, DLS, conductivity, and luminescence. Loading condition: 20 mM Tris pH 6.8, 50 mM NaCl. Elution condition: 20 mM Tris pH 6.8, 0.05-1 M NaCl gradient. Fractions collected are further analyzed by SDS-PAGE and AGE gel red staining. [0095] Figure 17 compares the recovery yield of lentivirus (LV) particles eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively, as detected by p24 ELISA.
  • LV lentivirus
  • Figure 18 compares the transduction unit (TU) recovery yield of lentivirus (LV) particles eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively, as detected by p24 ELISA.
  • Figure 19 compares the TU recovery yield of lentivirus (LV) particles in main elution fraction (a) and all elution fractions (b) eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively.
  • Figure 20 compares the p24 recovery yield of lentivirus (LV) particles in main elution fraction (a) and all elution fractions (b) eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively. 5.
  • DETAILED DESCRIPTION Provided herein are methods for purifying macromolecules, vesicles, cells, or viral particles using solid support, compositions and kits comprising solid support, and their uses thereof. The methods or compositions as disclosed herein can be used to produce purified macromolecules, vesicles, cells, or viral particles that are suitable for various uses (e.g., gene therapies).
  • the method comprises steps of contacting or eluting the vesicles, cells, or viral particles under certain pH or salt conditions (see Section 5.2.2 and 5.2.3) in the presence of solid support (see Section 5.2.4).
  • the solid support as used herein comprises an ionizable moiety (see Section 5.2.5).
  • the vesicles as used herein comprise nanovesicles, extracellular vesicles, or hybrid nanovesicles, including exosomes, liposomes, or hybridosomes (see Section 5.2.6).
  • the cells as used herein comprise source cells of extracellular vesicle, HEK293T (see Section 5.2.7).
  • the viral particles as used herein comprise enveloped viral particles, lentiviral particles, adeno- associated viral particles, or retroviral particles (see Section 5.2.8).
  • the method removes at least one or more impurities, such as a small molecule, a macromolecule, a different vesicle, a different cell, or a different viral particle in a sample (see Section 5.2.10). In one embodiment, the method recovers some or most of the vesicles, cells, or viral particles in an eluting step (5.2.11). [00101] In one embodiment, provided herein are methods for purifying macromolecules (see Section 5.2.9). Non-limiting examples of macromolecules that can be purified using the methods include polypeptides (e.g. enzyme, protein, antibody, or monoclonal antibody), virus-like particles, polysaccharide (e.g.
  • the method or composition as described here is suitable for small- scale purification of vesicles, particles, cells or macromolecules.
  • the method or composition as described here is suitable for large-scale industrial purification (e.g. greater than 1 L sample volume) of vesicles, particles, cell or macromolecules.
  • the sample is pretreated (see Section 5.2.10) before purification, which is particularly important for large-scale industrial purification.
  • the method as described herein may optionally comprises a washing step as described in Section 5.2.1, a step of preparing the solid support as described in Section 5.2.4 (i), a step of preparing the vesicles, cells or viral particles as described in Sections 5.2.6-5.2.8, a step of pretreating the sample as described in Section 5.2.10, or a detecting step as described in Section 5.2.12.
  • the solid support as used herein binds and releases the vesicles, cells, or viral particles in a pH-dependent manner (see Section 5.2.4).
  • the solid support comprises an ionizable moiety (see Section 5.2.5).
  • solid support is prepared from a cation exchange resin, an anion exchange resin, or a size-exclusion resin NAI-1540508512v1 -10- Attorney Docket No.14497-018-228 comprising reactive groups (5.2.4 (i)).
  • solid support is prepared by contacting a resin comprising reactive groups as described in Section 5.2.4 (ii) and a ligand as described in 5.2.4 (iii).
  • the solid support comprises the resin as described in Section 5.3.1.
  • the solid support is in the form of a membrane, a magnetic bead, or a chromatography column as described in Section 5.3.3.
  • the solid support is in the form of a chromatography column.
  • composition such as a resin of Formula (I) (see Section 5.3).
  • the composition comprises a polymer, a linker, and a tertiary amino group as defined in Section 5.3.
  • a method of preparing the composition see Section 5.3.2.
  • a solid support e.g. membrane, magnetic bead, or chromatography column
  • a kit comprising the composition as described in Section 5.3, a loading buffer as described in Section 5.4.1, and an elution buffer as described in Section 5.4.2.
  • the kit may optionally comprise a washing buffer as described in Section 5.4.3.
  • composition, solid support, or kit as described herein is used in the purification method as described in Section 5.2.
  • 5.1 Definitions [00108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. [00109] As used herein and unless otherwise specified, the term “about” means within plus or minus 10% of a given value or range.
  • alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated.
  • the alkyl group has, for example, from one to twenty-four carbon atoms (C 1 -C 24 alkyl), four to twenty carbon atoms (C 4 -C 20 alkyl), six to sixteen carbon atoms (C 6 - C16 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C 1 -C 6 alkyl) and which is attached to the rest of the molecule by a single bond.
  • alkyl groups examples include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n- butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, an alkyl group is optionally substituted. [00111] As used herein, and unless otherwise specified, the term “alkoxy” refers to -O-(alkyl), wherein alkyl is defined above.
  • aryloxy refers to -O-(aryl), wherein aryl is defined above.
  • cycloalkyl refers to a non- aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which is saturated. Cycloalkyl group may include fused, bridged, or spiro ring systems.
  • the cycloalkyl has, for example, from 3 to 15 ring carbon atoms (C3- C 15 cycloalkyl), from 3 to 10 ring carbon atoms (C 3 -C 10 cycloalkyl), or from 3 to 8 ring carbon atoms (C3-C8 cycloalkyl).
  • the cycloalkyl is attached to the rest of the molecule by a single bond.
  • monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • polycyclic cycloalkyl radicals include, but are not limited to, adamantyl, norbornyl, decalinyl, 7,7-dimethyl- bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified, a cycloalkyl group is optionally substituted.
  • heterocyclyl refers to a monocyclic and/or multicyclic non-aromatic group that contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorous, and sulfur. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom.
  • a heterocyclyl group can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other multicyclic ring system, wherein the multicyclic ring systems can be a fused, bridged or spiro ring system.
  • Heterocyclyl multicyclic ring systems can include one or more heteroatoms in one or more rings.
  • a heterocyclyl group can be saturated or partially unsaturated. Saturated heterocycloalkyl groups can be termed “heterocycloalkyl”.
  • heterocycloalkenyl if the heterocyclyl contains at least one double bond
  • heterocycloalkynyl if the heterocyclyl contains at least one triple bond.
  • the heterocyclyl has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms (5- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-membered heterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl).
  • heterocyclyl groups include, but are not limited to, imidazolidinyl, oxazolidinyl, NAI-1540508512v1 -12- Attorney Docket No.14497-018-228 thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuryl, and piperidinyl. Unless otherwise specified, a heterocyclyl group is optionally substituted. [00114] As used herein, and unless otherwise specified, the term “aryl” refers to a monocyclic aromatic group and/or multicyclic aromatic group that contain at least one aromatic hydrocarbon ring.
  • the aryl has from 6 to 18 ring carbon atoms (C6-C18 aryl), from 6 to 14 ring carbon atoms (C6-C14 aryl), or from 6 to 10 ring carbon atoms (C6-C10 aryl).
  • aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl.
  • aryl also refers to bicyclic, tricyclic, or other multicyclic hydrocarbon rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise specified, an aryl group is optionally substituted.
  • alkylene or “alkylene chain” refers to a straight or branched multivalent (e.g., divalent or trivalent) hydrocarbon chain linking the rest of the molecule to a radical group (or groups), consisting solely of carbon and hydrogen, which is saturated.
  • the alkylene has, for example, from one to twenty-four carbon atoms (C 1 -C 24 alkylene), one to fifteen carbon atoms (C 1 -C 15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (C1-C8 alkylene), one to six carbon atoms (C1- C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene).
  • alkylene groups include, but are not limited to, methylene, ethylene, propylene, n- butylene, and the like.
  • the alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond.
  • the points of attachment of the alkylene chain to the rest of the molecule and to the radical group(s) can be through one carbon or any two (or more) carbons within the chain.
  • an alkylene chain is optionally substituted.
  • amino refers to –N(R # )(R # ), wherein each R # independently can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above.
  • a -N(R # )(R # ) group When a -N(R # )(R # ) group has two R # other than hydrogen, they can be combined with the nitrogen atom to form a ring.
  • the ring is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring.
  • one or more ring atoms are heteroatoms independently selected from O, S, or N.
  • the NAI-1540508512v1 -13- Attorney Docket No.14497-018-228 term “amino” also includes N-oxide (–N + (R # )(R # )O-).
  • each R # or the ring formed by -N(R # )(R # ) independently may be unsubstituted or substituted with one or more substituents.
  • aminoalkyl refers to -(alkyl)- (amino), wherein alkyl and amino are defined above.
  • aminoalkoxy refers to -O-(alkyl)-(amino), wherein alkyl and amino are defined above.
  • alkylamino refers to -NH(alkyl) or -N(alkyl)(alkyl), wherein alkyl is defined above.
  • alkylamino groups include, but are not limited to, -NHCH3, -NHCH2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, - NH(CH 2 ) 4 CH 3 , -NH(CH 2 ) 5 CH 3 , -N(CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , -N((CH 2 ) 2 CH 3 ) 2 , -N((CH 2 ) 2 CH 3 ) 2 , -N(CH 3 )(CH 2 CH 3 ), and the like.
  • substituents include, but are not limited to, those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chloro, iodo, bromo, or fluoro); alkyl; alkenyl; alkynyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxyl amine; alkoxyamine
  • the term "resin” as used herein refers to the solid phase of chromatography, such as column chromatography.
  • resins include: beaded resin, gels, monoliths, membranes, non-woven supports, and combinations thereof.
  • the methods disclosed herein can be applied to any form of chromatography suitable for the purification of particles, e.g, NAI-1540508512v1 -14- Attorney Docket No.14497-018-228 vesicles.
  • the resin comprises an "affinity" chromatography resin, which refers to a chromatography resin that interacts with one or more molecules present in the mobile phase of the chromatography.
  • An affinity chromatography can be used in a "bind-and-elute” mode, wherein the desired molecules interact with the stationary phase until certain conditions are created that cause the desired molecules to release from the stationary phase and elute from the chromatography resin; or in a "pass through” mode, wherein one or more impurities present in the mobile phase, but not the desired molecules, interact with the chromatography resin, allowing the desired molecules to "pass through” the chromatography resin, while the impurities remain associated with the chromatography resin.
  • the chromatography resin comprises an anion exchange (AEX) resin, a cation exchange (CEX) resin, a pseudo affinity chromatography resin, a hydrophobic interaction resin, a ceramic hydroxyapatite resin, a fluoro hydroxyapatite resin, and any combination thereof.
  • the chromatography resin comprises a mixed-mode chromatography (MMC) resin.
  • MMC mixed-mode chromatography
  • such columns are in the form of conduits, tubes, or other structures having a relatively large ratio of flow length to flow cross section.
  • the column defines a cylindrically shaped hollow interior with a volume that can be characterized by its internal radius and/or flow length.
  • the column body is a tube having two open ends connected by an open channel, which can also be referred to as a through passageway.
  • the tube can be in any shape, including, but not limited to, cylindrical or frustoconical, and of any dimensions consistent with the function of the column as described herein.
  • the column body takes the form of a syringe, a pipette tip, or similar tubular bodies.
  • the syringe is modified to contain the chromatography resin.
  • the end of the column body wherein the bed of chromatography resin is placed can take any of a number of geometries, e.g., it can be tapered or cylindrical.
  • one of the open ends of the column is adapted for a particular use, for example, the outlet opening of the column can be adapted for attachment to a collection tube.
  • the column can be composed of any material that is sufficiently non-porous that it can retain chromatography resin and that is compatible with substances used in devices and methods described herein. A wide range of materials suitable for use in a column are known in the art.
  • plastics can be used as column materials, but other materials such as NAI-1540508512v1 -15- Attorney Docket No.14497-018-228 glass, ceramics, or metals are suitable for use in devices and methods described herein.
  • Non- limiting examples of materials include polysulfone, polypropylene, polyethylene, polyethylene terephthalate, polyethersulfone, polytetrafluoroethylene, cellulose, acrylonitrile PVC copolymer, polystyrene, polystyrene/acrylonitrile copolymer, polyvinylidene fluoride, polytetrafluoroethylene (PTFE) (e.g., TEFLON®) and similar materials, glass, poly ether ether ketone (PEEK), metal, silica, and combinations thereof.
  • PTFE polytetrafluoroethylene
  • PEEK poly ether ether ketone
  • chromatography refers to any kind of technique (e.g. a preparative technique) which separates a product of interest (e.g. macromolecules, vesicles, cells, or viral particles) from other molecules present in a mixture by differential partitioning between a mobile phase and a stationary phase.
  • the stationary phase is thereby preferably held in place, while the mobile phase moves in a definite direction. Partitioning is to be understood to occur repeatedly as the sample flows with the mobile phase along or through a dimension, e.g. length, of the stationary phase.
  • Cold chromatography is a technique in which a stationary phase material is contained in a container such as a tube or column having an inlet on one side of the container and an outlet on another side, preferably the opposite side.
  • the stationary phase material may for example be comprised of particles of a solid stationary phase material or a support coated with a liquid stationary phase. It may fill the whole inside volume of the container (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). In one embodiment, a packed column chromatography is preferred.
  • sample herein implies a complex mixture or complex composition (e.g.
  • a fluid that includes one or more different molecules such as macromolecules, vesicles, cells, or viral particles, nucleic acids, polypeptides, or small molecules.
  • a method of purifying vesicles e.g. vesicles described in Section 5.2.6.
  • a method of purifying cells e.g. cells described in Section 5.2.7.
  • a method of purifying viral particles viral particles described in Section 5.2.8.
  • a method for purifying macromolecules e.g. macromolecules described in Section 5.2.9).
  • the purification is achieved by an increase of pH from loading to elution. In one embodiment, the purification is achieved by an increase of salt concentration or NAI-1540508512v1 -16- Attorney Docket No.14497-018-228 conductivity from loading to elution.
  • the loading step uses a loading buffer described in Section 5.4.1. In one embodiment, the elution step uses an elution buffer described in Section 5.4.2. In one embodiment, the elution buffer has a higher pH than the loading buffer. In one embodiment, the elution buffer has a higher salt concentration or conductivity than the loading buffer.
  • the elution buffer and loading buffer has the same pH, and the elution buffer has a higher salt concentration or conductivity than the loading buffer.
  • a method of purifying small sample volume e.g. from 0.1 mL to 50 mL.
  • a method of purifying medium sample volume e.g. from 50 mL to 1 L.
  • a method of purifying large sample volume e.g. from 1 L to 2000 L. The method provides herein is applicable to both laboratory scale purification and industrial scale purification.
  • the sample is pretreated (e.g. as described in Section 5.2.10) before subjecting to the purification method described herein.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and c. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C 1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c.
  • a method of purifying macromolecules from a sample comprising the steps of: NAI-1540508512v1 -17- Attorney Docket No.14497-018-228 a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b.
  • a method of purifying macromolecules from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is - N(C 1-6 alkyl) 2 , and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c.
  • the method further comprises a washing step as described in Section 5.2.1.
  • the method further comprises one or more steps of preparing the solid support as described in Section 5.2.4 (i).
  • the method further comprises one or more steps of preparing the solid support with ligands to create an ionizable moiety as described in Section 5.2.4 (i).
  • the method further comprises one or more steps of preparing the vesicles, cells or viral particles as described in Sections 5.2.6, 5.2.7, or 5.2.8. In one embodiment, the method further comprises one or more steps of pretreating the sample as described in Section 5.2.10. In one embodiment, the method further comprises one or more detecting steps as described in Section 5.2.11. In one embodiment, the method further comprises one or more detecting steps as described in Section 5.2.12. In one embodiment, the method further comprises a combination of any of the steps as described in Sections 5.2.1, 5.2.4 (i), 5.2.6, 5.2.7, 5.2.8, 5.2.11 or 5.2.12. 5.2.1 Washing Step [00132] In one embodiment, the method further comprises a washing step.
  • the method further comprises two, three, four, or five washing steps. In one embodiment, the method further comprises a washing step at the first pH. In one embodiment, the washing step is performed at a third pH. In one embodiment, the third pH is lower than the first pH. In one NAI-1540508512v1 -18- Attorney Docket No.14497-018-228 embodiment, the third pH is higher than the first pH. In one embodiment, the third pH is the same as the first pH. In one embodiment, the third pH is between 4.5 to 7.4. In one embodiment, the third pH is between 5 to 7.4. In one embodiment, the third pH is between 6 to 7.4. In one embodiment, the third pH is between 6.5 to 7.4.
  • the third pH is 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.1, 7.2, 7.3 or 7.4.
  • the washing step is performed before the contacting step.
  • a washing step is used to equilibrate the solid support before loading of the sample.
  • the washing step is performed after the contacting step.
  • the washing step is performed before the eluting step.
  • a washing step is performed after the eluting step to re-equilibrate the solid support.
  • the washing step is performed with a washing buffer (for example, the washing buffer as described in Section 5.4.3).
  • the washing buffer has a pH between 4.5 to 7.4.
  • the washing buffer has a pH between 5 to 7.4.
  • the washing buffer has a pH between 6 to 7.4.
  • the washing buffer has a pH between 6.5 to 7.4.
  • the washing buffer has a pH of 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.1, 7.2, 7.3 or 7.4.
  • the washing buffer is saline.
  • the washing buffer is MOPSO buffer.
  • the washing buffer is Tris buffer. In one embodiment, the washing buffer is phosphate buffer. In one embodiment, the washing buffer is MOPS buffer. In one embodiment, the washing buffer is Bis- Tris buffer. In one embodiment, the washing buffer is ADA buffer. In one embodiment, the washing buffer is MES buffer. In one embodiment, the washing buffer is HEPES buffer. In one embodiment, the washing buffer is citric buffer. In one embodiment, the washing buffer is acetate buffer. [00137] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a.
  • the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; c. washing the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is the same or higher than the first pH.
  • a method of purifying macromolecules from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the macromolecules bind to the solid support; c. washing the solid support; and d. eluting the macromolecules from the solid support at a second pH, wherein the second pH is the same or higher than the first pH.
  • the first pH is lower than the pKa of the ionizable moiety. In one embodiment, the first pH is below 8. In one embodiment, the first pH of 8– 9. In one embodiment, the first pH is between 4.5 to 7.4. In one embodiment, the first pH is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the first pH is 6.5. In one embodiment, the first pH is 6.8.
  • the first pH is 7.0. In one embodiment, the first pH is 7.2. In one embodiment, the first pH is 7.4. In one embodiment, the first pH is 8. [00140] In one embodiment, the contacting is performed with a loading buffer (for example, the loading buffer as described in Section 5.4.1) having the first pH. In one embodiment, the loading buffer has a pH lower than the pKa of the ionizable moiety. In one embodiment, the loading buffer has a pH below 8. In one embodiment, the loading buffer has a pH from 6 to 9. In one embodiment, the loading buffer has a pH from 4.5 to 7.4.
  • a loading buffer for example, the loading buffer as described in Section 5.4.1
  • the loading buffer has a pH of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4.
  • the loading buffer has pH of 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, or 9.
  • the loading buffer is saline.
  • the loading buffer is MOPSO buffer.
  • the loading buffer is Tris buffer.
  • the loading buffer is phosphate buffer.
  • the loading buffer is MOPS buffer. In one embodiment, the loading buffer is Bis-Tris buffer. In one embodiment, the loading buffer is ADA buffer. In one embodiment, the loading buffer is MES buffer. In one embodiment, the loading buffer is HEPES buffer. In one embodiment, the loading buffer is citric buffer. In one embodiment, the loading buffer is acetate buffer. NAI-1540508512v1 -20- Attorney Docket No.14497-018-228 [00142] In one embodiment, the loading buffer comprises one or more salt. In one embodiment, the loading buffer is Tris buffer comprising sodium chloride. In one embodiment, the loading buffer is MOPS buffer comprising sodium chloride. In one embodiment, the loading buffer is HEPES buffer comprising sodium chloride.
  • the contacting is performed in the presence of one or more salt.
  • the salt is a non-chaotropic salt.
  • the salt is a monovalent salt.
  • the salt is an alkali metal salt, preferably an alkali metal halide.
  • the salt is NaCl, KCl, KPO 4 , NaPO 4 , CaCl 2 , Mg 2 SO 4 , ZnCl 2 , MnCl 2 , MnSO 4 , NaSCN, KSCN, LiCl, MgCl2, and any combination thereof.
  • the salt is sodium chloride.
  • the salt is potassium chloride.
  • the contacting is performed at a salt concentration from 0.001 M to 1 M. In one embodiment, the salt concentration is from 0.01 M to 1 M. In one embodiment, the salt concentration is from 0.01 M to 0.2 M. In one embodiment, the contacting is performed at 0.01 M to 0.2 M sodium chloride. In one embodiment, the contacting is performed at 0.01 M sodium chloride. In one embodiment, the contacting is performed in 0.2 M sodium chloride. [00145] In one embodiment, the contacting is performed at a conductivity from about 0.1 mS/cm to 50 mS/cm. In one embodiment, the conductivity is from about 0.1 mS/cm to about 25 mS/cm.
  • the conductivity is from about 1 mS/cm to about 25 mS/cm. 5.2.3 Second pH [00146] In one embodiment, the second pH is above 7. In one embodiment, the second pH is between 7.4 to 9. In one embodiment, the second pH is 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9. In one embodiment, the second pH is 7.2. In one embodiment, the second pH is 7.4. In one embodiment, the second pH is 8.3. In one embodiment, the second pH is 8. In one embodiment, the second pH is 8.8. In one embodiment, the second pH is higher than the pKa of the ionizable moiety (see Section 5.2.5).
  • the second pH is lower than the pKa of the ionizable moiety.
  • the elution is performed with an elution buffer (for example, the elution buffer as described in Section 5.4.2) having the second pH.
  • the elution buffer has a pH higher than the pKa of the ionizable moiety.
  • the elution buffer has a pH lower than the pKa of the ionizable moiety.
  • the elution buffer has a pH higher than 7.
  • the elution buffer has a pH between 7.4 to 9.
  • the elution buffer has a pH of 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, NAI-1540508512v1 -21- Attorney Docket No.14497-018-228 8.7, 8.8, 8.9 or 9.
  • the elution buffer is phosphate buffer.
  • the elution buffer is Tris buffer.
  • the elution buffer is Bicine buffer.
  • the elution buffer is Tricine buffer.
  • the elution buffer is HEPES buffer.
  • the elution buffer comprises one or more salt.
  • the elution buffer is Tris buffer comprising sodium chloride.
  • the elution is performed in the presence of one or more salt.
  • the salt is a non-chaotropic salt.
  • the salt is a monovalent salt.
  • the salt is an alkali metal salt, preferably an alkali metal halide.
  • the salt is selected from NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO 4 , NaSCN, KSCN, LiCl, MgCl 2 , and any combination thereof.
  • the salt is sodium chloride.
  • the salt is potassium chloride.
  • the elution is performed at a salt concentration from 0.001 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 1 M. In one embodiment, the salt concentration is from 0.1 M to 2 M. In one embodiment, the salt concentration is from 0.7 M to 2 M. In one embodiment, the elution is performed in a buffer comprising 0.7 M to 2 M sodium chloride. In one embodiment, the elution is performed in a buffer comprising 0.2 M sodium chloride. In one embodiment, the elution is performed in a buffer comprising 0.7 M sodium chloride. In one embodiment, the elution is performed in a buffer comprising 1 M sodium chloride.
  • the elution is performed under a salt gradient.
  • the salt gradient is from 0.001 M to 2 M. In one embodiment, the salt gradient is from 0.01 M to 2 M. In one embodiment, the salt gradient is from 0.01 M to 1 M. In one embodiment, the salt gradient is from 0.7 M to 1 M. In one embodiment, the salt gradient is from 0.7 M to 2 M.
  • the elution is performed under a sodium chloride gradient. In one embodiment, the elution is performed under a potassium chloride gradient. In one embodiment, the elution is performed under a sodium chloride gradient from 0.01 M to 1 M.
  • the elution is performed at a conductivity from about 10 mS/cm to about 250 mS/cm. In one embodiment, the conductivity is from about 25 mS/cm to about 200 mS/cm. In one embodiment, the conductivity is from about 55 mS/cm to about 140 mS/cm.
  • the conductivity is about 55 mS/cm, about 60 mS/cm, about 70 mS/cm, about 80 mS/cm, about 90 mS/cm, about 100 mS/cm, about 110 mS/cm, about 120 mS/cm, about 130 NAI-1540508512v1 -22- Attorney Docket No.14497-018-228 mS/cm, or about 140 mS/cm.
  • the elution is performed under a conductivity gradient.
  • the conductivity gradient is from about 10 mS/cm to about 250 mS/cm.
  • the conductivity gradient is from about 25 mS/cm to about 200 mS/cm. In one embodiment, the conductivity gradient is from about 55 mS/cm to about 140 mS/cm.
  • the first pH is between 4.5 to 7.4, and the second pH is between 7.4 to 9. In one embodiment, the first pH is between 6.5 to 7.4, and the second pH is between 7.4 to 9. In one embodiment, the first pH is 6.5, and the second pH is 7.4. In one embodiment, the first pH is 7.4, and the second pH is 8.3. In one embodiment, the first pH is 7, and the second pH is 8. In one embodiment, the first pH is 7, and the second pH is 8.8.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles bind to the solid support, wherein the first pH is between 4.5 to 7.4; and c.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b.
  • the eluting step is performed at a higher ionic strength than the contacting step.
  • the contacting is performed at a salt concentration between 0.01 to 0.1 M and the elution is performed at a salt concentration between 0.1 M to 1 M.
  • the contacting is performed at a salt concentration between 0.1 to 0.7 M and the NAI-1540508512v1 -23- Attorney Docket No.14497-018-228 elution is performed at a salt concentration between 0.7 M to 2 M.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b.
  • a solid support wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH, wherein the first pH is lower than the pKa of the ionizable moiety; and c. eluting the macromolecules from the solid support at a second pH, wherein the second pH is higher than the pKa of the ionizable moiety.
  • a method of purifying macromolecules from a sample comprising the steps of: a.
  • the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH, wherein the first pH is lower than the pKa of the ionizable moiety; and NAI-1540508512v1 -24- Attorney Docket No.14497-018-228 c. eluting the macromolecules from the solid support at a second pH, wherein the second pH is lower than the pKa of the ionizable moiety, and wherein the eluting is performed at a salt concentration from about 0.01 M to about 2 M.
  • the solid support comprises the resin as described in Section 5.3.1. In one embodiment, the solid support is the solid support as described in Section 5.3.3. In one embodiment, the solid support is a chromatography column as described in Section 5.3.3. In one embodiment, the solid support comprises the ionizable moieties as described in Section 5.2.5. In one embodiment, the solid support is prepared from a resin comprising the reactive groups (e.g. epoxy group, NHS group) as described in part (ii) of this section. In one embodiment, the solid support is prepared by contacting a resin comprising the reactive groups as described in part (ii) of this section with a ligand (e.g. amine) as described in part (iii) of this section.
  • a ligand e.g. amine
  • the solid support binds with the vesicle, cell or viral particle at the first pH and release it at the second pH.
  • the solid support has a binding affinity to the vesicle, cell or viral particle at the first pH that is at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times higher than the binding affinity to the vesicle, cell or viral particle at the second pH.
  • the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 50% at the first pH.
  • the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 60% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 70% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 80% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 90% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 95% at the first pH.
  • the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 50% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 40% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 30% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an NAI-1540508512v1 -25- Attorney Docket No.14497-018-228 efficiency of less than 20% at the first pH.
  • the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 10% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 5% at the second pH. [00166] In one embodiment, the solid support binds with the macromolecules at the first pH and release it at the second pH. In one embodiment, the solid support has a binding affinity to the macromolecules at the first pH that is at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times higher than the binding affinity to the vesicle, cell or viral particle at the second pH.
  • the solid support binds with the macromolecule at an efficiency of at least 50% at the first pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of at least 60% at the first pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of at least 70% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 80% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 90% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 95% at the first pH.
  • the solid support binds with the macromolecule at an efficiency of less than 50% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 40% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 30% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 20% at the first pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 10% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 5% at the second pH. [00168] In one embodiment, the solid support has an average pore size from about 10 nm to about 1000 nm.
  • the average pore size is from about 50 nm to about 800 nm. In one embodiment, the average pore size is from about 100 nm to about 600 nm. In one embodiment, the average pore size is from about 50 nm to about 200 nm. In one embodiment, the average pore size is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, or about 200 nm.
  • the method further comprises a step of preparing the solid support.
  • the preparation comprises one or more steps as described in Section 5.3.2.
  • the solid support is prepared from a commercially available resin.
  • the solid support is prepared from a synthetic resin.
  • the solid support is prepared from a natural resin.
  • the solid support is prepared from a resin comprising at least one reactive group on the surface.
  • the solid support is prepared from a cation exchange (CEX) resin.
  • the CEX resin is a weak acid cation exchange resin. In one embodiment, the CEX resin comprises a carboxylic acid or carboxylate group. In one embodiment, the CEX resin is a strong acid cation exchange resin. [00173] In one embodiment, the CEX resin comprises one or more sulfoisobutyl group, sulfonate group, sulfoethyl group, sulfopropyl group, or carboxymethyl group. In one embodiment, the CEX resin comprises a sulphonate group. In one embodiment, the CEX resin is a commercially available CEX resin.
  • Non-limiting examples of commercially available CEX resins include, those having a sulfoisobutyl group (e.g., Fractogel EMD SO3); a sulfonate group (e.g., SP Sepharose Fast Flow, Capto S, Macro-Prep High S); a sulfoethyl based group (e.g., Fractogel SE); a sulfopropyl based group (e.g., TSKGel SP 5PW, Poros HS-20, Poros HS-50); and a carboxymethyl based group (e.g., CM Sepharose Fast Flow, Macro-Prep CM, Toyopearl CM-650S, Toyopearl CM-650M , Toyopearl CM-650C).
  • a sulfoisobutyl group e.g., Fractogel EMD SO3
  • the solid support is prepared from an anion exchange (AEX) resin.
  • AEX resin is a weak base anion exchange resin.
  • the AEX resin comprises a tertiary amino group.
  • the AEX resin is a strong base anion exchange resin.
  • the AEX resin comprises a quaternary ammonium group.
  • the solid support is prepared from an AEX resin comprising one or more groups selected from a group consisting of diethylaminopropyl, diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, carboxymethyl, carboxylic acid, glutamic acid, aspartic acid, histidine, hydroxyl, phosphate, tertiary amines, quaternary amines, diethaminoethyl, dimethylaminoethyl, trimethylaminoethyl and amino acid.
  • the AEX resin is a commercially available AEX resin.
  • Non-limiting examples of commercially available AEX resins include, Q SEPHAROSETM FF, Q SEPHAROSETM HP, Q SEPHAROSETM BB, Q SEPHAROSETM NAI-1540508512v1 -27- Attorney Docket No.14497-018-228 XL, DEAE SEPHAROSETM FF, ANX SEPHAROSETM 4FF low sub, ANX SEPHAROSETM 4FF high sub, SOURCETM 15Q, SOURCETM 30Q, CAPTOTM Q, CAPTOTM DEAE, or CAPTOTM Q ImpRes, available from GE Healthcare; FRACTOGEL® EMD DEAE (M), FRACTOGEL® EMD TMAE (M), FRACTOGEL® EMD TMAE (S), FRACTOGEL® EMD TMAE Hicap (M), FRACTOGEL® EMD TMAE Medcap (M), ESHMUNO® Q or ESHMUNO® Q, available from Merck Millipore; TOYOPEAR
  • the solid support is prepared from a size-exclusion (SEC) resin.
  • SEC resin is a commercially available size exclusion resin.
  • commercially available SEC resin include, Superdex (e.g., Superdex 30, Superdex 75, Superdex 200), Sephacryl (e.g., Sephacryl S-100, Sephacryl S-100, Sephacryl S-100, Sephacryl S- 400, Sephacryl S-500), Sephadex (e.g., Sephadex G-25, Sephadex G-50, Sephadex G-100), Superose (e.g., Superose 6, Superose 12), Sepharose (e.g., Sepharose 2B, Sepharose CL-2B, Sepharose 4B, Sepharose 4 Fast Flow, Sepharose CL-4B, Sepharose 6B, Sepharose 6 Fast Flow, Sepharose CL-6B), Fractogel (e.g., Fractogel EMD),
  • Superdex e.g., Superd
  • the SEC resin is Sepharose CL-4B.
  • the solid support is prepared from a mixed mode resin. In one embodiment, the solid support is prepared from an affinity resin. In one embodiment, the solid support is prepared from a pseudo affinity resin. In one embodiment, the solid support is prepared from a hydrophobic interaction resin. In one embodiment, the solid support is prepared from a hydrophobic charge induction resin. In one embodiment, the solid support is prepared from a combination of two or more types of resins. (ii)Reactive Group [00178] In one embodiment, the solid support is prepared from a resin comprising one or more reactive group.
  • the solid support is prepared from a resin comprising one or more NAI-1540508512v1 -28- Attorney Docket No.14497-018-228 reactive group selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, cyanogen bromide, a carbodiimide and lysine.
  • a resin comprising one or more NAI-1540508512v1 -28- Attorney Docket No.14497-018-228 reactive group selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sul
  • the solid support is prepared from a resin comprising an epoxy group. In one embodiment, the solid support is prepared from a resin comprising an NHS ester. In one embodiment, the solid support is prepared from a resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from a resin comprising an imidoester ester. In one embodiment, the solid support is prepared from a resin comprising an aldehyde. In one embodiment, the solid support is prepared from a resin comprising a carbonate. In one embodiment, the solid support is prepared from a resin comprising an anhydride. In one embodiment, the solid support is prepared from a resin comprising a sulfonyl chloride.
  • the solid support is prepared from a resin comprising an isothiocyanate. In one embodiment, the solid support is prepared from a resin comprising an isocyanate. In one embodiment, the solid support is prepared from a resin comprising an acyl azide. In one embodiment, the solid support is prepared from a resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from a resin comprising a carbodiimide. In one embodiment, the solid support is prepared from a resin comprising a lysine. [00181] In one embodiment, the solid support is prepared from an AEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from an AEX resin comprising an NHS ester.
  • the solid support is prepared from an AEX resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from an AEX resin comprising an imidoester ester. In one embodiment, the solid support is prepared from an AEX resin comprising an aldehyde. In one embodiment, the solid support is prepared from an AEX resin comprising a carbonate. In one embodiment, the solid support is prepared from an AEX resin comprising an anhydride. In one embodiment, the solid support is prepared from an AEX resin comprising a sulfonyl chloride. In one embodiment, the solid support is prepared from an AEX resin comprising an isothiocyanate.
  • the solid support is prepared from an AEX resin comprising an isocyanate. In one embodiment, the solid support is prepared from an AEX resin comprising an acyl azide. In one embodiment, the solid support is prepared from an AEX resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from an AEX resin comprising a carbodiimide. In one embodiment, the solid support is prepared from an NAI-1540508512v1 -29- Attorney Docket No.14497-018-228 AEX resin comprising a lysine. [00182] In one embodiment, the solid support is prepared from a CEX resin comprising an epoxy group.
  • the solid support is prepared from a CEX resin comprising an NHS ester. In one embodiment, the solid support is prepared from a CEX resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from a CEX resin comprising an imidoester ester. In one embodiment, the solid support is prepared from a CEX resin comprising an aldehyde. In one embodiment, the solid support is prepared from a CEX resin comprising a carbonate. In one embodiment, the solid support is prepared from a CEX resin comprising an anhydride. In one embodiment, the solid support is prepared from a CEX resin comprising a sulfonyl chloride.
  • the solid support is prepared from a CEX resin comprising an isothiocyanate. In one embodiment, the solid support is prepared from a CEX resin comprising an isocyanate. In one embodiment, the solid support is prepared from a CEX resin comprising an acyl azide. In one embodiment, the solid support is prepared from a CEX resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from a CEX resin comprising a carbodiimide. In one embodiment, the solid support is prepared from a CEX resin comprising a lysine. [00183] In one embodiment, the solid support is prepared from an SEC resin comprising an epoxy group. In one embodiment, the solid support is prepared from an SEC resin comprising an NHS ester.
  • the solid support is prepared from an SEC resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from an SEC resin comprising an imidoester ester. In one embodiment, the solid support is prepared from an SEC resin comprising an aldehyde. In one embodiment, the solid support is prepared from an SEC resin comprising a carbonate. In one embodiment, the solid support is prepared from an SEC resin comprising an anhydride. In one embodiment, the solid support is prepared from an SEC resin comprising a sulfonyl chloride. In one embodiment, the solid support is prepared from an SEC resin comprising an isothiocyanate. In one embodiment, the solid support is prepared from an SEC resin comprising an isocyanate.
  • the solid support is prepared from an SEC resin comprising an acyl azide. In one embodiment, the solid support is prepared from an SEC resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from an SEC resin comprising a carbodiimide. In one embodiment, the solid support is prepared from an SEC resin comprising a lysine. [00184] In one embodiment, the solid support is prepared from a weak acid CEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from a CEX resin NAI-1540508512v1 -30- Attorney Docket No.14497-018-228 comprising a carboxylic acid group and an epoxy group.
  • the solid support is prepared from a CEX resin comprising a carboxylate group and an epoxy group. In one embodiment, the CEX resin is a strong acid CEX comprising an epoxy group. [00185] In one embodiment, the solid support is prepared from a weak base AEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from a weak base AEX resin comprising a tertiary amino group and an epoxy group. In one embodiment, the solid support is prepared from a strong base AEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from an AEX resin comprising a quaternary ammonium group and an epoxy group.
  • the solid support is prepared from a resin comprising epoxy groups at a density from about 100 ⁇ mol to about 2000 ⁇ mol per gram of the resin. In one embodiment, the solid support is prepared from a resin comprising epoxy groups at a density from about 100 ⁇ mol to about 2000 ⁇ mol per milliliter of the resin. [00187] In one embodiment, the solid support is prepared from an AEX resin comprising epoxy groups at a density from about 100 ⁇ mol to about 2000 ⁇ mol per gram of the resin. In one embodiment, the solid support is prepared from an CEX resin comprising epoxy groups at a density from about 100 ⁇ mol to about 2000 ⁇ mol per gram of the resin.
  • the solid support is prepared from a SEC resin comprising epoxy groups at a density from about 100 ⁇ mol to about 2000 ⁇ mol per gram of the resin.
  • the solid support is prepared from agarose comprising the reactive groups as described herein.
  • the solid support is prepared from polystyrene comprising the reactive groups as described herein.
  • the solid support is prepared from polymethacrylate comprising the reactive groups as described herein.
  • the solid support is prepared from polyacrylamide comprising the reactive groups as described herein.
  • the solid support is prepared from hydroxylated methacrylic polymer comprising the reactive groups as described herein.
  • the solid support is prepared from agarose comprising epoxy groups. In one embodiment, the solid support is prepared from polystyrene comprising epoxy groups. In one embodiment, the solid support is prepared from polymethacrylate comprising epoxy groups. In one embodiment, the solid support is prepared from polyacrylamide comprising epoxy groups. In one embodiment, the solid support is prepared from hydroxylated methacrylic polymer comprising epoxy groups. NAI-1540508512v1 -31- Attorney Docket No.14497-018-228 [00190] In one embodiment, the solid support is prepared from TOYOPEARL AF-Epoxy-650M. In one embodiment, the solid support is prepared from Epoxy-Activated Sepharose 6B.
  • the solid support is prepared from Epoxy-Activated Separopore® 4B-CL. In one embodiment, the solid support is prepared from Epoxy-Activated Agarose. In one embodiment, the solid support is prepared from ProfinityTM Epoxide Resin. In one embodiment, the solid support is prepared from POROSTM 20 EP Epoxide Activated Resin. In one embodiment, the solid support is prepared from Praesto® Epoxy resin. In one embodiment, the solid support is prepared from Praesto® Epoxy resin.
  • the solid support is prepared from a commercially available resin, wherein resin is modified to increase the density of the reactive group by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%.
  • the solid support is prepared from a commercially available epoxy resin, wherein resin is modified to increase the density of the epoxy groups.
  • the modification comprises a step of contacting the resin with a compound comprising at least one epoxy group.
  • the compound comprises two epoxy groups. In one embodiment, the compound comprises three epoxy groups. In one embodiment, the compound has a molecular weight from 100 to 5000 g/mol, from 100 to 2500 g/mol, or from 100 to 1000 g/mol. In one embodiment, the compound is a diglycidyl ether. In one embodiment, the compound is 1,4- Bis(2,3-epoxypropyloxy)butane (other names: 1,4-Butanediol diglycidyl ether, or BDDE). In one embodiment, the compound is 2,2-Bis[4-(glycidyloxy)phenyl]propane (other name: bisphenol A diglycidyl ether).
  • the compound is neopentyl glycol diglycidyl ether. In one embodiment, the compound is 1,3-butanediol diglycidyl ether. In one embodiment, the compound is bis[4-(glycidyloxy)phenyl]methane (other name: bisphenol F diglycidyl ether). In one embodiment, the compound is N, N-Diglycidyl-4-glycidyloxyaniline. In one embodiment, the compound is poly(ethylene glycol) diglycidyl ether. In one embodiment, the compound is poly(propylene glycol) diglycidyl ether. [00193] In one embodiment, the reactive group is covalently connected to the resin via a linker.
  • the linker is a hydrophilic linker. In one embodiment, the linker is a hydrophobic linker. In one embodiment, the linker has a molecular weight less than 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol or less than 500 g/mol. In some embodiments, the linker NAI-1540508512v1 -32- Attorney Docket No.14497-018-228 is a linear, branched or cyclic alkylene group comprising 1 to 20 carbon atoms. In some embodiments, the linker further comprises one or more heteroatoms, such as oxygen, nitrogen, sulfur, silicon and halogen (e.g. fluorine, chlorine, bromine) atoms.
  • heteroatoms such as oxygen, nitrogen, sulfur, silicon and halogen (e.g. fluorine, chlorine, bromine) atoms.
  • the linker is an oligomeric polyethylene glycol (PEG) group with 2 to 48 repeating units.
  • the epoxy group is capable of reacting with the ligand via a ring- opening reaction to form an ionizable moiety and a hydroxyl group.
  • the reactive group has a density of from about 100 ⁇ mol to about 2000 ⁇ mol per gram of the resin. In one embodiment, the reactive group has a density of from about 500 ⁇ mol to about 1000 ⁇ mol per gram of the resin.
  • the reactive group has a density of about 100 ⁇ mol, 200 ⁇ mol, 300 ⁇ mol, 400 ⁇ mol, 500 ⁇ mol, 600 ⁇ mol, 700 ⁇ mol, 800 ⁇ mol, 900 ⁇ mol or 1000 ⁇ mol per gram of the resin.
  • the reactive group on the resin can react with a ligand, wherein the ligand is an amine, an alcohol, a thiol, or a carboxylic acid. In one embodiment, the ligand is a thiol. In one embodiment, the ligand is a carboxylic acid. In one embodiment, the ligand is an amine. In one embodiment, the ligand is a secondary amine.
  • the ligand has a formula of R 1 -NH-R 2 , wherein R 1 , R 2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R 1 and R 2 together with the atoms they are attached to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is optionally substituted.
  • R 1 is alkyl
  • R 2 is alkyl.
  • R 1 is alkyl
  • R 2 is hydroxyalkyl.
  • R 1 is hydroxyalkyl, and R 2 is hydroxyalkyl. In one embodiment, R 1 is C1-C12 alkyl, and R 2 is C1-C12 alkyl. In one embodiment, R 1 is C1-C12 alkyl, and R 2 is C 1 -C 6 alkyl. In one embodiment, R 1 is C 1 -C 6 alkyl, and R 2 is C 1 -C 6 alkyl. In one embodiment, R 1 is C 1 -C 12 alkyl, and R 2 is C 1 -C 12 hydroxyalkyl. In one embodiment, R 1 is C 1 -C 12 alkyl, and R 2 is C1-C6 hydroxyalkyl.
  • R 1 is C1-C6 alkyl, and R 2 is C1-C6 hydroxyalkyl. In one embodiment, R 1 is C1-C12 hydroxyalkyl, and R 2 is C1-C12 hydroxyalkyl. In one embodiment, R 1 is C 1 -C 12 hydroxyalkyl, and R 2 is C 1 -C 6 hydroxyalkyl. In one embodiment, R 1 is C 1 -C 6 hydroxyalkyl, and R 2 is C1-C6 hydroxyalkyl. [00200] In one embodiment, R 1 and R 2 together with the atoms they are attached to form a 5- membered heterocyclyl.
  • R 1 and R 2 together with the atoms they are attached to form a 6-membered heterocyclyl.
  • the heterocyclyl contains only one ring NAI-1540508512v1 -33- Attorney Docket No.14497-018-228 nitrogen atom.
  • the heterocyclyl contains two ring nitrogen atoms.
  • the heterocyclyl contains a ring nitrogen atom and a ring oxygen atom.
  • R 1 is unsubstituted. In one embodiment, R 1 is substituted.
  • R 1 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano.
  • R 1 is substituted with one or more halogen.
  • R 1 is substituted with one or more hydroxyl.
  • R 1 is substituted with one or more oxo.
  • R 1 is substituted with one or more phenyl.
  • R 2 is unsubstituted. In one embodiment, R 2 is substituted.
  • R 2 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano.
  • R 2 is substituted with one or more halogen.
  • R 2 is substituted with one or more hydroxyl.
  • R 2 is substituted with one or more oxo.
  • R 2 is substituted with one or more phenyl.
  • the ligand has a pKa of from about 7 to about 16.
  • the ligand has a pKa of from about 8 to about 11. In one embodiment, the ligand has a pKa of from about 8.5 to about 10.5. In one embodiment, the ligand has a pKa of about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12. [00204] In one embodiment, the amine has a pKa of from about 7 to about 16. In one embodiment, the amine has a pKa of from about 8 to about 11. In one embodiment, the amine has a pKa of from about 8.5 to about 10.5.
  • the amine has a pKa of about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12. [00205]
  • the pKa of amine is determined by an acid-base titration experiment. In one embodiment, acid-base titration experiment uses hydrochloric acid.
  • the ligand is selected from a group consisting of diethylamine, diethanolamine, dimethylethanolamine, diisopropylamine, isopropylethylamine, diisopropanolamine, 2-methylethanolamine, 2-ethylethanolamine, 2-(isopropylamino)ethanol, 2- (butylamino)ethanol, 2-benzylaminoethanol, piperidine, morpholine, 2,6-dimethyl-morpholine, 4- (2-chloroethyl)morpholine, 4-morpholinecarbonyl chloride, piperazine, 4-methylpiperazine and 4- hydroxyethylpiperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide; wherein each piperidine, morpholine, piperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glut
  • the ligand is diethylamine. In one embodiment, the ligand is diethanolamine. In one embodiment, the ligand is dimethylethanolamine. In one embodiment, the ligand is diisopropylamine. In one embodiment, the ligand is isopropylethylamine. In one embodiment, the ligand is diisopropanolamine (DIPA). In one embodiment, the ligand is 2- methylethanolamine. In one embodiment, the ligand is 2-ethylethanolamine. In one embodiment, the ligand is 2-(isopropylamino)ethanol.
  • DIPA diisopropanolamine
  • the ligand is 2-(butylamino)ethanol. In one embodiment, the ligand is 2-benzylaminoethanol. In one embodiment, the ligand is 2,6- dimethyl-morpholine. In one embodiment, the ligand is morpholine. In one embodiment, the ligand is 4-(2-chloroethyl)morpholine. In one embodiment, the ligand is 4-morpholinecarbonyl chloride. In one embodiment, the ligand is piperazine. In one embodiment, the ligand is 4- methylpiperazine. In one embodiment, the ligand is 4-hydroxyethylpiperazine. In one embodiment, the ligand is pyrrole. In one embodiment, the ligand is triazole.
  • the ligand is succinimide. In one embodiment, the ligand is phthalimide. In one embodiment, the ligand is glutarimide. [00208] In one embodiment, the ligand is unsubstituted. In one embodiment, the ligand is substituted. In one embodiment, the ligand is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano. In one embodiment, the ligand is substituted with one or more halogen.
  • the ligand is substituted with one or more hydroxyl. In one embodiment, the ligand is substituted with one or more C1-C6 alkyl. In one embodiment, the ligand is substituted with one or more C 1 -C 6 alkoxy. 5.2.5 Ionizable Moiety [00209] In one embodiment, the solid support comprises an ionizable moiety. In one embodiment, the preparation of the solid support further comprises a step of contacting a reactive group with a ligand to form an ionizable moiety. [00210] In one embodiment, provided herein is a method of purifying vesicles, cells, or viral particles from a sample comprising the steps of: a.
  • the resin comprises at least one reactive group, wherein the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and a lysine; NAI-1540508512v1 -35- Attorney Docket No.14497-018-228 b.
  • the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester
  • a solid support by contacting the resin with a ligand to form an ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample at a first pH at which the vesicles, cells, or viral particles bind to the solid support; and d. eluting the vesicles, cells, or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying vesicles, cells, or viral particles from a sample comprising the steps of: a.
  • the resin comprises at least one reactive group, wherein the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and a lysine; b.
  • the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfon
  • a solid support by contacting the resin with a ligand to form an ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample in a loading buffer; and d. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer.
  • a method of purifying macromolecules from a sample comprising the steps of: a.
  • the resin comprises at least one reactive group, wherein the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and a lysine; b.
  • the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfon
  • the ionizable moiety comprises one or more tertiary amino groups. In one embodiment, the ionizable moiety comprises a monoamine.
  • the ionizable moiety does not comprises a diamine.
  • the ionizable moiety wherein R 1 , R 2 are each independently alkyl, hydroxyalkyl, (alkylamino) alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R 1 and R 2 together with the atoms they are attached to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is optionally substituted; wherein the attachment to the left is to the linker.
  • the ionizable moiety is -N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted. In one embodiment, each alkyl is independently optionally substituted with one or more halogen or OH. [00216] In one embodiment, the ionizable moiety , wherein each alkyl is independently optionally substituted with one or In one embodiment, the ionizable , wherein X is C(R a )2, NR a , O, or S; wherein R a , R3 and R4 are each independently or C1-C6 alkyl; wherein the alkyl is optionally substituted with one or more halogen or hydroxyl.
  • the ionizable moiety is selected from a group consisting of: OH OH , NAI-1540508512v1 -37- Attorney Docket No.14497-018-228 ; wherein the attachment to the left is to the linker.
  • the ionizable moiety has a pKa from about 6.5 to about 9. In one embodiment, the ionizable moiety has a pKa from about 7.6 to about 8.8.
  • the ionizable moiety has a pKa of about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9 or about 9.
  • the ionizable moiety has a pKa of about 7.6.
  • the ionizable moiety has a pKa of about 8.2. [00219]
  • the pKa of the ionizable moiety is determined by an acid-base titration experiment.
  • acid-base titration experiment uses hydrochloric acid.
  • the pKa of the ionizable moiety is estimated by using analogous molecules bearing the same or similar functional groups.
  • the pKa of the ionizable moiety is estimated by measuring the pKa of R 5 -L-NR 1 R 2 in water, wherein R 5 is hydrogen, hydroxyl, C 1 - C 3 alkyl, C 1 -C 3 alkoxyl, or epoxy.
  • the pKa of the ionizable moiety is estimated by measuring the pKa of R6-NR1R2 in water, wherein R6 is C1-C3 alkyl, or C1-C3 alkoxyl.
  • the ionizable moiety has a density of from about 50 ⁇ mol to about 1000 ⁇ mol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 50 ⁇ mol to about 800 ⁇ mol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 100 ⁇ mol to about 800 ⁇ mol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 250 ⁇ mol to about 800 ⁇ mol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 300 ⁇ mol to about 800 ⁇ mol per gram of the solid support.
  • the ionizable moiety has a density of from about 400 ⁇ mol to about 800 ⁇ mol per gram of the solid support. In one embodiment, the ionizable moiety has a density of about 50 ⁇ mol, about 100 ⁇ mol, about 150 ⁇ mol, about 200 ⁇ mol, about 250 ⁇ mol, about 300 ⁇ mol, about 350 ⁇ mol, about 400 ⁇ mol, about 450 ⁇ mol, about 500 ⁇ mol, about 550 ⁇ mol, about 600 ⁇ mol, about 650 ⁇ mol, about 700 ⁇ mol, about 750 ⁇ mol, or about 800 ⁇ mol per gram of the solid support.
  • the ionizable moiety is capable of binding and releasing the vesicles, cells or viral particles in a pH-dependent manner.
  • NAI-1540508512v1 -38- Attorney Docket No.14497-018-228 [00222]
  • the ionizable moiety binds with the vesicles, cells or viral particles when the pH is below the pKa of the ionizable moiety.
  • the ionizable moiety binds with the vesicles, cells or viral particles at the first pH.
  • the ionizable moiety binds with the vesicles, cells or viral particles when the pH is at or below 7.4.
  • the ionizable moiety binds with the vesicles, cells or viral particles when the pH is between 4.5 to 7.4. [00223] In one embodiment, the ionizable moiety releases the vesicles, cells or viral particles when the pH is above the pKa of the ionizable moiety. In one embodiment, the ionizable moiety releases the vesicles, cells or viral particles at the second pH. In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles when the pH is at or above 7.4. In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles when the pH is between 7.4 to 9.
  • the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the first pH. In one embodiment, the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 80% at the first pH.
  • the solid support is capable of releasing the vesicles, cells, or viral particles at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the second pH.
  • the solid support is capable of releasing the vesicles, cells, or viral particles at an efficiency of at least 50% at the second pH.
  • the solid support is capable of releasing the vesicles, cells, or viral particles at an efficiency of at least 80% at the second pH.
  • the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 80% at the first pH, and releasing the vesicles, cells, or viral particles at an efficiency of at least 80% at the second pH. In one embodiment, the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 80% at the first pH, and releasing the vesicles, cells, or viral particles at an efficiency of at least 50% at the second pH. [00227] In one embodiment, the ionizable moiety is capable of binding and releasing the macromolecules in a pH-dependent manner.
  • the ionizable moiety binds with the macromolecules when the pH is below the pKa of the ionizable moiety.
  • the NAI-1540508512v1 -39- Attorney Docket No.14497-018-228 ionizable moiety binds with the macromolecules at the first pH.
  • the ionizable moiety binds with the macromolecules when the pH is at or below 7.4.
  • the ionizable moiety binds with the macromolecules when the pH is between 4.5 to 7.4.
  • the ionizable moiety releases the macromolecules when the pH is above the pKa of the ionizable moiety.
  • the ionizable moiety releases the macromolecules at the second pH. In one embodiment, the ionizable moiety binds with the macromolecules when the pH is at or above 7.4. In one embodiment, the ionizable moiety binds with the macromolecules when the pH is between 7.4 to 9. [00228] In one embodiment, the solid support is capable of binding the macromolecules at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the first pH. In one embodiment, the solid support is capable of binding the macromolecules at an efficiency of at least 80% at the first pH.
  • the solid support is capable of releasing the macromolecules at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the second pH.
  • the solid support is capable of releasing the macromolecules at an efficiency of at least 50% at the second pH.
  • the solid support is capable of releasing the macromolecules at an efficiency of at least 80% at the second pH.
  • the solid support is capable of binding the macromolecules at an efficiency of at least 80% at the first pH, and releasing the macromolecules at an efficiency of at least 80% at the second pH.
  • the vesicle has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm.
  • the nanovesicle has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.
  • the vesicle is negatively charged at the first pH. In one NAI-1540508512v1 -40- Attorney Docket No.14497-018-228 embodiment, the vesicle is negatively charged at the second pH. In one embodiment, the vesicle is negatively charged at both the first pH and the second pH. In one embodiment, the vesicles are purified by an increase in salt concentration or conductivity. [00232] In one embodiment, the vesicle is a nanovesicle. In one embodiment, the vesicle is a microvesicle. In one embodiment, the vesicle is an artificial nanovesicle (e.g. synthetic nanovesicles).
  • the vesicle is a liposome. In one embodiment, the vesicle is a lipid nanoparticle. In one embodiment, the vesicle is an extracellular vesicle. In one embodiment, the vesicle is an exosome. In one embodiment, the vesicle is a hybrid nanovesicle. In one embodiment, the vesicle is hybridosome. [00233] In one embodiment, the vesicle as used herein comprises fusogenic lipid that enhances the cellular uptake of the vesicle through cell fusion or endocytosis. Without bound by the theory, a fusogenic lipid work by undergoing a change in structure or charge at low pH (e.g.
  • Fusogenic lipids may be anionic lipids, neutral lipids or pH sensitive lipids. In some embodiments, when the temperature is raised above the phase transition temperature, for example 25° C., the fusogenic lipid undergoes a change in structure such that it adopts a hexagonal or cone-forming structure. Additional fusogenic lipids of this type are known in the art and may be used in the vesicles as described herein. The fusogenic lipids may also include those referred to as “cone-forming” lipids in the art.
  • the vesicle as used herein comprises one or more payloads.
  • the payload is a biologically active molecule.
  • Non-limiting examples of payload that can be included in vesicles include polypeptides (e.g, an antibody, an antigen, an adjuvant, a ligand, a receptor, an immune modulator, and or any fragment thereof), a polynucleotide, a viral particle, a small molecule, or any combination thereof.
  • Payloads that can be introduced into a vesicle, such as an exosome, or its producer cell include nucleotides, nucleic acids (e.g, DNA or mRNA molecules that encode a polypeptide, or RNA molecules such as miRNA, dsDNA, IncRNA, siRNA, antisense oligonucleotide, or combinations thereof), amino acids (e.g, amino acids comprising a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g, enzymes, antibodies, monoclonal antibodies), lipids (e.g. phospholipids), carbohydrates, and small molecules (e.g, small molecule drugs and toxins).
  • nucleic acids e.g, DNA or mRNA molecules that encode a polypeptide, or RNA molecules such as miRNA, dsDNA, IncRNA, siRNA, antisense oligonucleotide, or combinations thereof
  • amino acids e.g, amino acids comprising a detectable moiety or a
  • the method further comprises a step of preparing the vesicles as NAI-1540508512v1 -41- Attorney Docket No.14497-018-228 described herein.
  • (i)Nanovesicle provided herein is a method of purifying nanovesicle.
  • the nanovesicle has an average diameter between 1 nm to 1,000 nm.
  • Techniques to determine the size of nanovesicle include, but are not limited to Dynamic Light Scattering (DLS), Electron Microscope (EM), Small-angle X-ray scattering, atomic force microscopy, and nanoparticle tracking analysis.
  • the size of nanovesicle is determined by DLS.
  • Nanovesicles as used herein may comprise one or more payloads (for examples, as described in Section 5.2.6), such as polypeptides, or nucleic acids, and may further comprise a targeting moiety or other molecules (e.g. a detecting label or radioisotope).
  • payloads for examples, as described in Section 5.2.6
  • a targeting moiety or other molecules e.g. a detecting label or radioisotope.
  • the present disclosure relates to a plurality of nanovesicles, e.g. a population of nanovesicles which may comprise thousands, millions, billions or even trillions of nanovesicles.
  • nanovesicles are present in concentrations such as 10 5 , 10 8 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 , or 10 30 particles per unit of volume (for instance per mL or per gram), or any other number larger, smaller or anywhere in between.
  • Individual nanovesicles when present in a plurality constitute a nanovesicle population.
  • the present disclosure pertains both to individual nanovesicles and populations comprising nanovesicles.
  • the method further comprises a step of preparing the nanovesicles as described herein.
  • Liposomes as used herein comprise lipophilic materials, such as lipids, that isolate the aqueous interior from an aqueous exterior.
  • liposomes as used herein are multilamellar vesicles (MLVs) having more than one lamellar phase of lipid bilayers.
  • a liposome as used herein is a unilamellar vesicle having one single layer of lipid membrane.
  • the unilamellar vesicles are large unilamellar vesicles (LUVs).
  • the LUVs as used herein have a diameter of between about 0.1 to 1 ⁇ m.
  • the unilamellar vesicles are small unilamellar vesicles (SUVs).
  • the “SUVs” as used herein have a diameter of less than 100 nm. Liposomes are useful for the transfer and delivery of active ingredients to the site of action.
  • the liposomal NAI-1540508512v1 -42- Attorney Docket No.14497-018-228 bilayer fuses with bilayer of the cellular membranes, thereby facilitating the delivery of therapeutic payloads on or inside the liposome.
  • the liposome used herein is SUV.
  • the liposome used herein is MLV.
  • the liposome used herein comprises one or more fusogenic lipids (for example, as in Section 5.2.6).
  • the liposome used herein comprises one or more payloads (for example, as in Section 5.2.6) [00240] In one embodiment, the liposome has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm.
  • the liposome has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.
  • the liposome is a neutral liposome.
  • the liposome is an anionic liposome.
  • the liposome is a cationic liposome.
  • a cationic liposome as used herein has a positive charge at physiological pH.
  • the cationic liposomes can also comprise co-lipids that are negatively charged or neutral, so long as the net charge of the liposome is positive at physiological pH.
  • cationic lipids are present in the cationic liposome at from about 10 mole % to about 100 mole %, from about 20 mole % to about 80 mole % and, or from about 20 mole % to about 60 mole % of total liposomal lipid.
  • the cationic liposome as used herein comprise neutral lipids at a concentration from about 0 mole % to about 90 mole %, from about 20 mole % to about 80 mole %, or from about 40 mole % to about 80 mole % of the total liposomal lipid. In one embodiment, the cationic liposome as used herein comprise anionic lipids at a concentration from about 0 mole % to about 49 mole %, from about 0 mole % to about 40 mole %, or from about 0 mole % to about 20 mole % of the total liposomal lipid.
  • the cationic liposome comprises a cationic lipid and the neutral lipid at a 1:1 molar ratio.
  • the cationic lipid comprises DOTAP.
  • the cationic lipid comprises cholesterol, or its derivatives.
  • the cationic liposome further comprises lipid-PEG conjugates.
  • the method further comprises a step of preparing the liposomes as described herein. NAI-1540508512v1 -43- Attorney Docket No.14497-018-228 [00243]
  • the payload comprises mRNA.
  • the payload is a negatively charged molecule (e.g., nucleic acid)
  • the lipid components of the LNP comprise at least one cationic lipid or ionizable lipid.
  • the cationic lipid or ionizable lipid can interact with the negatively charged payload molecules and facilitates incorporation and/or encapsulation of the payload into the LNP during LNP formation.
  • the EV is a microvesicle. In one embodiment, the EV is an apoptotic body. In one embodiment, the EV is a fusosome. In one embodiment, the EV is an outer membrane vesicle produced by gram negative bacteria. [00245] In one embodiment, the EV is exosome. In one embodiment, the exosome is an engineered exosome. In one embodiment, the exosome comprises one or more payloads (for example, as in Section 5.2.6). Said payload can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, or combinations thereof. Exosome payloads may be located within the internal space of the exosome.
  • Exosome payload may be in contact with the exterior or interior surface of the exosome, for example through a covalent bond or a non-covalent bond.
  • the phospholipid bilayer of the EV or provided herein may comprise one or more transmembrane proteins, wherein a portion of the one or more transmembrane membrane proteins is located within the internal space of the exosome.
  • the exosome has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm.
  • the exosome has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.
  • the method further comprises a step of preparing the EVs as NAI-1540508512v1 -44- Attorney Docket No.14497-018-228 described herein.
  • the vesicle is a hybrid nanovesicle.
  • a hybrid nanovesicle as provided herein can be derived from any mixture of two or more vesicles, including vesicles as described in Section 5.2.6(i), 5.2.6(ii), 5.2.6, or 5.2.6(iii).
  • a hybrid nanovesicle as used herein may derive from the fusing of two, three, four, five, six, seven, eight, nine or ten different nanovesicles.
  • the hybrid nanovesicle can further fuse with another nanovesicle to form a new hybrid nanovesicle.
  • the hybrid nanovesicle is derived from the fusing of an extracellular vesicle and a liposome.
  • the hybrid nanovesicle is derived from the fusing of two extracellular vesicles of different cell sources, such as the fusion of a platelet-derived extracellular vesicle and a macrophage-derived extracellular vesicle.
  • the hybrid nanovesicle is derived from the fusing of two exosomes of different cell sources. In one embodiment, the hybrid nanovesicle is derived from the fusing of an extracellular vesicle and a lipid nanoparticle. In one embodiment, the hybrid nanovesicle is derived from the fusing of an exosome and a lipid nanoparticle. In one embodiment, the hybrid nanovesicle is derived from the fusing of an exosome and a liposome. In one embodiment, the hybrid nanovesicle is hybridosome. [00250] In one embodiment, the method further comprises a step of preparing the hybrid nanovesicles as described herein.
  • hybridosome used herein comprises one or more fusogenic lipids (for example, as in Section 5.2.6).
  • the hybridosome comprises one or more payloads (for example, as in Section 5.2.6).
  • the payload is nucleic acid.
  • the payload is DNA.
  • the payload is RNA, or preferably mRNA.
  • the payload is polypeptide. In one embodiment, the payload is an antibody. In one embodiment, the payload is an enzyme. [00252] In one embodiment, the hybridosome results from the fusing of at least one exosome and at least one lipid nanoparticle. In one embodiment, the hybridosome results from the fusing of 1 to NAI-1540508512v1 -45- Attorney Docket No.14497-018-228 10 exosomes with one lipid nanoparticle. In one embodiment, the hybridosome results from the fusing of 1 to 10 lipid nanoparticles with one exosome. In one embodiment, the hybridosome results from the fusing of one exosome and one lipid nanoparticle.
  • the hybridosome results from the fusing of one exosome comprising payloads, and one lipid nanoparticle comprising fusogenic lipids. In one embodiment, the hybridosome results from the fusing of one lipid nanoparticle comprising payload, and one exosome comprising fusogenic lipid. In one embodiment, the hybridosome results from the fusing of one exosome comprising payload, and one lipid nanoparticle comprising fusogenic lipid. [00253] In one embodiment, the hybridosome comprises one or more detectable label. In one embodiment, the hybridosome comprises a gene encoding one or more detectable label. In one embodiment, the detectable label is a fluorophore.
  • the detectable label is a fluorescent lipid. In one embodiment, the detectable label is a fluorescent dye. In one embodiment, the detectable label is a fluorescent protein. In one embodiment, the hybridosome comprises a gene encoding fluorescent protein. In one embodiment, the detectable label is green fluorescent protein (GFP) or red fluorescent protein (RFP). In one embodiment, the hybridosome comprises a gene encoding luciferase. In one embodiment, the detectable label is a luciferase. In one embodiment, the detectable label is a radioisotope. [00254] In one embodiment, the method further comprises a step of preparing the hybridosome as described herein.
  • the hybridosome is prepared by fusing one or more lipid nanoparticle with one or more exosome.
  • a method of purifying hybridosome from a sample comprising the steps of: a. preparing the hybridosome by fusing one or more LNP with one or more exosome; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d.
  • the fusing is performed at an LNP/exosome ratio of from 5:1 to 1:1. In one embodiment, the LNP/exosome ratio is from 2:1 to 1:1. In one embodiment, the LNP/exosome ratio is 1, 1.01, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, NAI-1540508512v1 -46- Attorney Docket No.14497-018-228 1.9, or 2.0.
  • the fusing is performed at a pH below 6.5. In one embodiment, the fusing is performed at a pH between 2 and 6.5. In one embodiment, the fusing is performed at a pH between 3 and 6. In one embodiment, the fusing is performed at a pH between 4 and 6. In one embodiment, the fusing is performed at pH of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5. In one embodiment, the fusing is performed at pH of 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6. In one embodiment, the fusing is performed at pH of 5.5.
  • the fusing is performed in an acidic buffer.
  • the acidic buffer has a pH between below 6.5. In one embodiment, the acidic buffer has a pH of 5.5. In one embodiment, the acidic buffer is a MES buffer. In one embodiment, the acidic buffer further comprises one or more salts. In one embodiment, the acidic buffer further comprises NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, and any combination thereof. In one embodiment, the acidic buffer comprises NaCl and KCl.
  • the acidic buffer is a MES buffer comprising NaCl and KCl. In one embodiment, the acidic buffer has a salt concentration from about 5 mM to about 1 M. In one embodiment, the acidic buffer has a conductivity from about 1 mS/cm to about 100 mS/cm. [00259] In one embodiment of purifying hybridosome, the fusing is performed at a temperature between 0 °C and 60 °C. In one embodiment, the fusing is performed at a temperature between 10 °C and 50 °C. In one embodiment, the fusing is performed at a temperature between 20 °C and 40 °C. In one embodiment, the fusing is performed at 37 °C.
  • the fusing process takes from 5 min to 24 hours. In one embodiment, the fusing process takes from 5 min to 6 hours. In one embodiment, the fusing process takes from 20 min to 3 hours. [00261] In one embodiment of purifying hybridosome, the fusing is performed at a temperature between 20 °C and 40 °C for a period of from 5 min to 24 hours in an acid buffer. 5.2.7 Cells [00262] In one embodiment, provided herein is a method of purifying cells. In one embodiment, the cell is negatively charged at the first pH. In one embodiment, the cell is negatively charged at the second pH. In one embodiment, the cell is negatively charged at both the first pH and the second pH.
  • the cells are purified by an increase in salt concentration or conductivity.
  • NAI-1540508512v1 -47- Attorney Docket No.14497-018-228 [00263]
  • the cell is mammalian cell.
  • the cell is human cell.
  • the cell is engineered human cell.
  • the cell is animal cell.
  • the cell is mouse cell.
  • the cell is stem cell.
  • the cell is a source cell of extracellular vesicle (EV).
  • Source cells of EV may be selected from a wide range of cells and cell lines which may grow in suspension or adherent culture or being adapted to suspension growth.
  • EVs may be derived from essentially any cell source, including a primary cell source or an immortalized cell line.
  • the EV source cells may be any embryonic, fetal, and adult somatic stem cell types, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method.
  • the source cell may be allogeneic, autologous, or xenogeneic in nature.
  • the cell may be from a subject himself or from an unrelated, matched or unmatched donor.
  • the source cells are allogeneic cells.
  • the source cells are human umbilical cord endothelial cells (HUVECs).
  • the source cells are human embryonic kidney (HEK) cells (e.g.
  • the source cells are CHO cells, BHK cells, PER.C6 cells, Vero cells, HeLa cells, PC 12 cells, or Sf9 cells.
  • the source cells are endothelial cell lines (e.g. microvascular or lymphatic endothelial cells, erythrocytes, erythroid progenitors, chondrocytes).
  • the source cells are amnion cells, amnion epithelial (AE) cells, any cells obtained through amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, or epithelial cells.
  • the cell is mesenchymal stem cell (MSC).
  • the cell is bone marrow MSC (BM-MSC).
  • BM-MSC bone marrow MSC
  • the cell is human BM-MSC.
  • the cell is mouse BM-MSC.
  • the cell is umbilical cord mesenchymal stem cells (UC-MSCs).
  • the cell is adipose tissue-derived mesenchymal stem cells (AD-MSCs). In one embodiment, the cell is embryonic mesenchymal stem cells (ES-MSCs).
  • the cell is immune cell.
  • the cell is macrophage. In one embodiment, the cell is macrophage dendritic cell (DC).
  • the cell is T cell. In one embodiment, the cell is natural killer (NK) cell.
  • the cell is blood cell. In one embodiment, the cell is red blood cell. In one embodiment, the cell is human red blood cell. In one embodiment, the cell is platelet. [00268] In one embodiment, the cell is neural cell.
  • the cell is neuron, NAI-1540508512v1 -48- Attorney Docket No.14497-018-228 astrocyte, or microglia. 5.2.8 Viral Particles
  • the viral particle is negatively charged at the first pH.
  • the viral particle is negatively charged at the second pH.
  • the viral particle negatively charged at both the first pH and the second pH.
  • the viral particles are purified by an increase in salt concentration or conductivity.
  • a method of purifying viral particles from a sample comprising the steps of: a.
  • the viral particle comprises live virus.
  • the viral particle comprises fractions of virus (e.g.
  • the viral particle comprises clusters of virus.
  • the viral particle is infectious (e.g. capable of transferring nucleic acid contained in the particle into a cell upon contacting with the cell).
  • the viral particle is enveloped viral particle.
  • the viral particle is a restrovirus.
  • the viral particle is lentiviral particle.
  • the viral particle is herpes simplex particle.
  • the lentiviral particle comprises a lipid-bilayer envelope.
  • the lentiviral particle comprises an RNA genome.
  • the lentiviral particle is capable of invading a target host cell.
  • the viral particle is adenoviral particle.
  • the viral particle is adeno- associated viral particle.
  • the viral particle is retroviral particle.
  • the viral particle is lentiviral particle produced from yeast.
  • the viral particle is lentiviral particle produced from bacteria.
  • the NAI-1540508512v1 -49- Attorney Docket No.14497-018-228 viral particle is lentiviral particle produced from mammalian cells.
  • the viral particle has a diameter from about 10 nm to about 200 nm.
  • the viral particle has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, or about 200 nm. 5.2.9 Macromolecules [00275] In one embodiment, provided herein is a method of purifying macromolecules. [00276] In one embodiment, the macromolecule is a synthetic polymer. In one embodiment, the macromolecule is a biopolymer. Non-limiting examples of macromolecules that can be purified using the methods include polypeptides (e.g.
  • the macromolecule is a monoclonal antibody.
  • the macromolecule is a nucleic acid.
  • the macromolecule is a DNA.
  • the macromolecule is RNA.
  • the macromolecule has a molecular weight from 5 kDa to 500 kDa. In one embodiment, the macromolecule has a molecular weight from 20 kDa to 400 kDa. In one embodiment, the macromolecule has a molecular weight from 50 kDa to 350 kDa.
  • the method removes at least one impurity from a sample.
  • the sample comprises at least one impurity that is a small molecule, a macromolecule, a different vesicle, a different cell, or a different viral particle.
  • the impurity to be removed is a small molecule.
  • the impurity to be removed is a lipid.
  • the impurity to be removed is a metal cation.
  • the impurity to be removed is a macromolecule.
  • the impurity to be removed is nucleic acid.
  • the impurity to be removed is DNA.
  • the impurity to be removed is RNA. In one embodiment, the impurity to be removed is a cell. In one embodiment, the impurity to be removed is a cell organelle. In one embodiment, the impurity to be removed is a cell membrane. In one embodiment, the impurity to be removed is a viral particle. [00279] In one embodiment, at least one impurity is removed by a change in pH from loading to elution. In one embodiment, at least one impurity is removed by a change in salt concentration or conductivity from loading to elution.
  • the impurity is a different vesicle.
  • the impurity is a source cell of the vesicle.
  • the impurity is an unencapsulated payload of the vesicle.
  • the impurity is an excess component (e.g. lipid) forming part of the vesicle.
  • the impurity is LNP.
  • the impurity is exosome. In one embodiment of purifying hybridosomes, the impurity is LNP and exosome. [00282] In one embodiment of purifying viral particles, the impurity is host-cell protein (HCP). In one embodiment, at least 90% of HCP is removed. In one embodiment of purifying viral particles, the impurity is total protein. In one embodiment, at least 70% or at least 80% of total protein is removed. In one embodiment of purifying viral particles, the impurity is double-strand DNA (dsDNA). In one embodiment, at least 50% or at least 60% of dsDNA is removed. [00283] In one embodiment, the impurity is positively charged at the first pH.
  • HCP host-cell protein
  • dsDNA double-strand DNA
  • dsDNA double-strand DNA
  • the impurity is positively charged at the first pH.
  • the impurity is positively charged at the first pH, and the vesicle, cell or viral particle is negatively charged at the first pH.
  • the impurity has a binding efficiency with the solid support of less than 60% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 40% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 20% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 10% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 5% at the first pH.
  • the impurity has a binding efficiency with the solid support of less than 20% at the first pH, and the vesicle, cell or viral particle has a binding efficiency with the solid support of more than 80% at the first pH.
  • the impurity is removed during the loading step. In one embodiment, the impurity is removed during the washing step. [00287] In one embodiment, at least 50% of the impurity is removed from the sample. In one embodiment, at least 60% of the impurity is removed from the sample. In one embodiment, at least 70% of the impurity is removed from the sample. In one embodiment, at least 80% of the impurity is removed from the sample.
  • the sample has a volume from about 0.1 mL to about 2000 L. In one embodiment, the sample has a volume from about 0.5 mL to about 50 L. In one embodiment, the sample has a volume from about 0.5 mL to about 100 mL. In one embodiment, the sample has a volume from about 1 L to about 100 L. In one embodiment, the sample has a volume from about 10 L to about 2000 L.
  • the sample has a volume from about 100 L to about 2000 L. In one embodiment, the sample has a volume from about 100 L to about 1000 L. In one embodiment, the sample has a volume from about 500 L to about 2000 L. In some embodiments, the method can be applied to a sample with a volume larger than about 1 L, about 5 L, about 10 L, about 15 L about 20 L, about 25 L, about 50 L, about 100 L, about 200 L, about 250 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1000 L, about 1200 L, about 1400 L, about 1600 L, about 1800 L, or about 2000 L.
  • the total amount of sample that goes through the purification step for each batch is from about 500 L to about 15,000 L. In other embodiments, the total amount of sample that goes through the purification step for each batch is from about 5000 L to about 15,000 L. In one embodiment, each sample has a volume of about 500 L and the 500 L volume sample goes through the purification step (e.g., CEX and AEX, CEX, AEX, and MMC, or any other combinations) as described herein.
  • the 500 L volume sample goes through the purification step (e.g., CEX and AEX, CEX, AEX, and MMC, or any other combinations) as described herein.
  • the total amount of sample that goes through the purification step for each batch is at least about 5,000 L, at least about 6,000 L, at least about 7,000 L, at least about 8,000 L, at least about 9,000 L, at least about 10,000 L, at least about 11,000 L, at least about 12,000 L, at least about 13,000 L, at least about 14,000 L, or at least about 15,000 L.
  • the total amount of sample that goes through the purification step for each batch is at least about 10,000 L.
  • the total amount of sample that goes through the purification step for each batch is at least about 15,000 L.
  • the total amount of sample that goes through the purification step for each batch is at least about 20,000 L.
  • the sample is pretreated before contacting with the solid support.
  • the samples comprising macromolecules, vesicles, cells, or viral particles can be pretreated to make them suitable for purification on solid support.
  • pretreatment can make sample comprising macromolecules, vesicles, cells, or viral particles suitable for binding to the ionizable moieties of the solid support.
  • pretreatment can provide a pH, buffer, conductivity, polarity, or any combination thereof, that is desirable for the binding and release of macromolecules, vesicles, cells, or viral particles.
  • the pretreatment process of the sample comprises centrifugation, ultra-centrifugation, filtration, ultra-filtration, clarification, chromatography step, lyophilization, lysing, digestion, drying, dilution, concentration, heating, cooling, or any combination thereof.
  • the pretreatment of the sample comprises clarification, nuclease digestion, ultrafiltration, diafiltration, or any combination thereof.
  • pretreatment of the sample comprising EV comprises clarification.
  • the clarification comprises depth filtration, nuclease treatment, centrifugation, acoustic separation, flocculation, or any combination thereof.
  • pretreatment of the sample comprises clarification.
  • the clarification comprises depth filtration.
  • the depth filtration uses a porous filtration medium, such as a depth filter medium. Without bound by the theory, a depth filter media retains contaminants throughout the medium rather than just on the medium’s surface, and thus can retain a larger number of contaminants before becoming clogged.
  • the porous filtration medium has an average pore size from about 0.1 to about 100 ⁇ m, from about 0.5 to about 80 ⁇ m, or from about 1 to about 60 ⁇ m.
  • the porous filtration medium has an average pore size of about 0.2 ⁇ m, about 0.3 ⁇ m, about 0.4 ⁇ m, about 0.5 ⁇ m, about 0.6 ⁇ m, about 0.7 ⁇ m, about 0.8 ⁇ m, about 1 ⁇ m, about 2 ⁇ m, about 3 ⁇ m, about 4 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 20 ⁇ m, about 30 ⁇ m, about 40 ⁇ m, about 50 ⁇ m, about 60 ⁇ m, about 70 ⁇ m, about 80 ⁇ m, about 90 ⁇ m, or about 100 ⁇ m.
  • the sample comprising the product of interest e.g.
  • the depth filtration is performed with a pad, a panel, a deep bed sand filter, or a lenticular design comprising stacked design.
  • the depth filtration is performed with a thick filter wound around a perforated cylinder of depth filter medium that surrounds a central core. The type and condition of depth filtration can be selected and adjusted depending on the source, volume, purity, and the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) concentration of the sample.
  • the depth filtration comprises uncharged filter materials.
  • the uncharged filter material is one or more uncharged polymers.
  • the uncharged filter material is cellulose or its derivatives, polypropylene or its derivatives, polyethylene or its derivatives, polyethersulfone or its derivatives, nylon, polyvinylidene fluoride, glass fiber, polytetrafluorethylene, or methacrylate or its derivatives.
  • the pretreatment process comprises NAI-1540508512v1 -53- Attorney Docket No.14497-018-228 the use of a large-scale depth filtration system comprising multiple housings and cartridges.
  • the depth filtration is performed with one or more depth filter selected from the group consisting of Emphaze (3M), Zeta Plus S (3M), PDH4 (Pall), Polysep II (Millipore Sigma), X0SP (Millipore Sigma), D0SP (Millipore Sigma), C0SP (Millipore Sigma), CR40 (Millipore Sigma), ZetaPlus activated carbon (3M), V100 (Pall), Bio20 (Pall), Biol0 (Pall), glass fiber (Pall), X0HC (Millipore Sigma), A1HC (Millipore Sigma), GF+ (Sartorius), and P-series filters (Pall).
  • the depth filtration uses two or more depth filters.
  • the two or more depth filters are arranged in series.
  • a filter with a larger structure is used to remove cells and cell debris and a filter with smaller pore structure is used to remove smaller contaminants.
  • two or more depth filters are used sequentially or in parallel.
  • the depth filtration uses 3 to 12 depth filters.
  • the depth filter comprises a single layer.
  • the depth filter comprises a dual-layer.
  • the depth filter comprises more than two layers. [00298]
  • the depth filtration methods provided herein is used to purify the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) from a sample produced in a cell culture container or a bioreactor.
  • the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) in the sample are produced in a single-use bioreactor.
  • the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) in the sample are produced in a perfusion, ATF perfusion, or TFF perfusion bioreactor.
  • the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) in the sample are produced in a cell culture lasting from about 10 days to 25 days.
  • the sample is passed over a series of uncharged cellulose depths filters with decreasing pore sizes to remove cells and cell debris while allowing the product of interest (e.g.
  • pretreatment of the sample comprises contacting the sample with nuclease to digest nucleic acid associated with the product of interest (e.g. macromolecules, vesicles, cells, or viral particles).
  • nuclease as used herein generally refers to any enzyme that hydrolyzes nucleic acid sequences.
  • the sample is clarified prior to contacting the sample with nuclease.
  • the sample comprising the product of interest e.g. macromolecules, vesicles, cells, or viral particles
  • the nuclease is DNase, RNase, or their mixtures. In one embodiment, the nuclease is endonuclease, exonuclease, or their mixtures. NAI-1540508512v1 -54- Attorney Docket No.14497-018-228 In one embodiment, the nuclease comprises one or more of DNase I, Benzonase ® , DENARASE ® , Exonuclease I, Exonuclease III, Mung Bean Nuclease, Nuclease BAL 31, RNase I, Si Nuclease, Lambda Exonuclease, RecJ, T7 exonuclease, eoxynuclease II, micrococcal nuclease, RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, or RNase V.
  • the nuclease digestion process takes from about 1 hour to about 48 hours, preferably from about 1 hour to 24 hours. In one embodiment, the concentration of nuclease in the digestion process is from about 1 U/mL to about 500 U/mL, from about 5 U/mL to about 200 U/mL, or from about 25 U/mL to about 100 U/mL.
  • a cofactor is used together with the nuclease.
  • the cofactor is an organic cofactor (e.g. flavin).
  • the cofactor is an inorganic cofactor (e.g. Mg 2+ , Cu + , or Mn 2+ ). In one embodiment, the cofactor is used in the form of MgCl2.
  • the concentration of cofactor in the digestion process is from about 1 mM to about 500 mM, from about 1 mM to about 100 mM, or from about 1 mM to about 20 mM.
  • pretreatment of the sample comprises ultrafiltration.
  • the term “ultrafiltration” or “UF” refers to any technique for treating a solution or suspension with a semi-permeable membrane that retains macromolecules while allowing a solvent or small solute molecules to pass through.
  • the ultrafiltration increases the concentration of macromolecules, vesicles, cells or particles in the sample.
  • the ultrafiltration is Tangential Ultrafiltration (also known as Tangential Flow Filtration, or TFF).
  • the TFF unit comprises at least one housing and at least one cross-flow (tangential) filter positioned in the housing such that a large portion of the filter's surface is positioned parallel to the flow of the sample through the unit.
  • the TFF unit includes one filter.
  • the TFF unit includes two filters.
  • the TFF unit includes three filters.
  • the housing can include a first inlet/outlet and a second inlet/outlet positioned, e.g., to allow fluid to pass through the first inlet/outlet, cross the at least one cross-flow filter, and through the second inlet/outlet.
  • the TFF units can be connected in series or parallel to provide a fluid path of desired length.
  • the TFF unit comprises spiral membranes, flat membranes, or hollow fibers.
  • the hollow fiber comprises polyethersulfone or its derivatives, polysulfone or its derivatives, or cellulose ester or its derivatives.
  • the TFF filters have a molecular weight cut-off threshold from about 25 kDa to about 1500 kDa, from about 200 kDa to NAI-1540508512v1 -55- Attorney Docket No.14497-018-228 about 1000 kDa, or from about 500 kDa to about 750 kDa.
  • pretreatment of the sample comprises diafiltration.
  • diafiltration or “DF” refers to a specialized filtration category that dilutes a retentate with a solvent and refilters the retentate in order to reduce soluble permeate components. Diafiltration may induce or not induce an increase in the concentration of, for example, retained vesicles.
  • the diafiltration is continuous diafiltration, wherein the solvent is continuously added to the retentate at the same rate as the production rate of the filtrate.
  • the diafiltration is discontinuous or sequentially diluted diafiltration, wherein the ultrafiltration step involves the addition of a solvent to the retentate side, and wherein the volume of solvent added to the retentate is equal to or greater than the volume of the resulting filtrate.
  • the diafiltration filters have a molecular weight cut-off threshold from about 25 kDa to about 1000 kDa, from about 100 kDa to about 750 kDa, or from about 300 kDa to about 750 kDa.
  • pretreatment of the sample comprises a sequential process of ultrafiltration and diafiltration (UF/DF).
  • the UF/DF process is performed after the nuclease digestion process.
  • the UF/DF process is performed after the depth filtration process.
  • the UF/DF removes cells or cell debris from the sample comprising the product of interest (e.g. macromolecules, vesicles, cells, or viral particles).
  • the filtration is conducted with successive filtrations.
  • the successive filtrations occur through filters with decreasing porosity.
  • the process of UF/DF comprises filtration through one or more filters having a porosity above 0.1 ⁇ m, from about 0.1 ⁇ m to about 20 ⁇ m, or from about 0.5 ⁇ m ⁇ m to about 10 ⁇ m.
  • the process of UF/DF comprises a first filtration through a filter having a porosity of from about 5 ⁇ m to about 15 ⁇ m, and a second filtration through a filter having a porosity of from about 0.5 ⁇ m to about 1.5 ⁇ m.
  • the process of UF/DF comprises a third filtration through a filter having a porosity of from about 0.2 ⁇ m to about 0.8 ⁇ m.
  • the process of UF/DF comprises a fourth filtration through a filter having a porosity of from about 0.1 ⁇ m to about 0.4 ⁇ m.
  • the UF/DF filtration further comprises a pre-filtration step using a pre- filter.
  • the pre- filter comprises one or more cellulose acetate, polypropylene, or polyether sulfone. In one embodiment, the pre-filter has a porosity from about 1 ⁇ m to about 20 ⁇ m, from about 2 ⁇ m to about 15 ⁇ m, or from about 2 ⁇ m to about 5 ⁇ m.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: NAI-1540508512v1 -56- Attorney Docket No.14497-018-228 a. pretreating the sample with depth filtration; b.
  • a solid support wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a.
  • pretreating the sample with nuclease wherein the nuclease optionally comprises a cofactor
  • b Further pretreating the sample with ultrafiltration or diafiltration
  • c providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9
  • d contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with ultrafiltration or diafiltration; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with ultrafiltration or diafiltration; c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; NAI-1540508512v1 -57- Attorney Docket No.14497-018-228 d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; d.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. further pretreating the sample with ultrafiltration or diafiltration; d.
  • a solid support wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; e. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and f. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a.
  • a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with clarification; b. pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. optionally further pretreating the sample with ultrafiltration or diafiltration; d. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C 1-6 alkyl) 2 , and wherein each alkyl is independently optionally substituted; e.
  • a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c.
  • a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; b. Further pretreating the sample with ultrafiltration or diafiltration; NAI-1540508512v1 -59- Attorney Docket No.14497-018-228 c.
  • a solid support wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with ultrafiltration or diafiltration; b.
  • a solid support wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with ultrafiltration or diafiltration; c.
  • a solid support wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b.
  • nuclease optionally comprises a cofactor
  • nuclease optionally comprises a cofactor
  • c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; NAI-1540508512v1 -60- Attorney Docket No.14497-018-228
  • d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH.
  • a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. further pretreating the sample with ultrafiltration or diafiltration; d. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; e. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and f.
  • the eluting is performed at the second pH as described in Section 5.2.3. In one embodiment, the eluting is performed using an elution buffer as described in Section 5.4.2. In one embodiment, the eluting is performed using an elution buffer having a salt concentration or conductivity higher than the salt concentration or conductivity of loading buffer used in the loading step. [00319] In one embodiment, the vesicles (for example, vesicles as described in Section 5.2.6) are eluted at a yield of at least 50%.
  • the vesicles are eluted at a yield of at least 60%. In one embodiment, the vesicles are eluted at a yield of at least 70%. In one embodiment, the vesicles are eluted at a yield of at least 80%. In one embodiment, the vesicles are eluted at a yield of at least 90%. In one embodiment, the vesicles are eluted at a yield of at least 95%. In one embodiment, the vesicles are eluted at a yield of at least 99%. In one embodiment, the elution yield is not affected by the increase of the sample volume.
  • the elution yield is maintained when the sample volume is increase from less than 50 mL to above 1 L. In one embodiment, the elution yield at a sample volume of above 1 L is at least 75% of the elution yield at a sample volume of below 50 mL.
  • NAI-1540508512v1 -61- Attorney Docket No.14497-018-228 [00320]
  • the cells (for example, cells as described in Section 5.2.7) are eluted at a yield of at least 50%. In one embodiment, the cells are eluted at a yield of at least 60%. In one embodiment, the cells are eluted at a yield of at least 70%.
  • the cells are eluted at a yield of at least 80%. In one embodiment, the cells are eluted at a yield of at least 90%. In one embodiment the cells are eluted at a yield of at least 95%. In one embodiment, the cells are eluted at a yield of at least 99%.
  • the viral particles (for example, viral particles as described in Section 5.2.8) are eluted at a yield of at least 50%. In one embodiment, the viral particles are eluted at a yield of at least 60%. In one embodiment, the viral particles are eluted at a yield of at least 70%. In one embodiment, the viral particles are eluted at a yield of at least 80%.
  • the viral particles are eluted at a yield of at least 90%. In one embodiment, the viral particles are eluted at a yield of at least 95%. In one embodiment, the viral particles are eluted at a yield of at least 99%. In one embodiment, the recovery yield of viral particle is measured based on recovery of p24 capsid protein. In one embodiment, the recovery yield of viral particle is measured based on recovery of transduction unit (TU). [00322] In one embodiment, the macromolecules (for example, macromolecules as described in Section 5.2.9) are eluted at a yield of at least 50%. In one embodiment, the viral particles are eluted at a yield of at least 60%.
  • the viral particles are eluted at a yield of at least 70%. In one embodiment, the viral particles are eluted at a yield of at least 80%. In one embodiment, the viral particles are eluted at a yield of at least 90%. In one embodiment, the viral particles are eluted at a yield of at least 95%. In one embodiment, the viral particles are eluted at a yield of at least 99%.
  • the method further comprises a step of detecting the elution of the vesicles (for example, vesicles as described in Section 5.2.6).
  • the method further comprises a step of detecting the elution of the cells (for example, cells as described in Section 5.2.7). In one embodiment, the method further comprises a step of detecting the elution of the viral particles (for example, viral particles as described in Section 5.2.8). In one embodiment, the method further comprises a step of detecting the elution of the macromolecules (for example, macromolecules as described in Section 5.2.9). [00324] In one embodiment, the detecting involves the detection of optical signal from the vesicles. In one embodiment, the detecting involves the detection of UV-Vis signal from the NAI-1540508512v1 -62- Attorney Docket No.14497-018-228 vesicles.
  • the detecting involves the detection of fluorescence signal from the vesicles. In one embodiment, the detecting involves the detection of luminescence signal from the vesicles. In one embodiment, the detecting involves the detection of radioactivity from the vesicles. In one embodiment, the detecting involves the detection of vesicle density. In one embodiment, the detecting involves the detection vesicle size. In one embodiment, the detecting involves the detection of light scattering of the vesicles. [00325] In one embodiment, the detecting involves the detection of optical signal from the cells. In one embodiment, the detecting involves the detection of UV-Vis signal from the cells. In one embodiment, the detecting involves the detection of fluorescence signal from the cells.
  • the detecting involves the detection of luminescence signal from the cells. In one embodiment, the detecting involves the detection of radioactivity from the cell. In one embodiment, the detecting involves the detection of cell density. In one embodiment, the detecting involves the detection cell size. In one embodiment, the detecting involves the detection of light scattering of the cells. [00326] In one embodiment, the detecting involves the detection of optical signal from the viral particles. In one embodiment, the detecting involves the detection of UV-Vis signal from the viral particles. In one embodiment, the detecting involves the detection of fluorescence signal from the viral particles. In one embodiment, the detecting involves the detection of luminescence signal from the viral particles. In one embodiment, the detecting involves the detection of radioactivity from the viral particles.
  • the detecting involves the detection of viral particle density. In one embodiment, the detecting involves the detection of viral particle size. In one embodiment, the detecting involves the detection of light scattering of the viral particles. [00327] In one embodiment, the detecting involves the detection of optical signal from macromolecules. In one embodiment, the detecting involves the detection of UV-Vis signal from the macromolecules. In one embodiment, the detecting involves the detection of fluorescence signal from the macromolecules. In one embodiment, the detecting comprises the use of a fluorescence assay. In one embodiment, the detecting involves the detection of radioactivity from the macromolecules. In one embodiment, the detecting involves the detection of light scattering of the macromolecules.
  • the detecting involves the detection of circular dichroism of the macromolecules.
  • the signal is from a small-molecular a fluorescent dye.
  • the signal is from a fluorescent lipid.
  • the signal is from a NAI-1540508512v1 -63- Attorney Docket No.14497-018-228 fluorescent protein.
  • the signal is from a green fluorescent protein (GFP) or red fluorescent protein (RFP).
  • the signal is from a luciferase.
  • the signal is from a radioisotope.
  • the signal is light scattering.
  • the method further comprises a step of: f. detecting the elution of the vesicles, cells or viral particles.
  • the detecting involves the detection of optical signal from the hybridosome. In one embodiment, the detecting involves the detection of UV-Vis signal from the hybridosome. In one embodiment, the detecting involves the detection of fluorescence signal from the hybridosome. In one embodiment, the detecting involves the detection of luminescence signal from the hybridosome. In one embodiment, the detecting involves the detection of radioactivity from the hybridosome.
  • compositions for use as a solid support as described in Section 5.2.4.
  • the composition is used for the purification of vesicles (for example, vesicles as described in Section 5.2.6).
  • the composition is used for the purification of cells (for example, cells as described in Section 5.2.7).
  • the composition is used for the purification of viral particles (for example, viral particles as described in Section 5.2.8).
  • the composition is used for the purification of macromolecules (for example, macromolecules as described in Section 5.2.9).
  • a solid support comprising the resin as described herein.
  • provided herein is a chromatography column comprising the resin as described herein. NAI-1540508512v1 -64- Attorney Docket No.14497-018-228 In one embodiment, provided herein is a membrane comprising the resin as described herein. In one embodiment, provided herein is a bead comprising the resin as described herein.
  • L is C1-C48 alkylene. In one embodiment, L is C1-C36 alkylene. In one embodiment, L is C 1 -C 24 alkylene. In one embodiment, L is C 1 -C 16 alkylene. In one embodiment, L is C 1 -C 12 alkylene. In one embodiment, L is C 1 -C 6 alkylene. In one embodiment, L is methylene. In one embodiment, L is ethylene. In one embodiment, L is C3 alkylene. In one embodiment, L is C4 alkylene. In one embodiment, L is C5 alkylene. In one embodiment, L is C6 alkylene. [00334] In one embodiment, one or more -CH2- in L is replaced by -O-.
  • two -CH2- in L are each replaced by a -O-. In one embodiment, three -CH2- in L are each replaced by a -O-. In one embodiment, four -CH 2 - in L are each replaced by a -O-. In one embodiment, four - CH 2 - in L are each replaced by a -O-. In one embodiment, five -CH 2 - in L are each replaced by a - O-. In one embodiment, six -CH2- in L are each replaced by a -O-. In one embodiment, L comprises from 1 to 12 -O-CH 2 CH 2 - repeating unit.
  • L comprises from 1 to 12 NAI-1540508512v1 -65- Attorney Docket No.14497-018-228 -O-CH(CH 3 )CH2- repeating unit.
  • one or more -CH2- in L is replaced by -S-.
  • one or more -CH2- in L is replaced by -NH-.
  • one or more -CH2- in L is replaced by a phenyl.
  • two -CH2- in L are each replaced by a phenyl.
  • the phenyl is substituted with one or more halogen.
  • the phenyl is substituted with one or more C1-C6 alkyl.
  • the phenyl is substituted with one or more C1-C6 alkoxy.
  • L is substituted with one or more halogen.
  • L is substituted with one or more hydroxyl group. In one embodiment, L is substituted with one hydroxyl group. In one embodiment, L is substituted with two hydroxyl groups. In one embodiment, L is substituted with three hydroxyl groups. In one embodiment, L is substituted with four hydroxyl groups. In one embodiment, L is substituted with six hydroxyl groups. In one embodiment, L is substituted with one or more C 1 -C 6 alkyl. In one embodiment, L is substituted with one or more C1-C6 alkoxy. [00338] In one embodiment, L has a molecular weight less than 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol, or less than 500 g/mol.
  • L consists of carbon, hydrogen, and oxygen atoms. In one embodiment, L does not comprise nitrogen atom. In one embodiment, L does not comprise sulfur atom. , - Attorney Docket No.14497-018-228 O O wherein m is from 1 to 12. In one embodiment, L , wherein n is from 1 to 12. The attachment to the left is to the is to the tertiary amino group.
  • the polymer is agarose. In one embodiment, the polymer is cellulose. In one embodiment, the polymer is polystyrene. In one embodiment, the polymer is polymethacrylate. In one embodiment, the polymer is polyacrylamide. In one embodiment, the polymer is hydroxylated methacrylic polymer. [00342] In one embodiment, the polymer is unsubstituted. In one embodiment, the polymer is substituted. In one embodiment, the polymer is substituted with one or more a carboxylic acid or carboxylate group.
  • the polymer is substituted with one or more sulfoisobutyl group, sulfonate group, sulfoethyl group, sulfopropyl group, or carboxymethyl group.
  • the polymer is substituted with one or more diethylaminopropyl, diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, carboxymethyl, glutamic acid, aspartic acid, histidine, hydroxyl, phosphate, tertiary amines, quaternary amines, diethaminoethyl, dimethylaminoethyl, trimethylaminoethyl, or amino acid group.
  • R 1 is alkyl, and R 2 is alkyl. In one embodiment, R 1 is alkyl, and R 2 is hydroxyalkyl. In one embodiment, R 1 is hydroxyalkyl, and R 2 is hydroxyalkyl. In one embodiment, R 1 is C1-C12 alkyl, and R 2 is C1-C12 alkyl. In one embodiment, R 1 is C1-C12 alkyl, and R 2 is C 1 -C 6 alkyl. In one embodiment, R 1 is C 1 -C 6 alkyl, and R 2 is C 1 -C 6 alkyl. In one embodiment, R 1 is C 1 -C 12 alkyl, and R 2 is C 1 -C 12 hydroxyalkyl.
  • R 1 is C 1 -C 12 alkyl, and R 2 is C1-C6 hydroxyalkyl. In one embodiment, R 1 is C1-C6 alkyl, and R 2 is C1-C6 hydroxyalkyl. In one embodiment, R 1 is C1-C12 hydroxyalkyl, and R 2 is C1-C12 hydroxyalkyl. In one embodiment, R 1 is C 1 -C 12 hydroxyalkyl, and R 2 is C 1 -C 6 hydroxyalkyl. In one embodiment, R 1 is C 1 -C 6 hydroxyalkyl, and R 2 is C1-C6 hydroxyalkyl.
  • R 1 and R 2 together with the atoms they are attached to form a 5- membered heterocyclyl. In one embodiment, R 1 and R 2 together with the atoms they are attached to form a 6-membered heterocyclyl. In one embodiment, the heterocyclyl contains only one ring nitrogen atom. In one embodiment, the heterocyclyl contains two ring nitrogen atoms. In one embodiment, the heterocyclyl contains a ring nitrogen atom and a ring oxygen atom. [00345] In one embodiment, R 1 is unsubstituted. In one embodiment, R 1 is substituted.
  • R 1 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano.
  • R 1 is substituted with one or more halogen.
  • R 1 is substituted with one or more hydroxyl.
  • R 1 is substituted with one or more oxo.
  • R 1 is substituted with one or more phenyl.
  • R 2 is unsubstituted. In one embodiment, R 2 is substituted. In one embodiment, R 2 is substituted. In one embodiment, R 2 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano. In one embodiment, R 2 is substituted with one or more halogen. In one embodiment, R 2 is substituted with one or more hydroxyl. In one embodiment, R 2 is substituted with one or more oxo. In one embodiment, R 2 is substituted with one or more phenyl.
  • m is an integer from 1 to 24. In one embodiment, m is an integer from 1 to 12. In one embodiment, m is an integer from 1 to 6. In one embodiment, m is 1. In one embodiment, m is 2. In one embodiment, m is 3. In one embodiment, m is 4. In one embodiment, m is 5. In one embodiment, m is 6. In one embodiment, m is 7. In one embodiment, m is 8. In one embodiment, m is 9. In one embodiment, m is 10. In one embodiment, m is 11. In one embodiment, m is 12. In one embodiment, m is 14. In one embodiment, m is 16. In one embodiment, m is 18. In one embodiment, m is 20. In one embodiment, m is 22. In one embodiment, m is 24.
  • n is an integer from 1 to 24. In one embodiment, n is an integer from 1 to 12. In one embodiment, n is an integer from 1 to 6. In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is 3. In one embodiment, n is 4. In one embodiment, n is 5. In one embodiment, n is 6. In one embodiment, n is 7. In one embodiment, n is 8. In one embodiment, n is 9. In one embodiment, n is 10. In one embodiment, n is 11. In one embodiment, n is 12. In one embodiment, n is 14. In one embodiment, n is 16. In one embodiment, n is 18. In one embodiment, n is 20. In one embodiment, n is 22. In one embodiment, n is 24.
  • the tertiary amino group has a pKa from about 6.5 to about 9. In one embodiment, the tertiary amino group has a pKa from about 7.6 to about 8.8. In one embodiment, the tertiary amino group has a pKa of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9. In one embodiment, the tertiary amino group has a pKa of about 7.6. In one embodiment, the tertiary amino group has a pKa of about 8.2.
  • the tertiary amino each alkyl is independently optionally substituted with one or the NAI-1540508512v1 -69- Attorney Docket No.14497-018-228 attachment to the left is to the linker L.
  • the tertiary amino group is , wherein X is C(R a )2, NR a , O, or S; wherein R a , R3 and R4 are each independently or C 1 -C 6 alkyl, wherein the alkyl is optionally substituted with one or more halogen or hydroxyl, wherein the attachment to the left is to the linker L.
  • the tertiary amino group is selected from a group consisting of: OH OH , ; wherein the attachment to the left is to the linker L. OH
  • the tertiary amino group or ionizable one embodiment, the solid support or , wherein the attachment is to a polymer described
  • the pKa of the tertiary amino group of the resin is estimated by measuring the pKa of R5-L-NR1R2 in a solvent, wherein R5 is hydrogen, hydroxyl, C1-C3 alkyl, C1- C 3 alkoxyl, or epoxy.
  • the pKa of the tertiary amino group of the resin is estimated by measuring the pKa of R6-NR1R2 in a solvent, wherein R6 is C1-C3 alkyl, or C1-C3 NAI-1540508512v1 -70- Attorney Docket No.14497-018-228 alkoxyl.
  • the solvent is water.
  • the solvent is DMSO.
  • the pKa is determined by an acid-base titration experiment.
  • the acid-base titration experiment uses hydrochloric acid.
  • the resin has an average pore size from about 10 nm to about 1000 nm.
  • the average pore size is from about 50 nm to about 800 nm. In one embodiment, the average pore size is from about 100 nm to about 600 nm. In one embodiment, the average pore size is from about 50 nm to about 200 nm. In one embodiment, the average pore size is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, or about 200 nm. [00355] In one embodiment, the tertiary amino group has a density of from about 50 ⁇ mol to about 1000 ⁇ mol per gram of the resin.
  • the tertiary amino group has a density of from about 100 ⁇ mol to about 1000 ⁇ mol per gram of the resin. In one embodiment, the tertiary amino group has a density of from about 250 ⁇ mol to about 1000 ⁇ mol per gram of the resin. In one embodiment, the tertiary amino group has a density of from about 250 ⁇ mol to about 800 ⁇ mol per gram of the resin. In one embodiment, the tertiary amino group has a density of from about 300 ⁇ mol to about 1000 ⁇ mol per gram of the resin. In one embodiment, tertiary amino group has a density of from about 500 ⁇ mol to about 1000 ⁇ mol per gram of the resin.
  • the tertiary amino group has a density of about 50 ⁇ mol, about 100 ⁇ mol, about 150 ⁇ mol, about 200 ⁇ mol, about 250 ⁇ mol, about 300 ⁇ mol, about 350 ⁇ mol, about 400 ⁇ mol, about 500 ⁇ mol, about 600 ⁇ mol, about 700 ⁇ mol, about 800 ⁇ mol, about 900 ⁇ mol or about 1000 ⁇ mol per gram of the resin.
  • the resin is prepared using the method as described in Section 5.2.4 (i).
  • the resin is prepared using a source resin comprising the reactive groups as described in Section 5.2.4 (ii).
  • the resin is prepared by contacting a source resin comprising the reactive groups as described in Section 5.2.4 (ii), and a ligand as described in 5.2.4 (iii).
  • the source resin is a CEX resin, an AEX resin, or a SEC resin (for example, as described in Section 5.2.4 (i)).
  • the solid support is prepared from agarose comprising the reactive groups as described in in Section 5.2.4 (ii).
  • the solid support is prepared from NAI-1540508512v1 -71- Attorney Docket No.14497-018-228 polystyrene comprising the reactive groups as described in Section 5.2.4 (ii).
  • the solid support is prepared from polymethacrylate comprising the reactive groups as described in Section 5.2.4 (ii).
  • the solid support is prepared from polyacrylamide comprising the reactive groups as described in Section 5.2.4 (ii).
  • the solid support is prepared from hydroxylated methacrylic polymer comprising the reactive groups as described in Section 5.2.4 (ii).
  • the resin is prepared from a source resin comprising one or more reactive group selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, cyanogen bromide, a carbodiimide and lysine.
  • a source resin comprising one or more reactive group selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester,
  • the source resin is a commercially available resin.
  • the source resin is agarose comprising epoxy groups.
  • the source resin is polystyrene comprising epoxy groups.
  • the source resin is polymethacrylate comprising epoxy groups.
  • source resin is polyacrylamide comprising epoxy groups.
  • source resin is hydroxylated methacrylic polymer comprising epoxy groups.
  • the source resin is epoxy-activated agarose resin.
  • the source resin is TOYOPEARL ® AF-Epoxy-650M.
  • the source resin is epoxy-activated Sepharose TM 6B.
  • the source resin is Epoxy-Activated Separopore® 6B-CL. In one embodiment, the source resin is Epoxy-Activated Separopore® 4B- CL. In one embodiment, the source resin is Epoxy-Activated Agarose. In one embodiment, the source resin is ProfinityTM Epoxide Resin. In one embodiment, the source resin is POROSTM 20 EP Epoxide Activated Resin. In one embodiment, the source resin is Praesto® Epoxy resin. [00360] In one embodiment, the method further comprises a step of modifying the source resin to increase the density of reactive group (e.g. epoxy groups).
  • reactive group e.g. epoxy groups
  • the density of the reactive group is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%.
  • the modification comprises contacting the source resin with a compound, wherein the compound comprises the same reactive group of the resin.
  • the modification comprises contacting an epoxy resin with a compound comprising at least one epoxy group.
  • the modification comprises contacting an epoxy resin NAI-1540508512v1 -72- Attorney Docket No.14497-018-228 with a compound comprising two epoxy groups.
  • the modification comprises contacting an epoxy resin with a compound comprising an epoxy group and a leaving group, wherein the leaving group is halogen (e.g. Cl, Br, or I), or sulfonate ester (e.g. tosylate, mesylate, or triflate).
  • the resin is prepared by contacting a source resin as described above, with a ligand as described in 5.2.4 (iii).
  • the ligand is a thiol.
  • the ligand is a carboxylic acid. In one embodiment, the ligand is an amine. In one embodiment, the ligand is a secondary amine. In one embodiment, the ligand has a formula of R 1 - NH-R 2 , wherein R 1 , R 2 is defined herein or elsewhere.
  • the contacting is performed at a temperature from about 0 °C to about 200 °C. In one embodiment, the contacting is performed at a temperature from about 20 °C to about 100 °C. In one embodiment, the contacting is performed at room temperature. In one embodiment, the contacting is performed in the presence of a base, such an inorganic base (e.g. NaOH), or an organic base (e.g.
  • a base such an inorganic base (e.g. NaOH), or an organic base (e.g.
  • the contacting is performed in the presence of one or more solvents, such as an organic solvent (e.g. DMSO, DMF, or THF).
  • the contacting is performed in water.
  • the contacting is performed in a mixture of water and organic solvent.
  • the contacting is performed in one or more solvents, at a temperature from about 20 °C to about 100 °C, and in the presence of a base. 5.3.3 Solid Support [00362] Also provided herein is a solid support (e.g.
  • the solid support comprises a membrane comprising the resin as described in Section 5.3.1.
  • the solid support comprises a magnetic bead comprising the resin as described in Section 5.3.1.
  • the solid support comprises a pre-packed chromatography column comprising the resin as described in Section 5.3.1.
  • the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C 1-6 alkyl) 2 , and wherein each alkyl is independently optionally substituted.
  • the polymer is agarose, polystyrene, polymethacrylate, polyacrylamide, or hydroxylated methacrylic polymer.
  • the solid support comprises a magnetic bead, wherein the magnetic bead is prepared by binding the resin as described in Section 5.3.1 with an activated magnetic bead.
  • the activated magnetic bead binds to the polymer moiety of the resin. In one embodiment, the activated magnetic bead binds to the linker of the resin. In one embodiment, the activated magnetic bead binds to the tertiary amino group of the resin. In one embodiment, the activated magnetic bead binds to the unreacted reactive group (e.g. epoxy group) of the resin.
  • the solid support further comprises other materials, including glass, modified or functionalized glass, plastics (e.g.
  • the solid support is contained within a flow cell.
  • flow cell refers to a chamber including a solid support across which one or more fluid reagents can be flowed. Flow cells and related fluidic systems and detection platforms can be readily used as described herein.
  • the solid support as described herein is used for purification purpose, including the purification of vesicles, cells or viral particles as described in Section 5.2. In one embodiment, the solid support as described herein is used for the purification of small molecules. In one embodiment, the solid support as described herein is used for the purification of macromolecules, such as nucleic acids, polypeptides, or polysaccharides. [00368] In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 0.5 mL to about 100 L. In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 0.5 mL to about 100 mL.
  • the solid support as described herein is capable of purifying samples in an amount from about 0.1 L to about 1 L. In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 1 L to about 100 L. In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 50 L to about 500 L. [00369] In one embodiment, the solid support comprises the resin as described in Section 5.3.1 in an amount from about 0.05 gram to about 5000 gram. In one embodiment, the solid support comprises the resin in an amount from about 0.5 gram to about 1000 gram. In one embodiment, the solid support comprises the resin in an amount from about 0.5 gram to about 20 gram.
  • the solid support comprises the resin in an amount of about 0.5 gram, about 1 gram, about 2 gram, about 3 gram, about 4 gram, about 5 gram, about 6 gram, about 7 gram, about 8 gram, about 9 gram, about 10 gram, about 11 gram, about 12 gram, about 13 gram, about 14 gram, about NAI-1540508512v1 -74- Attorney Docket No.14497-018-228 15 gram, about 16 gram, about 17 gram, about 18 gram, about 19 gram, or about 20 gram.
  • the solid support comprises the resin in an amount from about 500 gram to about 5000 gram.
  • the solid support comprises the resin in an amount of about 500 gram, about 1000 gram, about 2000 gram, about 3000 gram, about 4000 gram, or about 5000 gram.
  • the chromatography column has a volume of from about 0.1 mL to about 5000 mL. In one embodiment, the chromatography column has a volume of from about 0.25 mL to about 2500 mL. In one embodiment, the chromatography column has a volume of from about 0.5 mL to about 20 mL.
  • the chromatography column has a volume of about 0.5 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, or about 20 mL. In one embodiment, the chromatography column has a volume of from about 1000 mL to about 5000 mL.
  • the chromatography column has a volume of about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L or about 100 L.
  • the chromatography column is a gravity-flow column.
  • the chromatography column is a spin column.
  • the chromatography column is a high-pressure liquid chromatography (HPLC) column.
  • the chromatography column is a low-pressure liquid chromatography column.
  • the chromatography column as described herein can be made of any material, including those described in Section 5.1.
  • the chromatography column is a disposable single-use column.
  • the chromatography column is a multi-use column.
  • Kits [00373] Also provided herein is a kit comprising the composition as described in Section 5.3, including the resin as described in Section 5.3.1, or the solid support (e.g. membrane, magnetic bead, or chromatography column) as described in Section 5.3.3. [00374] In one embodiment, the kit as described herein is used in a purification method, including the purification of vesicles, cells or viral particles as described in Section 5.2. In one embodiment, the kit as described herein is used for the purification of small molecules. In one embodiment, the kit as described herein is used for the purification of macromolecules, such as nucleic acids or polypeptides.
  • the kit further comprises one or more loading buffer (e.g. Section 5.4.1), one or more elution buffer (e.g. Section 5.4.2), or one or more washing buffer (e.g. Section 5.4.3).
  • a kit comprising a solid support described herein, a loading buffer, and an elution buffer, wherein the loading buffer has a pH between 4.5 to 7.4, and wherein the elution buffer has a pH between 7.4 to 9.
  • kits comprising a solid support described herein, a loading buffer, and an elution buffer, wherein the loading buffer has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer.
  • the loading buffer and the elution buffer have the same pH.
  • the kit further comprises one or more washing buffer.
  • the washing buffer has the same pH, salt concentration, or conductivity as the loading buffer.
  • the washing buffer is the same as the loading buffer. 5.4.1 Loading Buffer [00379]
  • the method described herein uses a loading buffer.
  • the kit described herein comprises a loading buffer.
  • the loading buffer has the first pH as described in Section 5.2.2. [00380] In one embodiment, the loading buffer has a pH of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the elution buffer has a pH of 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.
  • the loading buffer has a pH from 4.5 to 7.4, and the elution buffer has a pH from 7.4 to 9. In one embodiment, the loading buffer has a pH from 6.5 to 7.4, and the elution buffer has a pH from 7.4 to 9. In one embodiment, the loading buffer has a pH of 6.5, and the elution buffer has a pH of 7.4. In one embodiment, the loading buffer has a pH of 7.4, and the elution buffer has a pH of 8.3. In one embodiment, the loading buffer has a pH of 7, and the elution buffer has a pH of 8. In one embodiment, the loading buffer has a pH of 7, and the elution buffer has a pH 8.8.
  • the loading buffer has a pH of 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, or 9.
  • the loading buffer has a pH of 6–9
  • the elution buffer has a pH of 6–9.
  • the loading buffer has a pH of 7–8, and the elution buffer has a pH of 7–8.
  • the loading buffer has a pH of 6.8, and the elution buffer has a pH of 7.2.
  • the loading buffer has a pH of 7.2, and the elution buffer has a pH of 7.2.
  • the loading buffer has a pH of 8, and the elution buffer has a pH of 8.
  • the loading buffer is used in the method described in Section 5.2.
  • the loading buffer is used in the purification of vesicles described in Section 5.2.6, purification of cells described in Section 5.2.7, or purification of viral particles described in Section 5.2.8.
  • the loading buffer has the same pH as the elution buffer described in Section 5.4.2.
  • the loading buffer has a salt concentration or conductivity that is lower than the salt concentration or conductivity of the elution buffer described in Section 5.4.2.
  • the loading buffer is saline. In one embodiment, the loading buffer is MOPSO buffer. In one embodiment, the loading buffer is phosphate buffer. In one embodiment, the loading buffer is Tris buffer. In one embodiment, the loading buffer is MOPS buffer. In one embodiment, the loading buffer is Bis-Tris buffer. In one embodiment, the loading buffer is ADA buffer. In one embodiment, the loading buffer is MES buffer. In one embodiment, the loading buffer is HEPES buffer. In one embodiment, the loading buffer is citric buffer. In one embodiment, the loading buffer is acetate buffer. [00385] In one embodiment, the loading buffer is Tris buffer with a pH from 6.5 to 7.4. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4.
  • the loading buffer comprises one or more salt.
  • the salt is an alkali metal salt.
  • the salt is an alkali metal halide.
  • the salt is NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl 2 , or any combination thereof.
  • the salt is sodium chloride.
  • the salt is potassium chloride.
  • the salt is a mixture of sodium chloride and potassium chloride.
  • the salt has a concentration from 0.001 M to 1 M.
  • the salt has a concentration from 0.01 M to 1 M. In one embodiment, the salt has a concentration from 0.01 M to 0.2 M. In one embodiment, the salt is 0.01 M to 0.2 M sodium chloride. In one embodiment, the salt is at 0.01 M sodium chloride. In one embodiment, the salt is 0.2 M sodium chloride. [00387] In one embodiment, the loading buffer has a conductivity from about 0.1 mS/cm to 100 mS/cm at 22 °C. In one embodiment, the conductivity is from about 0.1 mS/cm to about 50 mS/cm at 22 °C. In one embodiment, the conductivity is from about 1 mS/cm to about 25 mS/cm at 22 °C.
  • the conductivity is from about 5 mS/cm to about 20 mS/cm at 22 °C.
  • the loading buffer is Tris buffer with a pH from 6.5 to 7.4 and a salt NAI-1540508512v1 -77- Attorney Docket No.14497-018-228 concentration from 0.01 M to 0.2 M.
  • the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4 and a salt concentration from 0.01 M to 0.2 M.
  • the loading buffer is HEPES buffer with a pH from 4.5 to 7.4 and a salt concentration from 0.01 M to 0.2 M.
  • the loading buffer is Tris buffer with a pH from 6.5 to 7.4 and NaCl concentration from 0.01 M to 0.2 M. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4 and NaCl concentration from 0.01 M to 0.2 M. In one embodiment, the loading buffer is HEPES buffer with a pH from 4.5 to 7.4 and NaCl concentration from 0.01 M to 0.2 M. [00389] In one embodiment, the loading buffer is Tris buffer with a pH from 6.5 to 7.4, and a conductivity from about 0.1 to about 50 mS/cm at 22 °C.
  • the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4 and a conductivity from about 0.1 to about 50 mS/cm at 22 °C. In one embodiment, the loading buffer is HEPES buffer with a pH from 4.5 to 7.4 and a conductivity from about 0.1 to about 50 mS/cm at 22 °C. 5.4.2 Elution Buffer [00390] In one embodiment, the method described herein uses an elution buffer. In one embodiment, the kit described herein comprises an elution buffer. In one embodiment, the elution buffer has the second pH as described in Section 5.2.3.
  • the kit comprises two, three, or four elution buffers having the same or different pH or salt concentrations.
  • the elution buffer is used in the method described in Section 5.2.
  • the elution buffer is used in the purification of vesicles described in Section 5.2.6, purification of cells described in Section 5.2.7, or purification of viral particles described in Section 5.2.8.
  • the elution buffer has a higher pH than the loading buffer described in Section 5.4.1.
  • the elution buffer has the same pH as the loading buffer described in Section 5.4.1.
  • the elution buffer has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer described in Section 5.4.1.
  • the elution buffer is saline.
  • the elution buffer is phosphate buffer.
  • the elution buffer is Tris buffer.
  • the elution buffer is Bicine buffer.
  • the elution buffer is Tricine buffer.
  • the elution buffer is HEPES buffer.
  • the elution buffer is phosphate buffer with a pH from 7.4 to 9.
  • the elution buffer comprises one or more salt.
  • the salt is an alkali metal salt.
  • the salt is an alkali metal halide.
  • the salt is NaCl, KCl, KPO 4 , NaPO 4 , CaCl 2 , Mg 2 SO 4 , ZnCl 2 , MnCl 2 , MnSO 4 , NaSCN, KSCN, LiCl, MgCl 2 , or any combination thereof.
  • the salt is sodium chloride.
  • the salt is potassium chloride. In one embodiment, the salt is a mixture of sodium chloride and potassium chloride. In one embodiment, the salt concentration is from 0.001 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 2 M. In one embodiment, the salt concentration is from 0.1 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 1 M. In one embodiment, the salt concentration is from 0.7 M to 2 M. In one embodiment, the elution buffer comprises 0.01 M to 2 M NaCl. In one embodiment, the elution buffer comprises 0.01 M to 1 M NaCl. In one embodiment, the elution buffer comprises 0.7 M to 2 M NaCl. In one embodiment, the elution buffer comprises 0.15 M NaCl.
  • the elution buffer comprises 0.2 M NaCl. In one embodiment, the elution buffer comprises 0.275 M NaCl. In one embodiment, the elution buffer comprises 0.5 M NaCl. In one embodiment, the elution buffer comprises 0.7 M NaCl. In one embodiment, the elution buffer comprises 1 M NaCl. [00396] In one embodiment, the elution buffer has a conductivity from about 10 mS/cm to about 250 mS/cm at 22 °C. In one embodiment, the conductivity is from about 25 mS/cm to about 200 mS/cm at 22 °C.
  • the conductivity is from about 55 mS/cm to about 140 mS/cm at 22 °C. In one embodiment, the conductivity is about 55 mS/cm, about 60 mS/cm, about 70 mS/cm, about 80 mS/cm, about 90 mS/cm, about 100 mS/cm, about 110 mS/cm, about 120 mS/cm, about 130 mS/cm, or about 140 mS/cm at 22 °C.
  • the elution buffer has a salt concentration that is at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 25 times, at least 50 times, or at least 100 times higher than the loading buffer as described in Section 5.4.1.
  • the elution buffer has a conductivity that is at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 25 times, at least 50 times, or at least 100 times higher than the conductivity of the loading buffer as described in Section 5.4.1 at the same temperature (e.g. at 22 °C).
  • each elution buffer independently has a salt concentration or conductivity that is at least 1.2 times, at least 1.4 times, at least 1.6 times, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, or at least 20 times higher than the salt NAI-1540508512v1 -79- Attorney Docket No.14497-018-228 concentration or conductivity of the loading buffer.
  • the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 2 M. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.1 M to 2 M.
  • the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 1 M. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.7 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.1 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 1 M.
  • the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.7 M to 2 M.
  • the elution buffer is Tris buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 2 M.
  • the elution buffer is Tris buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 1 M.
  • the elution buffer is Tris buffer with a pH from 7.4 to 9 and NaCl concentration from 0.7 M to 2 M.
  • the elution buffer is phosphate buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 2 M.
  • the elution buffer is phosphate buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 1 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and NaCl concentration from 0.7 M to 2 M. [00402] In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a conductivity from about 10 to about 250 mS/cm at 22 °C. In one embodiment, the loading buffer is phosphate buffer with a pH from 7.4 to 9 and a conductivity from about 10 to about 250 mS/cm at 22 °C.
  • the elution buffer is Tris buffer with a pH from 7.4 to 9 and a conductivity from about 55 to about 140 mS/cm. In one embodiment, the loading buffer is phosphate buffer with a pH from 7.4 to 9 and a conductivity from about 55 to about 140 mS/cm. [00403] In one embodiment, the loading buffer has a pH of 7.2 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 7.2 and a salt concentration of above 0.1 M. In one embodiment, the loading buffer has a pH of 7.2 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 7.2 and a salt concentration of above 0.65 M.
  • the loading buffer has a pH of 8 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 8 and a salt concentration of above 0.1 M.
  • the loading buffer has a pH of 8 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 8 and a salt concentration of above 0.65 M.
  • the kit further comprises a washing buffer. In one embodiment, the kit further comprises two, three, four, five, or six washing buffers. 5.4.3 Washing Buffer [00406] In one embodiment, the method described herein further uses a washing buffer.
  • the kit described herein further comprises a washing buffer. In one embodiment, the kit further comprises two, three, or four washing buffers. In one embodiment, the kit does not comprise a washing buffer.
  • the washing buffer is the same as the loading buffer as described in Section 5.4.1. In one embodiment, the washing buffer is different from loading buffer as described in Section 5.4.1. In one embodiment, the washing buffer is used in the washing step as described in Section 5.2.1. [00408] In one embodiment, a washing buffer is used before loading of the sample to equilibrate the solid support. In one embodiment, a washing buffer is used after loading of the sample to remove one or more impurities.
  • a washing buffer is used after elution to re- equilibrate the solid support.
  • the washing buffer has a pH between 4.5 to 8.5. In one embodiment, the washing buffer has a pH between 4.5 to 7.4. In one embodiment, the washing buffer has a pH between 5 to 7.4. In one embodiment, the washing buffer has a pH between 6 to 7.4. In one embodiment, the washing buffer has a pH between 6.5 to 7.4. In one embodiment, the washing buffer has a pH of 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the washing buffer is saline.
  • the washing buffer is MOPSO buffer. In one embodiment, the washing buffer is Tris buffer. In one embodiment, the washing buffer is PBS buffer. In one embodiment, the washing buffer is MOPS buffer. In one embodiment, the washing buffer is Bis-Tris buffer. In one embodiment, the washing buffer is ADA buffer. In one embodiment, the washing buffer is MES buffer. In one embodiment, the washing buffer is HEPES buffer. In one embodiment, the washing buffer is citric buffer. In one embodiment, the washing buffer is acetate buffer. [00410] In one embodiment, the washing buffer is Tris buffer with a pH from 6.5 to 7.4. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4. [00411] In one embodiment, the wash buffer comprises one or more salt.
  • the salt is an alkali metal salt.
  • the salt is an alkali metal halide.
  • the salt is NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, or any combination thereof.
  • the salt is sodium chloride.
  • the salt is potassium chloride.
  • the salt is a mixture of sodium chloride and potassium chloride.
  • the salt has a concentration from 0.001 M to 1 M.
  • the salt has a concentration from 0.01 M to 1 M. In one embodiment, the salt has a concentration from 0.01 M to 0.2 M. In one embodiment, the salt is 0.01 M to 0.2 M sodium chloride. In one embodiment, the salt is at 0.01 M sodium chloride. In one embodiment, the salt is 0.2 M sodium chloride. [00412] In one embodiment, the washing buffer has a conductivity from about 0.1 mS/cm to 100 mS/cm at 22 °C. In one embodiment, the conductivity is from about 0.1 mS/cm to about 50 mS/cm at 22 °C. In one embodiment, the conductivity is from about 1 mS/cm to about 25 mS/cm at 22 °C.
  • the conductivity is from about 5 mS/cm to about 20 mS/cm at 22 °C. 6.
  • ligands e.g. diethanolamine, diisopropanolamine (DIPA), 2-methylethanolamine, 2-ethylethanolamine, morpholine, 2,6-dimethylmorpholine, 4-methylpiperazine, or 4-hydroxyethylpiperazine.
  • the reaction proceeds under a ring opening reaction as shown below:
  • a detailed description about the preparation and purification of the new resins are described in Section 6.2.
  • Other resins are prepared and purified using similar method.
  • the resin was washed with 35 mL ice cold ultrapure water, centrifuged at 300 x g for 5 min at 4 °C, and water was removed. This washing step was repeated for 2 more times.
  • a fresh solution of 4 M diisopropanolamine (DIPA) in ultrapure water was prepared, and 30 mL of the solution was added to the 30 mL of washed beads at room temperature (e.g.23 °C).
  • the concentration of DIPA in the reaction is 2 M.
  • the amount of epoxy groups in the reaction mixture is about 4808 ⁇ mol.
  • the amount of DIPA is about 120 mmol, which is about 25 equivalents of the amount of the epoxy groups in the reaction.
  • a 2 cm magnetic stirrer was added for gentle mixing during the reaction (slow mixing at number 3 of IKA RCT basic magnetic stirrer).
  • the resin coupling was done in a round bottom flask at about 90 °C in an oil bath for 15 h. After reaction, the mixture was cooled to room temperature, and the resin was transferred into two 50 mL falcon tubes, giving about 15 mL resin volume in each tube. The resin was centrifuged at 300 g at 5 °C for 5 min to settle the beads and the liquid was removed. The resin was washed by filling up to 50 mL in each tube with ultrapure water, centrifuged at 300 x g at 25 °C for 5 min, and the liquid was removed. This washing step was repeated for 3 times.
  • the tubes were filled up with 1 M NaOH in water, incubated for 1 h at 25 °C (no shaking), centrifuged at 300 g at 25 °C for 5 min, and liquid was removed.
  • the resin was washed by filling up to 50 mL in each tube with ultrapure water, centrifuged at 300 g at 25 °C for 5 min, and liquid was removed. This washing step was repeated 3 times, and 25 mL of wet resin was obtained.
  • the tubes were then filled up with 1 M NaCl in water, and stored at 4 °C until further use. Long-term storage condition is 1 M NaCl with 20 % EtOH.
  • EVs were isolated and purified from the clarified conditioned media using a variety of methods, typically a combination of diafiltration/ultrafiltration with tangential flow filtration (TFF), including concentration of the EV containing supernatant approximately 10X with a tangential flow filtration (TFF) with a 300 kDa MWCO and diafiltered into 37 mM MES acid, 20 mM NaOH, 137 mM NaCl, pH 6.2.
  • the supernatant was additionally purified using a flow through based multimodal chromatography (Captocore 700). Purified EVs can optionally be frozen and stored for downstream analysis and application.
  • Hybridosomes prepared from different kinds of LNPs and exosomes were purified on commercial POROS D resin, which is a weak anion exchange rein, under different eluting conditions.
  • Hybridosomes prepared by the fusing of LNP comprising siRNA and Cy5 labelled lipid, and exosome were purified with POROS D resin under elution conditions of pH at 9.0 and 1 M NaCl. Recovery yield of Hybridosomes was 6.6% (see Figure 4).
  • Hybridosomes prepared by the fusing of LNP comprising polyA and Cy5 labelled lipid, and exosome (untargeted) were purified with POROS D resin under elution conditions of pH at 5.5 and 1 M NaCl. Recovery yield of Hybridosomes was 34.9%.
  • NAI-1540508512v1 -84- Attorney Docket No.14497-018-228 [00421]
  • Hybridosomes prepared by the fusing of LNP comprising polyA and Cy5 labelled lipid, and exosome (untargeted) were purified with POROS D resin under elution conditions of pH at 9.0 and 1 M NaCl. Recovery yield of Hybridosomes was 25.9 %.
  • Exosomes are strongly retained on the resins and are not released even at high pH (9-10) and high salt concentrations (NaCl up to 2M and phosphate up to 1M).
  • EXAMPLE 7 Purification of Exosomes Using New Resins Prepared [00426] Exosomes are purified using the resins prepared in EXAMPLE 1. The purification process is depicted in Figure 7. In general, exosomes are loaded to gravity-flow column comprising the resins at pH about 5.5.
  • cells are transfected with the vector plasmid (containing the E1a-GFP transgene cassette) and two helper plasmids (psPAX2 which contains Gag-Pol -Rev-Tat genes and the pMD2G which contains the VSV-G pseudotyped envelope) using the CaPO4 precipitate technique.
  • the culture medium is removed from the four CS5 and exchanged with the transfection medium; the cells are then incubated 6 to 15 hours at 37 +/- 1 °C and 5 +/- 1% CO2.
  • the transfection medium is then removed from the CS5 and replaced by fresh exchange medium (Advanced DMEM 1X, 1% Pen/Strep) prior to a 2-day incubation at 37 +/- 1 °C and 5 +/- 1% CO 2 .
  • the cells of the 4 CS5 transfected are then harvested.
  • the supernatant is kept and clarified by a low-speed centrifugation (10 min at 1300 rpm).
  • Vector titration [00431] Infectious particles of the lentivirus vector (LV) were titrated by digital quantitative polymerase chain reaction (dPCR) after infection of HeLa cells with the LV sample.
  • LV purification In order to increase virus particles binding to DIPA column - we have applied the workflow shown below. NOTE: Loading was based on NTA data and Waytt DLS, similar technologies to those used for exosomes quantification. Final LV measurement was done using P24 protein ELISA. [00433] LV purification workflow: Clarified harvest DNase TFF1* supernatant was centrifuged and filtered through 0.45 ⁇ m filter.
  • Benzonase treatment was performed by adding to the clarified harvest 500 IU/L in the presence of 2 mM magnesium chloride at 2-8°C DURATION.
  • the treated clarified harvest was used for tangential flow filtration tests.
  • Tangential flow filtration (TFF) was performed using Hollow Fiber Filter (Repligen D02-E300-05-N) with a molecular weight cut-off (MWCO) of 300 kD.
  • the TFF was performed as follows: (1) ultrafiltration to concentrate the vector solution with a target concentration factor of 50–60, (2) diafiltration for buffer exchange using 20 diavolumes (DV) of diafiltration buffer (20 mM Tris, 50 mM NaCl, 1 % sucrose, pH 6.8).
  • TMP constant transmembrane pressure
  • Table 4 Chromatography conditions for DIPA resin: Phase Composition Column Volumes Equilibration 20 mM Tris, 50 mM NaCl, pH 6.8 5-7 CV * Loading Lentivirus (load about 1.5E+11 particles/mL) 0.5 mL Washing 20 mM Tris, 50 mM NaCl, pH 6.8 7 CV Elution 20 mM Tris, 50-800 mM NaCl, pH 7.2 10-15 CV CIP** 1 M NaOH 5 CV Re-equilibration 20 mM Tris, 50 mM NaCl, pH 6.8 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place.
  • Table 5 Chromatography conditions for Capto DEAE Resin Phase Composition Column Volumes Equilibration 50 mM Tris, 130 mM NaCl, pH 8 3-10 CV * Loading Lentivirus (load about 1.5E+11 particles/mL) 0.5 mL Washing 50 mM Tris, 130 mM NaCl, pH 8 7 CV Elution 50 mM Tris, 130-650 mM NaCl, pH 8 10-15 CV Wash 50 mM Tris, 1.3 M NaCl, pH 8 7 CV CIP** 1 M NaOH 5 CV Re-equilibration 50 mM Tris, 130 mM NaCl, pH 8 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place.
  • Lentiviral particles purified by the resins are analyzed by p24 ELISA assay. As shown in Figure 17, under chromatography conditions (20 mM Tris, 70 mM NaCl, pH 7.2), the total recovery yield of p24 protein of lentiviral particles from the DIPA resin was 79.5 ⁇ 5.5%. For the commercial Capto DEAE resin, the total recovery yield of lentivirus particles, quantified using p24 ELISA, was 20 ⁇ 10 %. Thus, the new DIPA resin is more efficiency in the purification of lentiviral particles than the commercial resin.
  • EXAMPLE 9 Purification of Lentivirus Using New Resins prepared for Polishing Step Cell amplification, transfection, and harvesting NAI-1540508512v1 -88- Attorney Docket No.14497-018-228 [00438] HEK293T cells were seeded in T300 surface-treated cell culture flasks with vent caps and cultured with DMEM-F12 supplemented with 10% FBS and 1% Pen/Strep.
  • cells were transfected with the vector plasmid (containing the SFFV-eGFP transgene cassette) and three helper plasmids (one plasmid containing Gag-Pol genes, one plasmid containing the Rev gene, and one plasmid encoding the VSV-G pseudotype envelope gene) using the Jetprime transfection reagent.
  • the culture medium was removed from the cell culture flasks and replaced with fresh pre-warmed OptiMEM medium.
  • Transfected HEK293 cells were incubated at 37 °C and 5 % CO 2 .
  • the conditioned cell culture medium was harvested and replaced with fresh pre-warmed OptiMEM medium. The cells were incubated for another 24 hours at 37 °C and 5 % CO2. The harvested conditioned medium was clarified by low-speed centrifugation at 300 xg and 4 °C for 5 minutes in a tabletop centrifuge. After centrifugation the supernatant was filtered through a 0.45 ⁇ m filter into a sterile bottle and stored at 4 °C overnight. At 48 hours and 72 hours post transfection the conditioned cell culture medium was harvested and processed identically to the previously harvested conditioned cell culture medium. The conditioned cell culture medium from all harvest days was pooled.
  • Viral vector quantification by viral transduction assay Infectious lentivirus vector samples were titrated by quantification of transgene expression in HEK293T cells infected with the LV sample. Serial dilutions of lentiviral vectors encoding eGFP were prepared and mixed with HEK293T cells in wells of a 96 well surface-treated cell culture plate, followed by incubation at 37 °C and 5 CO 2 . GFP fluorescence of HEK293T cells was measured 48 h after infection with the LV samples using a flow cytometer.
  • LV purification [00440] The Lentivirus from harvested supernatant was processed as described in Example 8 until DIPA resin step. Lentivirus supernatant was centrifuged and filtered through 0.45 ⁇ m filter. The clarified supernatant was concentrated and dialyzed by tangential flow filtration (TFF) in combination with nuclease treatment.
  • TNF tangential flow filtration
  • TFF was performed using Hollow Fiber Filter (Repligen D02-E300-05-N) with a molecular weight cut-off (MWCO) of 300 kD.
  • the TFF was performed as follows: (1) ultrafiltration to concentrate the vector solution with a target concentration factor of NAI-1540508512v1 -89- Attorney Docket No.14497-018-228 50–60; (2) DENARASE® treatment by adding to the clarified harvest 25 IU/mL DENARASE® in the presence of 2 mM MgCl at 37 °C for 1 hour; (3) diafiltration for buffer exchange using 10 diavolumes (DV) of diafiltration buffer (20 mM Tris, 50 mM NaCl, pH 7.2).
  • DV diavolumes
  • the concentrated and re-buffered lentiviral vector solution was then purified using a chromatography step with 7 mL TOYOPEARL- DIPA resin or 7 mL Capto-DEAE resin as outlined in Table 6.
  • the DEAE chromatography was performed as described in Example 8, whereby Tris or Phosphate buffer were used. Lentivirus elution fractions were diluted 1:3 (v/v) with 20 mM Tris pH 7.2, 105 mM sucrose for DIPA resin, or 1:7 (v/v) with 20 mM buffer pH 7.2, 81.6 mM sucrose for DEAE resin.
  • Lentiviral particles purified by the resins were analyzed by viral transduction assay, p24 ELISA assay and contaminants assays. As shown in Figure 18, under chromatography conditions, the recovery yield of infectious lentivirus from the DIPA resin was 72.7 ⁇ 4.1%. For the commercial Capto DEAE resin, the recovery yield of infectious lentivirus was only 25.7 ⁇ 6.7 %.
  • Table 6 Chromatography conditions for DIPA resin: Phase Composition Column Volumes Equilibration 20 mM Tris, 50 mM NaCl, pH 7.2 6 CV * Loading Diafiltrated Lentivirus 2 mL (about 0.6E+8 to 1.1E+8 3 CV infectious particles per mL) Washing 20 mM Tris, 75 mM NaCl, pH 7.2 4 CV Elution 20 mM Tris, 275 mM NaCl, pH 7.2 4 CV Wash 20 mM Tris, 500 mM NaCl, pH 7.2 4 CV CIP** 1 M NaOH 2 CV Re-equilibration 20 mM Tris, 50 mM NaCl, pH 7.2 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place.
  • the host cell protein content was evaluated by HEK293 Host cell protein ELISA (Cygnus) according to the manufacturer’s manual, whereby samples were used undiluted and 1:100 diluted in the respective buffer matrix and 50 ⁇ L of sample was used per well. Total protein concentration was assessed by Bio-Rad protein assay according to the manufacturer’s manual, whereby a standard range of 50 ug/mL to 500 ug/mL bovine serum albumin was used for the clarified conditioned medium. For samples after dialysis the micro BCA assay QuantiPro (Sigma-Aldrich) with a standard curve range from 2.5 ⁇ g/mL to 40 ⁇ g/mL bovine serum albumin were used.
  • Host cell DNA was assessed by QuantiFluor dsDNA system (Promega) NAI-1540508512v1 -90- Attorney Docket No.14497-018-228 according to the manufacturer’s manual and 10 ⁇ L of sample was used per well.
  • Table 7 Contaminant clearance assessment Host cell protein P rocess step DIPA Clearance DEAE Clearance ( %) (%) C onditioned media TFF 97.23 ⁇ 2.39 96.48 AEX polishing 99.81 ⁇ 0.04 98.56 Total protein P rocess step DIPA Clearance DEAE Clearance ( %) (%) C onditioned media TFF 95.24 ⁇ 16.33 62 AEX polishing 97.87 ⁇ 0.94 82.25 ⁇ 3.61 dsDNA P rocess step DIPA Clearance DEAE Clearance ( %) (%) C onditioned media TFF 99.07 ⁇ 0.15 99 AEX polishing 99.34 ⁇ 0.27 98.9 ⁇ 0.71 6.9 EXAMPLE 10: Capturing and Purification of Lentivirus from Cell Culture Medium Using New Resins Prepared Cell expansion, transfection, harvesting, and clarification.
  • HEK293T cells expansion, transfection and LV cell culture harvest and clarification was executed as described in Example 9, although only 24 h and 48 h harvest was collected and pooled.
  • Viral vector quantification [00445] Infectious lentivirus vector l titer of the clarified harvests were determined by viral transduction assay as describe above. Total LV particles were quantified by p24 ELSIA (Origene) according to manufacturer’s protocol, whereby samples were diluted 1:500 to 1:5’000 in Dulbecco's phosphate buffered saline.
  • Lentiviral Vector purification from cell culture medium workflow NAI-1540508512v1 -91- Attorney Docket No.14497-018-228 Clarified harvest medium was supplemented with 25 U/mL DENARASE, 2 mM MgCl 2 , and incubated at 37 °C for 1 hour.
  • the DENARASE® treated LV supernatant was used in a chromatography step with TOYOPEARL-DIPA resin or Capto-DEAE resin.
  • the DENARASE® treated LV feed was adjusted to pH 7 and a conductivity of 10.2 mS/cm by addition of a Bis-Tris propane buffer (5.4 mM Bis Tris, pH 6.2).
  • Table 8 Chromatography conditions DIPA resin for capturing mode Phase Composition Column Volumes Equilibration 20 mM Tris, 50 mM NaCl, pH 7.0 5-7 CV * Loading Lentivirus (about 1.3E+9 infectious particles) Sample dependent Washing 20 mM Tris, 75 mM NaCl, pH 8 8 CV Elution 1 (main elution) 20 mM Tris, 150 mM NaCl, pH 8 10-15 CV Elution 2 20 mM Tris 275 mM, pH 8 10 CV Elution 3 20 mM Tris 1300 mM, pH 8 10 CV CIP** 1 M NaOH 5 CV Re-equilibration 20 mM Tris, 50 mM NaCl, pH 7 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place.
  • Table 9 Chromatography conditions for Capto DEAE Resin Phase Composition Column Volumes Equilibration 50 mM Tris, 130 mM NaCl, pH 8 3-10 CV * Loading Lentivirus (about 1.3E+9 infectious particles) Sample dependent Washing 50 mM Tris, 130 mM NaCl, pH 8 7-10 CV Elution 1 (main elution) 50 mM Tris, 130-650 mM NaCl, pH 8 10-15 CV NAI-1540508512v1 -92- Attorney Docket No.14497-018-228 Elution 2 50 mM Tris, 1.3 M NaCl, pH 8 10 CV CIP** 1 M NaOH 15 CV Re-equilibration 50 mM Tris, 130 mM NaCl, pH 8 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place.
  • Capturing mode chromatography was performed as summarized in tables 8 and 9 using an 5 mL TOYOPEARL-DIPA column or a HiTrap Capto-DEAE column.
  • DIPA resin elution fractions were diluted 1:2 with 20 mM Bis-Tris pH 6.2, 140 mM sucrose, while DEAE elution fractions were diluted 1:5 in sterile 5 % sucrose solution.
  • the clarified DENARASE treated lentivirus feed and collected chromatography fractions were analyzed for viral titer by cell transduction and p24 ELISA assay.
  • Transduction unit (TU) recovery in the DIPA chromatography Elution 1 compared to the starting material was 59.7 ⁇ 10.3% and 65.1 ⁇ 13% for all elution fractions, while the TU recovery for the main elution fractions of the Capto DEAE chromatography was 17.3 ⁇ 4.2 % and 23.8 ⁇ 6.9 % for all elution fractions ( Figure 19).
  • Contaminants clearance in the DIPA main elution fractions compared to the starting material was 98.0 ⁇ 0.6% for HCP, 91.7 ⁇ 1.5% for total protein, and 70.5 ⁇ 3.1% for dsDNA (Table 10).
  • DEAE contaminant clearance was 96.03 ⁇ 0.6% for HCP, 67.2 ⁇ 5.42% for total protein, and 35.4 ⁇ 15.2% for dsDNA in the DEAE main elution fraction compared to the starting material (Table 10).
  • Table 10 DIPA and DEAE chromatography contaminant clearance Host cell protein DIPA DEAE HCP Clearance HCP Process step (ug) (%) (ug) Clearance (%) S upernatant/Denarase 493 ⁇ 43 495 ⁇ 44 A EX main fraction 9.58 ⁇ 2.40 98.04 ⁇ 0.56 18.2 ⁇ 1.7 96.28 ⁇ 0.56 A EX all fractions 10.99 ⁇ 2.61 97.79 ⁇ 0.72 19.03 ⁇ 2.0 96.21 ⁇ 0.65 Total protein DIPA DEAE Total Protein Clearance Total Clearance Process step (mg) (%) Protein (%) NAI-1540508512v1 -93- Attorney Docket No.14497-018-228 (mg) S upernatant/Denarase 10.5 ⁇ 1.5 11.0 ⁇ 1.1 A EX main fraction 0.878 ⁇ 0.235 91.70 ⁇ 1.55 3.56 ⁇ 0.26 67.22 ⁇ 5.42 A EX all fractions *0.8

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Abstract

The present application relates to methods for the purification of macromolecules, vesicles, cells or viral particles with the use of solid support, such as membranes, magnetic beads, or chromatography columns, and pH gradients. The application further provides compositions and kits comprising the solid support for use in the purification of macromolecules, vesicles, cells or viral particles at both small scale and large scale.

Description

Attorney Docket No.14497-018-228 METHODS AND COMPOSITIONS FOR PURIFYING PARTICLES AND MACROMOLECULES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No.63/525,304 filed on July 6, 2023 and U.S. Provisional Application No.63/622,946 filed on January 19, 2024, the entirety of each of which is incorporated herein by reference. 1. FIELD [0002] The present disclosure relates to methods, compositions, and kits for purifying macromolecules, vesicles, cells, or viral particles, using solid support, compositions comprising such solid support, and uses thereof. The methods described herein can produce purified macromolecules, vesicles, cells, or viral particles that are suitable for various uses (e.g., gene therapies). 2. BACKGROUND [0003] Many types of macromolecules, vesicles, cells and viral particles have been developed for therapeutic purposes. However, reliable production and purification of these therapies have been challenging as compared to conventional small molecular therapies. For example, while extracellular vesicle offers many advantages for targeted gene delivery (e.g. low immune response), current purification methods do not offer sufficient selectivity to efficiently capture the EVs and remove significant amount of impurities, including undesirable nucleic acids, host cell proteins, carbohydrates and lipids. Several types of commercially available resins, such as anion exchange resins, have been used in the purification of vesicles, cells, or viral particles. However, current purification methods of macromolecules, vesicles, cells, or viral particle are limited in terms of recover yield and efficiency. For example, yields of hybridosome purification on various types of commercially available resin, including anion exchange resin, is low (<35 %). Those methods also have low reproducibility, and are unable to isolate different types of vesicles. Significant loss of vesicles, such as exosomes, has been observed using currently available methods, which is at least partially due to the high retention of the vesicles on the resin. Purification of viral particles, such as lentiviral particles, also comes with low yield due to their high sensitivity to external factors, such as shear force, acidity, and temperature. [0004] Thus, there is a need to improve the purification methods of macromolecules, vesicles, cells or viral particles, particularly for therapeutic purposes wherein efficient and large-scale NAI-1540508512v1 -1- Attorney Docket No.14497-018-228 production is necessary. The present application provides methods, compositions, and kits to improve the purification of vesicles, cells, or viral particles. 3. SUMMARY [0005] In one embodiment, provided herein are methods of purifying vesicles, cells or viral particles using solid support (see Section 5.2). In one embodiment, provided herein are compositions of solid support (see Section 5.3). In one embodiment, provided herein are kits for purifying vesicles, cells or viral particles (see Section 5.4). In one embodiment, provided herein are methods of purifying macromolecules. [0006] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at (see Section 5.2.2) which the vesicles bind to the solid support; and c. eluting the vesicles from the solid support at a second pH (see Section 5.2.3), wherein the second pH is higher than the first pH. [0007] In one embodiment, the method further comprises a washing step at the first pH. [0008] In one embodiment, the first pH is lower than the pKa of the ionizable moiety. [0009] In one embodiment, the first pH is below 8. [0010] In one embodiment, the first pH is between 4.5 to 7.4 [0011] In one embodiment, the second pH is above 7. [0012] In one embodiment, the second pH is between 7.4 to 9. [0013] In one embodiment, the first pH is between 6.5 to 7.4 and the second pH is between 7.4 to 9. [0014] In one embodiment, the second pH is 7.4. [0015] In one embodiment, the eluting step is performed at a conductivity from about 55 mS/cm to about 140 mS/cm. [0016] In one embodiment, the eluting step is performed at a salt concentration from about 0.01 M to about 2 M. [0017] In one embodiment, the solid support has a binding affinity to the vesicles, cells, or viral particles at the first pH that is at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times higher NAI-1540508512v1 -2- Attorney Docket No.14497-018-228 than the binding affinity to the vesicles, cells or viral particles at the second pH. [0018] In one embodiment, the solid support has an average pore size from about 10 nm to about 1000 nm. In one embodiment, the average pore size is from about 10 nm to about 200 nm. [0019] In one embodiment, the method further comprises a step of preparing the solid support. [0020] In one embodiment, the solid support is prepared by providing a resin comprising at least one reactive group on the surface. [0021] In one embodiment, the reactive group is covalently connected to the resin via a linker. [0022] In one embodiment, the resin is a cation exchange (CEX) resin, an anion exchange (AEX) resin, a mixed mode resin, an affinity resin, a pseudo affinity resin, a hydrophobic interaction resin, a hydrophobic charge induction resin, or any combination thereof. [0023] In one embodiment, the resin is an anion exchange (AEX) resin. [0024] In one embodiment, the reactive group can react with a ligand, wherein the ligand is an amine, an alcohol, a thiol, or a carboxylic acid. [0025] In one embodiment, the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and lysine. [0026] In one embodiment, the reactive group is an epoxy group. [0027] In one embodiment, the epoxy group is capable of reacting with the ligand via a ring- opening reaction to form an ionizable moiety and a hydroxyl group. [0028] In one embodiment, the reactive group has a density of from about 100 µmol to about 2000 µmol per gram of the resin. [0029] In one embodiment, the reactive group has a density of from about 500 µmol to about 1000 µmol per gram of the resin. [0030] In one embodiment, the ligand is an amine. In one embodiment, the amine is a secondary amine. In one embodiment, the secondary amine is R1-NH-R2, wherein R1, R2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the atoms they are attached to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is optionally substituted. [0031] In one embodiment, the amine has a pKa of from about 8 to about 11 as determined by titration experiment using hydrochloric acid. [0032] In one embodiment, the ligand is selected from a group consisting of diethylamine, NAI-1540508512v1 -3- Attorney Docket No.14497-018-228 diethanolamine, dimethylethanolamine, diisopropylamine, isopropylethylamine, diisopropanolamine, 2-methylethanolamine, 2-ethylethanolamine, 2-(isopropylamino)ethanol, 2- (butylamino)ethanol, 2-benzylaminoethanol, piperidine, morpholine, 2,6-dimethyl-morpholine, 4- (2-chloroethyl)morpholine, 4-morpholinecarbonyl chloride, piperazine, 4-methylpiperazine and 4- hydroxyethylpiperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide; wherein each piperidine, morpholine, piperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide is optionally substituted. [0033] In one embodiment, the preparation of the solid support further comprises a step of contacting the reactive group with the ligand to form an ionizable moiety. [0034] In one embodiment, the ionizable moiety comprises a tertiary amino group. [0035] In one embodiment, the ionizable moiety has a pKa from about 7.6 to 8.8. [0036] In one embodiment, the ionizable moiety has a density of from about 50 µmol to about 1000 µmol per gram of the solid support. [0037] In one embodiment, the ionizable moiety is capable of binding and releasing the vesicles, cells or viral particles in a pH-dependent manner. [0038] In one embodiment, the solid support is capable of binding the vesicles, cells or viral particles at an efficiency of at least 80% at the first pH and releasing the vesicles, cells or viral particles at an efficiency of at least 50% at the second pH. [0039] In one embodiment, the vesicle, cell or viral particle is negatively charged at the first pH. [0040] In one embodiment, the vesicle, cell or viral particle is negatively charged at the second pH. [0041] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [0042] In one embodiment, the polymer is agarose, polystyrene, polymethacrylate, NAI-1540508512v1 -4- Attorney Docket No.14497-018-228 polyacrylamide, or hydroxylated methacrylic polymer. OH [0043] In one embodiment, the ionizable . [0044] In one embodiment, the ionizable
Figure imgf000007_0001
of from about 50 µmol to about 1000 µmol per gram of the solid support, or from about 250 µmol to about 800 µmol per gram of the solid support. [0045] In one embodiment, the loading buffer and the one or more elution buffers have the same pH. [0046] In one embodiment, the one or more elution buffers each independently has a salt concentration or conductivity that is at least 1.2 times, at least 1.4 times, at least 1.6 times, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, or at least 20 times higher than the salt concentration or conductivity of the loading buffer. [0047] In one embodiment, the method further comprises one or more washing steps. [0048] In one embodiment of purifying vesicles, the vesicle is a nanovesicle. [0049] In one embodiment of purifying viral particles, the viral particle is lentiviral particle. [0050] In one embodiment, the nanovesicle has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm. [0051] In one embodiment, the vesicle is an extracellular vesicle. In one embodiment, the extracellular vesicle is exosome. [0052] In one embodiment, the nanovesicle is an artificial nanovesicle. [0053] In one embodiment, the artificial nanovesicle is a liposome. [0054] In one embodiment, the nanovesicle is a hybrid nanovesicle. In one embodiment, hybrid nanovesicle is hybridosome. [0055] In one embodiment of purifying hybridosome, the method further comprises a step of preparing the hybridosome by fusing one or more lipid nanoparticle with one or more exosome. [0056] In one embodiment, the fusing is performed at a pH below 6.5. [0057] In one embodiment, the sample comprises at least one impurity that is a small molecule, a macromolecule, a different vesicle, a different cell, or a different viral particle. [0058] In one embodiment, the sample comprises at least one impurity that is a different vesicle, wherein the different vesicle is a lipid nanoparticle, an exosome or a liposome. NAI-1540508512v1 -5- Attorney Docket No.14497-018-228 [0059] In one embodiment, the impurity has a binding efficiency with the solid support of less than 20% at the first pH. [0060] In one embodiment, the sample has a volume from about 0.1 mL to about 2000 L, from about 10 L to about 2000 L, or from about 100 L to about 1000 L. [0061] In one embodiment, the sample is pretreated before contacting with the solid support. [0062] In one embodiment, the sample is pretreated by chromatography step, centrifugation, ultra-centrifugation, filtration, ultra-filtration, diafiltration, clarification, lyophilization, lysing, digestion, drying, dilution, concentration, heating, cooling, or any combination thereof. [0063] In one embodiment, the sample is produced from a cell culture container or bioreactor. [0064] In one embodiment, the vesicle, cell or viral particle is eluted at a yield of at least 50%. [0065] In one embodiment, the vesicle, cell or viral particle is eluted at a yield of at least 80%. [0066] In one embodiment, the method further comprises a step of detecting the elution of the vesicle, cell or viral particle. [0067] In one embodiment, the detecting involves the detection of optical signal from the vesicle, cell or viral particle, wherein the optical signal is UV-Vis signal, fluorescence signal, luminescence signal, enzyme-linked immunosorbent assay (ELISA) or light scattering signal. [0068] In one embodiment, provided herein is a resin of formula (I): wherein is a polymer
Figure imgf000008_0001
of agarose, cellulose, polystyrene, polyacrylamide, and hydroxylated methacrylic polymer; wherein the polymer is optionally substituted; wherein R1 and R2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the -N- to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is independently substituted; wherein L is C1-C48 alkylene, wherein one or more -CH2- in the alkylene is independently optionally replaced by -O-, -S-, -NH-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, -NHC(=O)-, or phenyl, and wherein the alkylene or phenyl is optionally substituted with one or more halogen, hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; and wherein the tertiary amino group has a pKa from about 6.5 to about 9. NAI-1540508512v1 -6- Attorney Docket No.14497-018-228 [0069] In one embodiment, the resin has an average pore size of from about 10 nm to about 1000 nm; and [0070] In one embodiment, the density of the tertiary amino group on the resin is from about 50 µmol to about 1000 µmol per gram of the resin, preferably from about 250 µmol to about 800 µmol per gram of the resin. [0071] In one embodiment, provided herein is a solid support comprising the resin as described herein. In one embodiment, the solid support is in the form of membrane, bead, or chromatography column. In one embodiment, the solid support comprises the resin in an amount from about 0.05 gram to about 5000 gram. [0072] In one embodiment, provided herein is a kit comprising the solid support as described herein, a loading buffer and an elution buffer, wherein the loading buffer has a pH between 4.5 to 7.4, and the elution buffer has a pH between 7.4 to 9. [0073] In one embodiment, provided herein is a kit comprising the resin as described herein, a loading buffer, and an elution buffer, and wherein the loading buffer has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [0074] In one embodiment, the kit further comprises a washing buffer, wherein the washing buffer has a pH between 4.5 to 7.4. [0075] In one embodiment, the loading buffer is a MOPSO buffer, Tris buffer, phosphate buffer, MOPS buffer, Bis-Tris buffer, ADA buffer, MES buffer, HEPES buffer, citric buffer or acetate buffer. [0076] In one embodiment, the elution buffer is a phosphate buffer, HEPES buffer, Tris buffer, Bicine buffer or Tricine buffer. [0077] In one embodiment, the elution buffer has a conductivity from about 55 mS/cm to about 140 mS/cm. [0078] In one embodiment the elution buffer has a salt concentration from about 0.01 M to about 2 M. In one embodiment, the elution buffer has a salt concentration of greater than 0.65 M. 4. BRIEF DESCRIPTION OF THE DRAWINGS [0079] Figure 1 depicts a representative workflow of preparing hybridosomes, followed by loading and eluting from solid support in a pH-dependent manner. [0080] Figure 2 depicts the measurements of pKa values of ionizable moieties prepared from diethanolamine, DiIsoPropylAmine (DIPA), 2, 6-dimethylmopholine, or mopholine. [0081] Figure 3 depicts the measurements of pKa values of ionizable moieties prepared from 2- NAI-1540508512v1 -7- Attorney Docket No.14497-018-228 methylethanolamine, 2-ethylethanolamine, 4-methylpiperazine, or 4-hydroxyethylpiperazine. [0082] Figure 4 depicts a representative purification of hybridosomes on commercially available AEX resins (POROS D), which gives an elution yield of vesicles of 6.6%. Elution of vesicles was detected by UV-Vis and fluorescence. [0083] Figure 5 depicts a representative purification of hybridosomes to remove excess LNPs using different resins under various loading and eluting conditions. Elution of the vesicles was detected by DLS and fluorescence. [0084] Figure 6 depicts a representative purification of hybridosomes comprising pxDNA cargo using TOYOPEARL-DIPA resin, which was detected by UV-Vis, DLS, conductivity, fluorescence and luminescence. Loading condition: 50 mM HEPES, pH 7.0, 0.2 M NaCl. Elution condition: 50 mM Tris, pH 8.0, 0.01-1 M NaCl gradient. [0085] Figure 7 depicts a scheme of using pH gradient for the purification of exosomes. [0086] Figure 8 depicts the recovery yields of exosomes using different commercial resins under various elution conditions. The recovery yields are generally below 25%. [0087] Figure 9 depicts the recovery yields of exosomes using different resins prepared. Higher recovery yields are observed compared with the use of commercial resins. [0088] Figure 10 depicts a correlation between the elution pH of exosomes and the pKa of the ionizable moieties. [0089] Figure 11 depicts a representative purification process of exosomes using Agarose- Morpholine resin, which is detected by UV-Vis, DLS and conductivity. Loading condition: 50 mM NaOAc, 50 mM NaCl, pH 5.5. Elution condition: 50 mM Glycine, 50 mM NaCl, pH 10. [0090] Figure 12 depicts a representative purification process of exosomes using TOYOPEARL-DIPA resin packed in a 6 mL XK16/20 column, which was detected by UV-Vis, DLS, conductivity, and luminescence. Loading condition: 50 mM HEPES, pH 7.0, 200 mM NaCl. Wash condition: 50 mM Tris pH 8.0, 10 mM NaCl. Elution condition: 50 mM TRIS, pH 8.0, 0.01- 1 M NaCl gradient. [0091] Figure 13 depicts a representative purification process of exosomes using TOYOPEARL-Morpholine resin, which is detected by UV-Vis, DLS, conductivity, and luminescence. Loading condition: 20 mM Tris, pH 6.8, 50 mM NaCl. Elution condition: 20 mM Tris, pH 7.9, 150 mM NaCl. Fractions collected are further analyzed by SDS-PAGE and AGE gel red staining. [0092] Figure 14 depicts a representative purification process of exosomes using NAI-1540508512v1 -8- Attorney Docket No.14497-018-228 TOYOPEARL-DIPA resin in 1 mL column, which was detected by UV-Vis, DLS, conductivity, and luminescence. Loading condition: 20 mM Tris, pH 6.8, 50 mM NaCl. Elution condition: 20 mM Tris, pH 7.9, 240 mM NaCl. Fractions collected are further analyzed by SDS-PAGE and AGE gel red staining. [0093] Figure 15 depicts a representative purification process of exosomes using TOYOPEARL-Morpholine resin in 1 mL column, which was detected by UV-Vis, DLS, conductivity, and luminescence. Loading condition: 20 mM Tris, pH 6.8, 50 mM NaCl. Elution condition: 20 mM Tris, pH 7.9, 0.05-1 M NaCl gradient. Fractions collected are further analyzed by SDS-PAGE. [0094] Figure 16 depicts a representative purification process of exosomes using TOYOPEARL-DIPA resin in 1 mL column, which was detected by UV-Vis, DLS, conductivity, and luminescence. Loading condition: 20 mM Tris pH 6.8, 50 mM NaCl. Elution condition: 20 mM Tris pH 6.8, 0.05-1 M NaCl gradient. Fractions collected are further analyzed by SDS-PAGE and AGE gel red staining. [0095] Figure 17 compares the recovery yield of lentivirus (LV) particles eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively, as detected by p24 ELISA. [0096] Figure 18 compares the transduction unit (TU) recovery yield of lentivirus (LV) particles eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively, as detected by p24 ELISA. [0097] Figure 19 compares the TU recovery yield of lentivirus (LV) particles in main elution fraction (a) and all elution fractions (b) eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively. [0098] Figure 20 compares the p24 recovery yield of lentivirus (LV) particles in main elution fraction (a) and all elution fractions (b) eluted from TOYOPEARL-DIPA resin or commercial Capto DEAE resin, respectively. 5. DETAILED DESCRIPTION [0099] Provided herein are methods for purifying macromolecules, vesicles, cells, or viral particles using solid support, compositions and kits comprising solid support, and their uses thereof. The methods or compositions as disclosed herein can be used to produce purified macromolecules, vesicles, cells, or viral particles that are suitable for various uses (e.g., gene therapies). [00100] In one embodiment, provided herein are methods for purifying vesicles, cells, or viral NAI-1540508512v1 -9- Attorney Docket No.14497-018-228 particles (see Section 5.2). In one embodiment, the method comprises steps of contacting or eluting the vesicles, cells, or viral particles under certain pH or salt conditions (see Section 5.2.2 and 5.2.3) in the presence of solid support (see Section 5.2.4). In one embodiment, the solid support as used herein comprises an ionizable moiety (see Section 5.2.5). In one embodiment, the vesicles as used herein comprise nanovesicles, extracellular vesicles, or hybrid nanovesicles, including exosomes, liposomes, or hybridosomes (see Section 5.2.6). In one embodiment, the cells as used herein comprise source cells of extracellular vesicle, HEK293T (see Section 5.2.7). In one embodiment, the viral particles as used herein comprise enveloped viral particles, lentiviral particles, adeno- associated viral particles, or retroviral particles (see Section 5.2.8). In one embodiment, the method removes at least one or more impurities, such as a small molecule, a macromolecule, a different vesicle, a different cell, or a different viral particle in a sample (see Section 5.2.10). In one embodiment, the method recovers some or most of the vesicles, cells, or viral particles in an eluting step (5.2.11). [00101] In one embodiment, provided herein are methods for purifying macromolecules (see Section 5.2.9). Non-limiting examples of macromolecules that can be purified using the methods include polypeptides (e.g. enzyme, protein, antibody, or monoclonal antibody), virus-like particles, polysaccharide (e.g. galactogen, or inulin), and polynucleotide (e.g. DNA, or RNA) [00102] In one embodiment, the method or composition as described here is suitable for small- scale purification of vesicles, particles, cells or macromolecules. In one embodiment, the method or composition as described here is suitable for large-scale industrial purification (e.g. greater than 1 L sample volume) of vesicles, particles, cell or macromolecules. In one embodiment, the sample is pretreated (see Section 5.2.10) before purification, which is particularly important for large-scale industrial purification. [00103] The method as described herein may optionally comprises a washing step as described in Section 5.2.1, a step of preparing the solid support as described in Section 5.2.4 (i), a step of preparing the vesicles, cells or viral particles as described in Sections 5.2.6-5.2.8, a step of pretreating the sample as described in Section 5.2.10, or a detecting step as described in Section 5.2.12. [00104] In one embodiment, the solid support as used herein binds and releases the vesicles, cells, or viral particles in a pH-dependent manner (see Section 5.2.4). In one embodiment, the solid support comprises an ionizable moiety (see Section 5.2.5). In one embodiment, solid support is prepared from a cation exchange resin, an anion exchange resin, or a size-exclusion resin NAI-1540508512v1 -10- Attorney Docket No.14497-018-228 comprising reactive groups (5.2.4 (i)). In one embodiment, solid support is prepared by contacting a resin comprising reactive groups as described in Section 5.2.4 (ii) and a ligand as described in 5.2.4 (iii). In one embodiment, the solid support comprises the resin as described in Section 5.3.1. In one embodiment, the solid support is in the form of a membrane, a magnetic bead, or a chromatography column as described in Section 5.3.3. In one embodiment, the solid support is in the form of a chromatography column. [00105] Also provided herein is a composition, such as a resin of Formula (I) (see Section 5.3). In one embodiment, the composition comprises a polymer, a linker, and a tertiary amino group as defined in Section 5.3. Also provided herein is a method of preparing the composition (see Section 5.3.2). Also provided herein is a solid support (e.g. membrane, magnetic bead, or chromatography column) comprising the composition (see Section 5.3.3). [00106] Also provided herein is a kit comprising the composition as described in Section 5.3, a loading buffer as described in Section 5.4.1, and an elution buffer as described in Section 5.4.2. The kit may optionally comprise a washing buffer as described in Section 5.4.3. [00107] In one embodiment, the composition, solid support, or kit as described herein is used in the purification method as described in Section 5.2. 5.1 Definitions [00108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. [00109] As used herein and unless otherwise specified, the term “about” means within plus or minus 10% of a given value or range. [00110] As used herein, and unless otherwise specified, the term “alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated. In one embodiment, the alkyl group has, for example, from one to twenty-four carbon atoms (C1-C24 alkyl), four to twenty carbon atoms (C4-C20 alkyl), six to sixteen carbon atoms (C6- C16 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) and which is attached to the rest of the molecule by a single bond. Examples of NAI-1540508512v1 -11- Attorney Docket No.14497-018-228 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n- butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, an alkyl group is optionally substituted. [00111] As used herein, and unless otherwise specified, the term “alkoxy” refers to -O-(alkyl), wherein alkyl is defined above. As used herein, and unless otherwise specified, the term “aryloxy” refers to -O-(aryl), wherein aryl is defined above. [00112] As used herein, and unless otherwise specified, the term “cycloalkyl” refers to a non- aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which is saturated. Cycloalkyl group may include fused, bridged, or spiro ring systems. In one embodiment, the cycloalkyl has, for example, from 3 to 15 ring carbon atoms (C3- C15 cycloalkyl), from 3 to 10 ring carbon atoms (C3-C10 cycloalkyl), or from 3 to 8 ring carbon atoms (C3-C8 cycloalkyl). The cycloalkyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include, but are not limited to, adamantyl, norbornyl, decalinyl, 7,7-dimethyl- bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified, a cycloalkyl group is optionally substituted. [00113] As used herein, and unless otherwise specified, the term “heterocyclyl” refers to a monocyclic and/or multicyclic non-aromatic group that contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorous, and sulfur. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom. A heterocyclyl group can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other multicyclic ring system, wherein the multicyclic ring systems can be a fused, bridged or spiro ring system. Heterocyclyl multicyclic ring systems can include one or more heteroatoms in one or more rings. A heterocyclyl group can be saturated or partially unsaturated. Saturated heterocycloalkyl groups can be termed “heterocycloalkyl”. Partially unsaturated heterocycloalkyl groups can be termed “heterocycloalkenyl” if the heterocyclyl contains at least one double bond, or “heterocycloalkynyl” if the heterocyclyl contains at least one triple bond. In one embodiment, the heterocyclyl has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms (5- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-membered heterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl). Examples of heterocyclyl groups include, but are not limited to, imidazolidinyl, oxazolidinyl, NAI-1540508512v1 -12- Attorney Docket No.14497-018-228 thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuryl, and piperidinyl. Unless otherwise specified, a heterocyclyl group is optionally substituted. [00114] As used herein, and unless otherwise specified, the term “aryl” refers to a monocyclic aromatic group and/or multicyclic aromatic group that contain at least one aromatic hydrocarbon ring. In certain embodiments, the aryl has from 6 to 18 ring carbon atoms (C6-C18 aryl), from 6 to 14 ring carbon atoms (C6-C14 aryl), or from 6 to 10 ring carbon atoms (C6-C10 aryl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. The term “aryl” also refers to bicyclic, tricyclic, or other multicyclic hydrocarbon rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise specified, an aryl group is optionally substituted. [00115] As used herein, and unless otherwise specified, the term “alkylene” or “alkylene chain” refers to a straight or branched multivalent (e.g., divalent or trivalent) hydrocarbon chain linking the rest of the molecule to a radical group (or groups), consisting solely of carbon and hydrogen, which is saturated. In one embodiment, the alkylene has, for example, from one to twenty-four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (C1-C8 alkylene), one to six carbon atoms (C1- C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene). Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, n- butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group(s) can be through one carbon or any two (or more) carbons within the chain. Unless otherwise specified, an alkylene chain is optionally substituted. [00116] As used herein, and unless otherwise specified, the term “amino” refers to –N(R#)(R#), wherein each R# independently can be, but is not limited to, hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocyclyl, each of which is defined above. When a -N(R#)(R#) group has two R# other than hydrogen, they can be combined with the nitrogen atom to form a ring. In one embodiment, the ring is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring. In one embodiment, one or more ring atoms are heteroatoms independently selected from O, S, or N. The NAI-1540508512v1 -13- Attorney Docket No.14497-018-228 term “amino” also includes N-oxide (–N+(R#)(R#)O-). In certain embodiments, each R# or the ring formed by -N(R#)(R#) independently may be unsubstituted or substituted with one or more substituents. [00117] As used herein, and unless otherwise specified, the term “aminoalkyl” refers to -(alkyl)- (amino), wherein alkyl and amino are defined above. As used herein, and unless otherwise specified, the term “aminoalkoxy” refers to -O-(alkyl)-(amino), wherein alkyl and amino are defined above. [00118] As used herein, and unless otherwise specified, the term “alkylamino” refers to -NH(alkyl) or -N(alkyl)(alkyl), wherein alkyl is defined above. Examples of such alkylamino groups include, but are not limited to, -NHCH3, -NHCH2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, - NH(CH2)4CH3, -NH(CH2)5CH3, -N(CH3)2, -N(CH2CH3)2, -N((CH2)2CH3)2, -N(CH3)(CH2CH3), and the like. [00119] When the groups described herein are said to be “substituted,” they may be substituted with any appropriate substituent or substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chloro, iodo, bromo, or fluoro); alkyl; alkenyl; alkynyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxyl amine; alkoxyamine; aryloxyamine, aralkoxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxo (═O); B(OH)2, O(alkyl)aminocarbonyl; cycloalkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or a heterocyclyl, which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiazinyl); monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl) aryloxy; aralkyloxy; heterocyclyloxy; and heterocyclyl alkoxy. [00120] The term "resin" as used herein refers to the solid phase of chromatography, such as column chromatography. Non-limiting examples of such resins include: beaded resin, gels, monoliths, membranes, non-woven supports, and combinations thereof. The methods disclosed herein can be applied to any form of chromatography suitable for the purification of particles, e.g, NAI-1540508512v1 -14- Attorney Docket No.14497-018-228 vesicles. In some embodiments, the resin comprises an "affinity" chromatography resin, which refers to a chromatography resin that interacts with one or more molecules present in the mobile phase of the chromatography. An affinity chromatography can be used in a "bind-and-elute" mode, wherein the desired molecules interact with the stationary phase until certain conditions are created that cause the desired molecules to release from the stationary phase and elute from the chromatography resin; or in a "pass through" mode, wherein one or more impurities present in the mobile phase, but not the desired molecules, interact with the chromatography resin, allowing the desired molecules to "pass through" the chromatography resin, while the impurities remain associated with the chromatography resin. In some embodiments, the chromatography resin comprises an anion exchange (AEX) resin, a cation exchange (CEX) resin, a pseudo affinity chromatography resin, a hydrophobic interaction resin, a ceramic hydroxyapatite resin, a fluoro hydroxyapatite resin, and any combination thereof. In some aspects, the chromatography resin comprises a mixed-mode chromatography (MMC) resin. [00121] The term “column” or “chromatography column” refers to any enclosure or housing adapted for use in a chromatography process that is suitable for retaining all or part of a chromatography resin and enabling sample and/or buffer to contact the chromatography resin. Typically, such columns are in the form of conduits, tubes, or other structures having a relatively large ratio of flow length to flow cross section. In some examples, the column defines a cylindrically shaped hollow interior with a volume that can be characterized by its internal radius and/or flow length. The column body is a tube having two open ends connected by an open channel, which can also be referred to as a through passageway. The tube can be in any shape, including, but not limited to, cylindrical or frustoconical, and of any dimensions consistent with the function of the column as described herein. In some embodiments, the column body takes the form of a syringe, a pipette tip, or similar tubular bodies. In some embodiments, where the column body is a syringe, the syringe is modified to contain the chromatography resin. The end of the column body wherein the bed of chromatography resin is placed can take any of a number of geometries, e.g., it can be tapered or cylindrical. In some embodiments, one of the open ends of the column is adapted for a particular use, for example, the outlet opening of the column can be adapted for attachment to a collection tube. The column can be composed of any material that is sufficiently non-porous that it can retain chromatography resin and that is compatible with substances used in devices and methods described herein. A wide range of materials suitable for use in a column are known in the art. Various plastics can be used as column materials, but other materials such as NAI-1540508512v1 -15- Attorney Docket No.14497-018-228 glass, ceramics, or metals are suitable for use in devices and methods described herein. Non- limiting examples of materials include polysulfone, polypropylene, polyethylene, polyethylene terephthalate, polyethersulfone, polytetrafluoroethylene, cellulose, acrylonitrile PVC copolymer, polystyrene, polystyrene/acrylonitrile copolymer, polyvinylidene fluoride, polytetrafluoroethylene (PTFE) (e.g., TEFLON®) and similar materials, glass, poly ether ether ketone (PEEK), metal, silica, and combinations thereof. [00122] The term "chromatography" refers to any kind of technique (e.g. a preparative technique) which separates a product of interest (e.g. macromolecules, vesicles, cells, or viral particles) from other molecules present in a mixture by differential partitioning between a mobile phase and a stationary phase. The stationary phase is thereby preferably held in place, while the mobile phase moves in a definite direction. Partitioning is to be understood to occur repeatedly as the sample flows with the mobile phase along or through a dimension, e.g. length, of the stationary phase. “Column chromatography” as used herein is a technique in which a stationary phase material is contained in a container such as a tube or column having an inlet on one side of the container and an outlet on another side, preferably the opposite side. The stationary phase material may for example be comprised of particles of a solid stationary phase material or a support coated with a liquid stationary phase. It may fill the whole inside volume of the container (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). In one embodiment, a packed column chromatography is preferred. [00123] The term "sample" herein implies a complex mixture or complex composition (e.g. a fluid) that includes one or more different molecules such as macromolecules, vesicles, cells, or viral particles, nucleic acids, polypeptides, or small molecules. 5.2 Methods [00124] In one embodiment, provided herein is a method of purifying vesicles (e.g. vesicles described in Section 5.2.6). In one embodiment, provided herein is a method of purifying cells (e.g. cells described in Section 5.2.7). In one embodiment, provided herein is a method of purifying viral particles (viral particles described in Section 5.2.8). In one embodiment, provided herein is a method for purifying macromolecules (e.g. macromolecules described in Section 5.2.9). [00125] In one embodiment, the purification is achieved by an increase of pH from loading to elution. In one embodiment, the purification is achieved by an increase of salt concentration or NAI-1540508512v1 -16- Attorney Docket No.14497-018-228 conductivity from loading to elution. In one embodiment, the loading step uses a loading buffer described in Section 5.4.1. In one embodiment, the elution step uses an elution buffer described in Section 5.4.2. In one embodiment, the elution buffer has a higher pH than the loading buffer. In one embodiment, the elution buffer has a higher salt concentration or conductivity than the loading buffer. In one embodiment, the elution buffer and loading buffer has the same pH, and the elution buffer has a higher salt concentration or conductivity than the loading buffer. [00126] In one embodiment, provided herein is a method of purifying small sample volume (e.g. from 0.1 mL to 50 mL). In one embodiment, provided herein is a method of purifying medium sample volume (e.g. from 50 mL to 1 L). In one embodiment, provided herein is a method of purifying large sample volume (e.g. from 1 L to 2000 L). The method provides herein is applicable to both laboratory scale purification and industrial scale purification. In one embodiment, the sample is pretreated (e.g. as described in Section 5.2.10) before subjecting to the purification method described herein. [00127] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and c. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00128] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [00129] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: NAI-1540508512v1 -17- Attorney Docket No.14497-018-228 a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the macromolecules bind to the solid support; and c. eluting the macromolecules from the solid support at a second pH, wherein the second pH is higher than the first pH. [00130] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is - N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [00131] In one embodiment, the method further comprises a washing step as described in Section 5.2.1. In one embodiment, the method further comprises one or more steps of preparing the solid support as described in Section 5.2.4 (i). In one embodiment, the method further comprises one or more steps of preparing the solid support with ligands to create an ionizable moiety as described in Section 5.2.4 (i). In one embodiment, the method further comprises one or more steps of preparing the vesicles, cells or viral particles as described in Sections 5.2.6, 5.2.7, or 5.2.8. In one embodiment, the method further comprises one or more steps of pretreating the sample as described in Section 5.2.10. In one embodiment, the method further comprises one or more detecting steps as described in Section 5.2.11. In one embodiment, the method further comprises one or more detecting steps as described in Section 5.2.12. In one embodiment, the method further comprises a combination of any of the steps as described in Sections 5.2.1, 5.2.4 (i), 5.2.6, 5.2.7, 5.2.8, 5.2.11 or 5.2.12. 5.2.1 Washing Step [00132] In one embodiment, the method further comprises a washing step. In one embodiment, the method further comprises two, three, four, or five washing steps. In one embodiment, the method further comprises a washing step at the first pH. In one embodiment, the washing step is performed at a third pH. In one embodiment, the third pH is lower than the first pH. In one NAI-1540508512v1 -18- Attorney Docket No.14497-018-228 embodiment, the third pH is higher than the first pH. In one embodiment, the third pH is the same as the first pH. In one embodiment, the third pH is between 4.5 to 7.4. In one embodiment, the third pH is between 5 to 7.4. In one embodiment, the third pH is between 6 to 7.4. In one embodiment, the third pH is between 6.5 to 7.4. In one embodiment, the third pH is 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.1, 7.2, 7.3 or 7.4. [00133] In one embodiment, the washing step is performed before the contacting step. In one embodiment, a washing step is used to equilibrate the solid support before loading of the sample. [00134] In one embodiment, the washing step is performed after the contacting step. [00135] In one embodiment, the washing step is performed before the eluting step. In one embodiment, a washing step is performed after the eluting step to re-equilibrate the solid support. [00136] In one embodiment, the washing step is performed with a washing buffer (for example, the washing buffer as described in Section 5.4.3). In one embodiment, the washing buffer has a pH between 4.5 to 7.4. In one embodiment, the washing buffer has a pH between 5 to 7.4. In one embodiment, the washing buffer has a pH between 6 to 7.4. In one embodiment, the washing buffer has a pH between 6.5 to 7.4. In one embodiment, the washing buffer has a pH of 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the washing buffer is saline. In one embodiment, the washing buffer is MOPSO buffer. In one embodiment, the washing buffer is Tris buffer. In one embodiment, the washing buffer is phosphate buffer. In one embodiment, the washing buffer is MOPS buffer. In one embodiment, the washing buffer is Bis- Tris buffer. In one embodiment, the washing buffer is ADA buffer. In one embodiment, the washing buffer is MES buffer. In one embodiment, the washing buffer is HEPES buffer. In one embodiment, the washing buffer is citric buffer. In one embodiment, the washing buffer is acetate buffer. [00137] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; c. washing the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is the same or higher than the first pH. NAI-1540508512v1 -19- Attorney Docket No.14497-018-228 [00138] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the macromolecules bind to the solid support; c. washing the solid support; and d. eluting the macromolecules from the solid support at a second pH, wherein the second pH is the same or higher than the first pH. 5.2.2 First pH [00139] In one embodiment, the first pH is lower than the pKa of the ionizable moiety. In one embodiment, the first pH is below 8. In one embodiment, the first pH of 8– 9. In one embodiment, the first pH is between 4.5 to 7.4. In one embodiment, the first pH is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the first pH is 6.5. In one embodiment, the first pH is 6.8. In In one embodiment, the first pH is 7.0. In In one embodiment, the first pH is 7.2. In one embodiment, the first pH is 7.4. In one embodiment, the first pH is 8. [00140] In one embodiment, the contacting is performed with a loading buffer (for example, the loading buffer as described in Section 5.4.1) having the first pH. In one embodiment, the loading buffer has a pH lower than the pKa of the ionizable moiety. In one embodiment, the loading buffer has a pH below 8. In one embodiment, the loading buffer has a pH from 6 to 9. In one embodiment, the loading buffer has a pH from 4.5 to 7.4. In one embodiment, the loading buffer has a pH of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the loading buffer has pH of 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, or 9. [00141] In one embodiment, the loading buffer is saline. In one embodiment, the loading buffer is MOPSO buffer. In one embodiment, the loading buffer is Tris buffer. In one embodiment, the loading buffer is phosphate buffer. In one embodiment, the loading buffer is MOPS buffer. In one embodiment, the loading buffer is Bis-Tris buffer. In one embodiment, the loading buffer is ADA buffer. In one embodiment, the loading buffer is MES buffer. In one embodiment, the loading buffer is HEPES buffer. In one embodiment, the loading buffer is citric buffer. In one embodiment, the loading buffer is acetate buffer. NAI-1540508512v1 -20- Attorney Docket No.14497-018-228 [00142] In one embodiment, the loading buffer comprises one or more salt. In one embodiment, the loading buffer is Tris buffer comprising sodium chloride. In one embodiment, the loading buffer is MOPS buffer comprising sodium chloride. In one embodiment, the loading buffer is HEPES buffer comprising sodium chloride. [00143] In one embodiment, the contacting is performed in the presence of one or more salt. In one embodiment, the salt is a non-chaotropic salt. In one embodiment, the salt is a monovalent salt. In one embodiment, the salt is an alkali metal salt, preferably an alkali metal halide. In one embodiment, the salt is NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, and any combination thereof. In one embodiment, the salt is sodium chloride. In one embodiment, the salt is potassium chloride. [00144] In one embodiment, the contacting is performed at a salt concentration from 0.001 M to 1 M. In one embodiment, the salt concentration is from 0.01 M to 1 M. In one embodiment, the salt concentration is from 0.01 M to 0.2 M. In one embodiment, the contacting is performed at 0.01 M to 0.2 M sodium chloride. In one embodiment, the contacting is performed at 0.01 M sodium chloride. In one embodiment, the contacting is performed in 0.2 M sodium chloride. [00145] In one embodiment, the contacting is performed at a conductivity from about 0.1 mS/cm to 50 mS/cm. In one embodiment, the conductivity is from about 0.1 mS/cm to about 25 mS/cm. In one embodiment, the conductivity is from about 1 mS/cm to about 25 mS/cm. 5.2.3 Second pH [00146] In one embodiment, the second pH is above 7. In one embodiment, the second pH is between 7.4 to 9. In one embodiment, the second pH is 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9. In one embodiment, the second pH is 7.2. In one embodiment, the second pH is 7.4. In one embodiment, the second pH is 8.3. In one embodiment, the second pH is 8. In one embodiment, the second pH is 8.8. In one embodiment, the second pH is higher than the pKa of the ionizable moiety (see Section 5.2.5). In one embodiment, the second pH is lower than the pKa of the ionizable moiety. [00147] In one embodiment, the elution is performed with an elution buffer (for example, the elution buffer as described in Section 5.4.2) having the second pH. In one embodiment, the elution buffer has a pH higher than the pKa of the ionizable moiety. In one embodiment, the elution buffer has a pH lower than the pKa of the ionizable moiety. In one embodiment, the elution buffer has a pH higher than 7. In one embodiment, the elution buffer has a pH between 7.4 to 9. In one embodiment, the elution buffer has a pH of 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, NAI-1540508512v1 -21- Attorney Docket No.14497-018-228 8.7, 8.8, 8.9 or 9. In one embodiment, the elution buffer is phosphate buffer. In one embodiment, the elution buffer is Tris buffer. In one embodiment, the elution buffer is Bicine buffer. In one embodiment, the elution buffer is Tricine buffer. In one embodiment, the elution buffer is HEPES buffer. [00148] In one embodiment, the elution buffer comprises one or more salt. In one embodiment, the elution buffer is Tris buffer comprising sodium chloride. [00149] In one embodiment, the elution is performed in the presence of one or more salt. In one embodiment, the salt is a non-chaotropic salt. In one embodiment, the salt is a monovalent salt. In one embodiment, the salt is an alkali metal salt, preferably an alkali metal halide. In one embodiment, the salt is selected from NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, and any combination thereof. In one embodiment, the salt is sodium chloride. In one embodiment, the salt is potassium chloride. [00150] In one embodiment, the elution is performed at a salt concentration from 0.001 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 1 M. In one embodiment, the salt concentration is from 0.1 M to 2 M. In one embodiment, the salt concentration is from 0.7 M to 2 M. In one embodiment, the elution is performed in a buffer comprising 0.7 M to 2 M sodium chloride. In one embodiment, the elution is performed in a buffer comprising 0.2 M sodium chloride. In one embodiment, the elution is performed in a buffer comprising 0.7 M sodium chloride. In one embodiment, the elution is performed in a buffer comprising 1 M sodium chloride. [00151] In one embodiment, the elution is performed under a salt gradient. In one embodiment, the salt gradient is from 0.001 M to 2 M. In one embodiment, the salt gradient is from 0.01 M to 2 M. In one embodiment, the salt gradient is from 0.01 M to 1 M. In one embodiment, the salt gradient is from 0.7 M to 1 M. In one embodiment, the salt gradient is from 0.7 M to 2 M. In one embodiment, the elution is performed under a sodium chloride gradient. In one embodiment, the elution is performed under a potassium chloride gradient. In one embodiment, the elution is performed under a sodium chloride gradient from 0.01 M to 1 M. [00152] In one embodiment, the elution is performed at a conductivity from about 10 mS/cm to about 250 mS/cm. In one embodiment, the conductivity is from about 25 mS/cm to about 200 mS/cm. In one embodiment, the conductivity is from about 55 mS/cm to about 140 mS/cm. In one embodiment, the conductivity is about 55 mS/cm, about 60 mS/cm, about 70 mS/cm, about 80 mS/cm, about 90 mS/cm, about 100 mS/cm, about 110 mS/cm, about 120 mS/cm, about 130 NAI-1540508512v1 -22- Attorney Docket No.14497-018-228 mS/cm, or about 140 mS/cm. [00153] In one embodiment, the elution is performed under a conductivity gradient. In one embodiment, the conductivity gradient is from about 10 mS/cm to about 250 mS/cm. In one embodiment, the conductivity gradient is from about 25 mS/cm to about 200 mS/cm. In one embodiment, the conductivity gradient is from about 55 mS/cm to about 140 mS/cm. [00154] In one embodiment, the first pH is between 4.5 to 7.4, and the second pH is between 7.4 to 9. In one embodiment, the first pH is between 6.5 to 7.4, and the second pH is between 7.4 to 9. In one embodiment, the first pH is 6.5, and the second pH is 7.4. In one embodiment, the first pH is 7.4, and the second pH is 8.3. In one embodiment, the first pH is 7, and the second pH is 8. In one embodiment, the first pH is 7, and the second pH is 8.8. [00155] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles bind to the solid support, wherein the first pH is between 4.5 to 7.4; and c. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is between 7.4 to 9, and wherein the eluting is performed at a conductivity from about 55 to about 140 mS/cm. [00156] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles bind to the solid support, wherein the first pH is between 4.5 to 7.4; and c. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is between 7.4 to 9, and wherein the eluting is performed at a salt concentration from about 0.01 M to about 2 M. [00157] In one embodiment, the eluting step is performed at a higher ionic strength than the contacting step. In one embodiment, the contacting is performed at a salt concentration between 0.01 to 0.1 M and the elution is performed at a salt concentration between 0.1 M to 1 M. In one embodiment, the contacting is performed at a salt concentration between 0.1 to 0.7 M and the NAI-1540508512v1 -23- Attorney Docket No.14497-018-228 elution is performed at a salt concentration between 0.7 M to 2 M. [00158] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH, wherein the first pH is lower than the pKa of the ionizable moiety; and c. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the pKa of the ionizable moiety. [00159] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH, wherein the first pH is lower than the pKa of the ionizable moiety; and c. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is lower than the pKa of the ionizable moiety, and wherein the eluting is performed at a salt concentration from about 0.01 M to about 2 M. [00160] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH, wherein the first pH is lower than the pKa of the ionizable moiety; and c. eluting the macromolecules from the solid support at a second pH, wherein the second pH is higher than the pKa of the ionizable moiety. [00161] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH, wherein the first pH is lower than the pKa of the ionizable moiety; and NAI-1540508512v1 -24- Attorney Docket No.14497-018-228 c. eluting the macromolecules from the solid support at a second pH, wherein the second pH is lower than the pKa of the ionizable moiety, and wherein the eluting is performed at a salt concentration from about 0.01 M to about 2 M. 5.2.4 Solid Support [00162] In one embodiment, the solid support comprises the resin as described in Section 5.3.1. In one embodiment, the solid support is the solid support as described in Section 5.3.3. In one embodiment, the solid support is a chromatography column as described in Section 5.3.3. In one embodiment, the solid support comprises the ionizable moieties as described in Section 5.2.5. In one embodiment, the solid support is prepared from a resin comprising the reactive groups (e.g. epoxy group, NHS group) as described in part (ii) of this section. In one embodiment, the solid support is prepared by contacting a resin comprising the reactive groups as described in part (ii) of this section with a ligand (e.g. amine) as described in part (iii) of this section. [00163] In one embodiment, the solid support binds with the vesicle, cell or viral particle at the first pH and release it at the second pH. In one embodiment, the solid support has a binding affinity to the vesicle, cell or viral particle at the first pH that is at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times higher than the binding affinity to the vesicle, cell or viral particle at the second pH. [00164] In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 50% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 60% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 70% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 80% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 90% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 95% at the first pH. [00165] In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 50% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 40% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 30% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an NAI-1540508512v1 -25- Attorney Docket No.14497-018-228 efficiency of less than 20% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 10% at the second pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of less than 5% at the second pH. [00166] In one embodiment, the solid support binds with the macromolecules at the first pH and release it at the second pH. In one embodiment, the solid support has a binding affinity to the macromolecules at the first pH that is at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times higher than the binding affinity to the vesicle, cell or viral particle at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of at least 50% at the first pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of at least 60% at the first pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of at least 70% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 80% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 90% at the first pH. In one embodiment, the solid support binds with the vesicle, cell or viral particle at an efficiency of at least 95% at the first pH. [00167] In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 50% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 40% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 30% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 20% at the first pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 10% at the second pH. In one embodiment, the solid support binds with the macromolecule at an efficiency of less than 5% at the second pH. [00168] In one embodiment, the solid support has an average pore size from about 10 nm to about 1000 nm. In one embodiment, the average pore size is from about 50 nm to about 800 nm. In one embodiment, the average pore size is from about 100 nm to about 600 nm. In one embodiment, the average pore size is from about 50 nm to about 200 nm. In one embodiment, the average pore size is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, or about 200 nm. (i)Preparation of Solid Support NAI-1540508512v1 -26- Attorney Docket No.14497-018-228 [00169] In one embodiment, the method further comprises a step of preparing the solid support. In one embodiment, the preparation comprises one or more steps as described in Section 5.3.2. [00170] In one embodiment, the solid support is prepared from a commercially available resin. In one embodiment, the solid support is prepared from a synthetic resin. In one embodiment, the solid support is prepared from a natural resin. [00171] In one embodiment, the solid support is prepared from a resin comprising at least one reactive group on the surface. [00172] In one embodiment, the solid support is prepared from a cation exchange (CEX) resin. In one embodiment, the CEX resin is a weak acid cation exchange resin. In one embodiment, the CEX resin comprises a carboxylic acid or carboxylate group. In one embodiment, the CEX resin is a strong acid cation exchange resin. [00173] In one embodiment, the CEX resin comprises one or more sulfoisobutyl group, sulfonate group, sulfoethyl group, sulfopropyl group, or carboxymethyl group. In one embodiment, the CEX resin comprises a sulphonate group. In one embodiment, the CEX resin is a commercially available CEX resin. Non-limiting examples of commercially available CEX resins include, those having a sulfoisobutyl group (e.g., Fractogel EMD SO3); a sulfonate group (e.g., SP Sepharose Fast Flow, Capto S, Macro-Prep High S); a sulfoethyl based group (e.g., Fractogel SE); a sulfopropyl based group (e.g., TSKGel SP 5PW, Poros HS-20, Poros HS-50); and a carboxymethyl based group (e.g., CM Sepharose Fast Flow, Macro-Prep CM, Toyopearl CM-650S, Toyopearl CM-650M , Toyopearl CM-650C). [00174] In one embodiment, the solid support is prepared from an anion exchange (AEX) resin. In one embodiment, the AEX resin is a weak base anion exchange resin. In one embodiment, the AEX resin comprises a tertiary amino group. In one embodiment, the AEX resin is a strong base anion exchange resin. In one embodiment, the AEX resin comprises a quaternary ammonium group. [00175] In one embodiment, the solid support is prepared from an AEX resin comprising one or more groups selected from a group consisting of diethylaminopropyl, diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, carboxymethyl, carboxylic acid, glutamic acid, aspartic acid, histidine, hydroxyl, phosphate, tertiary amines, quaternary amines, diethaminoethyl, dimethylaminoethyl, trimethylaminoethyl and amino acid. In one embodiment, the AEX resin is a commercially available AEX resin. Non-limiting examples of commercially available AEX resins include, Q SEPHAROSE™ FF, Q SEPHAROSE™ HP, Q SEPHAROSE™ BB, Q SEPHAROSE™ NAI-1540508512v1 -27- Attorney Docket No.14497-018-228 XL, DEAE SEPHAROSE™ FF, ANX SEPHAROSE™ 4FF low sub, ANX SEPHAROSE™ 4FF high sub, SOURCE™ 15Q, SOURCE™ 30Q, CAPTO™ Q, CAPTO™ DEAE, or CAPTO™ Q ImpRes, available from GE Healthcare; FRACTOGEL® EMD DEAE (M), FRACTOGEL® EMD TMAE (M), FRACTOGEL® EMD TMAE (S), FRACTOGEL® EMD TMAE Hicap (M), FRACTOGEL® EMD TMAE Medcap (M), ESHMUNO® Q or ESHMUNO® Q, available from Merck Millipore; TOYOPEARL® DEAE-650C, TOYOPEARL® DEAE-650M, TOYOPEARL® DEAE-650S, TOYOPEARL® SuperQ-650C, TOYOPEARL® SuperQ-650M, TOYOPEARL® SuperQ-650S, TOYOPEARL® QAE-550C, TOYOPEARL® GIGACAP® Q-650M, TOYOPEARL® Q-600C AR, TOYOPEARL® GIGACAP® DEAE-650M, TOYOPEARL® GIGACAP® Q-650S, TOYOPEARL® NH2-750F, TSKGEL® SuperQ-5PW , or TSKGEL® SuperQ-5PW), available from Tosoh Bioscience; MACRO-PREP®. [00176] In one embodiment, the solid support is prepared from a size-exclusion (SEC) resin. In one embodiment, the SEC resin is a commercially available size exclusion resin. Non-limiting examples of commercially available SEC resin include, Superdex (e.g., Superdex 30, Superdex 75, Superdex 200), Sephacryl (e.g., Sephacryl S-100, Sephacryl S-100, Sephacryl S-100, Sephacryl S- 400, Sephacryl S-500), Sephadex (e.g., Sephadex G-25, Sephadex G-50, Sephadex G-100), Superose (e.g., Superose 6, Superose 12), Sepharose (e.g., Sepharose 2B, Sepharose CL-2B, Sepharose 4B, Sepharose 4 Fast Flow, Sepharose CL-4B, Sepharose 6B, Sepharose 6 Fast Flow, Sepharose CL-6B), Fractogel (e.g., Fractogel EMD), Toyopearl (e.g., Toyopearl HW-40, Toyopearl HW-50, Toyopearl HW-55, Toyopearl HW-65, Toyopearl HW-75), or Bio-Gel (e.g., Bio-Gel P-2, Bio-Gel P-4, Bio-Gel P-6, Bio-Gel P-10, Bio-Gel P-30, Bio-Gel P-60, Bio-Gel P-100). In one embodiment, the SEC resin is Sepharose CL-4B. [00177] In one embodiment, the solid support is prepared from a mixed mode resin. In one embodiment, the solid support is prepared from an affinity resin. In one embodiment, the solid support is prepared from a pseudo affinity resin. In one embodiment, the solid support is prepared from a hydrophobic interaction resin. In one embodiment, the solid support is prepared from a hydrophobic charge induction resin. In one embodiment, the solid support is prepared from a combination of two or more types of resins. (ii)Reactive Group [00178] In one embodiment, the solid support is prepared from a resin comprising one or more reactive group. [00179] In one embodiment, the solid support is prepared from a resin comprising one or more NAI-1540508512v1 -28- Attorney Docket No.14497-018-228 reactive group selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, cyanogen bromide, a carbodiimide and lysine. [00180] In one embodiment, the solid support is prepared from a resin comprising an epoxy group. In one embodiment, the solid support is prepared from a resin comprising an NHS ester. In one embodiment, the solid support is prepared from a resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from a resin comprising an imidoester ester. In one embodiment, the solid support is prepared from a resin comprising an aldehyde. In one embodiment, the solid support is prepared from a resin comprising a carbonate. In one embodiment, the solid support is prepared from a resin comprising an anhydride. In one embodiment, the solid support is prepared from a resin comprising a sulfonyl chloride. In one embodiment, the solid support is prepared from a resin comprising an isothiocyanate. In one embodiment, the solid support is prepared from a resin comprising an isocyanate. In one embodiment, the solid support is prepared from a resin comprising an acyl azide. In one embodiment, the solid support is prepared from a resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from a resin comprising a carbodiimide. In one embodiment, the solid support is prepared from a resin comprising a lysine. [00181] In one embodiment, the solid support is prepared from an AEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from an AEX resin comprising an NHS ester. In one embodiment, the solid support is prepared from an AEX resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from an AEX resin comprising an imidoester ester. In one embodiment, the solid support is prepared from an AEX resin comprising an aldehyde. In one embodiment, the solid support is prepared from an AEX resin comprising a carbonate. In one embodiment, the solid support is prepared from an AEX resin comprising an anhydride. In one embodiment, the solid support is prepared from an AEX resin comprising a sulfonyl chloride. In one embodiment, the solid support is prepared from an AEX resin comprising an isothiocyanate. In one embodiment, the solid support is prepared from an AEX resin comprising an isocyanate. In one embodiment, the solid support is prepared from an AEX resin comprising an acyl azide. In one embodiment, the solid support is prepared from an AEX resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from an AEX resin comprising a carbodiimide. In one embodiment, the solid support is prepared from an NAI-1540508512v1 -29- Attorney Docket No.14497-018-228 AEX resin comprising a lysine. [00182] In one embodiment, the solid support is prepared from a CEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from a CEX resin comprising an NHS ester. In one embodiment, the solid support is prepared from a CEX resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from a CEX resin comprising an imidoester ester. In one embodiment, the solid support is prepared from a CEX resin comprising an aldehyde. In one embodiment, the solid support is prepared from a CEX resin comprising a carbonate. In one embodiment, the solid support is prepared from a CEX resin comprising an anhydride. In one embodiment, the solid support is prepared from a CEX resin comprising a sulfonyl chloride. In one embodiment, the solid support is prepared from a CEX resin comprising an isothiocyanate. In one embodiment, the solid support is prepared from a CEX resin comprising an isocyanate. In one embodiment, the solid support is prepared from a CEX resin comprising an acyl azide. In one embodiment, the solid support is prepared from a CEX resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from a CEX resin comprising a carbodiimide. In one embodiment, the solid support is prepared from a CEX resin comprising a lysine. [00183] In one embodiment, the solid support is prepared from an SEC resin comprising an epoxy group. In one embodiment, the solid support is prepared from an SEC resin comprising an NHS ester. In one embodiment, the solid support is prepared from an SEC resin comprising a Sulfo-NHS ester. In one embodiment, the solid support is prepared from an SEC resin comprising an imidoester ester. In one embodiment, the solid support is prepared from an SEC resin comprising an aldehyde. In one embodiment, the solid support is prepared from an SEC resin comprising a carbonate. In one embodiment, the solid support is prepared from an SEC resin comprising an anhydride. In one embodiment, the solid support is prepared from an SEC resin comprising a sulfonyl chloride. In one embodiment, the solid support is prepared from an SEC resin comprising an isothiocyanate. In one embodiment, the solid support is prepared from an SEC resin comprising an isocyanate. In one embodiment, the solid support is prepared from an SEC resin comprising an acyl azide. In one embodiment, the solid support is prepared from an SEC resin comprising a cyanogen bromide. In one embodiment, the solid support is prepared from an SEC resin comprising a carbodiimide. In one embodiment, the solid support is prepared from an SEC resin comprising a lysine. [00184] In one embodiment, the solid support is prepared from a weak acid CEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from a CEX resin NAI-1540508512v1 -30- Attorney Docket No.14497-018-228 comprising a carboxylic acid group and an epoxy group. In one embodiment, the solid support is prepared from a CEX resin comprising a carboxylate group and an epoxy group. In one embodiment, the CEX resin is a strong acid CEX comprising an epoxy group. [00185] In one embodiment, the solid support is prepared from a weak base AEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from a weak base AEX resin comprising a tertiary amino group and an epoxy group. In one embodiment, the solid support is prepared from a strong base AEX resin comprising an epoxy group. In one embodiment, the solid support is prepared from an AEX resin comprising a quaternary ammonium group and an epoxy group. [00186] In one embodiment, the solid support is prepared from a resin comprising epoxy groups at a density from about 100 µmol to about 2000 µmol per gram of the resin. In one embodiment, the solid support is prepared from a resin comprising epoxy groups at a density from about 100 µmol to about 2000 µmol per milliliter of the resin. [00187] In one embodiment, the solid support is prepared from an AEX resin comprising epoxy groups at a density from about 100 µmol to about 2000 µmol per gram of the resin. In one embodiment, the solid support is prepared from an CEX resin comprising epoxy groups at a density from about 100 µmol to about 2000 µmol per gram of the resin. In one embodiment, the solid support is prepared from a SEC resin comprising epoxy groups at a density from about 100 µmol to about 2000 µmol per gram of the resin. [00188] In one embodiment, the solid support is prepared from agarose comprising the reactive groups as described herein. In one embodiment, the solid support is prepared from polystyrene comprising the reactive groups as described herein. In one embodiment, the solid support is prepared from polymethacrylate comprising the reactive groups as described herein. In one embodiment, the solid support is prepared from polyacrylamide comprising the reactive groups as described herein. In one embodiment, the solid support is prepared from hydroxylated methacrylic polymer comprising the reactive groups as described herein. [00189] In one embodiment, the solid support is prepared from agarose comprising epoxy groups. In one embodiment, the solid support is prepared from polystyrene comprising epoxy groups. In one embodiment, the solid support is prepared from polymethacrylate comprising epoxy groups. In one embodiment, the solid support is prepared from polyacrylamide comprising epoxy groups. In one embodiment, the solid support is prepared from hydroxylated methacrylic polymer comprising epoxy groups. NAI-1540508512v1 -31- Attorney Docket No.14497-018-228 [00190] In one embodiment, the solid support is prepared from TOYOPEARL AF-Epoxy-650M. In one embodiment, the solid support is prepared from Epoxy-Activated Sepharose 6B. In one embodiment, the solid support is prepared from Epoxy-Activated Separopore® 4B-CL. In one embodiment, the solid support is prepared from Epoxy-Activated Agarose. In one embodiment, the solid support is prepared from Profinity™ Epoxide Resin. In one embodiment, the solid support is prepared from POROS™ 20 EP Epoxide Activated Resin. In one embodiment, the solid support is prepared from Praesto® Epoxy resin. In one embodiment, the solid support is prepared from Praesto® Epoxy resin. [00191] In one embodiment, the solid support is prepared from a commercially available resin, wherein resin is modified to increase the density of the reactive group by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%. [00192] In one embodiment, the solid support is prepared from a commercially available epoxy resin, wherein resin is modified to increase the density of the epoxy groups. In one embodiment, the modification comprises a step of contacting the resin with a compound comprising at least one epoxy group. In one embodiment, the compound comprises two epoxy groups. In one embodiment, the compound comprises three epoxy groups. In one embodiment, the compound has a molecular weight from 100 to 5000 g/mol, from 100 to 2500 g/mol, or from 100 to 1000 g/mol. In one embodiment, the compound is a diglycidyl ether. In one embodiment, the compound is 1,4- Bis(2,3-epoxypropyloxy)butane (other names: 1,4-Butanediol diglycidyl ether, or BDDE). In one embodiment, the compound is 2,2-Bis[4-(glycidyloxy)phenyl]propane (other name: bisphenol A diglycidyl ether). In one embodiment, the compound is neopentyl glycol diglycidyl ether. In one embodiment, the compound is 1,3-butanediol diglycidyl ether. In one embodiment, the compound is bis[4-(glycidyloxy)phenyl]methane (other name: bisphenol F diglycidyl ether). In one embodiment, the compound is N, N-Diglycidyl-4-glycidyloxyaniline. In one embodiment, the compound is poly(ethylene glycol) diglycidyl ether. In one embodiment, the compound is poly(propylene glycol) diglycidyl ether. [00193] In one embodiment, the reactive group is covalently connected to the resin via a linker. [00194] In one embodiment, the linker is a hydrophilic linker. In one embodiment, the linker is a hydrophobic linker. In one embodiment, the linker has a molecular weight less than 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol or less than 500 g/mol. In some embodiments, the linker NAI-1540508512v1 -32- Attorney Docket No.14497-018-228 is a linear, branched or cyclic alkylene group comprising 1 to 20 carbon atoms. In some embodiments, the linker further comprises one or more heteroatoms, such as oxygen, nitrogen, sulfur, silicon and halogen (e.g. fluorine, chlorine, bromine) atoms. In one embodiment, the linker is an oligomeric polyethylene glycol (PEG) group with 2 to 48 repeating units. [00195] In one embodiment, the epoxy group is capable of reacting with the ligand via a ring- opening reaction to form an ionizable moiety and a hydroxyl group. [00196] In one embodiment, the reactive group has a density of from about 100 µmol to about 2000 µmol per gram of the resin. In one embodiment, the reactive group has a density of from about 500 µmol to about 1000 µmol per gram of the resin. In one embodiment, the reactive group has a density of about 100 µmol, 200 µmol, 300 µmol, 400 µmol, 500 µmol, 600 µmol, 700 µmol, 800 µmol, 900 µmol or 1000 µmol per gram of the resin. (iii)Ligand [00197] In one embodiment, the reactive group on the resin can react with a ligand, wherein the ligand is an amine, an alcohol, a thiol, or a carboxylic acid. In one embodiment, the ligand is a thiol. In one embodiment, the ligand is a carboxylic acid. In one embodiment, the ligand is an amine. In one embodiment, the ligand is a secondary amine. [00198] In one embodiment, the ligand has a formula of R1-NH-R2, wherein R1, R2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the atoms they are attached to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is optionally substituted. [00199] In one embodiment, R1 is alkyl, and R2 is alkyl. In one embodiment, R1 is alkyl, and R2 is hydroxyalkyl. In one embodiment, R1 is hydroxyalkyl, and R2 is hydroxyalkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C12 alkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C6 alkyl. In one embodiment, R1 is C1-C6 alkyl, and R2 is C1-C6 alkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C12 hydroxyalkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C6 hydroxyalkyl. In one embodiment, R1 is C1-C6 alkyl, and R2 is C1-C6 hydroxyalkyl. In one embodiment, R1 is C1-C12 hydroxyalkyl, and R2 is C1-C12 hydroxyalkyl. In one embodiment, R1 is C1-C12 hydroxyalkyl, and R2 is C1-C6 hydroxyalkyl. In one embodiment, R1 is C1-C6 hydroxyalkyl, and R2 is C1-C6 hydroxyalkyl. [00200] In one embodiment, R1 and R2 together with the atoms they are attached to form a 5- membered heterocyclyl. In one embodiment, R1 and R2 together with the atoms they are attached to form a 6-membered heterocyclyl. In one embodiment, the heterocyclyl contains only one ring NAI-1540508512v1 -33- Attorney Docket No.14497-018-228 nitrogen atom. In one embodiment, the heterocyclyl contains two ring nitrogen atoms. In one embodiment, the heterocyclyl contains a ring nitrogen atom and a ring oxygen atom. [00201] In one embodiment, R1 is unsubstituted. In one embodiment, R1 is substituted. In one embodiment, R1 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano. In one embodiment, R1 is substituted with one or more halogen. In one embodiment, R1 is substituted with one or more hydroxyl. In one embodiment, R1 is substituted with one or more oxo. In one embodiment, R1 is substituted with one or more phenyl. [00202] In one embodiment, R2 is unsubstituted. In one embodiment, R2 is substituted. In one embodiment, R2 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano. In one embodiment, R2 is substituted with one or more halogen. In one embodiment, R2 is substituted with one or more hydroxyl. In one embodiment, R2 is substituted with one or more oxo. In one embodiment, R2 is substituted with one or more phenyl. [00203] In one embodiment, the ligand has a pKa of from about 7 to about 16. In one embodiment, the ligand has a pKa of from about 8 to about 11. In one embodiment, the ligand has a pKa of from about 8.5 to about 10.5. In one embodiment, the ligand has a pKa of about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12. [00204] In one embodiment, the amine has a pKa of from about 7 to about 16. In one embodiment, the amine has a pKa of from about 8 to about 11. In one embodiment, the amine has a pKa of from about 8.5 to about 10.5. In one embodiment, the amine has a pKa of about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12. [00205] In one embodiment, the pKa of amine is determined by an acid-base titration experiment. In one embodiment, acid-base titration experiment uses hydrochloric acid. [00206] In one embodiment, the ligand is selected from a group consisting of diethylamine, diethanolamine, dimethylethanolamine, diisopropylamine, isopropylethylamine, diisopropanolamine, 2-methylethanolamine, 2-ethylethanolamine, 2-(isopropylamino)ethanol, 2- (butylamino)ethanol, 2-benzylaminoethanol, piperidine, morpholine, 2,6-dimethyl-morpholine, 4- (2-chloroethyl)morpholine, 4-morpholinecarbonyl chloride, piperazine, 4-methylpiperazine and 4- hydroxyethylpiperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide; wherein each piperidine, morpholine, piperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide is optionally substituted. NAI-1540508512v1 -34- Attorney Docket No.14497-018-228 [00207] In one embodiment, the ligand is diethylamine. In one embodiment, the ligand is diethanolamine. In one embodiment, the ligand is dimethylethanolamine. In one embodiment, the ligand is diisopropylamine. In one embodiment, the ligand is isopropylethylamine. In one embodiment, the ligand is diisopropanolamine (DIPA). In one embodiment, the ligand is 2- methylethanolamine. In one embodiment, the ligand is 2-ethylethanolamine. In one embodiment, the ligand is 2-(isopropylamino)ethanol. In one embodiment, the ligand is 2-(butylamino)ethanol. In one embodiment, the ligand is 2-benzylaminoethanol. In one embodiment, the ligand is 2,6- dimethyl-morpholine. In one embodiment, the ligand is morpholine. In one embodiment, the ligand is 4-(2-chloroethyl)morpholine. In one embodiment, the ligand is 4-morpholinecarbonyl chloride. In one embodiment, the ligand is piperazine. In one embodiment, the ligand is 4- methylpiperazine. In one embodiment, the ligand is 4-hydroxyethylpiperazine. In one embodiment, the ligand is pyrrole. In one embodiment, the ligand is triazole. In one embodiment, the ligand is succinimide. In one embodiment, the ligand is phthalimide. In one embodiment, the ligand is glutarimide. [00208] In one embodiment, the ligand is unsubstituted. In one embodiment, the ligand is substituted. In one embodiment, the ligand is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano. In one embodiment, the ligand is substituted with one or more halogen. In one embodiment, the ligand is substituted with one or more hydroxyl. In one embodiment, the ligand is substituted with one or more C1-C6 alkyl. In one embodiment, the ligand is substituted with one or more C1-C6 alkoxy. 5.2.5 Ionizable Moiety [00209] In one embodiment, the solid support comprises an ionizable moiety. In one embodiment, the preparation of the solid support further comprises a step of contacting a reactive group with a ligand to form an ionizable moiety. [00210] In one embodiment, provided herein is a method of purifying vesicles, cells, or viral particles from a sample comprising the steps of: a. providing a resin, wherein the resin comprises at least one reactive group, wherein the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and a lysine; NAI-1540508512v1 -35- Attorney Docket No.14497-018-228 b. preparing a solid support by contacting the resin with a ligand to form an ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample at a first pH at which the vesicles, cells, or viral particles bind to the solid support; and d. eluting the vesicles, cells, or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00211] In one embodiment, provided herein is a method of purifying vesicles, cells, or viral particles from a sample comprising the steps of: a. providing a resin, wherein the resin comprises at least one reactive group, wherein the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and a lysine; b. preparing a solid support by contacting the resin with a ligand to form an ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample in a loading buffer; and d. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [00212] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. providing a resin, wherein the resin comprises at least one reactive group, wherein the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and a lysine; b. preparing a solid support by contacting the resin with a ligand to form an ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample at a first pH at which the macromolecules bind to the solid support; and d. eluting the macromolecules from the solid support at a second pH, wherein the second pH is higher than the first pH. NAI-1540508512v1 -36- Attorney Docket No.14497-018-228 [00213] In one embodiment, the ionizable moiety comprises one or more tertiary amino groups. In one embodiment, the ionizable moiety comprises a monoamine. In one embodiment, the ionizable moiety does not comprises a diamine. [00214] In one embodiment, the ionizable moiety , wherein R1, R2 are each independently alkyl, hydroxyalkyl, (alkylamino)
Figure imgf000039_0001
alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the atoms they are attached to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is optionally substituted; wherein the attachment to the left is to the linker. [00215] In one embodiment, the ionizable moiety is -N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted. In one embodiment, each alkyl is independently optionally substituted with one or more halogen or OH. [00216] In one embodiment, the ionizable moiety , wherein each alkyl is independently optionally substituted with one or
Figure imgf000039_0002
In one embodiment, the ionizable , wherein X is C(Ra)2, NRa, O, or S; wherein Ra, R3 and R4 are each independently
Figure imgf000039_0003
or C1-C6 alkyl; wherein the alkyl is optionally substituted with one or more halogen or hydroxyl. [00217] In one embodiment, the ionizable moiety is selected from a group consisting of: OH OH ,
Figure imgf000039_0004
NAI-1540508512v1 -37- Attorney Docket No.14497-018-228 ; wherein the attachment to the left is to the linker.
Figure imgf000040_0001
embodiment, the ionizable moiety has a pKa from about 6.5 to about 9. In one embodiment, the ionizable moiety has a pKa from about 7.6 to about 8.8. In one embodiment, the ionizable moiety has a pKa of about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9 or about 9. In one embodiment, the ionizable moiety has a pKa of about 7.6. In one embodiment, the ionizable moiety has a pKa of about 8.2. [00219] In one embodiment, the pKa of the ionizable moiety is determined by an acid-base titration experiment. In one embodiment, acid-base titration experiment uses hydrochloric acid. In one embodiment, the pKa of the ionizable moiety is estimated by using analogous molecules bearing the same or similar functional groups. In one embodiment, the pKa of the ionizable moiety is estimated by measuring the pKa of R5-L-NR1R2 in water, wherein R5 is hydrogen, hydroxyl, C1- C3 alkyl, C1-C3 alkoxyl, or epoxy. In one embodiment, the pKa of the ionizable moiety is estimated by measuring the pKa of R6-NR1R2 in water, wherein R6 is C1-C3 alkyl, or C1-C3 alkoxyl. [00220] In one embodiment, the ionizable moiety has a density of from about 50 µmol to about 1000 µmol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 50 µmol to about 800 µmol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 100 µmol to about 800 µmol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 250 µmol to about 800 µmol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 300 µmol to about 800 µmol per gram of the solid support. In one embodiment, the ionizable moiety has a density of from about 400 µmol to about 800 µmol per gram of the solid support. In one embodiment, the ionizable moiety has a density of about 50 µmol, about 100 µmol, about 150 µmol, about 200 µmol, about 250 µmol, about 300 µmol, about 350 µmol, about 400 µmol, about 450 µmol, about 500 µmol, about 550 µmol, about 600 µmol, about 650 µmol, about 700 µmol, about 750 µmol, or about 800 µmol per gram of the solid support. [00221] In one embodiment, the ionizable moiety is capable of binding and releasing the vesicles, cells or viral particles in a pH-dependent manner. NAI-1540508512v1 -38- Attorney Docket No.14497-018-228 [00222] In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles when the pH is below the pKa of the ionizable moiety. In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles at the first pH. In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles when the pH is at or below 7.4. In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles when the pH is between 4.5 to 7.4. [00223] In one embodiment, the ionizable moiety releases the vesicles, cells or viral particles when the pH is above the pKa of the ionizable moiety. In one embodiment, the ionizable moiety releases the vesicles, cells or viral particles at the second pH. In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles when the pH is at or above 7.4. In one embodiment, the ionizable moiety binds with the vesicles, cells or viral particles when the pH is between 7.4 to 9. [00224] In one embodiment, the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the first pH. In one embodiment, the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 80% at the first pH. [00225] In one embodiment, the solid support is capable of releasing the vesicles, cells, or viral particles at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the second pH. In one embodiment, the solid support is capable of releasing the vesicles, cells, or viral particles at an efficiency of at least 50% at the second pH. In one embodiment, the solid support is capable of releasing the vesicles, cells, or viral particles at an efficiency of at least 80% at the second pH. [00226] In one embodiment, the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 80% at the first pH, and releasing the vesicles, cells, or viral particles at an efficiency of at least 80% at the second pH. In one embodiment, the solid support is capable of binding the vesicles, cells, or viral particles at an efficiency of at least 80% at the first pH, and releasing the vesicles, cells, or viral particles at an efficiency of at least 50% at the second pH. [00227] In one embodiment, the ionizable moiety is capable of binding and releasing the macromolecules in a pH-dependent manner. In one embodiment, the ionizable moiety binds with the macromolecules when the pH is below the pKa of the ionizable moiety. In one embodiment, the NAI-1540508512v1 -39- Attorney Docket No.14497-018-228 ionizable moiety binds with the macromolecules at the first pH. In one embodiment, the ionizable moiety binds with the macromolecules when the pH is at or below 7.4. In one embodiment, the ionizable moiety binds with the macromolecules when the pH is between 4.5 to 7.4. In one embodiment, the ionizable moiety releases the macromolecules when the pH is above the pKa of the ionizable moiety. In one embodiment, the ionizable moiety releases the macromolecules at the second pH. In one embodiment, the ionizable moiety binds with the macromolecules when the pH is at or above 7.4. In one embodiment, the ionizable moiety binds with the macromolecules when the pH is between 7.4 to 9. [00228] In one embodiment, the solid support is capable of binding the macromolecules at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the first pH. In one embodiment, the solid support is capable of binding the macromolecules at an efficiency of at least 80% at the first pH. In one embodiment, the solid support is capable of releasing the macromolecules at an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 99.5% at the second pH. In one embodiment, the solid support is capable of releasing the macromolecules at an efficiency of at least 50% at the second pH. In one embodiment, the solid support is capable of releasing the macromolecules at an efficiency of at least 80% at the second pH. In one embodiment, the solid support is capable of binding the macromolecules at an efficiency of at least 80% at the first pH, and releasing the macromolecules at an efficiency of at least 80% at the second pH. 5.2.6 Vesicles [00229] In one embodiment, provided herein is a method of purifying vesicles. [00230] In one embodiment, the vesicle has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm. In one embodiment, the nanovesicle has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. [00231] In one embodiment, the vesicle is negatively charged at the first pH. In one NAI-1540508512v1 -40- Attorney Docket No.14497-018-228 embodiment, the vesicle is negatively charged at the second pH. In one embodiment, the vesicle is negatively charged at both the first pH and the second pH. In one embodiment, the vesicles are purified by an increase in salt concentration or conductivity. [00232] In one embodiment, the vesicle is a nanovesicle. In one embodiment, the vesicle is a microvesicle. In one embodiment, the vesicle is an artificial nanovesicle (e.g. synthetic nanovesicles). In one embodiment, the vesicle is a liposome. In one embodiment, the vesicle is a lipid nanoparticle. In one embodiment, the vesicle is an extracellular vesicle. In one embodiment, the vesicle is an exosome. In one embodiment, the vesicle is a hybrid nanovesicle. In one embodiment, the vesicle is hybridosome. [00233] In one embodiment, the vesicle as used herein comprises fusogenic lipid that enhances the cellular uptake of the vesicle through cell fusion or endocytosis. Without bound by the theory, a fusogenic lipid work by undergoing a change in structure or charge at low pH (e.g. pH of about 5.5), when compared to their charge or structure at high pH (e.g. pH of about 7.4). Fusogenic lipids may be anionic lipids, neutral lipids or pH sensitive lipids. In some embodiments, when the temperature is raised above the phase transition temperature, for example 25° C., the fusogenic lipid undergoes a change in structure such that it adopts a hexagonal or cone-forming structure. Additional fusogenic lipids of this type are known in the art and may be used in the vesicles as described herein. The fusogenic lipids may also include those referred to as “cone-forming” lipids in the art. Many fusogenic lipids include amphipathic lipids generally having a hydrophobic moiety and a polar head group, and can form vesicles in aqueous solution. [00234] In one embodiment, the vesicle as used herein comprises one or more payloads. In one embodiment, the payload is a biologically active molecule. Non-limiting examples of payload that can be included in vesicles include polypeptides (e.g, an antibody, an antigen, an adjuvant, a ligand, a receptor, an immune modulator, and or any fragment thereof), a polynucleotide, a viral particle, a small molecule, or any combination thereof. Payloads that can be introduced into a vesicle, such as an exosome, or its producer cell include nucleotides, nucleic acids (e.g, DNA or mRNA molecules that encode a polypeptide, or RNA molecules such as miRNA, dsDNA, IncRNA, siRNA, antisense oligonucleotide, or combinations thereof), amino acids (e.g, amino acids comprising a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g, enzymes, antibodies, monoclonal antibodies), lipids (e.g. phospholipids), carbohydrates, and small molecules (e.g, small molecule drugs and toxins). [00235] In one embodiment, the method further comprises a step of preparing the vesicles as NAI-1540508512v1 -41- Attorney Docket No.14497-018-228 described herein. (i)Nanovesicle [00236] In one embodiment, provided herein is a method of purifying nanovesicle. In one embodiment, the nanovesicle has an average diameter between 1 nm to 1,000 nm. Techniques to determine the size of nanovesicle include, but are not limited to Dynamic Light Scattering (DLS), Electron Microscope (EM), Small-angle X-ray scattering, atomic force microscopy, and nanoparticle tracking analysis. In one embodiment, the size of nanovesicle is determined by DLS. Nanovesicles as used herein may comprise one or more payloads (for examples, as described in Section 5.2.6), such as polypeptides, or nucleic acids, and may further comprise a targeting moiety or other molecules (e.g. a detecting label or radioisotope). [00237] In one embodiment, the present disclosure relates to a plurality of nanovesicles, e.g. a population of nanovesicles which may comprise thousands, millions, billions or even trillions of nanovesicles. In one embodiment, nanovesicles are present in concentrations such as 105, 108, 1010, 1011, 1012, 1013, 1014, 1015, 1018, 1025, or 1030 particles per unit of volume (for instance per mL or per gram), or any other number larger, smaller or anywhere in between. Individual nanovesicles when present in a plurality constitute a nanovesicle population. The present disclosure pertains both to individual nanovesicles and populations comprising nanovesicles. [00238] In one embodiment, the method further comprises a step of preparing the nanovesicles as described herein. (ii)Liposome [00239] In one embodiment, provided herein is a method of purifying liposome. Liposomes as used herein comprise lipophilic materials, such as lipids, that isolate the aqueous interior from an aqueous exterior. In one embodiment, liposomes as used herein are multilamellar vesicles (MLVs) having more than one lamellar phase of lipid bilayers. In one embodiment, a liposome as used herein is a unilamellar vesicle having one single layer of lipid membrane. In one embodiment, the unilamellar vesicles are large unilamellar vesicles (LUVs). In one embodiment, the LUVs as used herein have a diameter of between about 0.1 to 1 µm. In one embodiment, the unilamellar vesicles are small unilamellar vesicles (SUVs). In one embodiment, the “SUVs” as used herein have a diameter of less than 100 nm. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Without bound by the theory, since liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal NAI-1540508512v1 -42-
Figure imgf000044_0001
Attorney Docket No.14497-018-228 bilayer fuses with bilayer of the cellular membranes, thereby facilitating the delivery of therapeutic payloads on or inside the liposome. In one embodiment, the liposome used herein is SUV. In some embodiment, the liposome used herein is MLV. In one embodiment, the liposome used herein comprises one or more fusogenic lipids (for example, as in Section 5.2.6). In one embodiment, the liposome used herein comprises one or more payloads (for example, as in Section 5.2.6) [00240] In one embodiment, the liposome has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm. In one embodiment, the liposome has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. [00241] In one embodiment, the liposome is a neutral liposome. In one embodiment, the liposome is an anionic liposome. In one embodiment, the liposome is a cationic liposome. A cationic liposome as used herein has a positive charge at physiological pH. The cationic liposomes can also comprise co-lipids that are negatively charged or neutral, so long as the net charge of the liposome is positive at physiological pH. In one embodiment, cationic lipids are present in the cationic liposome at from about 10 mole % to about 100 mole %, from about 20 mole % to about 80 mole % and, or from about 20 mole % to about 60 mole % of total liposomal lipid. In one embodiment, the cationic liposome as used herein comprise neutral lipids at a concentration from about 0 mole % to about 90 mole %, from about 20 mole % to about 80 mole %, or from about 40 mole % to about 80 mole % of the total liposomal lipid. In one embodiment, the cationic liposome as used herein comprise anionic lipids at a concentration from about 0 mole % to about 49 mole %, from about 0 mole % to about 40 mole %, or from about 0 mole % to about 20 mole % of the total liposomal lipid. In one embodiment, the cationic liposome comprises a cationic lipid and the neutral lipid at a 1:1 molar ratio. In one embodiment, the cationic lipid comprises DOTAP. In one embodiment, the cationic lipid comprises cholesterol, or its derivatives. In one embodiment, the cationic liposome further comprises lipid-PEG conjugates. [00242] In one embodiment, the method further comprises a step of preparing the liposomes as described herein. NAI-1540508512v1 -43- Attorney Docket No.14497-018-228 [00243] In one embodiment, the payload comprises mRNA. In one embodiment, wherein the payload is a negatively charged molecule (e.g., nucleic acid), and the lipid components of the LNP comprise at least one cationic lipid or ionizable lipid. Without being bound by the theory, it is contemplated that the cationic lipid or ionizable lipid can interact with the negatively charged payload molecules and facilitates incorporation and/or encapsulation of the payload into the LNP during LNP formation. (iii) Extracellular Vesicle [00244] In one embodiment, provided herein is a method of purifying extracellular vesicle (EV). EVs as used herein can be derived from a living or dead organism, explanted tissues or organs, or cultured cells. In one embodiment, the EV is a microvesicle. In one embodiment, the EV is an apoptotic body. In one embodiment, the EV is a fusosome. In one embodiment, the EV is an outer membrane vesicle produced by gram negative bacteria. [00245] In one embodiment, the EV is exosome. In one embodiment, the exosome is an engineered exosome. In one embodiment, the exosome comprises one or more payloads (for example, as in Section 5.2.6). Said payload can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, or combinations thereof. Exosome payloads may be located within the internal space of the exosome. Exosome payload may be in contact with the exterior or interior surface of the exosome, for example through a covalent bond or a non-covalent bond. The phospholipid bilayer of the EV or provided herein may comprise one or more transmembrane proteins, wherein a portion of the one or more transmembrane membrane proteins is located within the internal space of the exosome. [00246] In one embodiment, the exosome has a diameter from about 20 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, or more preferably from about 100 nm to about 200 nm. In one embodiment, the exosome has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm. [00247] In one embodiment, the method further comprises a step of preparing the EVs as NAI-1540508512v1 -44- Attorney Docket No.14497-018-228 described herein. (iv)Hybrid Nanovesicle [00248] In one embodiment, the vesicle is a hybrid nanovesicle. A hybrid nanovesicle as provided herein can be derived from any mixture of two or more vesicles, including vesicles as described in Section 5.2.6(i), 5.2.6(ii), 5.2.6, or 5.2.6(iii). A hybrid nanovesicle as used herein may derive from the fusing of two, three, four, five, six, seven, eight, nine or ten different nanovesicles. In one embodiment, the hybrid nanovesicle can further fuse with another nanovesicle to form a new hybrid nanovesicle. [00249] In one embodiment, the hybrid nanovesicle is derived from the fusing of an extracellular vesicle and a liposome. In one embodiment, the hybrid nanovesicle is derived from the fusing of two extracellular vesicles of different cell sources, such as the fusion of a platelet-derived extracellular vesicle and a macrophage-derived extracellular vesicle. In one embodiment, the hybrid nanovesicle is derived from the fusing of two exosomes of different cell sources. In one embodiment, the hybrid nanovesicle is derived from the fusing of an extracellular vesicle and a lipid nanoparticle. In one embodiment, the hybrid nanovesicle is derived from the fusing of an exosome and a lipid nanoparticle. In one embodiment, the hybrid nanovesicle is derived from the fusing of an exosome and a liposome. In one embodiment, the hybrid nanovesicle is hybridosome. [00250] In one embodiment, the method further comprises a step of preparing the hybrid nanovesicles as described herein. (v)Hybridosome [00251] In one embodiment, provided herein is a method of purifying hybridosome (e.g., hybridosome described in WO2015/110957, U.S. Patent No.10,561,610, U.S. Patent No. 11,484,500, the entirely of each of which is incorporated herein by reference). In one embodiment, the hybridosome used herein comprises one or more fusogenic lipids (for example, as in Section 5.2.6). In one embodiment, the hybridosome comprises one or more payloads (for example, as in Section 5.2.6). In one embodiment, the payload is nucleic acid. In one embodiment, the payload is DNA. In one embodiment, the payload is RNA, or preferably mRNA. In one embodiment, the payload is polypeptide. In one embodiment, the payload is an antibody. In one embodiment, the payload is an enzyme. [00252] In one embodiment, the hybridosome results from the fusing of at least one exosome and at least one lipid nanoparticle. In one embodiment, the hybridosome results from the fusing of 1 to NAI-1540508512v1 -45- Attorney Docket No.14497-018-228 10 exosomes with one lipid nanoparticle. In one embodiment, the hybridosome results from the fusing of 1 to 10 lipid nanoparticles with one exosome. In one embodiment, the hybridosome results from the fusing of one exosome and one lipid nanoparticle. In one embodiment, the hybridosome results from the fusing of one exosome comprising payloads, and one lipid nanoparticle comprising fusogenic lipids. In one embodiment, the hybridosome results from the fusing of one lipid nanoparticle comprising payload, and one exosome comprising fusogenic lipid. In one embodiment, the hybridosome results from the fusing of one exosome comprising payload, and one lipid nanoparticle comprising fusogenic lipid. [00253] In one embodiment, the hybridosome comprises one or more detectable label. In one embodiment, the hybridosome comprises a gene encoding one or more detectable label. In one embodiment, the detectable label is a fluorophore. In one embodiment, the detectable label is a fluorescent lipid. In one embodiment, the detectable label is a fluorescent dye. In one embodiment, the detectable label is a fluorescent protein. In one embodiment, the hybridosome comprises a gene encoding fluorescent protein. In one embodiment, the detectable label is green fluorescent protein (GFP) or red fluorescent protein (RFP). In one embodiment, the hybridosome comprises a gene encoding luciferase. In one embodiment, the detectable label is a luciferase. In one embodiment, the detectable label is a radioisotope. [00254] In one embodiment, the method further comprises a step of preparing the hybridosome as described herein. In one embodiment, the hybridosome is prepared by fusing one or more lipid nanoparticle with one or more exosome. [00255] In one embodiment, provided herein is a method of purifying hybridosome from a sample comprising the steps of: a. preparing the hybridosome by fusing one or more LNP with one or more exosome; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00256] In one embodiment of purifying hybridosome, the fusing is performed at an LNP/exosome ratio of from 5:1 to 1:1. In one embodiment, the LNP/exosome ratio is from 2:1 to 1:1. In one embodiment, the LNP/exosome ratio is 1, 1.01, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, NAI-1540508512v1 -46- Attorney Docket No.14497-018-228 1.9, or 2.0. [00257] In one embodiment of purifying hybridosome, the fusing is performed at a pH below 6.5. In one embodiment, the fusing is performed at a pH between 2 and 6.5. In one embodiment, the fusing is performed at a pH between 3 and 6. In one embodiment, the fusing is performed at a pH between 4 and 6. In one embodiment, the fusing is performed at pH of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or 6.5. In one embodiment, the fusing is performed at pH of 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6. In one embodiment, the fusing is performed at pH of 5.5. [00258] In one embodiment of purifying hybridosome, the fusing is performed in an acidic buffer. In one embodiment, the acidic buffer has a pH between below 6.5. In one embodiment, the acidic buffer has a pH of 5.5. In one embodiment, the acidic buffer is a MES buffer. In one embodiment, the acidic buffer further comprises one or more salts. In one embodiment, the acidic buffer further comprises NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, and any combination thereof. In one embodiment, the acidic buffer comprises NaCl and KCl. In one embodiment, the acidic buffer is a MES buffer comprising NaCl and KCl. In one embodiment, the acidic buffer has a salt concentration from about 5 mM to about 1 M. In one embodiment, the acidic buffer has a conductivity from about 1 mS/cm to about 100 mS/cm. [00259] In one embodiment of purifying hybridosome, the fusing is performed at a temperature between 0 °C and 60 °C. In one embodiment, the fusing is performed at a temperature between 10 °C and 50 °C. In one embodiment, the fusing is performed at a temperature between 20 °C and 40 °C. In one embodiment, the fusing is performed at 37 °C. [00260] In one embodiment of purifying hybridosome, the fusing process takes from 5 min to 24 hours. In one embodiment, the fusing process takes from 5 min to 6 hours. In one embodiment, the fusing process takes from 20 min to 3 hours. [00261] In one embodiment of purifying hybridosome, the fusing is performed at a temperature between 20 °C and 40 °C for a period of from 5 min to 24 hours in an acid buffer. 5.2.7 Cells [00262] In one embodiment, provided herein is a method of purifying cells. In one embodiment, the cell is negatively charged at the first pH. In one embodiment, the cell is negatively charged at the second pH. In one embodiment, the cell is negatively charged at both the first pH and the second pH. In one embodiment, the cells are purified by an increase in salt concentration or conductivity. NAI-1540508512v1 -47- Attorney Docket No.14497-018-228 [00263] In one embodiment, the cell is mammalian cell. In one embodiment, the cell is human cell. In one embodiment, the cell is engineered human cell. In one embodiment, the cell is animal cell. In one embodiment, the cell is mouse cell. In one embodiment, the cell is stem cell. [00264] In one embodiment, the cell is a source cell of extracellular vesicle (EV). Source cells of EV may be selected from a wide range of cells and cell lines which may grow in suspension or adherent culture or being adapted to suspension growth. Generally, EVs may be derived from essentially any cell source, including a primary cell source or an immortalized cell line. The EV source cells may be any embryonic, fetal, and adult somatic stem cell types, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method. The source cell may be allogeneic, autologous, or xenogeneic in nature. The cell may be from a subject himself or from an unrelated, matched or unmatched donor. In one embodiment, the source cells are allogeneic cells. In one embodiment, the source cells are human umbilical cord endothelial cells (HUVECs). In one embodiment, the source cells are human embryonic kidney (HEK) cells (e.g. HEK293 cells, HEK293T cells, serum free HEK293 cells, adherent or suspension HEK293 cells). In one embodiment, the source cells are CHO cells, BHK cells, PER.C6 cells, Vero cells, HeLa cells, PC 12 cells, or Sf9 cells. In one embodiment, the source cells are endothelial cell lines (e.g. microvascular or lymphatic endothelial cells, erythrocytes, erythroid progenitors, chondrocytes). In one embodiment, the source cells are amnion cells, amnion epithelial (AE) cells, any cells obtained through amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, or epithelial cells. [00265] In one embodiment, the cell is mesenchymal stem cell (MSC). In one embodiment, the cell is bone marrow MSC (BM-MSC). In one embodiment, the cell is human BM-MSC. In one embodiment, the cell is mouse BM-MSC. In one embodiment, the cell is umbilical cord mesenchymal stem cells (UC-MSCs). In one embodiment, the cell is adipose tissue-derived mesenchymal stem cells (AD-MSCs). In one embodiment, the cell is embryonic mesenchymal stem cells (ES-MSCs). [00266] In one embodiment, the cell is immune cell. In one embodiment, the cell is macrophage. In one embodiment, the cell is macrophage dendritic cell (DC). In one embodiment, the cell is T cell. In one embodiment, the cell is natural killer (NK) cell. [00267] In one embodiment, the cell is blood cell. In one embodiment, the cell is red blood cell. In one embodiment, the cell is human red blood cell. In one embodiment, the cell is platelet. [00268] In one embodiment, the cell is neural cell. In one embodiment, the cell is neuron, NAI-1540508512v1 -48- Attorney Docket No.14497-018-228 astrocyte, or microglia. 5.2.8 Viral Particles [00269] In one embodiment, provided herein is a method of purifying viral particles (other names: virus particle, or virus-like particle). In one embodiment, the viral particle is negatively charged at the first pH. In one embodiment, the viral particle is negatively charged at the second pH. In one embodiment, the viral particle negatively charged at both the first pH and the second pH. In one embodiment, the viral particles are purified by an increase in salt concentration or conductivity. [00270] In one embodiment, provided herein is a method of purifying viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is - N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c. eluting the viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [00271] In one embodiment, the viral particle comprises live virus. In one embodiment, the viral particle comprises fractions of virus (e.g. virus-like particles, mammalian retrovirus-like protein such as Arc or Peg10). In one embodiment, the viral particle comprises clusters of virus. In one embodiment, the viral particle is infectious (e.g. capable of transferring nucleic acid contained in the particle into a cell upon contacting with the cell). [00272] In one embodiment, the viral particle is enveloped viral particle. In one embodiment, the viral particle is a restrovirus. In one embodiment, the viral particle is lentiviral particle. In one embodiment, the viral particle is herpes simplex particle. In one embodiment, the lentiviral particle comprises a lipid-bilayer envelope. In one embodiment, the lentiviral particle comprises an RNA genome. In one embodiment, the lentiviral particle is capable of invading a target host cell. In one embodiment, the viral particle is adenoviral particle. In one embodiment, the viral particle is adeno- associated viral particle. In one embodiment, the viral particle is retroviral particle. [00273] In one embodiment, the viral particle is lentiviral particle produced from yeast. In one embodiment, the viral particle is lentiviral particle produced from bacteria. In one embodiment, the NAI-1540508512v1 -49- Attorney Docket No.14497-018-228 viral particle is lentiviral particle produced from mammalian cells. [00274] In one embodiment, the viral particle has a diameter from about 10 nm to about 200 nm. In one embodiment, the viral particle has a diameter of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, or about 200 nm. 5.2.9 Macromolecules [00275] In one embodiment, provided herein is a method of purifying macromolecules. [00276] In one embodiment, the macromolecule is a synthetic polymer. In one embodiment, the macromolecule is a biopolymer. Non-limiting examples of macromolecules that can be purified using the methods include polypeptides (e.g. enzyme, antibody, or monoclonal antibody), polysaccharide (e.g. galactogen, or inulin), and polynucleotide (e.g. DNA, or RNA). In one embodiment, the macromolecule is a monoclonal antibody. In one embodiment, the macromolecule is a nucleic acid. In one embodiment, the macromolecule is a DNA. In one embodiment, the macromolecule is RNA. [00277] In one embodiment, the macromolecule has a molecular weight from 5 kDa to 500 kDa. In one embodiment, the macromolecule has a molecular weight from 20 kDa to 400 kDa. In one embodiment, the macromolecule has a molecular weight from 50 kDa to 350 kDa. 5.2.10 Sample [00278] In one embodiment, the method removes at least one impurity from a sample. In one embodiment, the sample comprises at least one impurity that is a small molecule, a macromolecule, a different vesicle, a different cell, or a different viral particle. In one embodiment, the impurity to be removed is a small molecule. In one embodiment, the impurity to be removed is a lipid. In one embodiment, the impurity to be removed is a metal cation. In one embodiment, the impurity to be removed is a macromolecule. In one embodiment, the impurity to be removed is nucleic acid. In one embodiment, the impurity to be removed is DNA. In one embodiment, the impurity to be removed is RNA. In one embodiment, the impurity to be removed is a cell. In one embodiment, the impurity to be removed is a cell organelle. In one embodiment, the impurity to be removed is a cell membrane. In one embodiment, the impurity to be removed is a viral particle. [00279] In one embodiment, at least one impurity is removed by a change in pH from loading to elution. In one embodiment, at least one impurity is removed by a change in salt concentration or conductivity from loading to elution. NAI-1540508512v1 -50- Attorney Docket No.14497-018-228 [00280] In one embodiment of purifying vesicles, the impurity is a different vesicle. In one embodiment of purifying vesicles, the impurity is a source cell of the vesicle. In one embodiment of purifying vesicles, the impurity is an unencapsulated payload of the vesicle. In one embodiment of purifying vesicles, the impurity is an excess component (e.g. lipid) forming part of the vesicle. [00281] In one embodiment of purifying hybridosomes, the impurity is LNP. In one embodiment of purifying hybridosomes, the impurity is exosome. In one embodiment of purifying hybridosomes, the impurity is LNP and exosome. [00282] In one embodiment of purifying viral particles, the impurity is host-cell protein (HCP). In one embodiment, at least 90% of HCP is removed. In one embodiment of purifying viral particles, the impurity is total protein. In one embodiment, at least 70% or at least 80% of total protein is removed. In one embodiment of purifying viral particles, the impurity is double-strand DNA (dsDNA). In one embodiment, at least 50% or at least 60% of dsDNA is removed. [00283] In one embodiment, the impurity is positively charged at the first pH. In one embodiment, the impurity is positively charged at the first pH, and the vesicle, cell or viral particle is negatively charged at the first pH. [00284] In one embodiment, the impurity has a binding efficiency with the solid support of less than 60% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 40% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 20% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 10% at the first pH. In one embodiment, the impurity has a binding efficiency with the solid support of less than 5% at the first pH. [00285] In one embodiment, the impurity has a binding efficiency with the solid support of less than 20% at the first pH, and the vesicle, cell or viral particle has a binding efficiency with the solid support of more than 80% at the first pH. [00286] In one embodiment, the impurity is removed during the loading step. In one embodiment, the impurity is removed during the washing step. [00287] In one embodiment, at least 50% of the impurity is removed from the sample. In one embodiment, at least 60% of the impurity is removed from the sample. In one embodiment, at least 70% of the impurity is removed from the sample. In one embodiment, at least 80% of the impurity is removed from the sample. In one embodiment, at least 90% of the impurity is removed from the sample. In one embodiment, at least 95% of the impurity is removed from the sample. In one embodiment, at least 99% of the impurity is removed from the sample. NAI-1540508512v1 -51- Attorney Docket No.14497-018-228 [00288] In one embodiment, the sample has a volume from about 0.1 mL to about 2000 L. In one embodiment, the sample has a volume from about 0.5 mL to about 50 L. In one embodiment, the sample has a volume from about 0.5 mL to about 100 mL. In one embodiment, the sample has a volume from about 1 L to about 100 L. In one embodiment, the sample has a volume from about 10 L to about 2000 L. In one embodiment, the sample has a volume from about 100 L to about 2000 L. In one embodiment, the sample has a volume from about 100 L to about 1000 L. In one embodiment, the sample has a volume from about 500 L to about 2000 L. In some embodiments, the method can be applied to a sample with a volume larger than about 1 L, about 5 L, about 10 L, about 15 L about 20 L, about 25 L, about 50 L, about 100 L, about 200 L, about 250 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1000 L, about 1200 L, about 1400 L, about 1600 L, about 1800 L, or about 2000 L. [00289] In other embodiments, the total amount of sample that goes through the purification step for each batch is from about 500 L to about 15,000 L. In other embodiments, the total amount of sample that goes through the purification step for each batch is from about 5000 L to about 15,000 L. In one embodiment, each sample has a volume of about 500 L and the 500 L volume sample goes through the purification step (e.g., CEX and AEX, CEX, AEX, and MMC, or any other combinations) as described herein. In one embodiment, the total amount of sample that goes through the purification step for each batch is at least about 5,000 L, at least about 6,000 L, at least about 7,000 L, at least about 8,000 L, at least about 9,000 L, at least about 10,000 L, at least about 11,000 L, at least about 12,000 L, at least about 13,000 L, at least about 14,000 L, or at least about 15,000 L. In other embodiments, the total amount of sample that goes through the purification step for each batch is at least about 10,000 L. In other embodiments, the total amount of sample that goes through the purification step for each batch is at least about 15,000 L. In other embodiments, the total amount of sample that goes through the purification step for each batch is at least about 20,000 L. [00290] In one embodiment, the sample is pretreated before contacting with the solid support. [00291] In one embodiment, the samples comprising macromolecules, vesicles, cells, or viral particles can be pretreated to make them suitable for purification on solid support. In one embodiment, pretreatment can make sample comprising macromolecules, vesicles, cells, or viral particles suitable for binding to the ionizable moieties of the solid support. In one embodiment, pretreatment can provide a pH, buffer, conductivity, polarity, or any combination thereof, that is desirable for the binding and release of macromolecules, vesicles, cells, or viral particles. NAI-1540508512v1 -52- Attorney Docket No.14497-018-228 [00292] In one embodiment, the pretreatment process of the sample comprises centrifugation, ultra-centrifugation, filtration, ultra-filtration, clarification, chromatography step, lyophilization, lysing, digestion, drying, dilution, concentration, heating, cooling, or any combination thereof. [00293] In one embodiment, the pretreatment of the sample comprises clarification, nuclease digestion, ultrafiltration, diafiltration, or any combination thereof. [00294] In one embodiment, pretreatment of the sample comprising EV comprises clarification. In one embodiment, the clarification comprises depth filtration, nuclease treatment, centrifugation, acoustic separation, flocculation, or any combination thereof. [00295] In one embodiment, pretreatment of the sample comprises clarification. In one embodiment, the clarification comprises depth filtration. In one embodiment, the depth filtration uses a porous filtration medium, such as a depth filter medium. Without bound by the theory, a depth filter media retains contaminants throughout the medium rather than just on the medium’s surface, and thus can retain a larger number of contaminants before becoming clogged. In one embodiment, the porous filtration medium has an average pore size from about 0.1 to about 100 µm, from about 0.5 to about 80 µm, or from about 1 to about 60 µm. In one embodiment, the porous filtration medium has an average pore size of about 0.2 µm, about 0.3 µm, about 0.4 µm, about 0.5 µm, about 0.6 µm, about 0.7 µm, about 0.8 µm, about 1 µm, about 2 µm, about 3 µm, about 4 µm, about 5 µm, about 10 µm, about 20 µm, about 30 µm, about 40 µm, about 50 µm, about 60 µm, about 70 µm, about 80 µm, about 90 µm, or about 100 µm. [00296] In one embodiment, the sample comprising the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) is pretreated by clarification, wherein the clarification is depth filtration. In some embodiments, the depth filtration is performed with a pad, a panel, a deep bed sand filter, or a lenticular design comprising stacked design. In one embodiment, the depth filtration is performed with a thick filter wound around a perforated cylinder of depth filter medium that surrounds a central core. The type and condition of depth filtration can be selected and adjusted depending on the source, volume, purity, and the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) concentration of the sample. In one embodiment, the depth filtration comprises uncharged filter materials. In one embodiment, the uncharged filter material is one or more uncharged polymers. In one embodiment, the uncharged filter material is cellulose or its derivatives, polypropylene or its derivatives, polyethylene or its derivatives, polyethersulfone or its derivatives, nylon, polyvinylidene fluoride, glass fiber, polytetrafluorethylene, or methacrylate or its derivatives. In one embodiment of large-scale purification, the pretreatment process comprises NAI-1540508512v1 -53- Attorney Docket No.14497-018-228 the use of a large-scale depth filtration system comprising multiple housings and cartridges. [00297] In one embodiment, the depth filtration is performed with one or more depth filter selected from the group consisting of Emphaze (3M), Zeta Plus S (3M), PDH4 (Pall), Polysep II (Millipore Sigma), X0SP (Millipore Sigma), D0SP (Millipore Sigma), C0SP (Millipore Sigma), CR40 (Millipore Sigma), ZetaPlus activated carbon (3M), V100 (Pall), Bio20 (Pall), Biol0 (Pall), glass fiber (Pall), X0HC (Millipore Sigma), A1HC (Millipore Sigma), GF+ (Sartorius), and P-series filters (Pall). In one embodiment, the depth filtration uses two or more depth filters. In one embodiment, the two or more depth filters are arranged in series. In one embodiment, a filter with a larger structure is used to remove cells and cell debris and a filter with smaller pore structure is used to remove smaller contaminants. In one embodiment, two or more depth filters are used sequentially or in parallel. In one embodiment, the depth filtration uses 3 to 12 depth filters. In one embodiment, the depth filter comprises a single layer. In one embodiment, the depth filter comprises a dual-layer. In one embodiment, the depth filter comprises more than two layers. [00298] In one embodiment, the depth filtration methods provided herein is used to purify the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) from a sample produced in a cell culture container or a bioreactor. In one embodiment, the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) in the sample are produced in a single-use bioreactor. In one embodiment, the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) in the sample are produced in a perfusion, ATF perfusion, or TFF perfusion bioreactor. In one embodiment, the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) in the sample are produced in a cell culture lasting from about 10 days to 25 days. In one embodiment, after production the sample is passed over a series of uncharged cellulose depths filters with decreasing pore sizes to remove cells and cell debris while allowing the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) to pass through the filter. [00299] In one embodiment, pretreatment of the sample comprises contacting the sample with nuclease to digest nucleic acid associated with the product of interest (e.g. macromolecules, vesicles, cells, or viral particles). The term “nuclease” as used herein generally refers to any enzyme that hydrolyzes nucleic acid sequences. In one embodiment, the sample is clarified prior to contacting the sample with nuclease. In some embodiments, the sample comprising the product of interest (e.g. macromolecules, vesicles, cells, or viral particles) is clarified using depth filtration prior to contacting the sample with nuclease. In one embodiment, the nuclease is DNase, RNase, or their mixtures. In one embodiment, the nuclease is endonuclease, exonuclease, or their mixtures. NAI-1540508512v1 -54- Attorney Docket No.14497-018-228 In one embodiment, the nuclease comprises one or more of DNase I, Benzonase®, DENARASE®, Exonuclease I, Exonuclease III, Mung Bean Nuclease, Nuclease BAL 31, RNase I, Si Nuclease, Lambda Exonuclease, RecJ, T7 exonuclease, eoxynuclease II, micrococcal nuclease, RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, or RNase V. In one embodiment, the nuclease digestion process takes from about 1 hour to about 48 hours, preferably from about 1 hour to 24 hours. In one embodiment, the concentration of nuclease in the digestion process is from about 1 U/mL to about 500 U/mL, from about 5 U/mL to about 200 U/mL, or from about 25 U/mL to about 100 U/mL. [00300] In one embodiment, a cofactor is used together with the nuclease. In one embodiment, the cofactor is an organic cofactor (e.g. flavin). In one embodiment, the cofactor is an inorganic cofactor (e.g. Mg2+, Cu+, or Mn2+). In one embodiment, the cofactor is used in the form of MgCl2. In one embodiment, the concentration of cofactor in the digestion process is from about 1 mM to about 500 mM, from about 1 mM to about 100 mM, or from about 1 mM to about 20 mM. [00301] In one embodiment, pretreatment of the sample comprises ultrafiltration. As used herein, the term “ultrafiltration” or “UF” refers to any technique for treating a solution or suspension with a semi-permeable membrane that retains macromolecules while allowing a solvent or small solute molecules to pass through. In one embodiment, the ultrafiltration increases the concentration of macromolecules, vesicles, cells or particles in the sample. In one embodiment, the ultrafiltration is Tangential Ultrafiltration (also known as Tangential Flow Filtration, or TFF). In one embodiment, the TFF unit comprises at least one housing and at least one cross-flow (tangential) filter positioned in the housing such that a large portion of the filter's surface is positioned parallel to the flow of the sample through the unit. In one embodiment, the TFF unit includes one filter. In one embodiment, the TFF unit includes two filters. In one embodiment, the TFF unit includes three filters. In one embodiment, the housing can include a first inlet/outlet and a second inlet/outlet positioned, e.g., to allow fluid to pass through the first inlet/outlet, cross the at least one cross-flow filter, and through the second inlet/outlet. In the methods provided herein, the TFF units can be connected in series or parallel to provide a fluid path of desired length. In one embodiment, 4, 5, 6, 7, 8, 9 or 10 TFF units can be connected in parallel or series. In one embodiment, the TFF unit comprises spiral membranes, flat membranes, or hollow fibers. In one embodiment, the hollow fiber comprises polyethersulfone or its derivatives, polysulfone or its derivatives, or cellulose ester or its derivatives. In one embodiment, the TFF filters have a molecular weight cut-off threshold from about 25 kDa to about 1500 kDa, from about 200 kDa to NAI-1540508512v1 -55- Attorney Docket No.14497-018-228 about 1000 kDa, or from about 500 kDa to about 750 kDa. [00302] In one embodiment, pretreatment of the sample comprises diafiltration. As used herein, the term “diafiltration” or “DF” refers to a specialized filtration category that dilutes a retentate with a solvent and refilters the retentate in order to reduce soluble permeate components. Diafiltration may induce or not induce an increase in the concentration of, for example, retained vesicles. In one embodiment, the diafiltration is continuous diafiltration, wherein the solvent is continuously added to the retentate at the same rate as the production rate of the filtrate. In one embodiment, the diafiltration is discontinuous or sequentially diluted diafiltration, wherein the ultrafiltration step involves the addition of a solvent to the retentate side, and wherein the volume of solvent added to the retentate is equal to or greater than the volume of the resulting filtrate. In one embodiment, the diafiltration filters have a molecular weight cut-off threshold from about 25 kDa to about 1000 kDa, from about 100 kDa to about 750 kDa, or from about 300 kDa to about 750 kDa. [00303] In one embodiment, pretreatment of the sample comprises a sequential process of ultrafiltration and diafiltration (UF/DF). In one embodiment, the UF/DF process is performed after the nuclease digestion process. In one embodiment, the UF/DF process is performed after the depth filtration process. In one embodiment, the UF/DF removes cells or cell debris from the sample comprising the product of interest (e.g. macromolecules, vesicles, cells, or viral particles). In one embodiment, the filtration is conducted with successive filtrations. In one embodiment, the successive filtrations occur through filters with decreasing porosity. In one embodiment, the process of UF/DF comprises filtration through one or more filters having a porosity above 0.1 µm, from about 0.1 µm to about 20 µm, or from about 0.5 µm µm to about 10 µm. In one embodiment, the process of UF/DF comprises a first filtration through a filter having a porosity of from about 5 µm to about 15 µm, and a second filtration through a filter having a porosity of from about 0.5 µm to about 1.5 µm. In one embodiment, the process of UF/DF comprises a third filtration through a filter having a porosity of from about 0.2 µm to about 0.8 µm. In one embodiment, the process of UF/DF comprises a fourth filtration through a filter having a porosity of from about 0.1 µm to about 0.4 µm. In one embodiment, the UF/DF filtration further comprises a pre-filtration step using a pre- filter. In one embodiment, the pre- filter comprises one or more cellulose acetate, polypropylene, or polyether sulfone. In one embodiment, the pre-filter has a porosity from about 1 µm to about 20 µm, from about 2 µm to about 15 µm, or from about 2 µm to about 5 µm. [00304] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: NAI-1540508512v1 -56- Attorney Docket No.14497-018-228 a. pretreating the sample with depth filtration; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00305] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; b. Further pretreating the sample with ultrafiltration or diafiltration; c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00306] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with ultrafiltration or diafiltration; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00307] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with ultrafiltration or diafiltration; c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; NAI-1540508512v1 -57- Attorney Docket No.14497-018-228 d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00308] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00309] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. further pretreating the sample with ultrafiltration or diafiltration; d. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; e. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and f. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00310] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with depth filtration; NAI-1540508512v1 -58- Attorney Docket No.14497-018-228 b. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; c. contacting the solid support with the sample in a loading buffer; and d. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [00311] In one embodiment, provided herein is a method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. pretreating the sample with clarification; b. pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. optionally further pretreating the sample with ultrafiltration or diafiltration; d. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; e. contacting the solid support with the sample in a loading buffer; and f. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. [00312] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00313] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; b. Further pretreating the sample with ultrafiltration or diafiltration; NAI-1540508512v1 -59- Attorney Docket No.14497-018-228 c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00314] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with ultrafiltration or diafiltration; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and d. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00315] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with ultrafiltration or diafiltration; c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00316] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; NAI-1540508512v1 -60- Attorney Docket No.14497-018-228 d. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and e. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. [00317] In one embodiment, provided herein is a method of purifying macromolecules from a sample comprising the steps of: a. pretreating the sample with depth filtration; b. further pretreating the sample with nuclease, wherein the nuclease optionally comprises a cofactor; c. further pretreating the sample with ultrafiltration or diafiltration; d. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; e. contacting the solid support with the pretreated sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and f. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. 5.2.11 Eluting Step [00318] In one embodiment, the eluting is performed at the second pH as described in Section 5.2.3. In one embodiment, the eluting is performed using an elution buffer as described in Section 5.4.2. In one embodiment, the eluting is performed using an elution buffer having a salt concentration or conductivity higher than the salt concentration or conductivity of loading buffer used in the loading step. [00319] In one embodiment, the vesicles (for example, vesicles as described in Section 5.2.6) are eluted at a yield of at least 50%. In one embodiment, the vesicles are eluted at a yield of at least 60%. In one embodiment, the vesicles are eluted at a yield of at least 70%. In one embodiment, the vesicles are eluted at a yield of at least 80%. In one embodiment, the vesicles are eluted at a yield of at least 90%. In one embodiment, the vesicles are eluted at a yield of at least 95%. In one embodiment, the vesicles are eluted at a yield of at least 99%. In one embodiment, the elution yield is not affected by the increase of the sample volume. In one embodiment, the elution yield is maintained when the sample volume is increase from less than 50 mL to above 1 L. In one embodiment, the elution yield at a sample volume of above 1 L is at least 75% of the elution yield at a sample volume of below 50 mL. NAI-1540508512v1 -61- Attorney Docket No.14497-018-228 [00320] In one embodiment, the cells (for example, cells as described in Section 5.2.7) are eluted at a yield of at least 50%. In one embodiment, the cells are eluted at a yield of at least 60%. In one embodiment, the cells are eluted at a yield of at least 70%. In one embodiment, the cells are eluted at a yield of at least 80%. In one embodiment, the cells are eluted at a yield of at least 90%. In one embodiment the cells are eluted at a yield of at least 95%. In one embodiment, the cells are eluted at a yield of at least 99%. [00321] In one embodiment, the viral particles (for example, viral particles as described in Section 5.2.8) are eluted at a yield of at least 50%. In one embodiment, the viral particles are eluted at a yield of at least 60%. In one embodiment, the viral particles are eluted at a yield of at least 70%. In one embodiment, the viral particles are eluted at a yield of at least 80%. In one embodiment, the viral particles are eluted at a yield of at least 90%. In one embodiment, the viral particles are eluted at a yield of at least 95%. In one embodiment, the viral particles are eluted at a yield of at least 99%. In one embodiment, the recovery yield of viral particle is measured based on recovery of p24 capsid protein. In one embodiment, the recovery yield of viral particle is measured based on recovery of transduction unit (TU). [00322] In one embodiment, the macromolecules (for example, macromolecules as described in Section 5.2.9) are eluted at a yield of at least 50%. In one embodiment, the viral particles are eluted at a yield of at least 60%. In one embodiment, the viral particles are eluted at a yield of at least 70%. In one embodiment, the viral particles are eluted at a yield of at least 80%. In one embodiment, the viral particles are eluted at a yield of at least 90%. In one embodiment, the viral particles are eluted at a yield of at least 95%. In one embodiment, the viral particles are eluted at a yield of at least 99%. 5.2.12 Detecting Step [00323] In one embodiment, the method further comprises a step of detecting the elution of the vesicles (for example, vesicles as described in Section 5.2.6). In one embodiment, the method further comprises a step of detecting the elution of the cells (for example, cells as described in Section 5.2.7). In one embodiment, the method further comprises a step of detecting the elution of the viral particles (for example, viral particles as described in Section 5.2.8). In one embodiment, the method further comprises a step of detecting the elution of the macromolecules (for example, macromolecules as described in Section 5.2.9). [00324] In one embodiment, the detecting involves the detection of optical signal from the vesicles. In one embodiment, the detecting involves the detection of UV-Vis signal from the NAI-1540508512v1 -62- Attorney Docket No.14497-018-228 vesicles. In one embodiment, the detecting involves the detection of fluorescence signal from the vesicles. In one embodiment, the detecting involves the detection of luminescence signal from the vesicles. In one embodiment, the detecting involves the detection of radioactivity from the vesicles. In one embodiment, the detecting involves the detection of vesicle density. In one embodiment, the detecting involves the detection vesicle size. In one embodiment, the detecting involves the detection of light scattering of the vesicles. [00325] In one embodiment, the detecting involves the detection of optical signal from the cells. In one embodiment, the detecting involves the detection of UV-Vis signal from the cells. In one embodiment, the detecting involves the detection of fluorescence signal from the cells. In one embodiment, the detecting involves the detection of luminescence signal from the cells. In one embodiment, the detecting involves the detection of radioactivity from the cell. In one embodiment, the detecting involves the detection of cell density. In one embodiment, the detecting involves the detection cell size. In one embodiment, the detecting involves the detection of light scattering of the cells. [00326] In one embodiment, the detecting involves the detection of optical signal from the viral particles. In one embodiment, the detecting involves the detection of UV-Vis signal from the viral particles. In one embodiment, the detecting involves the detection of fluorescence signal from the viral particles. In one embodiment, the detecting involves the detection of luminescence signal from the viral particles. In one embodiment, the detecting involves the detection of radioactivity from the viral particles. In one embodiment, the detecting involves the detection of viral particle density. In one embodiment, the detecting involves the detection of viral particle size. In one embodiment, the detecting involves the detection of light scattering of the viral particles. [00327] In one embodiment, the detecting involves the detection of optical signal from macromolecules. In one embodiment, the detecting involves the detection of UV-Vis signal from the macromolecules. In one embodiment, the detecting involves the detection of fluorescence signal from the macromolecules. In one embodiment, the detecting comprises the use of a fluorescence assay. In one embodiment, the detecting involves the detection of radioactivity from the macromolecules. In one embodiment, the detecting involves the detection of light scattering of the macromolecules. In one embodiment, the detecting involves the detection of circular dichroism of the macromolecules. [00328] In one embodiment, the signal is from a small-molecular a fluorescent dye. In one embodiment, the signal is from a fluorescent lipid. In one embodiment, the signal is from a NAI-1540508512v1 -63- Attorney Docket No.14497-018-228 fluorescent protein. In one embodiment, the signal is from a green fluorescent protein (GFP) or red fluorescent protein (RFP). In one embodiment, the signal is from a luciferase. In one embodiment, the signal is from a radioisotope. In one embodiment, the signal is light scattering. [00329] In one embodiment, provided herein is a method of purifying hybridosomes from a sample comprising the steps of: a. preparing the hybridosomes by fusing one or more LNP with one or more exosome; b. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; c. contacting the solid support with the sample at a first pH at which the hybridosomes bind to the solid support; d. washing the solid support to remove excess LNP or exosome in the sample; and e. eluting the hybridosome from the solid support at a second pH, wherein the second pH is higher than the first pH; optionally wherein the method further comprises a step of: f. detecting the elution of the vesicles, cells or viral particles. [00330] In one embodiment, the detecting involves the detection of optical signal from the hybridosome. In one embodiment, the detecting involves the detection of UV-Vis signal from the hybridosome. In one embodiment, the detecting involves the detection of fluorescence signal from the hybridosome. In one embodiment, the detecting involves the detection of luminescence signal from the hybridosome. In one embodiment, the detecting involves the detection of radioactivity from the hybridosome. 5.3 Compositions [00331] In one embodiment, provided herein is a composition for use as a solid support as described in Section 5.2.4. In one embodiment, the composition is used for the purification of vesicles (for example, vesicles as described in Section 5.2.6). In one embodiment, the composition is used for the purification of cells (for example, cells as described in Section 5.2.7). In one embodiment, the composition is used for the purification of viral particles (for example, viral particles as described in Section 5.2.8). In one embodiment, the composition is used for the purification of macromolecules (for example, macromolecules as described in Section 5.2.9). In one embodiment, provided herein is a solid support comprising the resin as described herein. In one embodiment, provided herein is a chromatography column comprising the resin as described herein. NAI-1540508512v1 -64- Attorney Docket No.14497-018-228 In one embodiment, provided herein is a membrane comprising the resin as described herein. In one embodiment, provided herein is a bead comprising the resin as described herein. 5.3.1 Resin [00332] In one embodiment, provided herein is a resin of Formula (I):
Figure imgf000067_0001
wherein is a polymer selected from the group consisting of agarose, cellulose, polystyrene, polyacrylamide and hydroxylated methacrylic polymer; wherein the polymer is optionally substituted; wherein R1 and R2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the -N- to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is independently substituted; wherein L is C1-C48 alkylene, wherein one or more -CH2- in the alkylene is independently optionally replaced by -O-, -S-, -NH-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, -NHC(=O)-, or phenyl, and wherein the alkylene or phenyl is optionally substituted with one or more halogen, hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; and wherein the tertiary amino group has a pKa from about 6.5 to about 9. [00333] In one embodiment, L is C1-C48 alkylene. In one embodiment, L is C1-C36 alkylene. In one embodiment, L is C1-C24 alkylene. In one embodiment, L is C1-C16 alkylene. In one embodiment, L is C1-C12 alkylene. In one embodiment, L is C1-C6 alkylene. In one embodiment, L is methylene. In one embodiment, L is ethylene. In one embodiment, L is C3 alkylene. In one embodiment, L is C4 alkylene. In one embodiment, L is C5 alkylene. In one embodiment, L is C6 alkylene. [00334] In one embodiment, one or more -CH2- in L is replaced by -O-. In one embodiment, two -CH2- in L are each replaced by a -O-. In one embodiment, three -CH2- in L are each replaced by a -O-. In one embodiment, four -CH2- in L are each replaced by a -O-. In one embodiment, four - CH2- in L are each replaced by a -O-. In one embodiment, five -CH2- in L are each replaced by a - O-. In one embodiment, six -CH2- in L are each replaced by a -O-. In one embodiment, L comprises from 1 to 12 -O-CH2CH2- repeating unit. In one embodiment, L comprises from 1 to 12 NAI-1540508512v1 -65- Attorney Docket No.14497-018-228 -O-CH(CH3)CH2- repeating unit. [00335] In one embodiment, one or more -CH2- in L is replaced by -S-. In one embodiment, one or more -CH2- in L is replaced by -NH-. In one embodiment, one or more -CH2- in L is replaced by -C(=O)-. In one embodiment, one or more -CH2- in L is replaced by -C(=O)O- or -OC(=O)-. In one embodiment, one or more -CH2- in L is replaced by -C(=O)NH- or -NHC(=O)-. [00336] In one embodiment, one or more -CH2- in L is replaced by a phenyl. In one embodiment, two -CH2- in L are each replaced by a phenyl. In one embodiment, the phenyl is substituted with one or more halogen. In one embodiment, the phenyl is substituted with one or more C1-C6 alkyl. In one embodiment, the phenyl is substituted with one or more C1-C6 alkoxy. [00337] In one embodiment, L is substituted with one or more halogen. In one embodiment, L is substituted with one or more hydroxyl group. In one embodiment, L is substituted with one hydroxyl group. In one embodiment, L is substituted with two hydroxyl groups. In one embodiment, L is substituted with three hydroxyl groups. In one embodiment, L is substituted with four hydroxyl groups. In one embodiment, L is substituted with six hydroxyl groups. In one embodiment, L is substituted with one or more C1-C6 alkyl. In one embodiment, L is substituted with one or more C1-C6 alkoxy. [00338] In one embodiment, L has a molecular weight less than 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol, or less than 500 g/mol. In one embodiment, L consists of carbon, hydrogen, and oxygen atoms. In one embodiment, L does not comprise nitrogen atom. In one embodiment, L does not comprise sulfur atom. ,
Figure imgf000068_0001
- Attorney Docket No.14497-018-228 O O wherein m is from 1 to 12. In one embodiment, L , wherein n is from
Figure imgf000069_0001
1 to 12. The attachment to the left is to the is to the tertiary amino group. [00340] In one embodiment, provided herein is a resin of Formula (II-A), (II-B), (II-C), (II-D), (II-E), (II-F), or (II-G):
Figure imgf000069_0002
wherein is a polymer selected from the group consisting of agarose, cellulose, polystyrene, polyacrylamide and hydroxylated methacrylic polymer; wherein the polymer is optionally substituted; wherein R1 and R2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the -N- to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is independently substituted; wherein m and n are each independently an integer from 1 to 24; wherein the tertiary amino group has a pKa from about 6.5 to about 9; wherein the density of the tertiary amino group on the resin is from about 250 µmol to about 1000 µmol per gram of the resin; and NAI-1540508512v1 -67- Attorney Docket No.14497-018-228 wherein the resin has an average pore size of from about 10 nm to about 1000 nm. [00341] In one embodiment, the polymer is agarose. In one embodiment, the polymer is cellulose. In one embodiment, the polymer is polystyrene. In one embodiment, the polymer is polymethacrylate. In one embodiment, the polymer is polyacrylamide. In one embodiment, the polymer is hydroxylated methacrylic polymer. [00342] In one embodiment, the polymer is unsubstituted. In one embodiment, the polymer is substituted. In one embodiment, the polymer is substituted with one or more a carboxylic acid or carboxylate group. In one embodiment, the polymer is substituted with one or more sulfoisobutyl group, sulfonate group, sulfoethyl group, sulfopropyl group, or carboxymethyl group. In one embodiment, the polymer is substituted with one or more diethylaminopropyl, diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, carboxymethyl, glutamic acid, aspartic acid, histidine, hydroxyl, phosphate, tertiary amines, quaternary amines, diethaminoethyl, dimethylaminoethyl, trimethylaminoethyl, or amino acid group. [00343] In one embodiment, R1 is alkyl, and R2 is alkyl. In one embodiment, R1 is alkyl, and R2 is hydroxyalkyl. In one embodiment, R1 is hydroxyalkyl, and R2 is hydroxyalkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C12 alkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C6 alkyl. In one embodiment, R1 is C1-C6 alkyl, and R2 is C1-C6 alkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C12 hydroxyalkyl. In one embodiment, R1 is C1-C12 alkyl, and R2 is C1-C6 hydroxyalkyl. In one embodiment, R1 is C1-C6 alkyl, and R2 is C1-C6 hydroxyalkyl. In one embodiment, R1 is C1-C12 hydroxyalkyl, and R2 is C1-C12 hydroxyalkyl. In one embodiment, R1 is C1-C12 hydroxyalkyl, and R2 is C1-C6 hydroxyalkyl. In one embodiment, R1 is C1-C6 hydroxyalkyl, and R2 is C1-C6 hydroxyalkyl. [00344] In one embodiment, R1 and R2 together with the atoms they are attached to form a 5- membered heterocyclyl. In one embodiment, R1 and R2 together with the atoms they are attached to form a 6-membered heterocyclyl. In one embodiment, the heterocyclyl contains only one ring nitrogen atom. In one embodiment, the heterocyclyl contains two ring nitrogen atoms. In one embodiment, the heterocyclyl contains a ring nitrogen atom and a ring oxygen atom. [00345] In one embodiment, R1 is unsubstituted. In one embodiment, R1 is substituted. In one embodiment, R1 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano. In one embodiment, R1 is substituted with one or more halogen. In one embodiment, R1 is substituted with one or more hydroxyl. In one embodiment, R1 is substituted with one or more oxo. In one NAI-1540508512v1 -68- Attorney Docket No.14497-018-228 embodiment, R1 is substituted with one or more phenyl. [00346] In one embodiment, R2 is unsubstituted. In one embodiment, R2 is substituted. In one embodiment, R2 is substituted with one or more halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, dialkylamino, carboxy, nitro, oxo, or cyano. In one embodiment, R2 is substituted with one or more halogen. In one embodiment, R2 is substituted with one or more hydroxyl. In one embodiment, R2 is substituted with one or more oxo. In one embodiment, R2 is substituted with one or more phenyl. [00347] In one embodiment, m is an integer from 1 to 24. In one embodiment, m is an integer from 1 to 12. In one embodiment, m is an integer from 1 to 6. In one embodiment, m is 1. In one embodiment, m is 2. In one embodiment, m is 3. In one embodiment, m is 4. In one embodiment, m is 5. In one embodiment, m is 6. In one embodiment, m is 7. In one embodiment, m is 8. In one embodiment, m is 9. In one embodiment, m is 10. In one embodiment, m is 11. In one embodiment, m is 12. In one embodiment, m is 14. In one embodiment, m is 16. In one embodiment, m is 18. In one embodiment, m is 20. In one embodiment, m is 22. In one embodiment, m is 24. [00348] In one embodiment, n is an integer from 1 to 24. In one embodiment, n is an integer from 1 to 12. In one embodiment, n is an integer from 1 to 6. In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is 3. In one embodiment, n is 4. In one embodiment, n is 5. In one embodiment, n is 6. In one embodiment, n is 7. In one embodiment, n is 8. In one embodiment, n is 9. In one embodiment, n is 10. In one embodiment, n is 11. In one embodiment, n is 12. In one embodiment, n is 14. In one embodiment, n is 16. In one embodiment, n is 18. In one embodiment, n is 20. In one embodiment, n is 22. In one embodiment, n is 24. [00349] In one embodiment, the tertiary amino group has a pKa from about 6.5 to about 9. In one embodiment, the tertiary amino group has a pKa from about 7.6 to about 8.8. In one embodiment, the tertiary amino group has a pKa of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9. In one embodiment, the tertiary amino group has a pKa of about 7.6. In one embodiment, the tertiary amino group has a pKa of about 8.2. (C1-C6)Alkyl [00350] In one embodiment, the tertiary amino each alkyl is independently optionally substituted with one or
Figure imgf000071_0001
the NAI-1540508512v1 -69- Attorney Docket No.14497-018-228 attachment to the left is to the linker L. In one embodiment, the tertiary amino group is , wherein X is C(Ra)2, NRa, O, or S; wherein Ra, R3 and R4 are each independently
Figure imgf000072_0001
or C1-C6 alkyl, wherein the alkyl is optionally substituted with one or more halogen or hydroxyl, wherein the attachment to the left is to the linker L. [00351] In one embodiment, the tertiary amino group is selected from a group consisting of: OH OH ,
Figure imgf000072_0002
; wherein the attachment to the left is to the linker L.
Figure imgf000072_0003
OH [00352] In one embodiment, the tertiary amino group or ionizable
Figure imgf000072_0004
one embodiment, the solid support or , wherein the attachment is to a polymer described
Figure imgf000072_0005
[00353] In one embodiment, the pKa of the tertiary amino group of the resin is estimated by measuring the pKa of R5-L-NR1R2 in a solvent, wherein R5 is hydrogen, hydroxyl, C1-C3 alkyl, C1- C3 alkoxyl, or epoxy. In one embodiment, the pKa of the tertiary amino group of the resin is estimated by measuring the pKa of R6-NR1R2 in a solvent, wherein R6 is C1-C3 alkyl, or C1-C3 NAI-1540508512v1 -70- Attorney Docket No.14497-018-228 alkoxyl. In one embodiment, the solvent is water. In one embodiment, the solvent is DMSO. In one embodiment, the pKa is determined by an acid-base titration experiment. In one embodiment, the acid-base titration experiment uses hydrochloric acid. [00354] In one embodiment, the resin has an average pore size from about 10 nm to about 1000 nm. In one embodiment, the average pore size is from about 50 nm to about 800 nm. In one embodiment, the average pore size is from about 100 nm to about 600 nm. In one embodiment, the average pore size is from about 50 nm to about 200 nm. In one embodiment, the average pore size is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, or about 200 nm. [00355] In one embodiment, the tertiary amino group has a density of from about 50 µmol to about 1000 µmol per gram of the resin. In one embodiment, the tertiary amino group has a density of from about 100 µmol to about 1000 µmol per gram of the resin. In one embodiment, the tertiary amino group has a density of from about 250 µmol to about 1000 µmol per gram of the resin. In one embodiment, the tertiary amino group has a density of from about 250 µmol to about 800 µmol per gram of the resin. In one embodiment, the tertiary amino group has a density of from about 300 µmol to about 1000 µmol per gram of the resin. In one embodiment, tertiary amino group has a density of from about 500 µmol to about 1000 µmol per gram of the resin. In one embodiment, the tertiary amino group has a density of about 50 µmol, about 100 µmol, about 150 µmol, about 200 µmol, about 250 µmol, about 300 µmol, about 350 µmol, about 400 µmol, about 500 µmol, about 600 µmol, about 700 µmol, about 800 µmol, about 900 µmol or about 1000 µmol per gram of the resin. 5.3.2 Preparation of the Resin [00356] Also provided herein is a method of preparing the resin as described in Section 5.3.1, such as the resin of Formula (I), (II-A), (II-B), (II-C), (II-D), (II-E), (II-F), or (II-G). In one embodiment, the resin is prepared using the method as described in Section 5.2.4 (i). In one embodiment, the resin is prepared using a source resin comprising the reactive groups as described in Section 5.2.4 (ii). In one embodiment, the resin is prepared by contacting a source resin comprising the reactive groups as described in Section 5.2.4 (ii), and a ligand as described in 5.2.4 (iii). In one embodiment, the source resin is a CEX resin, an AEX resin, or a SEC resin (for example, as described in Section 5.2.4 (i)). [00357] In one embodiment, the solid support is prepared from agarose comprising the reactive groups as described in in Section 5.2.4 (ii). In one embodiment, the solid support is prepared from NAI-1540508512v1 -71- Attorney Docket No.14497-018-228 polystyrene comprising the reactive groups as described in Section 5.2.4 (ii). In one embodiment, the solid support is prepared from polymethacrylate comprising the reactive groups as described in Section 5.2.4 (ii). In one embodiment, the solid support is prepared from polyacrylamide comprising the reactive groups as described in Section 5.2.4 (ii). In one embodiment, the solid support is prepared from hydroxylated methacrylic polymer comprising the reactive groups as described in Section 5.2.4 (ii). [00358] In one embodiment, the resin is prepared from a source resin comprising one or more reactive group selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, cyanogen bromide, a carbodiimide and lysine. In one embodiment, the source resin is a commercially available resin. In one embodiment, the source resin is agarose comprising epoxy groups. In one embodiment, the source resin is polystyrene comprising epoxy groups. In one embodiment, the source resin is polymethacrylate comprising epoxy groups. In one embodiment, source resin is polyacrylamide comprising epoxy groups. In one embodiment, source resin is hydroxylated methacrylic polymer comprising epoxy groups. [00359] In one embodiment, the source resin is epoxy-activated agarose resin. In one embodiment, the source resin is TOYOPEARL® AF-Epoxy-650M. In one embodiment, the source resin is epoxy-activated SepharoseTM 6B. In one embodiment, the source resin is Epoxy-Activated Separopore® 6B-CL. In one embodiment, the source resin is Epoxy-Activated Separopore® 4B- CL. In one embodiment, the source resin is Epoxy-Activated Agarose. In one embodiment, the source resin is Profinity™ Epoxide Resin. In one embodiment, the source resin is POROS™ 20 EP Epoxide Activated Resin. In one embodiment, the source resin is Praesto® Epoxy resin. [00360] In one embodiment, the method further comprises a step of modifying the source resin to increase the density of reactive group (e.g. epoxy groups). In one embodiment, the density of the reactive group is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%. In one embodiment, the modification comprises contacting the source resin with a compound, wherein the compound comprises the same reactive group of the resin. In one embodiment, the modification comprises contacting an epoxy resin with a compound comprising at least one epoxy group. In one embodiment, the modification comprises contacting an epoxy resin NAI-1540508512v1 -72- Attorney Docket No.14497-018-228 with a compound comprising two epoxy groups. In one embodiment, the modification comprises contacting an epoxy resin with a compound comprising an epoxy group and a leaving group, wherein the leaving group is halogen (e.g. Cl, Br, or I), or sulfonate ester (e.g. tosylate, mesylate, or triflate). [00361] In one embodiment, the resin is prepared by contacting a source resin as described above, with a ligand as described in 5.2.4 (iii). In one embodiment, the ligand is a thiol. In one embodiment, the ligand is a carboxylic acid. In one embodiment, the ligand is an amine. In one embodiment, the ligand is a secondary amine. In one embodiment, the ligand has a formula of R1- NH-R2, wherein R1, R2 is defined herein or elsewhere. In one embodiment, the contacting is performed at a temperature from about 0 °C to about 200 °C. In one embodiment, the contacting is performed at a temperature from about 20 °C to about 100 °C. In one embodiment, the contacting is performed at room temperature. In one embodiment, the contacting is performed in the presence of a base, such an inorganic base (e.g. NaOH), or an organic base (e.g. triethylamine, or pyridine). In one embodiment, the contacting is performed in the presence of one or more solvents, such as an organic solvent (e.g. DMSO, DMF, or THF). In one embodiment, the contacting is performed in water. In one embodiment, the contacting is performed in a mixture of water and organic solvent. In one embodiment, the contacting is performed in one or more solvents, at a temperature from about 20 °C to about 100 °C, and in the presence of a base. 5.3.3 Solid Support [00362] Also provided herein is a solid support (e.g. membrane, chromatography column, or magnetic beads) comprising the resin as described in Section 5.3.1, such as resin of Formula (I), (II- A), (II-B), (II-C), (II-D), (II-E), (II-F), or (II-G). In one embodiment, the solid support comprises a membrane comprising the resin as described in Section 5.3.1. In one embodiment, the solid support comprises a magnetic bead comprising the resin as described in Section 5.3.1. In one embodiment, the solid support comprises a pre-packed chromatography column comprising the resin as described in Section 5.3.1. [00363] In one embodiment, the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is -N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted. In one embodiment, the polymer is agarose, polystyrene, polymethacrylate, polyacrylamide, or hydroxylated methacrylic polymer. [00364] In one embodiment, the solid support comprises a magnetic bead, wherein the magnetic bead is prepared by binding the resin as described in Section 5.3.1 with an activated magnetic bead. NAI-1540508512v1 -73- Attorney Docket No.14497-018-228 In one embodiment, the activated magnetic bead binds to the polymer moiety of the resin. In one embodiment, the activated magnetic bead binds to the linker of the resin. In one embodiment, the activated magnetic bead binds to the tertiary amino group of the resin. In one embodiment, the activated magnetic bead binds to the unreacted reactive group (e.g. epoxy group) of the resin. [00365] In one embodiment, the solid support further comprises other materials, including glass, modified or functionalized glass, plastics (e.g. acrylics, polystyrene, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefin copolymers, or polyimides), nylon, ceramics, resins, Zeonor, silica or silica-based materials, carbon, or metals. [00366] In one embodiment, the solid support is contained within a flow cell. The term “flow cell” as used herein refers to a chamber including a solid support across which one or more fluid reagents can be flowed. Flow cells and related fluidic systems and detection platforms can be readily used as described herein. [00367] In one embodiment, the solid support as described herein is used for purification purpose, including the purification of vesicles, cells or viral particles as described in Section 5.2. In one embodiment, the solid support as described herein is used for the purification of small molecules. In one embodiment, the solid support as described herein is used for the purification of macromolecules, such as nucleic acids, polypeptides, or polysaccharides. [00368] In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 0.5 mL to about 100 L. In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 0.5 mL to about 100 mL. In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 0.1 L to about 1 L. In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 1 L to about 100 L. In one embodiment, the solid support as described herein is capable of purifying samples in an amount from about 50 L to about 500 L. [00369] In one embodiment, the solid support comprises the resin as described in Section 5.3.1 in an amount from about 0.05 gram to about 5000 gram. In one embodiment, the solid support comprises the resin in an amount from about 0.5 gram to about 1000 gram. In one embodiment, the solid support comprises the resin in an amount from about 0.5 gram to about 20 gram. In one embodiment, the solid support comprises the resin in an amount of about 0.5 gram, about 1 gram, about 2 gram, about 3 gram, about 4 gram, about 5 gram, about 6 gram, about 7 gram, about 8 gram, about 9 gram, about 10 gram, about 11 gram, about 12 gram, about 13 gram, about 14 gram, about NAI-1540508512v1 -74- Attorney Docket No.14497-018-228 15 gram, about 16 gram, about 17 gram, about 18 gram, about 19 gram, or about 20 gram. In one embodiment, the solid support comprises the resin in an amount from about 500 gram to about 5000 gram. In one embodiment, the solid support comprises the resin in an amount of about 500 gram, about 1000 gram, about 2000 gram, about 3000 gram, about 4000 gram, or about 5000 gram. [00370] In one embodiment when the solid support is a chromatography column, the chromatography column has a volume of from about 0.1 mL to about 5000 mL. In one embodiment, the chromatography column has a volume of from about 0.25 mL to about 2500 mL. In one embodiment, the chromatography column has a volume of from about 0.5 mL to about 20 mL. In one embodiment, the chromatography column has a volume of about 0.5 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, or about 20 mL. In one embodiment, the chromatography column has a volume of from about 1000 mL to about 5000 mL. In one embodiment, the chromatography column has a volume of about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L or about 100 L. [00371] In one embodiment, the chromatography column is a gravity-flow column. In one embodiment, the chromatography column is a spin column. In one embodiment, the chromatography column is a high-pressure liquid chromatography (HPLC) column. In one embodiment, the chromatography column is a low-pressure liquid chromatography column. [00372] The chromatography column as described herein can be made of any material, including those described in Section 5.1. In one embodiment, the chromatography column is a disposable single-use column. In one embodiment, the chromatography column is a multi-use column. 5.4 Kits [00373] Also provided herein is a kit comprising the composition as described in Section 5.3, including the resin as described in Section 5.3.1, or the solid support (e.g. membrane, magnetic bead, or chromatography column) as described in Section 5.3.3. [00374] In one embodiment, the kit as described herein is used in a purification method, including the purification of vesicles, cells or viral particles as described in Section 5.2. In one embodiment, the kit as described herein is used for the purification of small molecules. In one embodiment, the kit as described herein is used for the purification of macromolecules, such as nucleic acids or polypeptides. NAI-1540508512v1 -75- Attorney Docket No.14497-018-228 [00375] In one embodiment, the kit further comprises one or more loading buffer (e.g. Section 5.4.1), one or more elution buffer (e.g. Section 5.4.2), or one or more washing buffer (e.g. Section 5.4.3). [00376] In one embodiment, provided herein is a kit comprising a solid support described herein, a loading buffer, and an elution buffer, wherein the loading buffer has a pH between 4.5 to 7.4, and wherein the elution buffer has a pH between 7.4 to 9. [00377] In one embodiment, provided herein is a kit comprising a solid support described herein, a loading buffer, and an elution buffer, wherein the loading buffer has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. In one embodiment, the loading buffer and the elution buffer have the same pH. [00378] In one embodiment, the kit further comprises one or more washing buffer. In one embodiment, the washing buffer has the same pH, salt concentration, or conductivity as the loading buffer. In one embodiment, the washing buffer is the same as the loading buffer. 5.4.1 Loading Buffer [00379] In one embodiment, the method described herein uses a loading buffer. In one embodiment, the kit described herein comprises a loading buffer. In one embodiment, the loading buffer has the first pH as described in Section 5.2.2. [00380] In one embodiment, the loading buffer has a pH of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the elution buffer has a pH of 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9. In one embodiment the loading buffer has a pH from 4.5 to 7.4, and the elution buffer has a pH from 7.4 to 9. In one embodiment, the loading buffer has a pH from 6.5 to 7.4, and the elution buffer has a pH from 7.4 to 9. In one embodiment, the loading buffer has a pH of 6.5, and the elution buffer has a pH of 7.4. In one embodiment, the loading buffer has a pH of 7.4, and the elution buffer has a pH of 8.3. In one embodiment, the loading buffer has a pH of 7, and the elution buffer has a pH of 8. In one embodiment, the loading buffer has a pH of 7, and the elution buffer has a pH 8.8. [00381] In one embodiment, the loading buffer has a pH of 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, or 9. In one embodiment, the loading buffer has a pH of 6–9, and the elution buffer has a pH of 6–9. In one embodiment, the loading buffer has a pH of 7–8, and the elution buffer has a pH of 7–8. In one embodiment, the loading buffer has a pH of 6.8, and the elution buffer has a pH of 7.2. In one embodiment, the loading buffer has a pH of 7.2, and the elution buffer has a pH of 7.2. NAI-1540508512v1 -76- Attorney Docket No.14497-018-228 In one embodiment, the loading buffer has a pH of 8, and the elution buffer has a pH of 8. [00382] In one embodiment, the loading buffer is used in the method described in Section 5.2. In one embodiment, the loading buffer is used in the purification of vesicles described in Section 5.2.6, purification of cells described in Section 5.2.7, or purification of viral particles described in Section 5.2.8. [00383] In one embodiment, the loading buffer has the same pH as the elution buffer described in Section 5.4.2. In one embodiment, the loading buffer has a salt concentration or conductivity that is lower than the salt concentration or conductivity of the elution buffer described in Section 5.4.2. [00384] In one embodiment, the loading buffer is saline. In one embodiment, the loading buffer is MOPSO buffer. In one embodiment, the loading buffer is phosphate buffer. In one embodiment, the loading buffer is Tris buffer. In one embodiment, the loading buffer is MOPS buffer. In one embodiment, the loading buffer is Bis-Tris buffer. In one embodiment, the loading buffer is ADA buffer. In one embodiment, the loading buffer is MES buffer. In one embodiment, the loading buffer is HEPES buffer. In one embodiment, the loading buffer is citric buffer. In one embodiment, the loading buffer is acetate buffer. [00385] In one embodiment, the loading buffer is Tris buffer with a pH from 6.5 to 7.4. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4. [00386] In one embodiment, the loading buffer comprises one or more salt. In one embodiment, the salt is an alkali metal salt. In one embodiment, the salt is an alkali metal halide. In one embodiment, the salt is NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, or any combination thereof. In one embodiment, the salt is sodium chloride. In one embodiment, the salt is potassium chloride. In one embodiment, the salt is a mixture of sodium chloride and potassium chloride. In one embodiment, the salt has a concentration from 0.001 M to 1 M. In one embodiment, the salt has a concentration from 0.01 M to 1 M. In one embodiment, the salt has a concentration from 0.01 M to 0.2 M. In one embodiment, the salt is 0.01 M to 0.2 M sodium chloride. In one embodiment, the salt is at 0.01 M sodium chloride. In one embodiment, the salt is 0.2 M sodium chloride. [00387] In one embodiment, the loading buffer has a conductivity from about 0.1 mS/cm to 100 mS/cm at 22 °C. In one embodiment, the conductivity is from about 0.1 mS/cm to about 50 mS/cm at 22 °C. In one embodiment, the conductivity is from about 1 mS/cm to about 25 mS/cm at 22 °C. In one embodiment, the conductivity is from about 5 mS/cm to about 20 mS/cm at 22 °C. [00388] In one embodiment, the loading buffer is Tris buffer with a pH from 6.5 to 7.4 and a salt NAI-1540508512v1 -77- Attorney Docket No.14497-018-228 concentration from 0.01 M to 0.2 M. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4 and a salt concentration from 0.01 M to 0.2 M. In one embodiment, the loading buffer is HEPES buffer with a pH from 4.5 to 7.4 and a salt concentration from 0.01 M to 0.2 M. In one embodiment, the loading buffer is Tris buffer with a pH from 6.5 to 7.4 and NaCl concentration from 0.01 M to 0.2 M. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4 and NaCl concentration from 0.01 M to 0.2 M. In one embodiment, the loading buffer is HEPES buffer with a pH from 4.5 to 7.4 and NaCl concentration from 0.01 M to 0.2 M. [00389] In one embodiment, the loading buffer is Tris buffer with a pH from 6.5 to 7.4, and a conductivity from about 0.1 to about 50 mS/cm at 22 °C. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4 and a conductivity from about 0.1 to about 50 mS/cm at 22 °C. In one embodiment, the loading buffer is HEPES buffer with a pH from 4.5 to 7.4 and a conductivity from about 0.1 to about 50 mS/cm at 22 °C. 5.4.2 Elution Buffer [00390] In one embodiment, the method described herein uses an elution buffer. In one embodiment, the kit described herein comprises an elution buffer. In one embodiment, the elution buffer has the second pH as described in Section 5.2.3. [00391] In one embodiment, the kit comprises two, three, or four elution buffers having the same or different pH or salt concentrations. In one embodiment, the elution buffer is used in the method described in Section 5.2. In one embodiment, the elution buffer is used in the purification of vesicles described in Section 5.2.6, purification of cells described in Section 5.2.7, or purification of viral particles described in Section 5.2.8. [00392] In one embodiment, the elution buffer has a higher pH than the loading buffer described in Section 5.4.1. In one embodiment, the elution buffer has the same pH as the loading buffer described in Section 5.4.1. In one embodiment, the elution buffer has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer described in Section 5.4.1. [00393] In one embodiment, the elution buffer is saline. In one embodiment, the elution buffer is phosphate buffer. In one embodiment, the elution buffer is Tris buffer. In one embodiment, the elution buffer is Bicine buffer. In one embodiment, the elution buffer is Tricine buffer. In one embodiment, the elution buffer is HEPES buffer. [00394] In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9. In NAI-1540508512v1 -78- Attorney Docket No.14497-018-228 one embodiment, the elution Tris buffer with a pH from 7.4 to 9. [00395] In one embodiment, the elution buffer comprises one or more salt. In one embodiment, the salt is an alkali metal salt. In one embodiment, the salt is an alkali metal halide. In one embodiment, the salt is NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, or any combination thereof. In one embodiment, the salt is sodium chloride. In one embodiment, the salt is potassium chloride. In one embodiment, the salt is a mixture of sodium chloride and potassium chloride. In one embodiment, the salt concentration is from 0.001 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 2 M. In one embodiment, the salt concentration is from 0.1 M to 2 M. In one embodiment, the salt concentration is from 0.01 M to 1 M. In one embodiment, the salt concentration is from 0.7 M to 2 M. In one embodiment, the elution buffer comprises 0.01 M to 2 M NaCl. In one embodiment, the elution buffer comprises 0.01 M to 1 M NaCl. In one embodiment, the elution buffer comprises 0.7 M to 2 M NaCl. In one embodiment, the elution buffer comprises 0.15 M NaCl. In one embodiment, the elution buffer comprises 0.2 M NaCl. In one embodiment, the elution buffer comprises 0.275 M NaCl. In one embodiment, the elution buffer comprises 0.5 M NaCl. In one embodiment, the elution buffer comprises 0.7 M NaCl. In one embodiment, the elution buffer comprises 1 M NaCl. [00396] In one embodiment, the elution buffer has a conductivity from about 10 mS/cm to about 250 mS/cm at 22 °C. In one embodiment, the conductivity is from about 25 mS/cm to about 200 mS/cm at 22 °C. In one embodiment, the conductivity is from about 55 mS/cm to about 140 mS/cm at 22 °C. In one embodiment, the conductivity is about 55 mS/cm, about 60 mS/cm, about 70 mS/cm, about 80 mS/cm, about 90 mS/cm, about 100 mS/cm, about 110 mS/cm, about 120 mS/cm, about 130 mS/cm, or about 140 mS/cm at 22 °C. [00397] In one embodiment, the elution buffer has a salt concentration that is at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 25 times, at least 50 times, or at least 100 times higher than the loading buffer as described in Section 5.4.1. [00398] In one embodiment, the elution buffer has a conductivity that is at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 25 times, at least 50 times, or at least 100 times higher than the conductivity of the loading buffer as described in Section 5.4.1 at the same temperature (e.g. at 22 °C). [00399] In one embodiment, each elution buffer independently has a salt concentration or conductivity that is at least 1.2 times, at least 1.4 times, at least 1.6 times, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, or at least 20 times higher than the salt NAI-1540508512v1 -79- Attorney Docket No.14497-018-228 concentration or conductivity of the loading buffer. [00400] In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 2 M. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.1 M to 2 M. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 1 M. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a salt concentration from 0.7 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.1 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.01 M to 1 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and a salt concentration from 0.7 M to 2 M. [00401] In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 2 M. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 1 M. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and NaCl concentration from 0.7 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 2 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and NaCl concentration from 0.01 M to 1 M. In one embodiment, the elution buffer is phosphate buffer with a pH from 7.4 to 9 and NaCl concentration from 0.7 M to 2 M. [00402] In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a conductivity from about 10 to about 250 mS/cm at 22 °C. In one embodiment, the loading buffer is phosphate buffer with a pH from 7.4 to 9 and a conductivity from about 10 to about 250 mS/cm at 22 °C. In one embodiment, the elution buffer is Tris buffer with a pH from 7.4 to 9 and a conductivity from about 55 to about 140 mS/cm. In one embodiment, the loading buffer is phosphate buffer with a pH from 7.4 to 9 and a conductivity from about 55 to about 140 mS/cm. [00403] In one embodiment, the loading buffer has a pH of 7.2 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 7.2 and a salt concentration of above 0.1 M. In one embodiment, the loading buffer has a pH of 7.2 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 7.2 and a salt concentration of above 0.65 M. [00404] In one embodiment, the loading buffer has a pH of 8 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 8 and a salt concentration of above 0.1 M. In one NAI-1540508512v1 -80- Attorney Docket No.14497-018-228 embodiment, the loading buffer has a pH of 8 and a salt concentration of below 0.1 M, and the elution buffer has a pH of 8 and a salt concentration of above 0.65 M. [00405] In one embodiment, the kit further comprises a washing buffer. In one embodiment, the kit further comprises two, three, four, five, or six washing buffers. 5.4.3 Washing Buffer [00406] In one embodiment, the method described herein further uses a washing buffer. In one embodiment, the kit described herein further comprises a washing buffer. In one embodiment, the kit further comprises two, three, or four washing buffers. In one embodiment, the kit does not comprise a washing buffer. [00407] In one embodiment, the washing buffer is the same as the loading buffer as described in Section 5.4.1. In one embodiment, the washing buffer is different from loading buffer as described in Section 5.4.1. In one embodiment, the washing buffer is used in the washing step as described in Section 5.2.1. [00408] In one embodiment, a washing buffer is used before loading of the sample to equilibrate the solid support. In one embodiment, a washing buffer is used after loading of the sample to remove one or more impurities. In one embodiment, a washing buffer is used after elution to re- equilibrate the solid support. [00409] In one embodiment, the washing buffer has a pH between 4.5 to 8.5. In one embodiment, the washing buffer has a pH between 4.5 to 7.4. In one embodiment, the washing buffer has a pH between 5 to 7.4. In one embodiment, the washing buffer has a pH between 6 to 7.4. In one embodiment, the washing buffer has a pH between 6.5 to 7.4. In one embodiment, the washing buffer has a pH of 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.1, 7.2, 7.3 or 7.4. In one embodiment, the washing buffer is saline. In one embodiment, the washing buffer is MOPSO buffer. In one embodiment, the washing buffer is Tris buffer. In one embodiment, the washing buffer is PBS buffer. In one embodiment, the washing buffer is MOPS buffer. In one embodiment, the washing buffer is Bis-Tris buffer. In one embodiment, the washing buffer is ADA buffer. In one embodiment, the washing buffer is MES buffer. In one embodiment, the washing buffer is HEPES buffer. In one embodiment, the washing buffer is citric buffer. In one embodiment, the washing buffer is acetate buffer. [00410] In one embodiment, the washing buffer is Tris buffer with a pH from 6.5 to 7.4. In one embodiment, the loading buffer is MOPSO buffer with a pH from 4.5 to 7.4. [00411] In one embodiment, the wash buffer comprises one or more salt. In one embodiment, NAI-1540508512v1 -81- Attorney Docket No.14497-018-228 the salt is an alkali metal salt. In one embodiment, the salt is an alkali metal halide. In one embodiment, the salt is NaCl, KCl, KPO4, NaPO4, CaCl2, Mg2SO4, ZnCl2, MnCl2, MnSO4, NaSCN, KSCN, LiCl, MgCl2, or any combination thereof. In one embodiment, the salt is sodium chloride. In one embodiment, the salt is potassium chloride. In one embodiment, the salt is a mixture of sodium chloride and potassium chloride. In one embodiment, the salt has a concentration from 0.001 M to 1 M. In one embodiment, the salt has a concentration from 0.01 M to 1 M. In one embodiment, the salt has a concentration from 0.01 M to 0.2 M. In one embodiment, the salt is 0.01 M to 0.2 M sodium chloride. In one embodiment, the salt is at 0.01 M sodium chloride. In one embodiment, the salt is 0.2 M sodium chloride. [00412] In one embodiment, the washing buffer has a conductivity from about 0.1 mS/cm to 100 mS/cm at 22 °C. In one embodiment, the conductivity is from about 0.1 mS/cm to about 50 mS/cm at 22 °C. In one embodiment, the conductivity is from about 1 mS/cm to about 25 mS/cm at 22 °C. In one embodiment, the conductivity is from about 5 mS/cm to about 20 mS/cm at 22 °C. 6. EXAMPLES 6.1 EXAMPLE 1: Preparation of Resins [00413] Different resins are prepared by reacting the epoxy-activated resins, such as TOYOPEARL AF-Epoxy-650M resin or Epoxy-Activated Agarose, with ligands (e.g. diethanolamine, diisopropanolamine (DIPA), 2-methylethanolamine, 2-ethylethanolamine, morpholine, 2,6-dimethylmorpholine, 4-methylpiperazine, or 4-hydroxyethylpiperazine). The reaction proceeds under a ring opening reaction as shown below: The ligands
Figure imgf000084_0001
groups with theoretical pKa of about 7-8. A detailed description about the preparation and purification of the new resins are described in Section 6.2. Other resins are prepared and purified using similar method. 6.2 EXAMPLE 2: Preparation and Purification of TOYOPEARL-DIPA Resin [00414] An amount of 6.01 g of dry powder of TOYOPEARL AF-Epoxy-650M resin (density of NAI-1540508512v1 -82- Attorney Docket No.14497-018-228 epoxy groups is about 800 μmol/g of the dry powder, methacrylate polymer beads) was hydrated with 50 mL ice cold ultrapure water (at least 5 times more water than powder), which was kept at 5 °C until further use. The resin was centrifuged at 300 x g for 5 min at about 5 °C to settle the resin, and the water was removed with a serological pipette. The resin was washed with 35 mL ice cold ultrapure water, centrifuged at 300 x g for 5 min at 4 °C, and water was removed. This washing step was repeated for 2 more times. A fresh solution of 4 M diisopropanolamine (DIPA) in ultrapure water was prepared, and 30 mL of the solution was added to the 30 mL of washed beads at room temperature (e.g.23 °C). The concentration of DIPA in the reaction is 2 M. The amount of epoxy groups in the reaction mixture is about 4808 μmol. The amount of DIPA is about 120 mmol, which is about 25 equivalents of the amount of the epoxy groups in the reaction. A 2 cm magnetic stirrer was added for gentle mixing during the reaction (slow mixing at number 3 of IKA RCT basic magnetic stirrer). The resin coupling was done in a round bottom flask at about 90 °C in an oil bath for 15 h. After reaction, the mixture was cooled to room temperature, and the resin was transferred into two 50 mL falcon tubes, giving about 15 mL resin volume in each tube. The resin was centrifuged at 300 g at 5 °C for 5 min to settle the beads and the liquid was removed. The resin was washed by filling up to 50 mL in each tube with ultrapure water, centrifuged at 300 x g at 25 °C for 5 min, and the liquid was removed. This washing step was repeated for 3 times. The tubes were filled up with 1 M NaOH in water, incubated for 1 h at 25 °C (no shaking), centrifuged at 300 g at 25 °C for 5 min, and liquid was removed. The resin was washed by filling up to 50 mL in each tube with ultrapure water, centrifuged at 300 g at 25 °C for 5 min, and liquid was removed. This washing step was repeated 3 times, and 25 mL of wet resin was obtained. The tubes were then filled up with 1 M NaCl in water, and stored at 4 °C until further use. Long-term storage condition is 1 M NaCl with 20 % EtOH. 6.3 EXAMPLE 3: Measurement of pKa of Ionizable Moiety on Resin [00415] The pKa of the ionizable moieties on the resins are estimated by measuring the pKa of analogous small molecules prepared by coupling the same ligand (e.g. secondary amines) to 1,4- Butanediol diglycidyl ether (BDDE) to mimic the epoxy-activated resin. The pKa values are determined by acid-base titration experiments using 1 M HCl in water (see Figure 2, Figure 3). A list of the measured pKa is shown in Table 1. [00416] Table 1. Measured pKa Values of Ionizable Moieties of the polymer Resins pKa NAI-1540508512v1 -83- Attorney Docket No.14497-018-228 BDDE coupled with diethanolamine 8.2 BDDE coupled with DIPA 7.6 BDDE coupled with 2-methylethanolamine 8.6 BDDE coupled with 2-ethylethanolamine 8.8 BDDE coupled with 2,6-dimethylmorpholine 6.7 BDDE coupled with morpholine 6.8 BDDE coupled with 4-methylpiperazine 8.5 BDDE coupled with 4-hydroxyethylpiperazine 8.3 EXAMPLE 4: Preparation of Exosomes [00417] HEK293 cells grown in a bioreactor were harvested and clarified with depth filtration media. The samples were then treated with Benzonase® endonuclease to digest nucleic acids. For larger scale productions, high density cultures were maintained in a stirred bioreactor in perfusion mode, whereby the harvested perfusion supernatant was pre-clarified and filtered by an alternating tangential flow system fitted with a 0.2 um hollow fiber filter. EVs were isolated and purified from the clarified conditioned media using a variety of methods, typically a combination of diafiltration/ultrafiltration with tangential flow filtration (TFF), including concentration of the EV containing supernatant approximately 10X with a tangential flow filtration (TFF) with a 300 kDa MWCO and diafiltered into 37 mM MES acid, 20 mM NaOH, 137 mM NaCl, pH 6.2. Optionally the supernatant was additionally purified using a flow through based multimodal chromatography (Captocore 700). Purified EVs can optionally be frozen and stored for downstream analysis and application. 6.4 EXAMPLE 5: Purification of Hybridosomes on Commercial Anion Exchange (AEX) Resins [00418] Hybridosomes prepared from different kinds of LNPs and exosomes were purified on commercial POROS D resin, which is a weak anion exchange rein, under different eluting conditions. [00419] Hybridosomes prepared by the fusing of LNP comprising siRNA and Cy5 labelled lipid, and exosome were purified with POROS D resin under elution conditions of pH at 9.0 and 1 M NaCl. Recovery yield of Hybridosomes was 6.6% (see Figure 4). [00420] Hybridosomes prepared by the fusing of LNP comprising polyA and Cy5 labelled lipid, and exosome (untargeted) were purified with POROS D resin under elution conditions of pH at 5.5 and 1 M NaCl. Recovery yield of Hybridosomes was 34.9%. NAI-1540508512v1 -84- Attorney Docket No.14497-018-228 [00421] Hybridosomes prepared by the fusing of LNP comprising polyA and Cy5 labelled lipid, and exosome (untargeted) were purified with POROS D resin under elution conditions of pH at 9.0 and 1 M NaCl. Recovery yield of Hybridosomes was 25.9 %. [00422] As observed above, yield of hybridosomes purification on commercially available anion exchange resins is low (<35%), and generally non-reproducible. There is significant problem of particle loss. As for hybridosomes, they are not released even at high pH (9) and high salt concentrations (NaCl up to 1M). Strong retention of hybridosomes on resin is possibly due to high avidity. There is a need of weak anion exchange resin where charge can be controlled and switched on/off. 6.5 EXAMPLE 6: Purification of Exosomes on Commercial AEX Resin [00423] Exosomes isolated from cells are purified on several commercial resins (e.g. POROS D resin, CAPTO Q resin, or CAPTO DEAE resin) under different eluting conditions (see Figure 8). The results are summarized in Table 2. [00424] Table 2: Purification of Exosomes on Commercial Resins Resin Elution Condition Elution Yield (%) CAPTO Q pH 7.4, 1 M NaCl 0.5 CAPTO DEAE pH 7.4, 1.1 M NaCl 8.8 CAPTO DEAE pH 7.4, 0.8 M NaCl 13.7 CAPTO DEAE pH 10.0, 0.3 M NaCl 24.4 POROS D pH 5.5, 1 M NaCl 8.8 POROS D pH 9.0, 1 M NaCl 23.2 [00425] As observed above, recovery yield of exosomes from commercially available anion exchange resins is generally low (<25%). Among them, weak exchangers (DEAE, D) perform better than strong exchangers, but the maximal yield is less than 25% and non-reproducible. Exosomes are strongly retained on the resins and are not released even at high pH (9-10) and high salt concentrations (NaCl up to 2M and phosphate up to 1M). 6.6 EXAMPLE 7: Purification of Exosomes Using New Resins Prepared [00426] Exosomes are purified using the resins prepared in EXAMPLE 1. The purification process is depicted in Figure 7. In general, exosomes are loaded to gravity-flow column comprising the resins at pH about 5.5. Sequential pH elution (from pH 6.5 to 9.0, intervals of 0.25) in low salt (50 mM NaCl) was performed. The final elution condition is at pH 9.5 and 1 M NaCl. [00427] The new resins allow exosome elution in a pH-dependent manner with much higher NAI-1540508512v1 -85- Attorney Docket No.14497-018-228 recovery yield than commercial resins (Figure 8, Table 3). At low salt, exosomes are eluted at a pH that is close to the pKa of the ionizable moieties resin as measured in EXAMPLE 3. As shown in Figure 10, there is a positive correlation between the elution pH of exosomes and the pKa of the ionizable moieties of the resins. Figure 11 and Figure 12 show that elution yields of exosome over 70 % were readily achieved using DIPA or morpholine modified resins. [00428] The method also allows separation of different particle species using the new resins under salt gradient condition (see Figure 13 to Figure 16). Different particle species characterized by different exosomal markers (e.g. CD9, CD63), Turbo luc, total protein or nucleic acids are isolated and collected in different fractions. [00429] Table 3: Purification of Exosomes on New Resins Resin Loading Elution Elution Yield (%) Agarose-Morpholine 50 mM NaOAc, pH 5.5, 50 mM Glycine, pH 10, 72% 50 mM NaCl 50 mM NaCl TOYOPEARL-Morpholine 20 mM Tris, pH 6.8, 50 20 mM Tris pH 7.9, 150 53% mM NaCl mM NaCl TOYOPEARL-DIPA 20 mM Tris, pH 6.8, 50 20 mM Tris, pH 7.9, 24% mM NaCl 240 mM NaCl TOYOPEARL-Morpholine 20 mM Tris, pH 6.8, 50 20 mM Tris, pH 6.8, 46% mM NaCl 0.05-1 M NaCl gradient TOYOPEARL-DIPA 50 mM HEPES, pH 7.0, 50 mM TRIS, pH 8.0, 80% 200 mM NaCl 0.01-1M NaCl gradient 6.7 EXAMPLE 8: Purification of Lentivirus Using New Resins Prepared Cell amplification, transfection, harvesting and supernatant PEG-precipitation [00430] The HEK293T cells seeded in CellStack Cells 5 chambers with vent caps (CS5) are cultured with DMEM supplemented with 10% FBS and 1% Pen/Strep. At approximately 60-70% of confluency, cells are transfected with the vector plasmid (containing the E1a-GFP transgene cassette) and two helper plasmids (psPAX2 which contains Gag-Pol -Rev-Tat genes and the pMD2G which contains the VSV-G pseudotyped envelope) using the CaPO4 precipitate technique. The culture medium is removed from the four CS5 and exchanged with the transfection medium; the cells are then incubated 6 to 15 hours at 37 +/- 1 °C and 5 +/- 1% CO2. The transfection medium is then removed from the CS5 and replaced by fresh exchange medium (Advanced DMEM 1X, 1% Pen/Strep) prior to a 2-day incubation at 37 +/- 1 °C and 5 +/- 1% CO2. The cells of the 4 CS5 transfected are then harvested. The supernatant is kept and clarified by a low-speed centrifugation (10 min at 1300 rpm). NAI-1540508512v1 -86- Attorney Docket No.14497-018-228 Vector titration [00431] Infectious particles of the lentivirus vector (LV) were titrated by digital quantitative polymerase chain reaction (dPCR) after infection of HeLa cells with the LV sample. HeLa cells were cultivated in 6-well plate with different volume of clarified LV, harvested 5 days post infection and measured by digital PCR targeting the GAG gene. Infectious LV particles titer is expressed in TU/mL. LV purification [00432] In order to increase virus particles binding to DIPA column - we have applied the workflow shown below. NOTE: Loading was based on NTA data and Waytt DLS, similar technologies to those used for exosomes quantification. Final LV measurement was done using P24 protein ELISA. [00433] LV purification workflow: Clarified harvest
Figure imgf000089_0001
DNase TFF1* supernatant was centrifuged and filtered through 0.45 µm filter. Benzonase
Figure imgf000089_0002
treatment was performed by adding to the clarified harvest 500 IU/L in the presence of 2 mM magnesium chloride at 2-8°C DURATION. The treated clarified harvest was used for tangential flow filtration tests. Tangential flow filtration (TFF) was performed using Hollow Fiber Filter (Repligen D02-E300-05-N) with a molecular weight cut-off (MWCO) of 300 kD. The TFF was performed as follows: (1) ultrafiltration to concentrate the vector solution with a target concentration factor of 50–60, (2) diafiltration for buffer exchange using 20 diavolumes (DV) of diafiltration buffer (20 mM Tris, 50 mM NaCl, 1 % sucrose, pH 6.8). All steps were performed at a NAI-1540508512v1 -87- Attorney Docket No.14497-018-228 constant transmembrane pressure (TMP) of about 3 psi. The TMP was either controlled by adjusting the flow of the feed pump or by regulating the retentate pressure with an adjustable valve. Finally, the concentrated and re-buffered lentiviral vector solution was recovered from the TFF skid. The resulting LV material was then purified using chromatography step. [00435] Table 4: Chromatography conditions for DIPA resin: Phase Composition Column Volumes Equilibration 20 mM Tris, 50 mM NaCl, pH 6.8 5-7 CV* Loading Lentivirus (load about 1.5E+11 particles/mL) 0.5 mL Washing 20 mM Tris, 50 mM NaCl, pH 6.8 7 CV Elution 20 mM Tris, 50-800 mM NaCl, pH 7.2 10-15 CV CIP** 1 M NaOH 5 CV Re-equilibration 20 mM Tris, 50 mM NaCl, pH 6.8 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place. [00436] Table 5: Chromatography conditions for Capto DEAE Resin Phase Composition Column Volumes Equilibration 50 mM Tris, 130 mM NaCl, pH 8 3-10 CV* Loading Lentivirus (load about 1.5E+11 particles/mL) 0.5 mL Washing 50 mM Tris, 130 mM NaCl, pH 8 7 CV Elution 50 mM Tris, 130-650 mM NaCl, pH 8 10-15 CV Wash 50 mM Tris, 1.3 M NaCl, pH 8 7 CV CIP** 1 M NaOH 5 CV Re-equilibration 50 mM Tris, 130 mM NaCl, pH 8 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place. [00437] Lentiviral particles purified by the resins are analyzed by p24 ELISA assay. As shown in Figure 17, under chromatography conditions (20 mM Tris, 70 mM NaCl, pH 7.2), the total recovery yield of p24 protein of lentiviral particles from the DIPA resin was 79.5 ± 5.5%. For the commercial Capto DEAE resin, the total recovery yield of lentivirus particles, quantified using p24 ELISA, was 20 ± 10 %. Thus, the new DIPA resin is more efficiency in the purification of lentiviral particles than the commercial resin. 6.8 EXAMPLE 9: Purification of Lentivirus Using New Resins prepared for Polishing Step Cell amplification, transfection, and harvesting NAI-1540508512v1 -88- Attorney Docket No.14497-018-228 [00438] HEK293T cells were seeded in T300 surface-treated cell culture flasks with vent caps and cultured with DMEM-F12 supplemented with 10% FBS and 1% Pen/Strep. At approximately 70-90% of confluency, cells were transfected with the vector plasmid (containing the SFFV-eGFP transgene cassette) and three helper plasmids (one plasmid containing Gag-Pol genes, one plasmid containing the Rev gene, and one plasmid encoding the VSV-G pseudotype envelope gene) using the Jetprime transfection reagent. At 5 hours after transfection, the culture medium was removed from the cell culture flasks and replaced with fresh pre-warmed OptiMEM medium. Transfected HEK293 cells were incubated at 37 °C and 5 % CO2. After a total of 24 hours post transfection the conditioned cell culture medium was harvested and replaced with fresh pre-warmed OptiMEM medium. The cells were incubated for another 24 hours at 37 °C and 5 % CO2. The harvested conditioned medium was clarified by low-speed centrifugation at 300 xg and 4 °C for 5 minutes in a tabletop centrifuge. After centrifugation the supernatant was filtered through a 0.45 µm filter into a sterile bottle and stored at 4 °C overnight. At 48 hours and 72 hours post transfection the conditioned cell culture medium was harvested and processed identically to the previously harvested conditioned cell culture medium. The conditioned cell culture medium from all harvest days was pooled. Viral vector quantification by viral transduction assay [00439] Infectious lentivirus vector samples were titrated by quantification of transgene expression in HEK293T cells infected with the LV sample. Serial dilutions of lentiviral vectors encoding eGFP were prepared and mixed with HEK293T cells in wells of a 96 well surface-treated cell culture plate, followed by incubation at 37 °C and 5 CO2. GFP fluorescence of HEK293T cells was measured 48 h after infection with the LV samples using a flow cytometer. From the percentage of GFP positive cells, only conditions of dilutions indicating single copy infection, namely 2-20% positive cells, were considered to determine the infectious LV particles titer. Infectious LV particles titer is expressed in TU/mL. LV purification [00440] The Lentivirus from harvested supernatant was processed as described in Example 8 until DIPA resin step. Lentivirus supernatant was centrifuged and filtered through 0.45 µm filter. The clarified supernatant was concentrated and dialyzed by tangential flow filtration (TFF) in combination with nuclease treatment. TFF was performed using Hollow Fiber Filter (Repligen D02-E300-05-N) with a molecular weight cut-off (MWCO) of 300 kD. The TFF was performed as follows: (1) ultrafiltration to concentrate the vector solution with a target concentration factor of NAI-1540508512v1 -89- Attorney Docket No.14497-018-228 50–60; (2) DENARASE® treatment by adding to the clarified harvest 25 IU/mL DENARASE® in the presence of 2 mM MgCl at 37 °C for 1 hour; (3) diafiltration for buffer exchange using 10 diavolumes (DV) of diafiltration buffer (20 mM Tris, 50 mM NaCl, pH 7.2). All steps were performed at a constant transmembrane pressure. Finally, the concentrated and re-buffered lentiviral vector solution was then purified using a chromatography step with 7 mL TOYOPEARL- DIPA resin or 7 mL Capto-DEAE resin as outlined in Table 6. The DEAE chromatography was performed as described in Example 8, whereby Tris or Phosphate buffer were used. Lentivirus elution fractions were diluted 1:3 (v/v) with 20 mM Tris pH 7.2, 105 mM sucrose for DIPA resin, or 1:7 (v/v) with 20 mM buffer pH 7.2, 81.6 mM sucrose for DEAE resin. Lentiviral particles purified by the resins were analyzed by viral transduction assay, p24 ELISA assay and contaminants assays. As shown in Figure 18, under chromatography conditions, the recovery yield of infectious lentivirus from the DIPA resin was 72.7 ± 4.1%. For the commercial Capto DEAE resin, the recovery yield of infectious lentivirus was only 25.7 ± 6.7 %. [00441] Table 6: Chromatography conditions for DIPA resin: Phase Composition Column Volumes Equilibration 20 mM Tris, 50 mM NaCl, pH 7.2 6 CV* Loading Diafiltrated Lentivirus 2 mL (about 0.6E+8 to 1.1E+8 3 CV infectious particles per mL) Washing 20 mM Tris, 75 mM NaCl, pH 7.2 4 CV Elution 20 mM Tris, 275 mM NaCl, pH 7.2 4 CV Wash 20 mM Tris, 500 mM NaCl, pH 7.2 4 CV CIP** 1 M NaOH 2 CV Re-equilibration 20 mM Tris, 50 mM NaCl, pH 7.2 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place. [00442] For contaminant clearance determination, the host cell protein content was evaluated by HEK293 Host cell protein ELISA (Cygnus) according to the manufacturer’s manual, whereby samples were used undiluted and 1:100 diluted in the respective buffer matrix and 50 µL of sample was used per well. Total protein concentration was assessed by Bio-Rad protein assay according to the manufacturer’s manual, whereby a standard range of 50 ug/mL to 500 ug/mL bovine serum albumin was used for the clarified conditioned medium. For samples after dialysis the micro BCA assay QuantiPro (Sigma-Aldrich) with a standard curve range from 2.5 µg/mL to 40 µg/mL bovine serum albumin were used. Host cell DNA was assessed by QuantiFluor dsDNA system (Promega) NAI-1540508512v1 -90- Attorney Docket No.14497-018-228 according to the manufacturer’s manual and 10 µL of sample was used per well. [00443] Table 7: Contaminant clearance assessment Host cell protein Process step DIPA Clearance DEAE Clearance (%) (%) Conditioned media TFF 97.23 ± 2.39 96.48 AEX polishing 99.81 ± 0.04 98.56 Total protein Process step DIPA Clearance DEAE Clearance (%) (%) Conditioned media TFF 95.24 ± 16.33 62 AEX polishing 97.87 ± 0.94 82.25 ± 3.61 dsDNA Process step DIPA Clearance DEAE Clearance (%) (%) Conditioned media TFF 99.07 ± 0.15 99 AEX polishing 99.34 ± 0.27 98.9 ± 0.71 6.9 EXAMPLE 10: Capturing and Purification of Lentivirus from Cell Culture Medium Using New Resins Prepared Cell expansion, transfection, harvesting, and clarification. [00444] HEK293T cells expansion, transfection and LV cell culture harvest and clarification was executed as described in Example 9, although only 24 h and 48 h harvest was collected and pooled. Viral vector quantification [00445] Infectious lentivirus vector l titer of the clarified harvests were determined by viral transduction assay as describe above. Total LV particles were quantified by p24 ELSIA (Origene) according to manufacturer’s protocol, whereby samples were diluted 1:500 to 1:5’000 in Dulbecco's phosphate buffered saline. [00446] Lentiviral Vector purification from cell culture medium workflow: NAI-1540508512v1 -91- Attorney Docket No.14497-018-228 Clarified harvest
Figure imgf000094_0001
Figure imgf000094_0002
Figure imgf000094_0003
medium was supplemented with 25 U/mL DENARASE, 2 mM MgCl2, and incubated at 37 °C for 1 hour. The DENARASE® treated LV supernatant was used in a chromatography step with TOYOPEARL-DIPA resin or Capto-DEAE resin. The DENARASE® treated LV feed was adjusted to pH 7 and a conductivity of 10.2 mS/cm by addition of a Bis-Tris propane buffer (5.4 mM Bis Tris, pH 6.2). [00448] Table 8: Chromatography conditions DIPA resin for capturing mode Phase Composition Column Volumes Equilibration 20 mM Tris, 50 mM NaCl, pH 7.0 5-7 CV* Loading Lentivirus (about 1.3E+9 infectious particles) Sample dependent Washing 20 mM Tris, 75 mM NaCl, pH 8 8 CV Elution 1 (main elution) 20 mM Tris, 150 mM NaCl, pH 8 10-15 CV Elution 2 20 mM Tris 275 mM, pH 8 10 CV Elution 3 20 mM Tris 1300 mM, pH 8 10 CV CIP** 1 M NaOH 5 CV Re-equilibration 20 mM Tris, 50 mM NaCl, pH 7 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place. [00449] Table 9: Chromatography conditions for Capto DEAE Resin Phase Composition Column Volumes Equilibration 50 mM Tris, 130 mM NaCl, pH 8 3-10 CV* Loading Lentivirus (about 1.3E+9 infectious particles) Sample dependent Washing 50 mM Tris, 130 mM NaCl, pH 8 7-10 CV Elution 1 (main elution) 50 mM Tris, 130-650 mM NaCl, pH 8 10-15 CV NAI-1540508512v1 -92- Attorney Docket No.14497-018-228 Elution 2 50 mM Tris, 1.3 M NaCl, pH 8 10 CV CIP** 1 M NaOH 15 CV Re-equilibration 50 mM Tris, 130 mM NaCl, pH 8 7-10 CV *CV: Column Volume; **CIP: Cleaning In Place. [00450] Capturing mode chromatography was performed as summarized in tables 8 and 9 using an 5 mL TOYOPEARL-DIPA column or a HiTrap Capto-DEAE column. DIPA resin elution fractions were diluted 1:2 with 20 mM Bis-Tris pH 6.2, 140 mM sucrose, while DEAE elution fractions were diluted 1:5 in sterile 5 % sucrose solution. The clarified DENARASE treated lentivirus feed and collected chromatography fractions were analyzed for viral titer by cell transduction and p24 ELISA assay. Transduction unit (TU) recovery in the DIPA chromatography Elution 1 compared to the starting material was 59.7 ± 10.3% and 65.1 ± 13% for all elution fractions, while the TU recovery for the main elution fractions of the Capto DEAE chromatography was 17.3 ± 4.2 % and 23.8 ± 6.9 % for all elution fractions (Figure 19). Quantification of p24 recovery followed the same trend, with 59.8 ± 4.3% p24 recovery in the DIPA main elution fractions (71.1 ± 11% for all DIPA elution fractions), and 22.8 ± 2.5 % in the main DEAE elution fractions (28.8 ± 3.6 % for all elution fractions) (Figure 20). [00451] Contaminant clearance in the chromatography fractions were evaluated by Host cell protein (HCP) ELISA, QuantiFluor double stranded (ds) DNA assay, and Bio-Rad protein assay described in Example 9. Contaminants clearance in the DIPA main elution fractions compared to the starting material was 98.0 ± 0.6% for HCP, 91.7 ± 1.5% for total protein, and 70.5 ± 3.1% for dsDNA (Table 10). DEAE contaminant clearance was 96.03± 0.6% for HCP, 67.2 ± 5.42% for total protein, and 35.4 ± 15.2% for dsDNA in the DEAE main elution fraction compared to the starting material (Table 10). [00452] Table 10: DIPA and DEAE chromatography contaminant clearance Host cell protein DIPA DEAE HCP Clearance HCP Process step (ug) (%) (ug) Clearance (%) Supernatant/Denarase 493 ± 43 495 ± 44 AEX main fraction 9.58 ± 2.40 98.04 ± 0.56 18.2 ± 1.7 96.28 ± 0.56 AEX all fractions 10.99 ± 2.61 97.79 ± 0.72 19.03 ± 2.0 96.21 ± 0.65 Total protein DIPA DEAE Total Protein Clearance Total Clearance Process step (mg) (%) Protein (%) NAI-1540508512v1 -93- Attorney Docket No.14497-018-228 (mg) Supernatant/Denarase 10.5 ± 1.5 11.0 ± 1.1 AEX main fraction 0.878 ± 0.235 91.70 ± 1.55 3.56 ± 0.26 67.22 ± 5.42 AEX all fractions *0.878 ± 0.235 *91.70 ± 1.55 *3.56 ± 0.26 *67.22 ± 5.42 dsDNA DIPA DEAE DNA Clearance DNA Clearance Process step (ug) (%) (ug) (%) Supernatant/Denarase 5.93 ± 1.44 7.04 ± 1.59 AEX main fraction 1.78 ± 0.60 70.5 ± 3.1 4.11 ± 0.34 40.0 ± 10.4 AEX all fractions 2.27 ± 1.15 63.6 ± 9.7 4.67 ± 1.04 33.3 ± 8.7 *fractions <LOD NAI-1540508512v1 -94-

Claims

Attorney Docket No.14497-018-228 CLAIMS What is claimed is: 1. A method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises at least one ionizable moiety, wherein the ionizable moiety has a pKa from about 6.5 to about 9; b. contacting the solid support with the sample at a first pH at which the vesicles, cells or viral particles bind to the solid support; and c. eluting the vesicles, cells or viral particles from the solid support at a second pH, wherein the second pH is higher than the first pH. 2. The method of claim 1, wherein the method further comprises a washing step at the first pH. 3. The method of claim 1 or 2, wherein the first pH is lower than the pKa of the ionizable moiety. 4. The method of any one of claims 1 to 3, wherein the first pH is below 8. 5. The method of any one of claims 1 to 4, wherein the first pH is between 4.5 to 7.4. 6. The method of any one of claims 1 to 5, wherein the second pH is above 7. 7. The method of any one of claims 1 to 6, wherein the second pH is between 7.4 to 9. 8. The method of any one of claims 1 to 7, wherein the first pH is between 6.5 to 7.4 and the second pH is between 7.4 to 9. 9. The method of any one of claims 1 to 8, wherein the second pH is 7.4. 10. The method of any one of claims 1 to 9, wherein the eluting step is performed at a conductivity from about 55 mS/cm to about 140 mS/cm. 11. The method of any one of claims 1 to 10, wherein the eluting step is performed at a salt concentration from about 0.01 M to about 2 M. 12. The method of any one of claims 1 to 11, wherein the solid support has a binding affinity to the vesicles, cells, or viral particles at the first pH that is at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or NAI-1540508512v1 95 Attorney Docket No.14497-018-228 at least 1000 times higher than the binding affinity to the vesicles, cells, or viral particles at the second pH. 13. The method of any one of claims 1 to 12, wherein the solid support has an average pore size from about 10 nm to about 1000 nm. 14. The method of claim 13, wherein the average pore size is from about 10 nm to about 200 nm. 15. The method of any one of claims 1 to 14, further comprising a step of preparing the solid support. 16. The method of claim 15, wherein the solid support is prepared by providing a resin comprising at least one reactive group on the surface. 17. The method of claim 16, wherein the reactive group is covalently connected to the resin via a linker. 18. The method of claim 16, wherein the resin is a cation exchange (CEX) resin, an anion exchange (AEX) resin, a mixed mode resin, an affinity resin, a pseudo affinity resin, a hydrophobic interaction resin, a hydrophobic charge induction resin, or any combination thereof. 19. The method of claim 18, wherein the resin is an anion exchange (AEX) resin. 20. The method of any one of claim 16 to 19, wherein reactive group can react with a ligand, wherein the ligand is an amine, an alcohol, a thiol, or a carboxylic acid. 21. The method of any one of claim 16 to 20, wherein the reactive group is selected from a group consisting of an epoxy group, a N-hydroxysuccinimide (NHS) ester, a Sulfo-NHS ester, an imidoester, a fluorophenyl ester, an aldehyde, a carbonate, an anhydride, a sulfonyl chloride, an isothiocyanate, an isocyanate, an acyl azide, a thiol, a disulfide, a cyanogen bromide, a carbodiimide and lysine. 22. The method of claim 21, wherein the reactive group is an epoxy group. 23. The method of claim 21, wherein the epoxy group is capable of reacting with the ligand via a ring-opening reaction to form an ionizable moiety and a hydroxyl group. 24. The method of any one of claims 16 to 23, wherein the reactive group has a density of from about 100 µmol to about 2000 µmol per gram of the resin. NAI-1540508512v1 96 Attorney Docket No.14497-018-228 25. The method of claim 24, wherein the reactive group has a density of from about 500 µmol to about 1000 µmol per gram of the resin. 26. The method of claim 20, wherein the ligand is an amine. 27. The method of claim 26, wherein the amine is a secondary amine. 28. The method of claim 27, wherein the secondary amine is R1-NH-R2, wherein R1, R2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the atoms they are attached to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is optionally substituted. 29. The method of claim 26, wherein the amine has a pKa of from about 8 to about 11 as determined by titration experiment using hydrochloric acid. 30. The method of claim 20, wherein the ligand is selected from a group consisting of diethylamine, diethanolamine, dimethylethanolamine, diisopropylamine, isopropylethylamine, diisopropanolamine, 2-methylethanolamine, 2-ethylethanolamine, 2-(isopropylamino)ethanol, 2- (butylamino)ethanol, 2-benzylaminoethanol, piperidine, morpholine, 2,6-dimethyl-morpholine, 4- (2-chloroethyl)morpholine, 4-morpholinecarbonyl chloride, piperazine, 4-methylpiperazine and 4- hydroxyethylpiperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide; wherein each piperidine, morpholine, piperazine, pyrrole, pyrrolidine, imidazole, pyrazole, triazole, succinimide, phthalimide and glutarimide is optionally substituted. 31. The method of any one of claims 16 to 30, wherein the preparation of the solid support further comprises a step of contacting the reactive group with the ligand to form an ionizable moiety. 32. The method of any one of claims 1 to 31, wherein the ionizable moiety comprises a tertiary amino group. 33. The method of any one of claim 1 to 32, wherein the ionizable moiety has a pKa from about 7.6 to about 8.8. 34. The method of any one of claims 1 to 33, wherein the ionizable moiety has a density of from about 50 µmol to about 1000 µmol per gram of the solid support. 35. The method of any one of claims 1 to 34, wherein the ionizable moiety is capable of binding and releasing the vesicles, cells or viral particles in a pH-dependent manner. NAI-1540508512v1 97 Attorney Docket No.14497-018-228 36. The method of claim 35, wherein the solid support is capable of binding the vesicles, cells or viral particles at an efficiency of at least 80% at the first pH and releasing the vesicles, cells or viral particles at an efficiency of at least 50% at the second pH. 37. The method of any one of claims 1 to 36, wherein the vesicle, cell or viral particle is negatively charged at the first pH. 38. The method of any one of claims 1 to 37, wherein the vesicle, cell or viral particle is negatively charged at the second pH. 39. A method of purifying vesicles, cells or viral particles from a sample comprising the steps of: a. providing a solid support, wherein the solid support comprises a polymer, wherein the polymer is functionalized with an ionizable moiety, wherein the ionizable moiety is - N(C1-6 alkyl)2, and wherein each alkyl is independently optionally substituted; b. contacting the solid support with the sample in a loading buffer; and c. eluting the vesicles, cells or viral particles from the solid support with one or more elution buffers, wherein each elution buffer independently has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. 40. The method of claim 39, wherein the polymer is agarose, polystyrene, polymethacrylate, polyacrylamide, or hydroxylated methacrylic polymer. OH 41. The method of claim 39 or 40, wherein the ionizable . 42. The method of any one of claims 39 to 41, wherein
Figure imgf000100_0001
density of from about 50 µmol to about 1000 µmol per gram of the solid support, or from about 250 µmol to about 800 µmol per gram of the solid support. 43. The method of any one of claims 39 to 42, wherein the loading buffer and the one or more elution buffers have the same pH. 44. The method of any one of claims 39 to 43, wherein the one or more elution buffers each independently has a salt concentration or conductivity that is at least 1.2 times, at least 1.4 times, at NAI-1540508512v1 98 Attorney Docket No.14497-018-228 least 1.6 times, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, or at least 20 times higher than the salt concentration or conductivity of the loading buffer. 45. The method of any one of claims 39 to 44, further comprising one or more washing steps. 46. The method of any one of claims 1 to 45 for purifying vesicles, wherein the vesicle is a nanovesicle. 47. The method of any one of claims 1 to 45 for purifying viral particles, wherein the viral particle is lentiviral particle. 48. The method of claim 46, wherein the nanovesicle has a diameter of from about 20 nm to about 1000 nm, from about 50 nm to about 400 nm, or from about 100 nm to about 200 nm. 49. The method of any one of claims 1 to 45 for purifying vesicles, wherein the vesicle is an extracellular vesicle. 50. The method of claim 49, wherein the extracellular vesicle is exosome. 51. The method of claim 46, wherein the nanovesicle is an artificial nanovesicle. 52. The method of claim 51, wherein the artificial nanovesicle is a liposome. 53. The method of claim 46, wherein the nanovesicle is a hybrid nanovesicle. 54. The method of claim 53, wherein the hybrid nanovesicle is hybridosome. 55. The method of claim 54, further comprising a step of preparing the hybridosome by fusing one or more lipid nanoparticle with one or more exosome. 56. The method of claim 55, wherein the fusing is performed at a pH below 6.5. 57. The method of any one of claims 1 to 56, wherein the sample comprises at least one impurity that is a small molecule, a macromolecule, a different vesicle, a different cell, or a different viral particle. 58. The method of claim 57, wherein the sample comprises at least one impurity that is a different vesicle, wherein the different vesicle is a lipid nanoparticle, an exosome or a liposome. 59. The method of claim 57 or 58, wherein the impurity has a binding efficiency with the solid support of less than 20% at the first pH. NAI-1540508512v1 99 Attorney Docket No.14497-018-228 60. The method of any one of claims 1 to 59, wherein the sample has a volume of from about 0.1 mL to about 2000 L, from about 10 L to about 2000 L, or from about 100 L to about 1000 L. 61. The method of any one of claims 1 to 60, wherein the sample is pretreated before contacting with the solid support. 62. The method of claim 61, wherein the pretreatment comprises chromatography step, centrifugation, ultra-centrifugation, filtration, ultra-filtration, diafiltration, clarification, lyophilization, lysing, digestion, drying, dilution, concentration, heating, cooling, or any combination thereof. 63. The method of any one of claims 1 to 62, wherein the sample is produced from a cell culture container or bioreactor. 64. The method of any one of claims 1 to 63, wherein the vesicle, cell or viral particle is eluted at a yield of at least 50%. 65. The method of claim 64, wherein the vesicle, cell or viral particle is eluted at a yield of at least 80%. 66. The method of any one of claims 1 to 65, further comprising a step of detecting the elution of the vesicle, cell or viral particle. 67. The method of claim 66, wherein the detecting involves the detection of optical signal from the vesicle, cell or viral particle, wherein the optical signal is UV-Vis signal, fluorescence signal, luminescence signal, enzyme-linked immunosorbent assay (ELISA), or light scattering signal. 68. A resin of Formula (I):
Figure imgf000102_0001
, wherein is a polymer selected from the group consisting of agarose, cellulose, polystyrene, polyacrylamide, and hydroxylated methacrylic polymer; wherein the polymer is optionally substituted; NAI-1540508512v1 100 Attorney Docket No.14497-018-228 wherein R1 and R2 are each independently alkyl, hydroxyalkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, cycloalkyl or heterocyclyl; or R1 and R2 together with the -N- to form a ring; wherein each alkyl, alkoxy, cycloalkyl and heterocyclyl is independently substituted; wherein L is C1-C48 alkylene, wherein one or more -CH2- in the alkylene is independently optionally replaced by -O-, -S-, -NH-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH-, -NHC(=O)-, or phenyl, and wherein the alkylene or phenyl is optionally substituted with one or more halogen, hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; and wherein the tertiary amino group has a pKa of from about 6.5 to about 9. 69. The resin of claim 68, wherein the resin has an average pore size of from about 10 nm to about 1000 nm. 70. The resin of claim 68 or 69, wherein the density of the tertiary amino group is from about 50 µmol to about 1000 µmol per gram of the resin, preferably from about 250 µmol to about 800 µmol per gram of the resin. 71. A solid support comprising the resin of any one of claims 68 to 70. 72. The solid support of claim 71, wherein the solid support is in the form of membrane, bead, or chromatography column. 73. The solid support of claim 71, wherein the solid support comprises the resin in an amount from about 0.05 gram to about 5000 gram. 74. A kit comprising the resin of any one of claims 68 to 70 or the solid support of any one of claims 71 to 73, a loading buffer, and an elution buffer, wherein the loading buffer has a pH between 4.5 to 7.4, and wherein the elution buffer has a pH between 7.4 to 9. 75. A kit comprising the resin of any one of claims 68 to 70 or the solid support of any one of claims 71 to 73, a loading buffer, and an elution buffer, wherein the loading buffer has a salt concentration or conductivity that is higher than the salt concentration or conductivity of the loading buffer. 76. The kit of claim 74 or 75, further comprising a washing buffer, wherein the washing buffer has a pH between 4.5 to 7.4. NAI-1540508512v1 101 Attorney Docket No.14497-018-228 77. The kit of claim 74 or 75, wherein the loading buffer is a MOPSO buffer, Tris buffer, phosphate buffer, MOPS buffer, Bis-Tris buffer, ADA buffer, MES buffer, HEPES buffer, citric buffer, or acetate buffer. 78. The kit of claim 74 or 75, wherein the elution buffer is a phosphate buffer, HEPES buffer, Tris buffer, Bicine buffer or Tricine buffer. 79. The kit of any one of claims 74 to 78, wherein the elution buffer has a conductivity from about 55 mS/cm to about 140 mS/cm. 80. The kit of any one of claims 74 to 78, wherein the elution buffer has a salt concentration from about 0.01 M to about 2 M. 81. The kit of claim 80 , wherein the elution buffer has a salt concentration of greater than 0.65 M. NAI-1540508512v1 102
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