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WO2025114932A1 - Purification de molécules d'adn à extrémité fermée - Google Patents

Purification de molécules d'adn à extrémité fermée Download PDF

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Publication number
WO2025114932A1
WO2025114932A1 PCT/IB2024/061963 IB2024061963W WO2025114932A1 WO 2025114932 A1 WO2025114932 A1 WO 2025114932A1 IB 2024061963 W IB2024061963 W IB 2024061963W WO 2025114932 A1 WO2025114932 A1 WO 2025114932A1
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cedna
cells
optionally
cell lysate
lysate
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Khushdeep KAUR MANGAT
Ronit GHOSH
Wei-Chiang Chen
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Sanofi SA
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Sanofi SA
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • Gene therapy is a cutting-edge technology for treating diseases caused by dysfunction in gene expression.
  • Some approaches of gene therapy involve delivery of a therapeutic gene that encodes a protein deficient in the patient.
  • Viral vectors are commonly used for such delivery.
  • viral vectors are often limited by their cargo capacity.
  • adeno-associated viral (AAV) vectors typically deliver a cargo of no more than about 5 kb in size.
  • viral vectors which contain viral proteins, e.g., in the form of viral capsids, can provoke immune responses against the vectors in patients, limiting the redosing potential of gene therapy.
  • ceDNA Closed-ended DNA circumvents the limitations of viral-based gene delivery.
  • CeDNA is linear, double-stranded DNA (dsDNA) and is more resistant to nucleases, therefore more stable than conventional dsDNA and RNA.
  • ceDNA encapsulated in lipid nanoparticles (LNPs) presents many advantages over existing viral gene delivery systems. Unlike viral capsids, LNPs typically do not encounter pre-existing antibodies against them in the patients, enhancing redosing potential of ceDNA-LNP therapy. Additionally, ceDNA-LNP has a much larger genetic capacity, able to accommodate more than 10 kb of genetic material, expanding the range of genetic diseases that can be targeted by gene therapy. Furthermore, the stability and self-replication of ceDNA will render a single ceDNA dose sufficient for a much larger therapeutic window than existing AAV-based therapy.
  • ceDNA is a promising tool for gene therapy, its production remains challenging. CeDNA is prone to truncation, nicking, and folding. These problems create hurdles for large-scale production. Thus, there remains a need for efficient and scalable means of producing and purifying large amounts of ceDNA molecules for clinical use.
  • the present disclosure provides methods for obtaining a purified preparation of closed-ended DNA (ceDNA) from ceDNA-producing cells.
  • the method comprises: incubating the cells in an alkaline buffer to lyse the cells to generate a cell lysate, wherein the alkaline buffer does not contain a detergent and has a pH of 10 or higher; and isolating the ceDNA from the lysate.
  • the method comprises, before the isolating step, neutralizing the cell lysate with an acidic salt.
  • the method further comprises, before the isolating step, pre-clarifying the neutralized cell lysate by adding sodium bicarbonate and separating the resultant flocculants from the cell lysate to generate a pre-clarified cell lysate.
  • the present disclosure provides a method for obtaining a purified preparation of closed-ended DNA (ceDNA) from ceDNA-producing cells, comprising: obtaining a lysate of the cells; pre-clarifying the cell lysate by adding sodium bicarbonate and separating the resultant flocculants from the cell lysate to generate a pre-clarified cell lysate; and isolating the ceDNA from the lysate.
  • the lysate is obtained by incubating the cells in an alkaline buffer to lyse the cells, wherein the alkaline buffer does not contain a detergent and has a pH of 10 or higher.
  • the cell lysate is neutralized with an acidic salt.
  • the method of the present disclosure further comprises, before the isolating step, removal of RNA by calcium chloride precipitation, optionally wherein the removal of RNA is conducted by: subjecting the pre-clarified cell lysate to filtration to generate a clarified cell lysate, and subjecting the clarified cell lysate to calcium chloride precipitation to remove RNA; in further embodiments, the clarified lysate undergoes ultrafiltration before addition of calcium chloride, and/or the lysate undergoes ultrafiltration and diafiltration after calcium chloride precipitation to reduce the concentration of calcium chloride.
  • the method of the present disclosure further comprises, after the isolating step, removal of RNA by calcium chloride precipitation; in further embodiments, the nucleic acid preparation undergoes diafiltration after calcium chloride precipitation to reduce the concentration of calcium chloride.
  • Calcium chloride may be added to achieve a concentration of, for example, about 1-3 M, optionally about 2 M.
  • the isolated ceDNA is polished (further purified) (e.g., after removal of RNA).
  • the polishing step may be performed with one or both of (i) hydrophobic interaction chromatography (HIC), optionally wherein the HIC is performed with monolith or perfusive resin; or (ii) multimodal core shell resin, optionally wherein the multimodal core shell resin comprises resin beads with a size-exclusion outer shell, further optionally wherein the size-exclusion out shell has a molecular weight cutoff (MWCO) of 400 or 700 kDa.
  • HIC hydrophobic interaction chromatography
  • multimodal core shell resin optionally wherein the multimodal core shell resin comprises resin beads with a size-exclusion outer shell, further optionally wherein the size-exclusion out shell has a molecular weight cutoff (MWCO) of 400 or 700 kDa.
  • MWCO molecular weight cutoff
  • the isolated ceDNA preparation undergoes viral filtration, e.g., with a 35 nm filter.
  • the alkaline buffer for cell lysis contains sodium hydroxide, optionally at a final concentration of about 100-300 mM, further optionally at about 150 mM, after being added to the cells.
  • the incubating step for cell lysis does not last more than five minutes, e.g., lasts about 2.5 minutes or about 3.5 minutes.
  • the incubating step for cell lysis is performed in a continuous in-line system.
  • the acidic salt for neutralizing the cell lysate is potassium acetate, e.g., at about 2.5-3.5 M (e.g., about 3.1 M).
  • the sodium bicarbonate is added in the pre-clarifying step to reach a concentration from about 5 to about 50 (e.g., about 10) g/L.
  • the flocculants are removed by a filter with pore sizes of about 7.5-60 pm.
  • the incubation time for pre-clarification is about two hours.
  • the isolated ceDNA is subjected to tangential flow filtration, e.g., with an MWCO of 10 and/or 100 kDa.
  • the ceDNA-producing cells are insect cells infected with a recombinant baculovirus expression vector.
  • the recombinant baculovirus expression vector comprises a heterologous nucleic acid sequence comprising a transgene flanked by inverted terminal repeats (ITRs).
  • the ITRs are parvoviral ITRs (e.g., ITRs from AAV such as AAV2).
  • the heterologous nucleic acid encodes a therapeutic protein.
  • the ceDNA- producing cells are transgenic insect cells whose genome comprises a coding sequence for the ceDNA.
  • the ceDNA preparation methods herein comprise in-process monitoring of ceDNA purity by measuring levels of ceDNA and impurities through ion exchange ultra-performance liquid chromatography in a sample taken before, during, or after the isolating step.
  • ceDNA preparations obtained by the present methods and the use of ceDNA for therapeutic purposes are also provided herein.
  • FIG. 1 is a diagram illustrating a ceDNA molecule containing sequences derived from parvoviral (e.g., AAV) inverted terminal repeats (ITRs).
  • parvoviral e.g., AAV
  • ITRs inverted terminal repeats
  • FIG. 2 is a diagram illustrating three insect cell systems for producing ceDNA containing parvoviral ITRs.
  • the illustrated transgene encodes coagulation factor VIII.
  • Other transgenes that do not encode factor VIII may also be incorporated into the cells in the same manner.
  • “One-Bac” a system using one baculoviral vector.
  • “Two-Bac” a system using two baculoviral vectors.
  • PCL a producer cell line containing stably integrated copies of a transgene cassette (e.g., a FVIII expression cassette comprising parvoviral ITRs).
  • FIG. 3 is an agarose gel electrophoresis image showing the integrity of ceDNA isolated from ceDNA-producing insect (Sf9) cells lysed under the indicated lysis conditions.
  • Agarose gel electrophoresis was run with ceDNA samples that were untreated or treated with T5 exonuclease, which degrades nicked ceDNA and has no impact on non-nicked ceDNA.
  • the lysis methods were performed using either lysis buffer containing 66.7 mM NaOH and 0.33% SDS or lysis buffer containing 150 mM NaOH.
  • FIG. 4 is a bar graph comparing the integrity of ceDNA obtained from two lysis methods. The comparison was made by both densitometry of electrophoresis agarose gel image (as shown in FIG. 3) and quantitative PCR. Bars on the left in each group of two: ceDNA-producing Sf9 cells were resuspended in PBS. Bars on the right in each group of two: ceDNA-producing Sf9 cells were resuspended in a buffer containing 100 mM Tris and lO mM EDTA.
  • FIGs. 5A-B are agarose gel electrophoresis images showing the integrity of ceDNA (in the presence or absence of T5 exonuclease) isolated from ceDNA-producing insect (Sf9) cells lysed under the indicated lysis conditions.
  • FIG. 6A is an agarose gel electrophoresis image showing the integrity of ceDNA (in the presence or absence of T5 exonuclease) isolated from ceDNA-producing insect (Sf9) cells lysed with 150 mM NaOH for the indicated lysis hold times (2.5 minutes, 5 minutes, 7.5 minutes, or 15 minutes).
  • FIG. 6B is a bar graph showing the titers of ceDNA (left bar), bacDNA (middle bar), and sf9DNA (right bar), and the percent purity of ceDNA isolated from ceDNA- producing insect (Sf9) cells lysed with 150 mM NaOH for the indicated lysis hold times (2.5 minutes, 5 minutes, 7.5 minutes, or 15 minutes).
  • FIG. 7 is a diagram showing a continuous in-line system for lysis and neutralization of the ceDNA-expressing cell paste.
  • FIG. 8 is an agarose gel electrophoresis image showing the integrity of ceDNA (in the presence or absence of T5 exonuclease) isolated from ceDNA-producing insect (Sf9) cells lysed with 150 mM NaOH in a continuous in-line system under the indicated conditions. The control was performed in batch mode using 150 mM NaOH lysis buffer with a hold time of 5 min.
  • FIG. 9 is an agarose gel electrophoresis image comparing the impact of ammonium hydrogen bicarbonate (AHC) and sodium hydrogen bicarbonate (NaHC) as a preclarification salt on the integrity of ceDNA after T5 exonuclease treatment.
  • AHC ammonium hydrogen bicarbonate
  • NaHC sodium hydrogen bicarbonate
  • FIG. 10A is a diagram showing a pre-clarification process using sodium hydrogen carbonate (NaHC).
  • FIG. 10B is a pair of photographs showing the separation of flocculants at 5 minutes and 2 hours after addition of NaHC at (from left to right in each photograph) 20 g/L, 15 g/L, 10 g/L, and 5 g/L.
  • FIG. 11 is a bar graph comparing the recovery, throughput, and turbidity across different filters used in the cell lysate clarification step. For each group of two, the left bar indicates % recovery, and the right bar indicates % throughput. Turbidity values are associated with the bars with a line.
  • FIG. 12 is a pair of graphs for evaluating the impact of feed flux using NaHC- treated neutralized cell lysates on filter throughput.
  • FIG. 13A is a diagram showing the study design for evaluating the incorporation of calcium chloride precipitation into the ceDNA purification process to remove residual RNA (rRNA).
  • UF ultrafiltration.
  • DF diafiltration.
  • FIG. 13B is an agarose gel electrophoresis image showing the yield and percent purity of ceDNA isolated following several different calcium chloride precipitation methods as described in FIG. 13A.
  • FIG. 13C is a bar graph comparing the ceDNA yield (left bar) and the rRNA impurities (right bar) of the four different rRNA removal processes shown in FIG. 13A.
  • the control in this experiment is absent any CaCh treatment.
  • FIG. 14A is a chromatograph showing the elution peaks of the ceDNA product after application to a Sartobind® Q column with varying concentrations of NaCl in the load.
  • FIG. 14B is an agarose gel electrophoresis image of the eluted fractions obtained in the process of FIG. 14A.
  • FIG. 15 is a chromatograph showing the elution peaks of the ceDNA product after application to a Sartobind® Q column.
  • FIG. 16A is a chromatograph showing the profile of step elution of the ceDNA product after application to a C4 HLD monolith column.
  • FIG. 16B is an agarose gel electrophoresis image of the eluted fractions obtained in the process of FIG. 16A.
  • FIG. 17A is a chromatograph showing the elution profile of the ceDNA product after application to a C4 HLD monolith with a reverse ammonium sulfate step gradient operated in a bind-and-elute approach.
  • FIG. 17B is an agarose gel electrophoresis image of the eluted fractions obtained in the process of FIG. 17A.
  • FIG. 18A is a chromatograph showing the elution profile of the ceDNA product after application to a C4 HLD monolith operated in a flow-through mode.
  • FIG. 18B is an agarose gel electrophoresis image of the eluted fractions obtained in the process of FIG. 18A.
  • FIG. 19A is a bar graph showing the percent recovery and percent purity of the ceDNA product recovered from several different hydrophobic interaction chromatography (HIC) media.
  • HIC Load % qPCR purity.
  • Poros Benzyl ultra % ceSDS purity.
  • Poros Ethyl “Poros Benzyl,” and “HIC Monolith”: bars from left to right are % recovery, % qPCR purity, and % ceSDS purity, respectively.
  • FIG. 19B is an agarose gel electrophoresis image of the eluted fractions obtained in in the process of FIG. 19A.
  • FIG. 20 is a diagram showing the study design for using the POROSTM Benzyl Ultra HIC column in either a bind-and-elute mode or a flow-through mode.
  • FIG 21A is a set of chromatographs showing the elution profile of the ceDNA product after polishing using a POROSTM Benzyl Ultra HIC column in either a bind-and- elute mode or a flow-through mode as described in FIG. 20.
  • FIG. 21B is an agarose gel electrophoresis image of the eluted fractions obtained in the process of FIG. 21 A.
  • FIG. 22A is a chromatograph showing the elution profile of the ceDNA product after polishing using a CaptoTM Core 400 or CaptoTM Core 700 core shell resin.
  • FIG. 22B is an agarose gel electrophoresis image of the filtrate obtained from applying the ceDNA product to core shell resins as in FIG. 22A.
  • FIG. 23 is an agarose gel electrophoresis image of the filtrate obtained from applying purified ceDNA to a PlanovaTM 35N viral removal filter.
  • FIG. 24 is an agarose gel electrophoresis image comparing 10 kDa and 30kDa molecular weight cutoff (MWCO) tangential flow filtration (TFF) cassettes used to concentrate ceDNA material containing about 1.5- 1.7 M ammonium sulfate.
  • MWCO molecular weight cutoff
  • TFF tangential flow filtration
  • FIG. 25 is a schematic diagram illustrating a “three-column” ceDNA purification process.
  • FIG. 26 is a panel of agarose gel electrophoresis images and table showing the ce- SDS Lab Chip and agarose gel electrophoresis results of eluate, product, strip, wash, and regeneration fractions from all column chromatography runs in a three-column purification approach. Product fractions from each polishing column runs are star-marked.
  • FIG. 27 is a schematic diagram illustrating two polishing strategies employing different orders of CaptoTM Core shell-based and HIC adsorbents.
  • FIG. 28 is an agarose gel and table showing the presence and intensity of the ceDNA product band and impurities based on agarose gel densitometry or next-generation sequencing short-read sequencing analysis.
  • FIG. 29 is an agarose gel and table showing the purify of ceDNA product using a combination of Lab Chip, agarose gel densitometry, and next-generation sequencing shortread sequencing analyses.
  • FIG. 30 is an overlaid ion exchange (LEX) chromatogram of four samples (“Load,” “FT,” “Wash,” and “Elution”) taken from the Sartobind® Q ceDNA purification step.
  • rHCP residual host cell protein.
  • RFP red fluorescent protein (an introduced cell marker).
  • rRNA residual RNA.
  • FT flow-through.
  • the present disclosure provides a scalable and robust manufacturing process to purify ceDNA in eukaryotic cells (e.g., insect cells).
  • the ceDNA may comprise a sequence of interest (e.g., a coding sequence for a therapeutic protein). Once purified, the ceDNA may be encapsulated in lipid nanoparticles for delivery in patients.
  • the ceDNA preparations of the present disclosure have reduced amounts of nucleic acid impurities, such as open-ended double-stranded DNA, and are expected to have an improved safety profile, including causing less anti-drug immune response when delivered to patients.
  • the present disclosure is based on discoveries associated with purifying ceDNA from eukaryotic cells such as insect cells.
  • the present purification process comprises: (i) harvesting and resuspending the producer cells to form a cell paste, optionally wherein the cells are harvested by continuous centrifugation; (ii) lysing the cells for a brief period of time in an alkaline buffer that does not contain a detergent (e.g., SDS) and contains, for example, sodium hydroxide, and neutralizing the cell lysate with an acidic salt (e.g., potassium acetate), where a continuous in-line system is used for the lysis and neutralization; (iii) chemically pre-clarifying the neutralized cell lysate by adding in salt that does not include ammonium salts (e.g., ammonium bicarbonate) and contains, for example, sodium bicarbonate, and removing flocculants by filtration; (iv) removing RNA by salt precipitation (e.g., calcium
  • CeDNA may be characterized by having no exposed ends and containing loop structures at its ends.
  • ceDNA has covalently linked ends, i.e., the 5’ end of the sense strand is covalently linked to the 3’ end of the antisense strand, and the 3’ end of the sense strand is covalently linked to the 5’ end of the antisense strand.
  • the ceDNA contains self-annealed loop structures at both ends of its both strands.
  • the ceDNA contains viral-derived inverted terminal repeat (ITR) sequences such that each end of the DNA strands is self-annealed into a hairpin-like structure (FIG. 1).
  • ITR sequences are derived from parvoviruses such as adeno-associated viruses (AAV) and bocaviruses.
  • the ITR sequences may be wildtype viral sequences, or contain mutations relative to wildtype viral sequences.
  • the ITR sequences may be from AAV2.
  • the ITR sequences flank a cargo sequence such as a transgene expression cassette; for example, an expression cassette for a therapeutic protein (e.g., an enzyme, an antibody, a cell surface receptor, a transcription factor, a hormone, or a cytokine).
  • the expression cassette may contain a promoter (e.g., constitutive or inducible) and other regulatory elements (e.g., enhancers, insulators, polyadenylation sites, etc.) for directing expression of the coding sequence in the host cells.
  • the promoter may be a promiscuous promoter that is active in multiple tissues, or may be a tissue-specific promoter. By way of example, the promoter may be specific for the liver, the lung, muscles, cells in the peripheral or central nervous system, cells in the cardiovascular system, cells in the ocular system, or cells in the immune system.
  • CeDNA such as ceDNA containing viral ITRs may be produced in recombinant eukaryotic host cells.
  • the ceDNA may be produced in mammalian host cells such as HEK293 cells, HeLa cells, and CHO cells.
  • the ceDNA may be produced in non-mammalian host cells such as insect cells.
  • the ceDNA may be produced in insect cells such as Sf21 , Sf9, S2, Tni-Hi5, Super9, and ExpresSF+.
  • the insect cells derived from Spodoptera frugiperda such as Sf21 and Sf9 cells are free of rhabdovirus (Sf-rhabdovirus-negative).
  • the ceDNA may be produced in stable cell lines (e.g., mammalian or insect cells) that are engineered to contain copies of a transgene expression cassette flanked by parvoviral ITRs.
  • the ITR-specific replicase protein encoded by the Rep gene recognizes the ITR sequences at the terminal resolution sites (TRS) and generates copies of the transgene-containing ceDNA in the producing cells.
  • the Rep gene may be stably integrated into the genome of the host cells, or may be transiently expressed from an episomal vector such as a baculoviral vector in the case of insect producing cells.
  • the transgenic host cell lines are derived from insect cells such as Sf21 , Sf9, S2, Tni-Hi5, Super9, and ExpresSF+.
  • a parvoviral (e.g., AAV) Rep gene may be introduced into the insect cells transiently via a baculoviral vector.
  • the template for the ceDNA is carried on the same baculoviral vector (“one- bac” system).
  • the template for the ceDNA is carried on a separate baculoviral vector (“two-bac” system). In the “two-bac” system, the two baculoviral vectors may be introduced into the insect host cells simultaneously or sequentially.
  • a producing cell line (PCL) is established with stably integrated copies of the ceDNA template, and a baculoviral vector carrying the Rep gene is introduced into the cell line transiently.
  • PCL producing cell line
  • ceDNA purification process can be implemented across multiple scales, including shake flasks, mini bioreactors (e.g., 100-250 mL), benchtop bioreactors (e.g., 50 L), and large bioreactors (e.g., 500 L, 1000 L, and 10,000 L).
  • mini bioreactors e.g., 100-250 mL
  • benchtop bioreactors e.g., 50 L
  • large bioreactors e.g., 500 L, 1000 L, and 10,000 L.
  • the host cell genome and baculoviral vector DNAs pose the major challenges for downstream purification of transgene-specific full-length ceDNA.
  • This production system also includes the intermediate replicative species of ceDNA that are not full-length and potentially interfere with purification of the transgene-specific full-length ceDNA. Additionally, the process is complicated by the presence of RNA impurities, including those from baculovirus and viruses endogenous to the cell lines (e.g., rhabdovirus).
  • the present disclosure provides an improved method of purifying ceDNA from producing host cells that is efficient and scalable for commercial production. The steps of this method are described in detail below.
  • the ceDNA-producing cells are harvested by centrifugation, e.g., continuous flow centrifugation.
  • continuous flow centrifugation large volumes of material are centrifuged at high centrifugal forces while the supernatant is simultaneously extracted through a drain line.
  • a fixed volume of cell-containing culture liquid is collected in a bowl while maintaining a pre-determined centrifugal speed and the supernatant continuously flows out of the bowl into a collection vessel. Once the fixed volume is pumped into the bowl, the concentrated cells are dispensed through a collection line for further processing while the supernatant is discarded. The number of cycles is determined according to the pre-established cell concentration factor needed for the process.
  • the concentrated cells may then be resuspended in a buffer to generate a cell paste.
  • a buffer may be used.
  • the pH of the buffer may range from about 6.5 to about 8.5.
  • the buffer may contain sodium salts, potassium salts, and/or buffering agents.
  • the buffer contains Tris, EDTA, and a polyol (e.g., sucrose) may be used.
  • a polyol e.g., sucrose
  • the buffer contains 100 mM Tris, 10 mM EDTA, and 50 mM sucrose, pH 8.
  • the buffer is a phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the PBS may contain about 100-150 mM NaCl, about 1.5-3.0 mM KC1, and about 10-15 mM phosphate, pH 7.4.
  • the PBS contains about 135 mM NaCl, about 2.7 mM KCL, and about 11 mM phosphate (e.g., 10 mM Na2HPO4 and 1.8 mM KH2PO4,), pH about 7.4.
  • the cell paste is lysed with an alkaline buffer that does not contain any detergent such as SDS, or contains a detergent at a very low concentration (e.g., SDS as a concentration no greater than 0.1%).
  • SDS a detergent at a very low concentration
  • the lysis buffer comprises an alkaline agent such as NaOH.
  • the lysis buffer comprises NaOH at a stock concentration of about 25 mM to 500 mM, e.g., about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mm, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, or about 500 mM.
  • the lysis buffer comprises NaOH at a stock concentration of about 300 mM.
  • the effective concentration of NaOH decreases once the lysis buffer is added to the cells.
  • the effective NaOH concentration of the lysis buffer in the cell mixture is about 150 mM.
  • the alkaline lysis buffer has a pH of about 9-14.
  • the lysis buffer comprises a pH of about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, about 10.0, about 10.2, about 10.4, about 10.5, about 10.6, about 10.8, about 11.0, about 11.2, about 11.4, about 11.5, about 11.6, about 11.8, about 12.0, about 12.2, about 12.4, about 12.5, about 12.6, about 12.8, about 13.0, about 13.2, about 13.4, about 13.5, about 13.6, about 13.8, or about 14.0.
  • the lysis buffer comprises a pH of about 12.5 or higher.
  • the lysis buffer is applied to the cell paste for a predetermined amount of time. This amount time is also termed herein “lysis hold time.” In some embodiments, the lysis hold time is no greater than ten minutes, e.g., no greater than five minutes. A longer lysis hold time results in a longer exposure to high pH and consequently irreversible denaturation of the ceDNA product. In some embodiments, the lysis hold time may be about 0.5 minutes, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, or about 5 minutes.
  • the cells are lysed in an alkaline lysis buffer comprising a NaOH concentration of about 300 mM, pH of about 12.5 or higher, and once the cell solution is mixed with the lysis buffer in equal volume, the effective NaOH concentration is halved to become about 150 mM; and the lysis hold time is about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, or about 5 minutes.
  • the lysate is neutralized to return the pH of the lysate to achieve acidic conditions (e.g., a pH of about 5.0 to 6.5, or about 5.5 to 6.0).
  • the lysate is neutralized with an acidic salt such as potassium acetate.
  • the alkaline lysate is neutralized with potassium acetate by mixing with a potassium acetate solution of about 0.5 M to about 5 M.
  • the acidic salt such as potassium acetate may be provided at a concentration of about 0.5 M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M, about 3.0 M, about 3.5 M. about 4.0 M, about 4.5 M, or about 5.0 M.
  • the potassium acetate is provided at a concentration of about 3.0 M (e.g., 3.1 M), with a pH of about 5.2.
  • the cells may be lysed in an continuous in-line lysis operation.
  • FIG. 7 illustrates such an operation.
  • concentrated cells (cell paste) and the alkaline lysis buffer are continuously fed through the apparatus at predetermined rates and volume ratio (e.g., 1 : 1 volume ratio) by controlling pump speed for each buffer/lysate performed.
  • the cells travel through a tube in which lysis occurs.
  • the lysis hold time is determined by the length of the tube and the feed rates.
  • the mixture is then mixed with a continuous stream of the neutralization buffer and the neutralized cell lysate continues into a collection tank.
  • continuous cell lysis may be used to process 10 L or more cell paste with a lysis hold time of about 2.5 minutes (e.g., using a static mixer).
  • a gentle mixing speed is preferred.
  • the cell lysate may be pre-clarified, e.g., chemically, to remove impurities such as high molecular weight (BMW) genomic DNA, host cell proteins (HCP), and other cellular components.
  • BMW high molecular weight
  • HCP host cell proteins
  • the present inventors have discovered that chemical pre-clarification demonstrates superior results over physical pre-clarification (e.g., by batch or continuous centrifugation).
  • the neutralized cell lysate is treated with a pre-clarifying salt.
  • a pre-clarifying salt Conventionally, such operation is performed with ammonium bicarbonate. But ammonium bicarbonate generates noxious ammonia gas and may become an environmental hazard. The present inventors have discovered that sodium bicarbonate not only is environmentally friendly, but also generates satisfactory pre-clarification results.
  • the pre-clarifying salt is sodium bicarbonate and may be added to reach a concentration of about 5-50 g/L.
  • the pre-clarifying salt may be present at a concentration of about 5 g/L, about 7.5 g/L, about 10 g/L, about 12.5 g/L, about 15 g/L, about 17.5 g/L, about 20 g/L, about 22.5 g/L, about 25 g/L, about 27.5 g/L, about 30 g/L, about 32.5 g/L, about 35 g/L, about 37.5 g/L, about 40 g/L, about 42.5 g/L, about 45 g/L, about 47.5 g/L, or about 50 g/L.
  • the pre-clarifying salt is present at a concentration of 10 g/L.
  • the neutralized cell lysate is treated with the pre-clarifying salt for a pre-determined amount of time. In some embodiments, the neutralized cell lysate is treated with the pre-clarifying salt for a period of about 0.5-8 hours. For example, the neutralized cell lysate may be treated with the pre-clarifying salt for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, or about 8 hours.
  • the neutralized cell lysate may be treated with the pre-clarifying salt for about 2 hours or about 4 hours. In some embodiments, the neutralized cell lysate is mixed with the pre-clarifying salt through mild agitation for efficient solubilization.
  • the pre-clarified, neutralized cell lysate may be clarified through depth filtration to remove cell debris and particles from feed stream.
  • the choice of clarification filter may be driven by cell density, cell types, and harvest viscosities.
  • Depth filtration may be performed by using, e.g., a polypropylene filter, a cellulose filter, a silica filter, a polyacrylic filter, or a filter with mixed materials.
  • the filter may have pore sizes of 0.6-60 pm, e.g., 7.5-60 pm, or 0.6-8.0 pm. Examples of suitable depth filtration systems are Clarisolve® 60HX and D0HC filters (Millipore).
  • RNase typically is sourced from animals, which not only increases costs, but also poses safety issues for therapeutic products.
  • the present inventors have discovered that calcium chloride achieve excellent results in removing RNA as well as other impurities such as genomic DNA fragments, baculoviral DNA, and HCP, from the cell lysate.
  • the clarified cell lysate is treated with CaCh, wherein the calcium chloride salt is present at a concentration of about 0.5-10.0 M.
  • the salt may be present at a concentration of about 0.5 M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M, about 3.0 M, about 3.5 M, about 4.0 M, about 4.5 M, about 5.0 M, about 5.5 M, about 6.0 M, about 6.5 M, about 7.0 M, about 7.5 M, about 8.0 M, about 8.5 M, about 9.0 M, about 9.5 M, or about 10.0 M.
  • the salt may be present at a concentration of about 2 M.
  • the clarified cell lysate undergoes ultrafiltration (e.g., TFF) before the calcium chloride treatment, to concentrate the lysate.
  • the clarified cell lysate is concentrated by a factor of about 2-1 OX, e.g., by a factor of about 2X, about 3X, about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, or about 10X.
  • the lysate may undergo ultrafiltration and diafiltration in the presence of a buffer suitable for subsequent chromatography.
  • the diafiltration may be done in the presence of a buffer comprising Tris, EDTA, and sodium chloride, pH about 8 (e.g., 50 mM Tris, 10 mM EDTA, and 0.3 M NaCl).
  • a buffer comprising Tris, EDTA, and sodium chloride, pH about 8 (e.g., 50 mM Tris, 10 mM EDTA, and 0.3 M NaCl).
  • the clarified cell lysate is treated with calcium chloride salt to remove RNA prior to the isolation of the ceDNA product from the lysate.
  • the removal of RNA from the clarified lysate via calcium chloride treatment occurs after the ceDNA is isolated from the cell lysate (e.g., through anion exchange), prior to polishing of the ceDNA product.
  • calcium chloride precipitation is implemented after the capture/isolation step, the material may not need to be filtered, and the capture eluate (isolation step product) may undergo diafiltration after calcium chloride precipitation and before going through polishing.
  • the ceDNA product may be isolated from the cell lysate through chromatography.
  • the present method comprises one or more, two or more, or three or more chromatography steps of the same or different chromatography modes.
  • the clarified cell lysate is treated with a load adjustment salt to remove impurities during chromatography.
  • the load adjustment salt is NaCl.
  • the load adjustment salt is present in the cell lysate load at a concentration of about 50-500 mM.
  • the clarified cell lysate laod may contain NaCl at a concentration of about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, or about 600 mM.
  • the clarified cell lysate load may comprise NaCl at a concentration of about 300 mM.
  • the ceDNA is isolated by anion exchange chromatography.
  • Suitable anion exchange chromatography systems include strong basic anion ligand exchanger with quaternary ammonium (R-CH2-N + (CH3)3) or weak basic anion ligand exchanger with diethyl amino ethyl (DEAE) (R-C2H4-N + H(C2H5)2.
  • the anion exchange chromatography system may be the Sartobind® Q system (Sartorius), Natrix® HD Q (Millipore Sigma), CIMmultus® DEAE (Sartorius), CIMmultus® Q (Sartorius), MustangTM Q (Pall), ReadytoProcess Adsorber Q (Cytiva), POROSTM 50D (Thermo Fisher), POROSTM 50HQ (Thermo Fisher), Sartorius STIC® PA (Sartorius), or an equivalent thereof.
  • the capture chromatography media is a Membrane or Monolith or resin.
  • the anion exchange chromatography step comprises flow- through of impurities with a load adjustment of NaCl at 50-500 mM (e.g., 300 mM), a wash step to further remove trace impurities, and then elution of ceDNA with an elution buffer.
  • the wash buffer comprises a salt, such as NaCl, at a concentration of about 0.15-0.8 M.
  • the wash buffer may comprise a salt, such as NaCl, at a concentration of about 0.15 M, about 0.2 M, about 0.25 M, about 0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, about 0.5 M, about 0.55 M, about 0.6 M, about 0.65 M, about 0.7 M, about 0.75 M, or about 0.8 M.
  • the wash buffer comprises a salt, such as NaCl, at a concentration of 0.6 M.
  • the elution buffer comprises a salt, such as NaCl, at a concentration of about 0.5-3 M.
  • the elution buffer may comprise a salt, such as NaCl, at a concentration of about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2.0 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, or about 3.0 M.
  • the elution buffer comprises a salt, such as NaCl, present at a concentration of about 0.9 M.
  • the elution buffer comprises a different salt such as sodium sulfate, sodium acetate, or ammonium acetate.
  • the above isolated ceDNA preparation may be further polished by one or more additional chromatography steps that may be based on, e.g., hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • the HIC media may be monolith columns or resins and may contain additional operation modes such as multimodal core shell resinbased purification.
  • the ceDNA preparation is treated with an HIC load adjustment salt prior to the HIC polishing step.
  • the HIC load adjustment salt is ammonium sulfate (AS).
  • AS ammonium sulfate
  • the HIC load adjustment salt is present at a concentration of about 1-4 M.
  • the clarified cell lysate may be treated with salt at a concentration of about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M, about 3.0 M, about 3.5 M, or about 4.0 M.
  • the clarified cell lysate is treated with an HIC load adjustment salt present at a concentration of about 3.0 M.
  • the HIC step comprises an elution step selected from a linear gradient or a step gradient may be operated in a bind-and-elute mode or a flow-through mode.
  • the linear gradient elution step comprises a reverse salt gradient containing AS at a linear gradient of salt concentration from, e.g., about 4-0 M or about 3-0 M.
  • the step gradient elution step comprises a step-wise reverse salt gradient.
  • the step-wise reverse salt gradient comprises AS at concentration from about 10-0 M, e.g., 2-0 M.
  • the AS salt in the elution buffer may be present at a concentration of about 0.5 M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M, about 3.0 M, about 3.5 M, or about 4.0 M. In further embodiments, the bind-and-elute salt may be present at a concentration of about 1.74 M.
  • the AS salt in the load may be present at a concentration of about 0.75-2.5 M.
  • the HIC flow-through purification step may be performed at an AS load concentration of about 0.75 M, about 1 M, about 1.5 M, about 2 M, or about 2.5 M.
  • the AS concentration in the load is about 1.5 M.
  • the HIC step comprises a channel.
  • the HIC channel has a size of about 0.5-10 pm.
  • the HIC channel size may be about 0.5 pm, about 1.0 pm, about 1.5 pm, about 2.0 pm, about 2.5 pm, about 3.0 pm, about 3.5 pm, about 4.0 pm, about 4.5 pm, about 5.0 pm, about 5.5 pm, about 6.0 pm, about 6.5 pm, about 7.0 pm, about 7.5 pm, about 8.0 pm, about 8.5 pm, about 9.0 pm, about 9.5 pm, or about 10.0 pm.
  • the HIC channel has a size of about 2.0 pm.
  • the HIC channel has a size of about 6.0 pm.
  • HIC systems include: C4 HLD Monolith resin (Sartorius), POROSTM Ethyl perfusive resin (Thermo Scientific), POROSTM Benzyl perfusive resin (Thermo Scientific), or POROSTM Benzyl Ultra perfusive resin (Thermo Scientific), CaptoTM PlasmidSelect (Cytiva), CaptoTM Phenyl (Cytiva), or Sartobind® Phenyl (Sartorius).
  • the polishing step includes mixed-mode resins combining size exclusion chromatography with anionic and hydrophobic interaction chromatographic properties.
  • the ceDNA preparation is treated with a load adjustment salt, prior to application to the size exclusion column.
  • the load adjustment salt is NaCl.
  • the load adjustment salt is AS.
  • the load adjustment salt is present at a concentration of about 0.1-4.0 M.
  • the load adjustment salt may be present at a concentration of about 0.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2.0 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3.0 M, about 3.1 M, about 3.2 M, about 3.3 M, about 3.4 M, about 3.5 M, about 3.6 M, about 3.7 M, about 3.8 M,. about 3.9 M, or about 4.0 M.
  • the load adjustment salt is NaCl present at a concentration of about 0.15-0.9 M.
  • the load adjustment salt is AS present a concentration of about 1.5-2.5 M.
  • size exclusion chromatography systems include CaptoTM Core 700 and CaptoTM Core 400 multimodal resin (Cytiva), which operates by both size exclusion and HIC.
  • the size exclusion chromatography has a MWCO of 400 to 900 kDa, e.g., 750 kDa.
  • CaptoTM Core 400 may be used for ceDNA products that are about 3 to 5 kb in length.
  • the polishing step utilizes two different chromatography methods.
  • the ceDNA isolated by Sartobind® may be subject to HIC (e.g., C4 HLD HIC monolith in bind-and-elute mode, C4 HLD HIC monolith in flow-through mode, POROSTM Benzyl Ultra HIC resin in bind-and-elute mode, or POROSTM Benzyl Ultra HIC resin in flow-through mode), and then subject to a multimodal core shell resin (e.g., CaptoTM Core 400 or 700); or in a reverse order.
  • HIC e.g., C4 HLD HIC monolith in bind-and-elute mode, C4 HLD HIC monolith in flow-through mode, POROSTM Benzyl Ultra HIC resin in bind-and-elute mode, or POROSTM Benzyl Ultra HIC resin in flow-through mode
  • a multimodal core shell resin e.g., CaptoTM Core 400 or 700
  • the ceDNA preparation undergoes viral filtration to remove any viral contaminants.
  • viral filtration is performed using a filter with a surface area of about 0.001-1.0 m 2 .
  • the filter may be present with a surface area of about 0.001 m 2 , about 0.01 m 2 , about 0.12 m 2 , about 0.3 m 2 , or about 1.0 m 2 .
  • viral filtration is performed using a filter with a surface area of about 0.001 m 2 .
  • Examples of viral filtration systems include PlanovaTM 35N viral removal filter (Asahi Kasei Bioprocess).
  • a purified ceDNA preparation is concentrated by a tangential flow filtration (TFF) cassette.
  • the TFF cassette may comprise a MWCO of about 1-100 kDa.
  • the TFF cassette may comprise a molecular MWCO of about 1 kDa, about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, or about 100 kDa.
  • the TFF comprises a MWCO of about 10 kDa.
  • the ceDNA product is concentrated by a TFF by a factor of about 2-20X.
  • the ceDNA product may be concentrated by a tangential flow filtration cassette by a factor of about 2X, about 3X, about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 11X, about 12X, about 13X, about 14X, about 15X, about 16X, about 17X, about 18X, about 19X, or about 20X.
  • the purified ceDNA product is concentrated by a TFF cassette by a factor of about 10X.
  • compositions of intermediate products of the ceDNA manufacturing may be monitored.
  • samples taken from different stages of the purification process such as samples from the loading material, the flow-through material, the wash product, and the elution product, may be analyzed to assess amounts of the ceDNA and impurities (e.g., host cell proteins and DNA/RNA fragments) present in the samples.
  • impurities e.g., host cell proteins and DNA/RNA fragments
  • the analysis may be done by ion exchange (IEX) ultraperformance liquid chromatography (UPLC) to separate proteins and nucleic acids based on their charges.
  • IEX ion exchange
  • UPLC ultraperformance liquid chromatography
  • the levels of the various components in the analyzed sample may be determined by spectrophotometry at wavelengths of 254 nm (for nucleic acids) and 280 nm (for proteins).
  • IEX columns are those comprising non-porous particles (e.g., polymethacrylate particles) coated by networks of ion exchange groups (e.g., sulfopropyl, carboxymethyl, and/or quaternary ammonium groups), such as Protein-Pak Hi Res Q by WaterTM and TSKgel DNA-STAT by Tosoh.
  • the ceDNA-producing cells may contain an exogenously introduced expression cassette for expressing a fluorescent protein (e.g., a red, blue, yellow, green, or cyan fluorescent protein).
  • a fluorescent protein e.g., a red, blue, yellow, green, or cyan fluorescent protein.
  • fluorescence spectrophotometry may be additionally used to monitor the amount of the fluorescent protein in the samples as one indicator for the presence of host cell proteins.
  • the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
  • the ceDNA-producing Sf9 cells were harvested and centrifuged. The cell pellet was resuspended in a resuspension buffer. The initial process development runs were performed by resuspending the cells in a 50 mM Tris/10 mM EDTA buffer (pH 8), which is similar to conditions of commercial plasmid DNA purification kits. The resuspension buffer was modified by increasing the Tris concentration to 100 mM Tris (pH 8) and adding sucrose to a final concentration of 50 mM. The data show that inclusion of sucrose in the resuspension buffer prevented abrupt osmotic shock to the Sf9 cells and mitigated the loss of cell viability during the cell concentration phase using UniFuge®.
  • the lysates were neutralized, clarified, and finally purified using an ion exchange column (Sartobind® Q). Titer assessment of ceDNA was performed using qPCR and nicking impact on ceDNA was evaluated by treating the purified ceDNA with T5 exonuclease and further running T5 non-treated and treated samples side by side on an agarose gel (FIG. 3). T5 treatment works on the principle that a nicked DNA will be completely digested by T5 exonuclease and would not show up as intact DNA on agarose gel electrophoresis. T5 has no impact on non-nicked ceDNA. For T5 treatment, the DNA sample was incubated with the enzyme at 37°C for 30 minutes.
  • the commercial plasmid DNA extraction kit from Invitrogen uses 0.1 M NaOH + 0.5% SDS as the lysis buffer recipe.
  • a high throughput study was implemented to identify a suitable lysis condition for ceDNA-producing Sf9 cells. During this study, varying concentrations of NaOH (from about 0 mM to about 100 mM) and SDS (from about 0% to about 1%) were evaluated.
  • a qPCR assay shows that 66.7mM NaOH+0.33% SDS resulted in high titers of ceDNA and less genomic DNA.
  • Lysis study was performed by changing the NaOH concentration from 50 mM to 200 mM within a pH range of 10-12.6. Arginine was used as one of the conditions corresponding to pH 10. The data show that all lysis conditions ranging from 50 mM to 200 mM NaOH resulted in fully intact and non-nicked ceDNA (FIG. 5A). Furthermore, using a lysis buffer containing only NaOH resulted in higher yields of ceDNA than using a lysis buffer containing a detergent (FIG. 5B). Based on this study, highly alkaline 150 mM NaOH was chosen as the final lysis buffer condition for further downstream studies. The 150 mM NaOH lysis condition demonstrated consistent and reproducible intact ceDNA across multiple harvests/runs. We hypothesized that the absence of SDS may mitigate nuclease activity, and the presence of highly alkaline condition (pH 10-12.6) might be beneficial towards denaturation of the nucleases present in the cell lysate.
  • the lysis hold time i.e., the incubation time of the cells with a lysis buffer
  • T5 digestion was used to indicate the presence of nicks in ceDNA.
  • the experiment was designed to evaluate different lysis time: 2.5, 5, 7.5, or 12.5 minutes in batch mode followed by purifying ceDNA using Sartobind® Q in high throughput format. Based on the agarose gel electrophoresis, the ceDNA band showed decrease in intensity and homogeneity with increase in time.
  • the study shows that a lysis hold time of 2.5 to 5 minutes gave rise to the best yield of intact ceDNA (FIG. 6A) while reducing impurities such as baculovirus and insect cell DNA (FIG. 6B).
  • Flowrates 1, 2, and 3 were the same; two flowrates were tested, 15 mL/min and 27 mL/min, dependent on shear rate - 165/sec and 308/sec. Multiple parameters were studied in the continuous in-line cell lysis system. The residence time of lysis duration tested was 3.7, 2.5, and 1.5 minutes. Different neutralization mechanisms were also tested: static mixer, T-mixer, and batch mode (in-bottle). The data show that continuous cell lysis was successfully scaled up to 10L with 2.5-minute lysis duration using static mixer and neutralization step using T-mixer (FIG. 8).
  • a salt-based pre-clarification approach was then developed for efficient removal of flocculants and improved filtration capacity.
  • the initial attempt in this approach was performed by using ammonium hydrogen carbonate (i.e., ammonium bicarbonate (NH4HCO3); “AHC”).
  • NH4HCO3 ammonium bicarbonate
  • AHC ammonium bicarbonate
  • the use of this salt was based on the mechanism that after addition of ammonium hydrogen carbonate to the neutralized lysate, flocculants are lifted to the surface of solution by the release of carbon dioxide and ammonia.
  • a range of AHC concentrations of 2.5 to 25 g/L was evaluated for optimal phase separation.
  • NaHCCh sodium bicarbonate
  • a comparison study was done to compare the effectiveness of cell debris separation using AHC and NaHC. Four arms were designed: 15 g/L AHC, 20 g/L AHC, 15 g/L NaHC, and 20 g/L NaHC.
  • the Sartobind® Q membrane was selected for further studies. After evaluating the elution profile and then spiking in different concentrations of NaCl in the loading material, we found that 150 mM NaCl and 300 mM NaCl in the load shifted the impurity peak (composed primarily of low molecular weight species) to the flow-through fraction (FIGs. 14A-B). Droplet digital PCR and qPCR titer assays confirmed that the addition of NaCl to the load samples did not affect the titer of ceDNA in the eluate fractions (Table 2). Optimizing the load conductivity can direct low molecular weight impurities to the flowthrough or wash fractions. This strategy potentially increases the binding capacity for the target molecule by reducing chances of impurities competing or available binding sites on the membrane (FIG. 15).
  • the ceDNA capture process with the Sartobind® Q membrane was evaluated with two different upstream processes: the one-bac system (using a producing cell line (PCL) that stably expressed a factor VIII transgene and was transiently transfected with a baculoviral vector for expressing AAV Rep proteins); and the two-bac system (Sf9 cells transiently transfected with two baculovirus vectors encoding AAV Rep proteins and factor VIII, respectively.
  • PCL producing cell line
  • Sf9 cells transiently transfected with two baculovirus vectors encoding AAV Rep proteins and factor VIII, respectively.
  • the capture purification using the Sartobind® Q membrane provided good separation of HCP and RNA impurities in the flow-through and wash fractions. Additionally, Sartobind® Q resulted in a 20-30% reduction of Sf9 DNA and baculoviral DNA. However, complete removal of nucleic acid impurities was not observed across the capture step; some RNA, Sf9 DNA, and baculoviral DNA co-eluted with the ceDNA product.
  • the purified ceDNA from the capture step was also composed of ceDNA fragments (size ⁇ 4.5 kb).
  • the C4 HLD monolith columns (Sartorius) have high ligand density and are butyl- modified. They were evaluated as a potential polishing medium for ceDNA purification. Binding of DNA to C4 HLD HIC monolith columns requires a high amount of ammonium sulfate (AS). Thus, the HIC load was adjusted to 3 M AS to induce hydrophobic interactions. Elution was then achieved by performing a linear descending ammonium sulfate salt gradient from 3 M to 0 M over 60 CV. Under these conditions, it was observed that the ceDNA product eluted over a wide elution range (2.25 M to 0 M AS) with typical four-peak elution behavior.
  • AS ammonium sulfate
  • LMW lower molecular weight
  • a step elution process was implemented for the ceDNA polishing process. Since full length ceDNA was observed to elute between 2.25 M to 1.5 M AS, a step elution was performed at a 1.74 M AS concentration to recover pure and concentrated full length ceDNA. The step gradient also included steps at 0.9 M, 0.45 M, 0.24 M and 0 M AS to capture ceDNA and other nucleic acid fragments. It was observed that implementing a step elution at 1.74 M AS resulted in recovery of roughly 77% of ceDNA product (FIGs. 16A-B and Table 4).
  • POROSTM resins were evaluated for efficiency of product separation.
  • POROSTM resins have wide pore sizes in the order between 100-400 nm, making them ideal resin candidates for large biomolecule purification without any significant impact on binding capacity.
  • Three POROSTM HIC candidates were tested to evaluate if the nature of HIC ligands (ethyl or benzyl) combined with perfusive flow properties would provide improved resolution between linear ceDNA product and process- and product-related impurities.
  • POROSTM HIC resins behaved similarly, however POROSTM Ethyl exhibited highest binding capacity compared to Benzyl or Benzyl Ultra, where it as able to accommodate a load challenge of 2.5 mg/mL resin.
  • the POROSTM HIC adsorbents demonstrated an inverse relationship in binding capacities for ceDNA material as a function of increasing hydrophobic strength of the HIC ligands (Ethyl, Benzyl, and Benzyl Ultra). Typically, a stronger HIC ligand is expected to exhibit higher binding capacities for biomolecules compared to a ligand with lower hydrophobicity strength.
  • the POROSTM Benzyl Ultra HIC column was observed to have a binding capacity with breakthrough observed close to 0.7 mg/mL-resin load challenge.
  • the bind-and- elute approach exhibited greater than 90% reduction of baculoviral and Sf9 DNA, with ceDNA product recovery of 42% due to loss of ceDNA in flow-through as a result of column breakthrough (FIGs. 21A-B).
  • the POROSTM Benzyl Ultra HIC column was also assessed for the efficiency of flow-through operation.
  • ceDNA was observed to elute between 2 M-1.5 M during the linear gradient run on the POROSTM Benzyl Ultra HIC column.
  • the load was first adjusted to a final AS concentration of 1.8 M and loaded onto the POROSTM Benzyl Ultra HIC column.
  • the ceDNA product was resolved in the flow-through section and only nucleic acid impurities like Sf9, baculoviral DNA, and RNA bound onto the column and was subsequently removed in a strip step performed with a buffer containing 50 mM Tris and 10 mM EDTA, pH 8, and no salts.
  • the flow-through approach resulted in 92% ceDNA product recovery with 62% reduction in process-related impurities. Evaluation of the ceDNA purity via agarose gel indicates similar purity for both bind-and-elute and flow-through modes operation.
  • CaptoTM Core resin is based on a core shell or core bead concept with each resin bead having a ligand activated core and an outer inactive layer without ligands.
  • the outer layer has size exclusion properties which prevents large targets from entering the beads, whereas smaller protein and DNA impurities are able to enter into the core where they bind to the hydrophobic and positively charged octylamine ligands.
  • CaptoTM Core resin has not been previously used as a polishing approach in DNA purification process.
  • the CaptoTM Core 700 resin has a larger pore structure and a larger MWCO than the CaptoTM Core 400 resin (750 kDa vs. 400 kDa). [0157] Our studies show that the CaptoTM Core 700 resin was more effective in reducing Sf9 and baculoviral DNA impurities (>70% reduction), as compared to the CaptoTM Core 400 resin ( ⁇ 50% reduction) (Table 8).
  • Chromatographic overlay of the flow-through blocks shows a higher peak height for CaptoTM Core 400 over 700, exhibiting lower removal of impurities in the flow-through product portion (FIG. 22A).
  • Agarose gel analysis of the flow-through fraction from these columns exhibit less smearing of ceDNA product with CaptoTM Core 700 resin compared to CaptoTM Core 400 resin (FIG. 22B).
  • the CaptoTM Core 700 resin performed well in a polishing step in ceDNA purification.
  • Baculovirus is endogenously present in Sf9 cells due to the use of baculoviral virus for transduction and protein expression. Additionally, the original Sf9 cell line is known to be contaminated with rhabdovirus. Thus, clearance of viral contaminants is necessary in the present ceDNA purification scheme.
  • a challenge of using viral filtration for DNA purification is the size of the product. The radius of gyration of the ceDNA monomer (about 150 nm) makes it difficult to distinguish ceDNA from baculoviral DNA by size-exclusion separation due to the size limitation.
  • PlanovaTM 35N size-based virus removal filter To evaluate the feasibility of viral removal in ceDNA purification, we utilized the PlanovaTM 35N size-based virus removal filter.
  • the experimental trains comprised three different feed streams: the Sartobind® Q eluate (50 mM Tris pH 8.0, 10 mM EDTA, 0.9 M NaCl), the C4 HLD load (50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M AS) and the C4 HLD product (flow-through and wash volume) (50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M AS).
  • the Sartobind® Q eluate 50 mM Tris pH 8.0, 10 mM EDTA, 0.9 M NaCl
  • the C4 HLD load 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M AS
  • the C4 HLD product flow-through and wash volume
  • the pressure was controlled at 12 psi, and the tested throughput was 50 L/m 2 .
  • the data show that the ceDNA recovery from the Sartobind® Q train and the C4HLD product trains were each >50% based on A260 absorbance (Table 9). Additionally, analysis of ceDNA product purity by agarose gel electrophoresis show a reduction of contaminating species from the C4 HLD product train, indicating that the PlanovaTM 35N viral filter represented a feasible size-based viral removal step in ceDNA purification (FIG. 23).
  • the 10 kDa MWCO TFF membrane resulted in no loss of ceDNA product in permeate and obtained 100% recovery based on qPCR as well as band intensity on an agarose gel (FIG. 24).
  • the 10 kDa MWCO TFF membrane could therefore be used as a final purification step when the polished ceDNA product contains high molar concentrations of ammonium sulfate.
  • a three-column purification approach was tested for ceDNA purification (FIG. 25).
  • ceDNA was purified using the Sartobind® Q capture column.
  • the Sartobind® Q eluate was further polished using four different polishing approaches comprising of C4 HLD HIC monolith in bind-and-elute mode, C4 HLD HIC monolith in flow-through mode, POROSTM Benzyl Ultra HIC resin in bind-and-elute mode, and POROSTM Benzyl Ultra HIC resin in flow-through mode.
  • the eluate from the bind- and-elute secondary column approach was combined and purified further using the CaptoTM Core multimodal core shell resin.
  • ceDNA product quality attribute for all these runs were analyzed using agarose gel electrophoresis along with ce-SDS lab chip.
  • the agarose gel and lab chip results are shown in FIG. 26.
  • the data show that ceDNA intact purity from the initial purification process (Sartobind® Q - HIC polishing) ranged between 27-34%.
  • the ce-SDS purity of intact ceDNA increased to 80% and greater after the incorporating CaCh precipitation and two-column polishing process comprising of HIC and CaptoTM Core polishing purification steps.
  • Polishing Strategy 1 CaptoTM Core followed by HIC adsorbent
  • Polishing Strategy 2 HIC adsorbent followed by CaptoTM Core resin
  • Process conditions were optimized to facilitate the direct loading of eluate from the capture column onto the intermediate polishing column, and subsequently onto the final polishing column with minimal adjustments. This enables a fully integrated continuous approach for the capture, intermediate polishing, and final polishing steps.
  • the final purified ceDNA material is recovered in the eluate (for bind-and-elute mode) or the flow-through and wash fractions (flow-through mode) in 1.5-2 M AS in 50 mM Tris, 10 mM EDTA, 0.45 M NaCl, pH 8.
  • Sf9 insect producer cells previously infected with two baculoviral expression vectors were concentrated by a factor of 4-6X using a continuous centrifugation system at 2000 x g with a flow rate of 3 L/min (Minifuge/UniFuge®, CARR Biosystems UniFuge®).
  • the concentrated Sf9 cells were resuspended in IX PBS, pH 7.4 (2.7 mM KC1, 11 mM phosphate, 135 mM NaCl) wash buffer to produce a cell paste.
  • the resuspended cell paste was immediately lysed through alkaline lysis by addition of 0.3 M NaOH to the suspension for a final concentration of 150 mM NaOH.
  • the cell paste was lysed via continuous in-line lysis with a flow rate of 15 mL/min.
  • the cell paste was lysed under alkaline conditions for no more than five minutes, preferably no more than 2.5 minutes.
  • the flocculant cell lysate was treated with 10 g/L of sodium bicarbonate and stirred for ⁇ 30 seconds. The reaction then was allowed to proceed for about two hours. This pre-clarification treatment caused a phase separation between the flocculants and the cleared cell lysate.
  • the neutralized cell lysate was passed through either a 23 cm 2 Clarisolve 60HX (Millipore) or 23 cm 2 D0HC (Millipore) depth filter at a flow rate of 50 L/m 2 /hr.
  • the clarified cell lysate was subjected to a TFF system to concentrate the cell lysate six- to ten-fold.
  • the tangential flow filtration step was conducted in 50 mM Tris, pH 8.0, 10 mM EDTA, 0.3 M NaCl.
  • the clarified cell lysate was supplemented with 5 M CaCh for a final concentration of 2 M CaCh in solution. The precipitation reaction was allowed to proceed at room temperature for 30 minutes. The clarified cell lysate was passed through a second tangential flow filtration system to exchange the buffer into 50 mM Tris, pH 8.0, 10 mM EDTA, 0.3 M NaCl. The buffer was exchanged six times.
  • the clarified cell lysate was passed through a Sartobind® Q anion exchange chromatography column. After binding of the ceDNA to the column, the column was washed with 50 mM Tris, pH 8.0, 10 mM EDTA. The column was washed a second time with 50 mM Tris pH 8.0, 10 mM EDTA, 0.6 M NaCl. Following the washing steps, the ceDNA was eluted from the Sartobind® Q column with 50 mM Tris pH 8.0, 10 mM EDTA, 0.9 M NaCl. The anion exchange chromatography step proceeded with a flow rate of 3 matrix volumes/min.
  • the eluate was applied to an HIC chromatography column.
  • the HIC column was either a C4 HLD Monolith 2 pm channel column (Sartorius) or POROSTM perfusive resin (Thermo Scientific).
  • the Monolith C4 HLD column the ceDNA product was isolated via either a bind-and-elute or flow-through method.
  • the bind-and-elute method the Sartobind® Q-eluted ceDNA was first supplemented to 3M ammonium sulfate to induce hydrophobic interactions.
  • the C4 HLD monolith was washed with 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate.
  • the bound ceDNA was eluted from the column by a step-wise reverse salt gradient of ammonium sulfate (3 M, 1.74 M, 0.9 M, 0.45 M, 0.24 M, 0 M) in 50 mM Tris pH 8.0, 10 mM EDTA.
  • the Sartobind® Q-eluted ceDNA was first supplemented to 1.5 M ammonium sulfate.
  • the unbound ceDNA product was recovered from the column in the flow-through pool using 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate. Some additional ceDNA product was removed during the wash step using 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate.
  • the Sartobind® Q-eluted ceDNA was first supplemented to 3 M ammonium sulfate. Following sample loading, the POROSTM HIC resin was washed with 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate. The bound ceDNA was eluted from the column using a reverse linear salt gradient of 3-0 M ammonium sulfate in 50 mM Tris pH 8.0, 10 mM EDTA.
  • the Sartobind® Q-eluted ceDNA was first supplemented to 1.8 M AS.
  • the unbound ceDNA was recovered from the column in the flow-through pool using 50 mM Tris pH 8.0, 10 mM EDTA, 0.9 M NaCl, 1.8 M ammonium sulfate.
  • Some additional ceDNA product was recovered during the wash step using 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate.
  • CaptoTM Core-based shell multimodal resin-based purification was implemented to remove trace amounts of nucleic acid fragments from the ceDNA product.
  • CaptoTM Core can be employed as either a secondary polishing chromatography column subsequent to the Sartobind® Q capture step, or it can alternatively serve as a tertiary polishing column following the second column polishing step involving Hydrophobic Interaction Chromatography (HIC).
  • HIC Hydrophobic Interaction Chromatography
  • the ceDNA product obtained from the HIC step was directly loaded onto the CaptoTM Core resin under conditions characterized by ammonium sulfate concentrations ranging from 1.7 M to 2 M in 50 mM Tris (pH 8.0) and 10 mM EDTA. Subsequently, the resin was subjected to a washing step using conditions identical to the loading condition, namely 1.7-2 M ammonium sulfate in 50 mM Tris (pH 8.0) and 10 mM EDTA.
  • the Sartobind® Q eluate in 50 mM Tris pH 8.0, 10 mM EDTA, 0.9 M NaCl composition or the HIC polishing product in 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate composition was filtered through a 0.001 m 2 Planova® 35N viral filter (Asahi Kasei Bioprocess) in dead-end filtration mode. The filtration was performed under constant operating pressure of 12 psi with a target throughput of 50 L/m 2 .
  • a post-recovery flush of 10 L/m 2 was performed with the ceDNA product composition buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 0.9 M NaCl, or 50 mM Tris (pH 8.0), 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate).
  • the ceDNA product composition buffer 50 mM Tris (pH 8.0), 10 mM EDTA, 0.9 M NaCl, or 50 mM Tris (pH 8.0), 10 mM EDTA, 0.45 M NaCl, 1.5 M ammonium sulfate).
  • the purified ceDNA composition was passed through two stages of TFF, both with a MWCO of 10 kDa. Both TFF stages were operated at a constant TMP of 8 psi, cross flow flux of 240 LMH; Stage 1 had a throughput of 20-30 L/m 2 and Stage 2 had a throughput of 40 L/m 2 . Stage 1 had an ultrafiltration step to concentrate the ceDNA product 10-20 times, followed by a diafiltration step where buffer was exchanged ten times. This tangential flow filtration step exchanged the ceDNA product buffer into 10 mM Tris (pH 8.0), 1 mM EDTA. Stage 2 only had an ultrafiltration step to further concentrate ceDNA product 10 times to the desired concentration of 2 mg/mL.
  • the “elution” sample contained predominantly ceDNA, with about 2500-fold reduction in the red fluorescent protein, a marker protein expressed by the ceDNA-producing cells.
  • the principle of IEX UPLC is to use the difference in charge property of molecules to achieve the separation.
  • the IEX analysis here combines different wavelength of the detectors, UV and fluorescence. The above results show that IEX chromatography is a sensitive and convenient in-process method to monitor the contents of impurities and ceDNA during ceDNA purification.

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Abstract

La présente invention concerne un procédé efficace et évolutif de purification d'ADN à extrémité fermée (ceDNA) à usage thérapeutique.
PCT/IB2024/061963 2023-11-30 2024-11-27 Purification de molécules d'adn à extrémité fermée Pending WO2025114932A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019051255A1 (fr) * 2017-09-08 2019-03-14 Generation Bio Co. Adn à extrémité fermée (cedna) modifié
WO2020154645A1 (fr) * 2019-01-24 2020-07-30 Generation Bio Co. Adn à extrémité fermée (cedna) et utilisation dans des procédés de réduction de la réponse immunitaire liée à une thérapie génique ou à acide nucléique
WO2020168222A1 (fr) * 2019-02-15 2020-08-20 Generation Bio Co. Modulation de l'activité de la protéine rep dans la production d'adn à extrémité fermée
WO2020181182A1 (fr) * 2019-03-06 2020-09-10 Generation Bio Co. Adn à extrémité fermée (cedna) et composés de modulation immunitaire
CN111886343A (zh) * 2018-02-22 2020-11-03 世代生物公司 使用闭合端dna（cedna）载体控制转基因的表达

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Publication number Priority date Publication date Assignee Title
WO2019051255A1 (fr) * 2017-09-08 2019-03-14 Generation Bio Co. Adn à extrémité fermée (cedna) modifié
CN111886343A (zh) * 2018-02-22 2020-11-03 世代生物公司 使用闭合端dna（cedna）载体控制转基因的表达
WO2020154645A1 (fr) * 2019-01-24 2020-07-30 Generation Bio Co. Adn à extrémité fermée (cedna) et utilisation dans des procédés de réduction de la réponse immunitaire liée à une thérapie génique ou à acide nucléique
WO2020168222A1 (fr) * 2019-02-15 2020-08-20 Generation Bio Co. Modulation de l'activité de la protéine rep dans la production d'adn à extrémité fermée
WO2020181182A1 (fr) * 2019-03-06 2020-09-10 Generation Bio Co. Adn à extrémité fermée (cedna) et composés de modulation immunitaire

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