[go: up one dir, main page]

WO2025238197A1 - Échafaudage polymère tridimensionnel (3d) fonctionnalisé pour chromatographie d'affinité - Google Patents

Échafaudage polymère tridimensionnel (3d) fonctionnalisé pour chromatographie d'affinité

Info

Publication number
WO2025238197A1
WO2025238197A1 PCT/EP2025/063502 EP2025063502W WO2025238197A1 WO 2025238197 A1 WO2025238197 A1 WO 2025238197A1 EP 2025063502 W EP2025063502 W EP 2025063502W WO 2025238197 A1 WO2025238197 A1 WO 2025238197A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
polymer scaffold
biological entity
scaffold
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/063502
Other languages
English (en)
Inventor
Herbert Stadler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IBA Lifesciences GmbH
Original Assignee
IBA Lifesciences GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by IBA Lifesciences GmbH filed Critical IBA Lifesciences GmbH
Publication of WO2025238197A1 publication Critical patent/WO2025238197A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • 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
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3823—Affinity chromatography of other types, e.g. avidin, streptavidin or biotin
    • 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
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • 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
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3225—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product
    • B01J20/3227—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating involving a post-treatment of the coated or impregnated product by end-capping, i.e. with or after the introduction of functional or ligand groups
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing

Definitions

  • the present invention relates to a functionalized three-dimensional (3D) polymer scaffold which comprises immobilized proteins or protein fragments on its surface, wherein these immobilized proteins or protein fragments are used for binding a target biological entity during affinity chromatography as well as to a method of manufacturing such 3D polymer scaffold.
  • the present invention further relates to the use of said 3D polymer scaffold as stationary phase in affinity chromatography for the isolation of a target biological entity as well as to a method for isolation of a target biological entity using said 3D polymer scaffold.
  • Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures. It is based on highly specific biological interactions between two molecules, such as interactions between enzyme and substrate, receptor and ligand, or antibody and antigen. These interactions, which are typically reversible, are used for purification by placing one of the interacting molecules, referred to as affinity ligand, onto a solid matrix to create a stationary phase while the target molecule is in the mobile phase. The capture step is generally followed by washing and elution, resulting in recovery of highly purified molecules. Highly selective interactions allow for a fast, often single step, process, with potential for purification in the order of several hundred to thousand-fold.
  • affinity chromatography uses of affinity chromatography include the ability to concentrate substances present at low concentration and the ability to separate proteins based on their biological function where an active form can be separated from the inactive form or a form with different biological function (Urh et al., Methods Enzymol 463 (2009), 417-438).
  • biopharmaceutical-based therapies highlights the demand for efficient chromatographic methods that can be used to purify the desired biomolecules (e.g., nucleic acids, enzymes, or monoclonal antibodies) which are presently under consideration in clinical trials or approved by the Food and Drug Administration, or even cells or exosomes.
  • desired biomolecules e.g., nucleic acids, enzymes, or monoclonal antibodies
  • These molecules present distinct chemical and structural properties, which are critical cues for the development and production of adequate chromatographic supports.
  • the present invention relates to the embodiments as characterized in the claims and the description and illustrated in the Examples.
  • the present invention relates to three- dimensional (3D) polymer scaffolds for use in affinity chromatography of a target biological entity, i.e., for use in purification and enrichment of a target biological entity via affinity chromatography, wherein the 3D polymer scaffolds comprise immobilized proteins or protein fragments on their surface, which can be used to isolate the target biological entity during affinity chromatography.
  • the protein or protein fragment may either be an affinity ligand being immobilized to the surface of the 3D polymer scaffold and used for capturing biological entities, wherein such an affinity ligand can be for example streptavidin or a functional analog thereof like Strep-Tactin® and Strep-Tactin®XT (IB A lifesciences, Gottingen, Germany) and can thus be used to capture Strep-tagged biological entities, or the protein or protein fragment may be used as an anchor being immobilized to the surface of the 3D polymer scaffold for binding affinity ligands, such as oligonucleotides, or proteins or protein fragments comprising an antigen binding domain like Fab fragments, which can be used for capturing the target biological entity.
  • affinity ligands are preferably Strep-tagged affinity ligands.
  • Biological entities like cells, exosomes, proteins, e.g., enzymes or antibodies, and oligonucleotides present distinct chemical and structural properties, which are critical cues for the development and production of adequate chromatographic supports.
  • Three-dimensional printing (3DP) is in the early stage of its use in the production of chromatographic supports as a fast, very precise, and reproducible methodology.
  • 3DP allowing to produce 3D polymer scaffolds, can provide excellent performance properties to the chromatographic structures, it cannot, per se, lead to high-quality pharmaceutical products, but the coupling of specific ligands is crucial to enable the attainment of high-purity yields of the desired biological product.
  • amino acids have been successfully applied as affinity ligands to the purification of biomolecules, especially DNA, by chromatography since this kind of ligand can mimic the natural interaction phenomena that occur between nucleic acids and proteins in biological environments.
  • this approach has been applied to chromatographic supports that have been produced by 3DP, i.e., a novel chromatographic matrix has been generated by coupling arginine to such 3D scaffolds and extensive studies have been performed to explore the capacity of these novel chromatographic matrices to interact with the given biomolecules.
  • Example 1 it has been shown that it is possible to couple Strep-tagged Fab fragments (aCD3-Fab fragments) via StrepTactin®-Tetramers to a polymer matrix that has been generated by 3DP.
  • aCD3-Fab fragments Strep-tagged Fab fragments
  • StrepTactin®-Tetramers Such a functionalized 3D polymer scaffold has been successfully used for purification of CD3+ cells from buffy coat; see Example 2.
  • the present invention relates to a 3D polymer scaffold (polymer matrix that has been generated by 3DP) for use in purification and enrichment of a target biological entity via affinity chromatography, wherein the 3D polymer scaffold is functionalized with proteins or protein fragments, meaning that the protein or protein fragments are immobilized on the surface of the scaffold and are thus either used as affinity ligands for capturing the target biological entity or as anchors to bind affinity ligands, which are used for capturing the target biological entity.
  • a 3D polymer scaffold which is functionalized with proteins or protein fragments encompasses a 3D polymer scaffold which comprises (i) a protein or protein fragment as affinity ligand, (ii) a protein or protein fragment as anchor for an affinity ligand, or (iii) a protein or protein fragment as anchor for an affinity ligand including the affinity ligand (which can also be a protein or protein fragment, but also an oligonucleotide).
  • a protein fragment refers to any peptide or protein that results from cleavage or destruction of a larger protein.
  • Oligonucleotides, including aptamers are polymeric sequences of nucleotides - RNA, DNA and their analogs, typically within a range of 20 - 100 bases. Although they can be formed by bond cleavage of (longer)segments, they are more commonly synthesized by polymerizing individual nucleotide precursors,
  • the protein or protein fragment, either used as affinity ligand or as anchor for an affinity ligand is in one particular embodiment streptavidin or a functional analog thereof like Strep-Tactin® and Strep-Tactin®XT (IB A lifesciences, Gottingen, Germany), which are specially engineered streptavidin molecules.
  • the polymer scaffolds which are functionalized with these streptavidin molecules can be used for purification of biological entities, like proteins, which comprise a streptavidin binding peptide, like biotin or a Strep-tag®, in particular Strep-tag®II which consists of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, SEQ ID NO: 3), or Twin- Strep-tag® (TST) which includes this motif two times in series connected by a linker and is accordingly composed of 28 amino acids, e.g.., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys- (GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 9), or Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-T
  • the affinity ligand which is preferably immobilized to the 3D polymer scaffold via an anchor, i.e., via a protein or protein fragment, like streptavidin or a functional analog thereof, comprises an antigen-binding domain.
  • a protein or protein fragment can be for example an antibody, an antibody fragment, or a receptor, preferably a Fab fragment.
  • the polymer scaffolds that are functionalized with these kinds of proteins or protein fragments, i.e., proteins or protein fragments which comprise an antigen-binding domain can be used for purification of biological entities, like cells or exosomes which comprise antigens on their surface or proteins, which bind to the antigen-binding domain.
  • proteins or protein fragments which comprise an antigen-binding domain are coupled to the polymer scaffold via a streptavidin or a functional analog thereof, i.e., streptavidin or a functional analog thereof serves as anchor for the affinity ligand and is coupled to the polymer scaffold, and the protein or protein fragment which comprises an antigen-binding domain like an antibody, an antibody fragment, or a receptor, preferably a Fab fragment, comprises a streptavidin-binding peptide which enables coupling of said protein or protein fragment to the streptavidin.
  • the affinity ligand which is preferably immobilized to the 3D polymer scaffold via an anchor, z.e., via a protein or protein fragment, like streptavidin or a functional analog thereof, is an oligonucleotide.
  • the oligonucleotide is coupled to the polymer scaffold via a streptavidin or a functional analog thereof, z.e., streptavidin or a functional analog thereof serves as anchor for the affinity ligand and is coupled to the polymer scaffold, and the oligonucleotide comprises a streptavidin-binding peptide which enables coupling of said oligonucleotide to the streptavidin.
  • Immobilization of protein or protein fragments on the surface of the 3D polymer scaffold has been performed via functional groups as shown in Example 1.
  • the 3D polymer scaffold has been chemically coated with functional groups, z.e., the 3D polymer scaffold comprises on its surface functional groups for immobilization of the protein or protein fragment, wherein such functional groups allow (covalently) anchoring of the protein or protein fragment.
  • such functional group is an amino group, an aldehyde group, or a carboxyl group.
  • the surface of the 3D polymer scaffold is modified to become hydrophilic.
  • the 3D polymer scaffold is designed to provide for a large surface area, for example in the form of lattices or filters with large surface areas and desired flow properties resembling long-known chromatography matrices made for example of agarose beads.
  • a large surface area-to-volume ratio is advantageous for effective ligand immobilization and affinity purification.
  • a polymer scaffold made from different polymers like Polylactide (PLA) is suitable for the intended purpose, z.e., for functionalizing the polymer scaffold with the ligands and affinity chromatography.
  • PLA Polylactide
  • Such an affinity matrix is chemically and physically stable during the purification process, so that the support material itself as well as the attached ligand does not react to the solvents used in the process, nor does it degrade or become damaged by enzymes and microbes that might be present in the sample.
  • the present invention further relates to the use of the 3D polymer scaffold as stationary phase in affinity chromatography for the isolation/purification of a biological entity as well as to an affinity chromatographic process using said 3D polymer scaffold.
  • the present invention also relates to a method of manufacturing a 3D polymer scaffold comprising:
  • proteins or protein fragments can be coupled to polymer matrices that have been generated by 3DP opens a new field of efficient (in terms of time and costs) affinity chromatography of various biological entities, like proteins (e.g. antibodies), cells, and exosomes.
  • Such matrices which are used as stationary phase during affinity chromatography can be produced at low costs and in uniform and reproducible quality which was not possible with standard stationary phases like agarose, since agarose is expensive, and shows variation of beads in size and pores, and the beads need to be packed for every column which may lead to variations in different experimental runs.
  • the present invention also relates to a method for purification/isolation/enrichment of a target biological entity using the above-described 3D polymer scaffold, preferably a polymer scaffold which has been functionalized with streptavidin or a functional analog thereof or with an oligonucleotide, or protein or protein fragment comprising an antigen-binding domain, like a Fab fragment, immobilized to the polymer surface via streptavidin or a functional analog thereof, and wherein the biological entity comprises a streptavidin binding peptide and/or a surface antigen, preferably wherein the biological entity is a protein, a cell, or an exosome, or wherein the biological entity is an oligonucleotide.
  • the method comprises applying the biological entity to the 3D polymer scaffold under conditions allowing complex formation between the biological entity and the affinity ligand, z.e., the immobilized protein or protein fragment used as affinity ligand and the affinity ligand immobilized via a protein or protein fragment (anchor), respectively, and isolating/purifying/enriching the biological entity by releasing the biological entity from the affinity ligand.
  • the protein or protein fragment used as affinity ligand comprises streptavidin or a functionally analog or derivative thereof, preferably the ligand is Strep- Tactin® and the biological entity to be purified comprises a streptavidin binding peptide, preferably wherein the streptavidin-binding peptide is a Strep®-Tag.
  • Strep-Tactin® systems including for example the Strep-Tactin®XT system or the Strep-Tactin®XTS system (IBA GmbH, Gottingen, Germany).
  • the protein or protein fragment used as anchor comprises streptavidin or a functionally analog or derivative thereof, preferably the anchor is Strep-Tactin® and the affinity ligand to be coupled to said protein or protein fragment (anchor) comprises a streptavidin binding peptide, preferably wherein the streptavidin-binding peptide is a Strep®-Tag.
  • Strep-Tactin® systems including for example the Strep-Tactin®XT system or the Strep-Tactin®XTS system (IBA GmbH, Gottingen, Germany).
  • the affinity ligand is a protein or protein fragment comprising an antigen-binding domain and the biological entity to be purified comprises the corresponding antigen and is bound to the ligand via said antigen.
  • the affinity ligand comprises an oligonucleotide which comprises a specific sequence of nucleotides and the biological entity to be purified comprises their respective complementary oligonucleotides, DNA, or RNA and binds to said oligonucleotide of the affinity ligand by hybridization.
  • the affinity ligands comprise a streptavidin- binding peptide (in particular a Strep-Tag®), and the affinity ligand is preferably a strep-tagged oligonucleotide or a strep-tagged Fab-fragment.
  • Strep-Tactin® systems are for example described in detail in the international applications WO 02/077018 Al, WO 2014/076277 Al and WO 2017/186669 Al and in the European patent application EP 0 835 934 A2, all of which are herein expressly incorporated by reference. These systems are based on muteins of streptavidin that reversibly bind to biotin or the analog or derivative thereof and the corresponding binding peptides, respectively, as explained further below.
  • 3D polymer scaffolds have been developed and successfully tested that are useful alternatives as carriers in affinity chromatography and can replace some of the classical chromatography material like agarose or Sephadex, providing several important advantages over them.
  • the 3D polymer scaffold has fast flow properties as compared to conventional chromatography material resulting from the scaffold design.
  • the 3D polymer scaffold has a large surface area allowing for the isolation of a reasonable yield of the target biological material and unbound material can be rapidly removed, which altogether allows for a substantial time saving. Due to the free choice of the scaffold design, it can be adapted to desired flow characteristics, different liquid properties and it is scalable to any size.
  • the 3D polymer scaffold is also pressure stable and in contrast to the use of for example agarose beads, no column packing is necessary. Furthermore, the manufacturing costs are low since scaffold manufacturing can be carried out on simple 3D printers so that parallelization is possible and simple, inexpensive and sustainable plastic material can be used.
  • common chromatographic columns are based on non-ordered / granulated bed structures as mostly beads, for example classic agarose beads are used and the benefit of 3DP is the ability to design flow channels and pore structures tailored to the target product. Therefore, it is possible to optimize the stationary phase in a controlled and replicable manner to achieve the best binding and elution conditions.
  • Fig. 1 Example of a 3D-printed polymer scaffold.
  • Fig- 2 FACS analysis of CD3 positive cells (CD3+) in buffy coat before (starting material) and after (eluate) CD3 affinity chromatography by means of a Strep-Tactin® functionalized 3D-printed scaffold (surface modification: Amino-groups); A) FACS plot showing the percentage portion of CD3+ cells measured in the starting material; B) FACS plot showing the percentage portion of CD3+ cells measured in the eluate. Both plots also depict CD3+ cells which are additionally positive for CD4 (CD4+). C) Graphical illustration of enrichment of CD3+ cells by means of ST-scaffold.
  • the present invention relates to the embodiments as characterized in the claims, disclosed in the description and illustrated in the Examples and Figures further below.
  • the stationary phase needs a surface with immobilized proteins like amino acids, antibodies, antibody fragments, receptors, or affinity tags for purifying the target molecules or cells, i.e., the target biological entities.
  • immobilized proteins like amino acids, antibodies, antibody fragments, receptors, or affinity tags for purifying the target molecules or cells, i.e., the target biological entities.
  • Conventional chromatography in biotechnology and pharma usually uses agarose- or dextran-based materials in the form of beads, wherein these beads can be used for affinity chromatography with ligands attached to the surface of beads.
  • additive manufacturing of polymer material allows design of complex 3D structures ("scaffolds") in the form of lattices with large surface areas and desired flow properties resembling chromatography matrices.
  • surface of these scaffolds is usually hydrophobic and may not be used directly for chromatography of biologic material.
  • scaffolds with large surface areas per ccm have been created and their surfaces have been modified to become hydrophilic by plasma treatment and then coated chemically with amino groups that allow covalent anchoring of proteins or protein fragments which are then used as ligands or anchors for ligands for the isolation of target biological entities.
  • Strep- Tactin® anchor allows one the one hand the purification and enrichment of recombinant proteins or other molecules comprising a Strep-Tag®, and on the other hand allows the anchoring of further protein and protein fragments comprising a Strep-Tag®, for example a “Strep-tagged” Fab-fragment or other proteins or fragments comprising an antigen-binding domain which are used as ligands for the purification and enrichment of cells, exosomes, or other molecules comprising an antigen on their surface.
  • the present invention relates to a three-dimensional (3D) polymer scaffold, e.g., a polymer scaffold which has been produced by three-dimensional printing (3DP), for use as stationary phase in affinity chromatography of a target biological entity, wherein the surface of the 3D polymer scaffold comprises an immobilized protein or protein fragment, which is capable of capturing the target biological entity or which is capable of binding an affinity ligand which is capable of capturing the target biological entity.
  • the protein or protein fragment immobilized to the surface of the 3D polymer scaffold either serves as affinity ligand or as anchor to bind an affinity ligand.
  • the affinity chromatography employing the 3D polymer scaffold is useful for the isolation of a target biological entity.
  • Such 3D polymer scaffold which comprises an immobilized protein or protein fragment either as ligand, i.e., for binding to a target biological entity, or as anchor, i.e., for binding an affinity ligand for binding the target biological entity, or which comprises an immobilized protein or protein fragment as anchor including the bound affinity ligand, is also referred to a as functionalized scaffold.
  • the target biological entity is enriched in a sample that is obtained as a result of the use of the polymer scaffold of the present invention during affinity chromatography compared to the content (concentration) of the sample prior to the isolation of the biological entity.
  • the target biological entity might be enriched in a sample, for example, from about a content of about 0.1% of the entire amount of biological entities in a sample to about 10% or more, or 20% or more, 30% or more, 40% or more, in a sample collected after running a method according to the invention.
  • isolated also means that the sample, e.g., eluate or fraction obtained contains the target biological entity as essentially only kind of, e.g., a cell (cell population), for example, the isolated biological entities represents more than 75%, or more than 80%, or more than 85%, or more than 90%, or more than 95% or more than 97% or more than 99% of the biological entities present in a sample after the isolation procedure.
  • the term “about” refers to a value that is ⁇ 10% of the recited value.
  • the present invention relates to a 3D polymer scaffold, wherein the scaffold comprises on its surface functional groups for immobilization of the protein or protein fragment.
  • the surface of the 3D polymer scaffold is chemically treated to comprise functional groups that allow (covalently) anchoring of the protein or protein fragment.
  • Such a polymer scaffold is also referred to as activated polymer scaffold.
  • the functional group to be coated onto the surface of the 3D polymer scaffold can be an amino group, i.e., the polymer scaffold is activated with an amino group.
  • every functional group that can be coated onto polymer surfaces and that can be used to immobilize proteins and protein fragments is applicable for this approach.
  • Exemplarily other functional groups that are suitable for the manufacturing of the functionalized 3D polymer scaffold of the present invention are aldehyde or carboxyl groups, i.e., the polymer scaffold can be activated with carboxyl or aldehyde groups, wherein the coating can be performed by standard methods as for example described in WO 2006/100480 Al; Nishimori et al., Langmuir 34 (2016), 6396-6404; Wei and Haag, Mater. Horiz. 2 (2015), 567; Shimomura et al., Langmuir 29 (2013), 932-938.
  • the polymer scaffold per se is usually not suitable for being coated with functional groups due to its hydrophobic surface, but the surface needs to be modified to become hydrophilic so that the functional groups can be bound to the hydrophilic surface of the 3D polymer scaffold.
  • this is done by a combination of plasma surface activation (plasma treatment) and chemical modification to allow for subsequent conjugation of the relevant molecules, for example carboxyl- or aldehyde groups, for coupling of StrepTactin®- Tetramers.
  • the combination is also called plasma-induced grafting, which is described e.g. in Karam et al., J. Mater. Environ. Sci.
  • any method can be applied which provides a hydrophilic surface.
  • the surface modification can also be achieved by plasma polymerization leading to the deposition of very thin polymer films through reaction of the plasma with an organic monomer gas, either pure or mixed with a carrier gas like argon (see, Karam et al., 2013, pages 809/810; Bertin et al., Plasma Process Polym. (2024), doi.org/10.1002/ppap.202300208).
  • the surface of the 3D polymer scaffold is modified to become hydrophilic so that the functional groups are bound to the hydrophilic surface of the 3D polymer scaffold, wherein the modification is preferably performed by plasma treatment, in particular plasma surface activation or plasma polymerization.
  • the material of the 3D polymer scaffold of the present invention can be any polymer suitable for chromatographic approaches.
  • printed chromatography media must maintain highly porous characteristics, display large surface areas to maximise interaction with the analytes and thus enable high separation capacities and for certain applications must have excellent mechanical properties to withstand the high pressures typical of HPLC and UHPLC operations.
  • the material must be compatible and processable by 3D printers and must at the same time be suitable for chromatographic operations.
  • a summary of suitable materials is suggested for example in Salmean and Dimartino, Chromatographia 82 (2019), 443-463, which content is herein incorporated by reference.
  • polymers have been tested and shown to be suitable as scaffold materials, e.g., polylactide (PLA), polypropylene (PP) and poly caprolactone (PCL), and polyethylene terephthalate/glycol filament (PET/PETG) should also be feasible.
  • the polymer scaffold of the present invention is made out of any one of these materials.
  • all thermoplastics suitable for 3D printing can be used and selected due to their properties (i.e., heat or chemical or irradiation resistance are interesting for sanitisation /sterilisation procedures).
  • PCL has the advantage that it is biodegradable and one of the preferred polymers for extrusion-based 3DP.
  • PLA is also called bioplastic and it thus also biodegradable.
  • the term "scaffold” generally refers to a matrix which serves as anchoring platform for biological entities. In terms of chromatography, it thus refers to a stationary phase to which a biological entity can be temporarily bound (ie., anchored) for isolation purposes.
  • the polymer scaffold of the present invention can have different shapes, particle arraignments, and alignments dependent on its actual use and the kind of chromatography.
  • the 3DP process allows the manufacturing of various morphologies and even complex shapes and particle arrangements can be realized and thus, the polymer scaffold in principle can have any shape, alignment, and particle arrangement suitable for chromatographic approaches and is not limited.
  • the polymer scaffold of the present invention for example have a cylindric shape like the one used in the Examples or is manufactured in the form of lattices or filters with large surface areas and desired flow properties resembling chromatography matrices.
  • the polymer scaffold in form of a filter is for example particularly suitable for biochemical applications like particle filtration and size fractionation of all kinds.
  • the polymer scaffold of the present invention is produced by 3DP, but also the whole chromatography column, which is preferably of greater diameter than height, because this will allow to save time, increase binding efficacy, and decrease washing time.
  • the 3D polymer scaffold of the present invention can be produced with or without shell and the preferred layer hight of the 3D polymer scaffold of the present invention is 0.08 - 0.12 mm, even though a layer hight of > 0.12 mm will work as well).
  • the preferred filament distance is about >0.2 mm.
  • the 3D polymer scaffold of the present invention has a cylindric shape, either with or without a shell, but preferably with chamfer and shell (height 15 mm, diameter 13 mm) and preferably with the following slicing parameters: layer hight 0.08 - 0.12 mm, preferably 0.08 mm; infill type: connected lines; filament distance: 0.2 to 0.25 mm, preferably 0.25 mm; angle rotation: 0/90°.
  • the chromatography approach is most efficient if the stationary phase has a large surface area, z.e., if the polymer scaffold of the present invention has a large surface area to volume ratio. Accordingly, in one embodiment, the 3D polymer scaffold of the present invention has large surface area and a large surface area to volume ratio, respectively.
  • the present invention relates to a functionalized 3D polymer scaffold meaning a 3D polymer scaffold to which proteins or protein fragments are bound as affinity ligands to capture the target biological entity, or as anchors to bind affinity ligands to capture the target biological entity, or to which proteins or protein fragments are bound as anchors including the affinity ligands to capture the target biological entity.
  • the protein or protein fragment comprises streptavidin or a functional analog thereof.
  • the protein or protein fragment is streptavidin or a functional analog thereof, which is preferably Strep-Tactin®.
  • the affinity ligand which is preferably bound to the 3D polymer scaffold via an anchor (the protein or protein fragment as mentioned above) is a protein or protein fragment which comprises an antigen binding domain, like an antibody, an antibody fragment (Fab fragment), or a receptor, which is preferably bound to the surface of the 3D polymer scaffold via streptavidin or a functional analog thereof.
  • the affinity ligand which is preferably bound to the 3D polymer scaffold via an anchor (the protein or protein fragment as mentioned above) is an oligonucleotide, which is preferably bound to the surface of the 3D polymer scaffold via streptavidin or a functional analog thereof.
  • Strep-Tactin® is a recombinant protein derived from streptavidin that binds besides biotin also a peptide called Strep-Tag® via non-covalent binding), an affinity tag widely used for purification of proteins, cells, and other biomolecules, i.e., various biological entities.
  • Strep-Tags® exist, a mono-tag with relatively low affinity for Strep-Tactin®, and a tag called TST with high affinity for Strep-Tactin®.
  • streptactin called XT has been created.
  • Strep-Tactin® systems are for example described in detail in the international applications WO 02/077018 Al, WO 2014/076277 Al and WO 2017/186669 Al and in the European patent application EP 0 835 934 A2, all of which are herein expressly incorporated by reference.
  • Strep-Tactin® can be covalently bound.
  • a Strep-tagged biomolecule is bound to the immobilized Strep-Tactin® or a (non-Strep-tagged) biomolecule is bound to the protein comprising an antigen-binding domain (e.g., Fab fragment) which itself is bound to the immobilized Strep-Tactin® via a Strep-Tag®, out from a complex mixture of biomolecules, non-bound molecules are washed away and the bound (Strep-tagged) biomolecule can be eluted with biotin.
  • an antigen-binding domain e.g., Fab fragment
  • the 3D polymer scaffold of the present invention has exemplary been manufactured by covalently binding a Strep-Tactin®-Tetramer to its surface and used in a cell isolation method, wherein CD3+ cells have been isolated from buffy coat by using aCD3-Fab fragment with a twin-Strep-Tag®; see Examples 1 and 2.
  • the ligand or anchor which is immobilized to the surface of the 3D polymer scaffold comprises or is streptavidin or a functional analog (mutein) thereof, including the streptavidin mutein (analog) Val 44 -Thr 45 -Ala 46 -Arg 47 (SEQ ID NO: 12) or the streptavidin mutein (analog) Ile 44 -Gly 45 -Ala 46 -Arg 47 (SEQ ID NO: 13), both of which are described, e.g., in US patent 6,103,493 and are commercially available under the trademark Strep-Tactin®.
  • Such multimeric streptavidin muteins may also be referred to as multimerized Strep-Tactin® or Strep-Tactin®-Tetramer.
  • the ligand is based on the Strep-Tactin®XT system or the Strep-Tactin®XTS system (IBA GmbH, Gottingen, Germany). Those are described in detail in the international applications WO 02/077018 Al, WO 2014/076277 Al and WO 2017/186669 Al all of which are herein expressly incorporated by reference.
  • a target biological entity can be isolated via binding to an affinity ligand, which can be a further protein or protein fragment, like a receptor or a Fab fragment, or an oligonucleotide, which comprises a streptavidin binding partner, namely a streptavidin-binding peptide, and can thus be coupled and immobilized to the 3D polymer scaffold (via the above-described streptavidin/functional derivative thereof), or a target biological entity can be isolated which comprises itself a corresponding binding partner, namely a streptavidin-binding peptide, which preferably comprises or consists of one of the following sequences: a) -Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- (SEQ ID NO: 1), wherein Xaa is any amino
  • Trp-Arg-His-Pro- Gln-Phe-Gly-Gly SEQ ID NO: 2
  • Trp-Ser-His-Pro-Gln-Phe-Glu-Lys SEQ ID NO: 3
  • Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro- Gln-Phe-Glu-Lys SEQ ID NO: 9
  • Trp-Ser-His-Pro-Gln-Phe-Glu-Lys- (GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys SEQ ID NO: 10
  • streptavidin-binding peptides are described in WO 02/077018 Al, WO 2014/076277 Al and WO 2017/186669 Al and in the European patent application EP 0 835 934 A2, all of which are herein expressly incorporated by reference.
  • the affinity ligand which is immobilized to the surface of the 3D polymer scaffold of the invention preferably via protein or protein fragment used as anchor, more preferably via the above-described streptavidin and corresponding muteins/analogs thereof, respectively, is a protein or protein fragment which comprises an antigen binding domain.
  • the protein or protein fragment comprises the corresponding streptavidin binding partner.
  • the antigen binding domain comprising protein or protein fragment is capable of recognizing an antigen, in particular a surface antigen of a biological entity and may for instance be an antibody or an immunoglobulin, a functional fragment of an antibody or an immunoglobulin, or a proteinaceous binding molecule with immunoglobulin/antibody-like functions, or an antigen receptor.
  • Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single-chain Fv fragments (scFv), a bivalent antibody fragment such as an (Fab)2'-fragment, diabodies, triabodies (Iliades, P., etal., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L.J., etal., Trends Biotechnol. (2003), 21, 11, 484-490) like nanobodies.
  • the protein may be a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein that is also known as "duocalin", a proteinaceous binding molecule with antibody-like binding properties or an MHC molecule.
  • a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein that is also known as "duocalin”
  • a proteinaceous binding molecule with antibody-like binding properties or an MHC molecule a bivalent proteinaceous artificial binding molecule
  • a Fab fragment directed against a CD3 receptor can efficiently recognize the corresponding surface antigen on target cells and therefore, in one embodiment, the protein or protein fragment is an antigenbinding fragment, such as a Fab.
  • a proteinaceous binding molecule with antibody -like functions is a mutein based on a polypeptide of the lipocalin family (see for example, WO 03/029462, Beste et al., Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 1898-1903).
  • Lipocalins such as the bilin-binding protein, the human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D or human tear lipocalin possess natural ligand-binding sites that can be modified so that they bind a given target.
  • a proteinaceous binding molecule with antibody-like binding properties that can be used as protein or protein fragment bound to the surface of the 3D polymer scaffold of the present invention that specifically binds to the surface antigen
  • glubodies see e.g. international patent application WO 96/23879
  • proteins based on the ankyrin scaffold Mosavi, L.K., etal., Protein Science (2004) 13, 6, 1435- 1448
  • crystalline scaffold e.g., international application WO 01/04144
  • Avimers including multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains, contain so called A-domains that occur as strings of multiple domains in several surface receptors (Silverman, J., et al., Nature Biotechnology (2005) 23, 1556-1561).
  • Adnectins derived from a domain of human fibronectin, contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current Opinion in Biotechnology (2006) 17, 653-658).
  • Tetranectins derived from the respective human homotrimeric protein, likewise contain loop regions in a C-type lectin domain that can be engineered for desired binding (ibid.).
  • Peptoids which can act as protein ligands, are oligo(N-alkyl) glycines that differ from peptides in that the side chain is connected to the amide nitrogen rather than the a carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have a much higher cell permeability than peptides (see e.g. Kwon, Y.-U, and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509).
  • suitable proteinaceous binding molecules are an EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a Gia domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an immunoglobulin domain or a an immunoglobulin-like domain (for example, domain antibodies or camel heavy chain antibodies), a C-type lectin domain, a MAM domain, a von Willebrand
  • a nanobody a microbody, an affilin, an affibody, a knottin, ubiquitin, a zinc-finger protein, an autofluorescent protein or a leucine-rich repeat protein.
  • An example of a nucleic acid molecule with antibody-like functions is an aptamer. An aptamer folds into a defined three-dimensional motif and shows high affinity for a given target structure.
  • the affinity ligand which is immobilized to the surface of the 3D polymer scaffold of the invention preferably via a protein or protein fragment used as anchor, more preferably via the above-described streptavidin and corresponding muteins/analogs thereof, respectively, is an oligonucleotide.
  • the oligonucleotide comprises the corresponding streptavidin binding partner, i.e., it is covalently coupled to streptavidin binding partner, preferably to a Strep-Tag®; see for example Singh et al, Chemical Society Reviews 39 (2010), 2054-2070 which describes methods for respective modification of oligonucleotides allowing coupling of various tags and dyes).
  • Such an oligonucleotide is either a DNA-oligonucleotide or an RNA-oligonucleotide, including but not limited to so-called Aptamer oligonucleotides with a length of 25-80 nucleotides (Jayasena, Clinical Chemistry 45 (1999), 1628-1650; Perret & Boschetti, Biochimie 145 (2016), 98el l2).
  • Aptamer oligonucleotides with a length of 25-80 nucleotides
  • the target biological entity is to be understood to encompass proteins, nucleic acids, oligonucleotides, small molecules, cells and all other vesicles such as cell organelles, viruses, bacteria, exosomes, liposomes, synaptic vesicles and the like, i.e. the target is for example any biological entity in which a membrane (which can also be a lipid bilayer) separates the interior from the outside environment (ambience) and which comprise one or more kinds of specific surface antigens.
  • a biological entity or a population of biological entities is isolated from a sample that, for example, may include a variety of different proteins, oligonucleotides, cells or cell populations.
  • Virtually any biological entity that has at least one common surface antigen, i.e. antigen on its surface, or which can be modified to comprise an affinity tag, can be separated from other components contained in a sample.
  • the biological entity is a prokaryotic cell, such as a bacterial cell or archaeon.
  • the biological entity is a virus, an organelle, such as a mitochondrion, a chloroplast, a Golgi apparatus or a nucleus or an extracellular vesicle such as a microsome, an exosome, or a lysosome, or a synaptic vesicle.
  • the biological entity is a cell nucleus.
  • the biological entity is a eukaryotic cell, such as a plant cell, a fungal cell, a yeast cell, a protozoon or an animal cell.
  • the biological entity is a mammalian cell, including a cell of a rodent species, or an amphibian cell, e.g. of the subclass Lissamphibia that includes, e.g., frogs, toads, salamanders or newts.
  • Examples of a mammalian cell include, but are not limited to a blood cell, a semen cell or a tissue cell, e.g., a hepatocyte or a stem cell, e.g., CD34-positive peripheral stem cells or Nanog or Oct-4 expressing stem cells derived from a suitable source, a hematopoietic stem cell from bone marrow or cord blood.
  • a blood cell is, for instance, a leukocyte or an erythrocyte.
  • a leukocyte is, for example, a neutrophil, an eosinophil, a basophil, a monocyte, a lymphocyte, a macrophage or a dendritic cell.
  • a respective lymphocyte is, for example, a T cell, including a CMV-specific CD8 + T- lymphocyte, a cytotoxic T-cell a, memory T-cell (an illustrative example of memory T-cells are CD62L + CD8 + specific central memory T-cells) or a regulatory T-cell (an illustrative example of Treg are CD4 + CD25 + CD45RA + Treg cells), a T-helper cell, for example, a CD4 + T-helper cell, a B cell or a natural killer cell, to mention only a few illustrative examples.
  • CD3+ cells have been isolated using the 3D polymer scaffold of the present invention and thus, in one embodiment, the biological entity is a CD3+ cell. In one embodiment, the biological entity is a protein.
  • mammals include, but are not limited to a human, a rat, a mouse, a rabbit, a guinea pig, a squirrel, a hamster, a hedgehog, a cat, a platypus, an American pika, an armadillo, a dog, a lemur, a goat, a pig, an opossum, a horse, an elephant, a bat, a woodchuck, an orangutan, a rhesus monkey, a woolly monkey, a macaque, a chimpanzee, a tamarin (saguinus oedipus), a marmoset and a human.
  • the cell may, for instance, be a cell of a tissue, such as an organ or a portion thereof.
  • a tissue such as an organ or a portion thereof.
  • a respective organ include, without being limited thereto, adrenal tissue, bone, blood, bladder, brain, cartilage, colon, eye, heart, kidney, liver, lung, muscle, nerve, ovary, pancreas, prostate, skin, small intestine, spleen, stomach, testicular, thymus, tumor, vascular or uterus tissue, or connective tissue.
  • the cell is a stem cell.
  • a sample from which the biological entity is to be isolated may be of any origin. It may for instance, but not limited to, be derived from humans, animals, plants, bacteria, fungi, or protozoae. Accordingly, any of the following samples selected from, but not limited to, the group consisting of a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a sewage sample, a ground water sample, an abrasion sample, an archaeological sample, a food sample, a blood sample (including whole blood), a serum sample, a plasma sample, an urine sample, a stool sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, a mouth swab sample, a throat swab sample, a nasal swab sample, a bronchoalveolar lavage sample, a
  • the target biological entity is an oligonucleotide, for example an RNA or DNA oligonucleotide.
  • the oligonucleotide may be a single stranded or a double stranded oligonucleotide.
  • the oligonucleotide is designed to bind to any RNA or DNA and can be used to act on gene expression and targets pre-mRNA, mRNA, or non-coding RNA (e.g., microRNA (miRNA) or long non-coding RNA), to induce degradation, modulate splicing events, or interferes with protein translation.
  • pre-mRNA mRNA
  • non-coding RNA e.g., microRNA (miRNA) or long non-coding RNA
  • the oligonucleotide can also be a small activating RNA (saRNA) which is used for transcriptional activation through direct interaction with gene promoters.
  • saRNA small activating RNA
  • the oligonucleotides may also be chemically optimized and comprise for example modifications of the phosphodiester bonds and/or of the sugar groups to improve oligonucleotide stability in plasma by increasing their resistance to nucleases and their affinity for serum proteins as well as their specificity for their target sequence.
  • the oligonucleotide may be an antisense oligonucleotide acting as gene-expression inhibitors or splicing modulators; a small interfering RNA (siRNA), which is a double-stranded RNA molecule capable of hybridizing specifically to their target RNA via Watson-Crick base pairings and acting as inhibitor of gene expression; a miRNA, or a saRNA (Mounme et al., Pharmaceutics. 14 (2022), 260.
  • the oligonucleotide may also be conjugated to various moieties that promote intracellular uptake, target the drug to specific cells/tissues or reduce clearance from the circulation.
  • lipids for example, cholesterol that facilitates interactions with lipoprotein particles in the circulation
  • peptides for cell targeting and/or cell penetration
  • aptamers for cell targeting and/or cell penetration
  • sugars for example, /'/-acetylgalactosamine (GalNAc)
  • the target biological entity is preferably a biological entity which comprises a streptavidin binding peptide and/or a surface antigen, and more preferably a protein or oligonucleotide (which preferably comprises a streptavidin binding peptide), or a cell, or an exosome.
  • the present invention further relates to a method of manufacturing the 3D polymer scaffold of the present invention which includes a step of 3D printing of the polymer scaffold, wherein different materials as referred to above can be used.
  • the method further includes a step of chemical modification (activation) of the scaffold surface, preferably wherein the surface of the scaffold is coated with function groups, preferably with amino groups, aldehyde groups, or carboxy groups, most preferably with amino groups.
  • the method further comprises a step of treating the polymer scaffold, which is usually hydrophobic, to become hydrophile before chemically modifying the surface, i.e., before coating the surface with the functional groups.
  • the method further includes as step of coupling a protein or protein fragment, which is capable of binding the target biological entity or which is capable of binding an affinity ligand which is capable of binding the target biological entity, to the activated surface, preferably wherein the protein or protein fragment is streptavidin or a functional analog/mutein thereof, preferably Strep-Tactin® and/or the affinity ligand is a protein or protein fragment comprising an antigen binding domain, preferably a Fab fragment, or an oligonucleotide, optionally comprising a streptavidin binding peptide, preferably a Strep-Tag®.
  • the protein or protein fragment which is immobilized on the surface of the 3D polymer scaffold is streptavidin or a functional analog/mutein thereof, preferably Strep-Tactin® and the method further includes a step of coupling an affinity ligand, preferably a protein or protein fragment, which comprises an antigen binding domain, or an oligonucleotide to the streptavidin on the surface of the 3D polymer scaffold, preferably wherein the protein/protein fragment/oligonucleotide comprises a streptavidin binding peptide (for example a Strep-Tag®) which is coupled (here, by not-covalent binding) to the streptavidin.
  • an affinity ligand preferably a protein or protein fragment, which comprises an antigen binding domain, or an oligonucleotide
  • the protein/protein fragment/oligonucleotide comprises a streptavidin binding peptide (for example a Strep-Tag®) which is coupled (here, by not-covalent binding) to
  • the present invention further relates to a method for purification/isolation/enrichment of a target biological entity using the 3D polymer scaffold of the present invention.
  • the target biological entity is a biological entity as defined hereinbefore.
  • the method comprises a step of applying a sample comprising the target biological entity to the 3D polymer scaffold of the present invention, z.e., a scaffold which has been functionalized, for example via applying the sample to a syringe or any other container which assembles the shape of or is capable of holding the 3D polymer scaffold, or to the 3D polymer scaffold which has been manufactured/printed including a corresponding surrounding, under conditions allowing complex formation between the biological entity and the immobilized protein or protein fragment or the affinity ligand which is immobilized via a protein or protein fragment, and a step of eluting the target biological entity from the scaffold.
  • the method comprises a step of applying a sample comprising the target biological entity, which is preferably a biological entity comprising an affinity tag, preferably a protein comprising an affinity tag, preferably a Strep-Tag®, to the 3D polymer scaffold, and allowing the sample to flow through at least and preferably once, a step of washing the scaffold, and a step of eluting the target biological entity, preferably via loading biotin onto the scaffold, and an optional step of collecting the target biological entity.
  • the scaffold is preferably a 3D polymer scaffold which has been functionalized with streptavidin or a functional analog/mutein thereof.
  • the method comprises a step of applying a sample comprising the target biological entity, which is preferably a biological entity comprising a surface antigen, preferably a cell or an exosome, or an oligonucleotide to the 3D polymer scaffold and allowing the sample to flow through at least and preferably once, an optional step of washing the scaffold, and a step of eluting the target biological entity, preferably via loading biotin onto the scaffold, and a step of collecting the target biological entity.
  • a sample comprising the target biological entity which is preferably a biological entity comprising a surface antigen, preferably a cell or an exosome, or an oligonucleotide
  • the scaffold is preferably a 3D polymer scaffold which has been functionalized with streptavidin or a functional analog/mutein thereof to which an oligonucleotide or a protein or protein fragment comprising an antigen binding domain, like a Fab fragment has been bound.
  • the method comprises a step of applying a protein or protein fragment comprising an antigen binding domain, preferably a Fab fragment, and an affinity tag, preferably a Strep-Tag®, or an oligonucleotide comprising an affinity tag, preferably a Strep- Tag® to the 3D polymer scaffold of the present invention, a step of incubation under conditions allowing binding of the tagged protein/protein fragment/oligonucleotide to the scaffold surface, z.e., to the protein or protein fragment immobilized on the surface, which is preferably a streptavidin or a functional analog/mutein thereof, an optional washing step, a step of applying a sample comprising the target biological entity, which is preferably a biological entity comprising a surface antigen, preferably a cell or an exosome, or an oligonucleotide to the 3D polymer scaffold and allowing the sample to flow through at least and preferably once, an optional step of washing the scaffold, and a step of eluti
  • 3D polymer scaffolds have been developed and successfully tested that are useful alternatives as carriers in affinity chromatography and can replace some of the classical chromatography material like agarose or Sephadex, providing several important advantages over them, for example their cost-efficient manufacturing and the improved performance of chromatographic operations due to the ordered morphology of the 3D polymer scaffolds.
  • Example 1 Production of a functionalized three-dimensional (3D) polymer scaffold Production of the 3D polymer scaffold
  • CAD-based 3D printing was carried out by BellaSeno GmbH (Leipzig, Germany) using their software (Rhino3D, in combination with Ultimaker Cura)), wherein various materials, printing parameters, and geometries were tested. Finally, a scaffold of cylindric shape with chamfer and shell (height 15 mm, diameter 13 mm; material: Polylactide (PLA, Renkforce transparent 3D filament, 1.75 mm)) with the following slicing parameters was produced: layer hight 0.08 mm; infill type: connected lines; filament distance: 0.25 mm; angle rotation: 0/90°. An exemplarily 3D-printed scaffold is shown in Figure 1.
  • PCL poly caprolactone
  • PP polypropylene
  • the layer hight can be adjusted to 0.08 - 0.12 mm (others > 0.12 mm will work as well).
  • the scaffold can be produced with or without a shell and the filament distance can be >0.2 mm.
  • the Scaffold surfaces were chemically modified by SuSoS AG, 8600 Diibendorf, Switzerland, with the AziGrip4TM Amine coating (AziGrip4TM is based on azide groups that are activated by UV-light or temperature to form highly reactive nitrenes, which are capable of undergoing C- H and/or N-H insertion reactions with neighboring molecules, generating new chemical bonds; see also the corresponding description in EP 2 236 524 Al).
  • the scaffolds underwent dip coating with AziGrip4TM Amine for 30 minutes, rinsed 3x times in ethanol, blow dried with N2 and left in a laboratory fluid bed (LFB) to dry out; afterwards, scaffolds were UV treated at 4.5 mW/cm 2 for 5 min on each side.
  • the coated scaffolds were stored at 6-8°C until coupling of Strep-Tactin®-Tetramers.
  • the amine-coated scaffold was biotinylated by using N-Hydroxy-succinimide (NHS) esters of biotin (e.g. EZ LinkTM NHS Biotin, Thermo Scientific).
  • NHS N-Hydroxy-succinimide
  • EZ LinkTM NHS Biotin EZ LinkTM NHS Biotin, Thermo Scientific
  • the scaffold was incubated with 5 mg of freshly dissolved NHS Biotin (pH 7.55) for 1 h (RT) and after thorough washing (5x 50 ml PBS) incubated with Img Strep- Tactin®-Tetramers for 30 min (RT).
  • the Strep- Tactin®-functionalized scaffold (ST-scaffold) was ready for use, e.g. binding of strep-tagged Fab fragments for cell isolation; see below.
  • the Strep-Tactin®-Tetramers are strongly bound on one side to biotin, which is immobilized on the surface of the scaffold filaments, but have on the other side free binding sites for e.g. strep-tagged or biotinylated proteins.
  • carboxyl groups are coated onto the surface of the scaffold, the carboxyl groups are activated by the use of EDC (N-(3-dimethylaminopropyl)-N’ -ethylcarbodiimide hydrochloride) and NHS (N-hydroxy succinimide) followed by covalent attachment of Strep-Tactin® by its primary amine and deactivation of remaining esters using ethanolamine.
  • EDC N-(3-dimethylaminopropyl)-N’ -ethylcarbodiimide hydrochloride
  • NHS N-hydroxy succinimide
  • the Strep-Tactin® functionalized scaffold, ST-scaffold was placed into an 10 ml syringe (B. Braun with Luer Lock, #4606108V) and incubated for 15 min with low affinity aCD3-Fab fragment with a twin-Strep-tag (aCD3 -Fab-Strep specific for human CD3, IBA lifesciences; 50 pg Fab in 4 ml PBS buffer at RT). After washing of the aCD3-Fab loaded ST-scaffold (4x 10ml buffer Cl: PBS, ImM EDTA, 0.5% BSA), 6 ml of diluted buffy coat (1: 1 dilution with PBS) was loaded slowly onto the scaffold, allowed to flow through once and then discarded.
  • CD3 positive cells CD3+
  • buffy coat FACS analysis of CD3 positive cells (CD3+) in buffy coat was performed before (starting material) and after (eluate) the CD3 affinity chromatography and a clear enrichment of CD3+ cells was observed showing that the 3D printed polymer scaffold is suitable as stationary phase during affinity chromatography.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Materials Engineering (AREA)
  • Manufacturing & Machinery (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un échafaudage polymère tridimensionnel (3D) fonctionnalisé qui comprend des protéines immobilisées ou des fragments de protéine sur sa surface, qui sont capables de se lier à une entité biologique cible pendant une chromatographie d'affinité, ainsi qu'un procédé de fabrication d'un tel échafaudage polymère 3D.
PCT/EP2025/063502 2024-05-16 2025-05-16 Échafaudage polymère tridimensionnel (3d) fonctionnalisé pour chromatographie d'affinité Pending WO2025238197A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24176280 2024-05-16
EP24176280.6 2024-05-16

Publications (1)

Publication Number Publication Date
WO2025238197A1 true WO2025238197A1 (fr) 2025-11-20

Family

ID=91129794

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/063502 Pending WO2025238197A1 (fr) 2024-05-16 2025-05-16 Échafaudage polymère tridimensionnel (3d) fonctionnalisé pour chromatographie d'affinité

Country Status (1)

Country Link
WO (1) WO2025238197A1 (fr)

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996023879A1 (fr) 1995-01-30 1996-08-08 Terrapin Technologies, Inc. Corps agglutinants - multiplicite de proteines capables de lier diverses petites molecules
EP0835934A2 (fr) 1996-10-10 1998-04-15 Institut für Bioanalytik GmbH Mutéines de streptavidine
WO2001004144A2 (fr) 1999-07-13 2001-01-18 Scil Proteins Gmbh Fabrication de proteines a feuillet plisse beta et a proprietes de liaison specifiques
WO2002077018A1 (fr) 2001-03-21 2002-10-03 Iba Gmbh Modules de liaison avec la streptavidine disposes de maniere sequentielle, utilises comme etiquettes d'affinite
WO2003029462A1 (fr) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteines de la lipocaline neutrophile humaine associee a la gelatinase et de proteines apparentees
WO2006100480A1 (fr) 2005-03-24 2006-09-28 University Of Durham Méthode de production d'un revêtement contenant un aldéhyde
EP2236524A1 (fr) 2009-03-30 2010-10-06 SuSoS AG Promoteur d'adhésion à base d'une macromolécule functionalisée comprenant des groupes photoréactifs
WO2014076277A1 (fr) 2012-11-16 2014-05-22 Iba Gmbh Mutéines de streptavidine et procédés d'utilisation associés
US20170052176A1 (en) * 2014-04-30 2017-02-23 Iba Gmbh Method of isolating a target cell
CN107190418A (zh) * 2017-07-12 2017-09-22 航天特种材料及工艺技术研究所 基于3d打印pla材料的可吸附‑脱附蛋白质的纤维膜装置及其制备方法
WO2017186669A1 (fr) 2016-04-25 2017-11-02 Iba Gmbh Mutéines de streptavidine et méthodes d'utilisation associées
US20180126298A1 (en) * 2015-05-22 2018-05-10 Merck Patent Gmbh Device for substance separation
US11065601B2 (en) * 2015-12-18 2021-07-20 University Of Canterbury Separation medium
WO2022112416A1 (fr) * 2020-11-25 2022-06-02 Universität Für Bodenkultur Wien Nouveau lit de chromatographie
WO2023017115A1 (fr) * 2021-08-11 2023-02-16 Iba Lifesciences Gmbh Fractionnement de cellules sur la base d'une protéine de marquage

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996023879A1 (fr) 1995-01-30 1996-08-08 Terrapin Technologies, Inc. Corps agglutinants - multiplicite de proteines capables de lier diverses petites molecules
EP0835934A2 (fr) 1996-10-10 1998-04-15 Institut für Bioanalytik GmbH Mutéines de streptavidine
US6103493A (en) 1996-10-10 2000-08-15 Institut Fur Bioanalytic Streptavidin muteins
WO2001004144A2 (fr) 1999-07-13 2001-01-18 Scil Proteins Gmbh Fabrication de proteines a feuillet plisse beta et a proprietes de liaison specifiques
WO2002077018A1 (fr) 2001-03-21 2002-10-03 Iba Gmbh Modules de liaison avec la streptavidine disposes de maniere sequentielle, utilises comme etiquettes d'affinite
WO2003029462A1 (fr) 2001-09-27 2003-04-10 Pieris Proteolab Ag Muteines de la lipocaline neutrophile humaine associee a la gelatinase et de proteines apparentees
WO2006100480A1 (fr) 2005-03-24 2006-09-28 University Of Durham Méthode de production d'un revêtement contenant un aldéhyde
EP2236524A1 (fr) 2009-03-30 2010-10-06 SuSoS AG Promoteur d'adhésion à base d'une macromolécule functionalisée comprenant des groupes photoréactifs
WO2014076277A1 (fr) 2012-11-16 2014-05-22 Iba Gmbh Mutéines de streptavidine et procédés d'utilisation associés
US20170052176A1 (en) * 2014-04-30 2017-02-23 Iba Gmbh Method of isolating a target cell
US20180126298A1 (en) * 2015-05-22 2018-05-10 Merck Patent Gmbh Device for substance separation
US11065601B2 (en) * 2015-12-18 2021-07-20 University Of Canterbury Separation medium
WO2017186669A1 (fr) 2016-04-25 2017-11-02 Iba Gmbh Mutéines de streptavidine et méthodes d'utilisation associées
CN107190418A (zh) * 2017-07-12 2017-09-22 航天特种材料及工艺技术研究所 基于3d打印pla材料的可吸附‑脱附蛋白质的纤维膜装置及其制备方法
WO2022112416A1 (fr) * 2020-11-25 2022-06-02 Universität Für Bodenkultur Wien Nouveau lit de chromatographie
WO2023017115A1 (fr) * 2021-08-11 2023-02-16 Iba Lifesciences Gmbh Fractionnement de cellules sur la base d'une protéine de marquage

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
BERTIN ET AL., PLASMA PROCESS POLYM., 2024
BESTE ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 96, 1999, pages 1898 - 1903
GILL, D.S.DAMLE, N.K., CURRENT OPINION IN BIOTECHNOLOGY, vol. 17, 2006, pages 653 - 658
HOLLIGER ET AL., PNAS USA, vol. 90, 1993, pages 6444 - 6448
HOLT, L.J. ET AL., TRENDS BIOTECHNOL., vol. 21, no. 11, 2003, pages 484 - 490
ILIADES, P. ET AL., FEBS LETT, vol. 409, 1997, pages 437 - 441
ILL. ET AL., PROTEIN ENG, vol. 10, 1997, pages 949 - 57
JAYASENA, CLINICAL CHEMISTRY, vol. 45, 1999, pages 1628 - 1650
KARAM ET AL., J. MATER. ENVIRON. SCI., vol. 4, no. 5, 2013, pages 798 - 821
KWON, Y.-U.KODADEK, T., J. AM. CHEM. SOC., vol. 129, 2007, pages 1508 - 1509
MARTIN ET AL., EMBO J, vol. 13, 1994, pages 5303 - 5309
MOLEIRINHO MAFALDA G ET AL: "3D-printed ordered bed structures for chromatographic purification of enveloped and non-enveloped viral particles", SEPARATION AND PURIFICATION TECHNOLOGY, ELSEVIER SCIENCE, AMSTERDAM, NL, vol. 254, 2 September 2020 (2020-09-02), XP086294775, ISSN: 1383-5866, [retrieved on 20200902], DOI: 10.1016/J.SEPPUR.2020.117681 *
MOSAVI, L.K. ET AL., PROTEIN SCIENCE, vol. 13, no. 6, 2004, pages 1435 - 1448
NISHIMORI ET AL., LANGMUIR, vol. 34, 2018, pages 6396 - 6404
PERRETBOSCHETTI, BIOCHIMIE, vol. 145, 2018, pages 98 - 112
ROBERT ET AL., NATURE REVIEWS DRUG DISCOVERY, vol. 19, 2020, pages 673 - 694
SALMEANDIMARTINO, CHROMATOGRAPHIA, vol. 82, 2019, pages 443 - 463
SHIMOMURA ET AL., LANGMUIR, vol. 29, 2013, pages 932 - 938
SILVERMAN, J. ET AL., NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1556 - 1561
SIMON URSULA ET AL: "Direct 3D printing of monolithic ion exchange adsorbers", JOURNAL OF CHROMATOGRAPHY A, vol. 1587, 13 December 2018 (2018-12-13), AMSTERDAM, NL, pages 119 - 128, XP055802245, ISSN: 0021-9673, Retrieved from the Internet <URL:https://www.pure.ed.ac.uk/ws/files/77730643/Simon_Direct_3D_printing_of_monolithic_ion_exchange_adsorber.pdf> DOI: 10.1016/j.chroma.2018.12.017 *
SINGH ET AL., CHEMICAL SOCIETY REVIEWS, vol. 39, 2010, pages 2054 - 2070
SKERRA, J., MOL. RECOGNIT., vol. 13, 2000, pages 167 - 187
STONE, E. ET AL., JOURNAL OF IMMUNOLOGICAL METHODS, vol. 318, 2007, pages 88 - 94
TRAUNECKER ET AL., EMBO J, vol. 10, 1991, pages 3655 - 3659
TRAUNECKER ET AL., INT J CANCER, 1992, pages 51 - 52
URH ET AL., METHODS ENZYMOL, vol. 463, 2009, pages 417 - 438
VALENTE ET AL., PHARMACEUTICS, vol. 14, 2022, pages 2266
WEIHAAG, MATER. HORIZ., vol. 2, 2015, pages 567

Similar Documents

Publication Publication Date Title
JP7710427B2 (ja) 細胞および他の複雑な生物学的材料のクロマトグラフィーによる単離
JP2017514481A (ja) 標的細胞を単離する方法
US20240329039A1 (en) Methods of isolating a biological entity
CN102321616B (zh) 利用微流控芯片筛选蛋白质的核酸适体的方法
WO2025238197A1 (fr) Échafaudage polymère tridimensionnel (3d) fonctionnalisé pour chromatographie d&#39;affinité
CA2705009A1 (fr) Procede de purification d&#39;au moins une substance cible a mettre en evidence
KR101086026B1 (ko) 비스파틴에 특이적으로 결합하는 ｄｎａ 앱타머 및 그 제조방법
AU2007322660A1 (en) Nucleic acid molecule capable of binding to rabbit-derived IgG antibody
US20170137773A1 (en) Cell strainer with functionalized mesh to separate biological components
JP2017149810A (ja) 共重合体、これを用いたコーティング剤、及びコーティング方法