Purification of closed DNA molecules

Abstract

The present disclosure provides an efficient and scalable method for purifying closed DNA (ceDNA) for therapeutic use.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to EP Application No. 23307105.9, filed November 30, 2023; U.S. Patent Application No. 63 / 654,761, filed May 31, 2024; U.S. Patent Application No. 63 / 685,618, filed August 21, 2024; and U.S. Patent Application No. 63 / 711,988, filed October 25, 2024. The disclosures of the aforementioned priority applications are incorporated herein by reference in their entirety. Background Technology

[0003] Gene therapy is a cutting-edge technology for treating diseases caused by dysfunction of gene expression. Some approaches to gene therapy involve the delivery of therapeutic genes encoding proteins that are deficient in patients. Viral vectors are commonly used for this delivery. However, viral vectors often have limitations on the delivery capacity. For example, adeno-associated virus (AAV) vectors typically deliver targets that are approximately 5 kb or smaller in size. Additionally, viral vectors containing viral proteins, for example in the form of a viral capsid, can induce an immune response to the vector in patients, which may limit the possibility of re-administering gene therapy.

[0004] Closed DNA (ceDNA) overcomes the limitations of virus-based gene delivery. ceDNA is linear double-stranded DNA (dsDNA) and is more stable than conventional dsDNA and RNA due to its stronger resistance to nucleases. Furthermore, ceDNA encapsulated in lipid nanoparticles (LNPs) exhibits many advantages over existing viral gene delivery systems. Unlike viral capsids, LNPs generally do not encounter existing antibodies against them in patients, which increases the likelihood of re-administration of ceDNA-LNP therapy. Additionally, ceDNA-LNPs possess a much larger genetic capacity and can accommodate genetic material exceeding 10 kb, thereby expanding the range of genetic diseases that can be treated with gene therapy. Moreover, the stability and self-replication of ceDNA will provide a single ceDNA dose sufficient for a much larger therapeutic window than existing AAV-based therapies.

[0005] Although ceDNA is a promising tool for gene therapy, its production remains a challenge. ceDNA is prone to cutting, nicking, and folding. These issues hinder large-scale production. Therefore, efficient and scalable means are still needed to produce and purify large quantities of ceDNA molecules for clinical use.

[0006] The present disclosure provides a method for obtaining a purified preparation of ceDNA from closed DNA (ceDNA)-producing cells. In one embodiment, the method comprises the step of incubating the cells in an alkaline buffer to lyse the cells and produce a cell lysate, wherein the alkaline buffer does not contain detergent and has a pH of 10 or higher; and the step of isolating ceDNA from the lysate. In some embodiments, the method comprises the step of neutralizing the cell lysate with an acidic salt prior to the isolation step. In some embodiments, the method further comprises the step of pre-clarifying the neutralized cell lysate by adding sodium bicarbonate and separating the resulting aggregate from the cell lysate prior to the isolation step to produce a pre-clarified cell lysate.

[0007] In another aspect, the present disclosure provides a method for obtaining a purified preparation of ceDNA from closed DNA (ceDNA)-producing cells, comprising the steps of: obtaining a lysate of said cells; pre-clarifying said cell lysate by adding sodium bicarbonate and separating the resulting aggregate from said cell lysate to produce a pre-clarified cell lysate; and isolating ceDNA from said lysate. In some embodiments, said lysate is obtained by lysing said cells by incubating said cells in an alkaline buffer, said alkaline buffer being detergent-free and having a pH of 10 or higher. In further embodiments, said cell lysate is neutralized with an acidic salt prior to said pre-clarification step.

[0008] In the present method, the isolation step can be performed, for example, by anion exchange chromatography.

[0009] In some embodiments, the method of the present disclosure further comprises, prior to the isolation step, a step of removing RNA by calcium chloride precipitation, optionally said removal of RNA is performed by: filtering the pre-clarified cell lysate to produce a clarified cell lysate, and treating the clarified cell lysate with calcium chloride precipitation to remove RNA; in additional embodiments, said lysate is subjected to ultrafiltration before the addition of calcium chloride, and / or said lysate is subjected to ultrafiltration and volume filtration after calcium chloride precipitation to reduce the calcium chloride concentration. In other embodiments, the method of the present disclosure further comprises, after the isolation step, a step of removing RNA by calcium chloride precipitation; in additional embodiments, said nucleic acid preparation is subjected to volume filtration after calcium chloride precipitation to reduce the calcium chloride concentration. Calcium chloride may be added to achieve, for example, a concentration of about 1 to 3 M, optionally about 2 M.

[0010] In some embodiments of the present method, the isolated ceDNA is polished (further purified, e.g., after RNA removal). The polishing step may be performed using one or both of (i) hydrophobic interaction chromatography (HIC) (optional, the HIC is performed with a monolithic or perfusive resin); or (ii) a multimode core-shell resin (optional, the multimode core-shell resin comprises resin beads having a size-exclusion sheath, and optionally, the size-exclusion sheath has a molecular weight cutoff (MWCO) of 400 or 700 kDa).

[0011] In some embodiments of the present method, the isolated ceDNA preparation is subjected to viral filtration using, for example, a 35 nm filter.

[0012] In some embodiments, the alkaline buffer for cell lysis contains sodium hydroxide at a final concentration of about 100 to 300 mM, optionally about 150 mM, after optionally being added to the cells. In some embodiments, the incubation step for cell lysis does not last longer than 5 minutes, for example, about 2.5 minutes or about 3.5 minutes. In some embodiments, the incubation step for cell lysis is performed in a continuous inline system.

[0013] In some embodiments, the acidic salt for neutralizing the cell lysate is, for example, potassium acetate of about 2.5 to 3.5 M (e.g., about 3.1 M).

[0014] In some embodiments, sodium bicarbonate is added during the pre-clarification step to a concentration of about 5 to about 50 (e.g., about 10) g / L. In some embodiments, aggregates are removed by a filter having a pore size of about 7.5 to 60 μm. In some embodiments, the incubation time for pre-clarification is about 2 hours.

[0015] In some embodiments, the isolated ceDNA undergoes tangential flow filtration, for example, with an MWCO of 10 and / or 100 kDa.

[0016] In some embodiments, the ceDNA-producing cell is an insect cell infected with a recombinant baculovirus expression vector. In some embodiments, the recombinant baculovirus expression vector comprises a heterologous nucleic acid sequence containing a transplanted gene flanked by an inversion terminal repeat (ITR). In some embodiments, the ITR is a parvovirus ITR (e.g., an ITR of AAV origin such as AAV2). In some embodiments, the heterologous nucleic acid encodes a therapeutic protein. In some embodiments, the ceDNA-producing cell is a transplanted insect cell containing a sequence encoding for ceDNA in its genome.

[0017] In some embodiments, the method for producing ceDNA according to the present specification includes the step of monitoring ceDNA purity during the process by measuring the levels of ceDNA and impurities through ion-exchange ultra-high performance liquid chromatography in a sample taken before, during, or after the isolation step.

[0018] In addition, the ceDNA preparation obtained by the present method and the use of ceDNA for therapeutic purposes are provided in this specification.

[0019] Other features, objects, and advantages of the present invention are clearly evident from the following detailed description. However, it should be understood that the detailed description illustrates embodiments and aspects of the invention, but is provided only as an example and not as a limitation. Various modifications and variations within the scope of the invention will be apparent to those skilled in the art from the detailed description. Brief explanation of the drawing

[0020] Fig. 1 This is a diagram illustrating a ceDNA molecule containing a sequence derived from an inversion terminal repeat (ITR) of a parvovirus (e.g., AAV). Fig. 2 This is a diagram illustrating three insect cell systems for producing ceDNA containing parvovirus ITR. In the diagram, the illustrated transplant gene encodes coagulation factor VIII. Other transplant genes that do not encode factor VIII can also be incorporated into cells in the same manner. "One-Bac": A system using a single baculovirus vector. "Two-Bac": A system using two baculovirus vectors. "PCL": A producer cell line containing a stably incorporated copy of a transplant gene cassette (e.g., an FVIII expression cassette containing parvovirus ITR). Fig. 3This 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 performed using ceDNA samples treated with or untreated with T5 exonuclease, which lyses nicked ceDNA and does not affect unnicked ceDNA. The lysis method was performed using a lysis buffer containing 66.7 mM NaOH and 0.33% SDS or a lysis buffer containing 150 mM NaOH. Fig. 4 is This is a bar graph comparing the integrity of ceDNA obtained from two lysis methods. Concentration measurement of electrophoretic agarose gel images ( Fig. 3 Comparisons were performed using both (as shown in [figure]) and quantitative PCR. Left bar of each group in pairs: ceDNA-producing Sf9 cells were resuspended in PBS. Right bar of each group in pairs: ceDNA-producing Sf9 cells were resuspended in a buffer containing 100 mM Tris and 10 mM EDTA. Figures 5a and 5b is an agarose gel electrophoresis image showing the integrity of ceDNA isolated from ceDNA-producing insect (Sf9) cells lysed under the indicated lysis conditions (in the presence or absence of T5 exonuclease). Fig. 6a is an agarose gel electrophoresis image showing the integrity of ceDNA isolated from ceDNA-producing insect (Sf9) cells dissolved in 150 mM NaOH for the indicated lysis retention times (2.5 min, 5 min, 7.5 min, or 15 min) (in the presence or absence of T5 exonuclease). Fig. 6b This is a bar graph showing the titer of ceDNA (left bar), the titer of bacDNA (middle bar), the titer of sf9DNA (right bar), and the purity (%) of ceDNA isolated from ceDNA-producing insect (Sf9) cells dissolved in 150 mM NaOH for the indicated lysis holding times (2.5 min, 5 min, 7.5 min, or 15 min). Fig. 7 This is a diagram showing a continuous inline system for the dissolution and neutralization of ceDNA-expressing cell paste. Fig. 8 This is an agarose gel electrophoresis image showing the integrity of ceDNA isolated from ceDNA-producing insect (Sf9) cells dissolved in 150 mM NaOH in a continuous inline system under the indicated conditions (in the presence or absence of T5 exonuclease). A control was performed in batch mode using 150 mM NaOH lysis buffer with a retention time of 5 minutes. Fig. 9 This is an agarose gel electrophoresis image comparing the effects of ammonium bicarbonate (AHC) and sodium bicarbonate (NaHC) as pre-clarifying salts on the integrity of ceDNA after T5 exonuclease treatment. Fig. 10a This is a diagram showing a pre-clarification process using sodium bicarbonate (NaHC). Fig. 10b This is a pair of photos showing the separation of aggregates after 5 minutes and 2 hours after the addition of 20 g / L, 15 g / L, 10 g / L, and 5 g / L NaHC (from left to right in each photo). Fig. 11 This is a bar graph comparing the recovery rate, throughput, and turbidity for various filters used in the cell lysate clarification step. For each group of two, the left bar represents the recovery rate %, and the right bar represents the throughput %. Turbidity values ​​are linked to the bars via lines. Fig. 12 This is a pair of graphs to evaluate the effect of feed flow rate using NaHC-treated neutralized cell lysate on filter throughput. Fig. 13a This is a diagram showing the study design to evaluate the introduction of calcium chloride precipitate into the ceDNA purification process to remove residual RNA (rRNA). UF: Ultrafiltration. DF: Normal filtration. Fig. 13b Is Fig. 13aThis is an agarose gel electrophoresis image showing the yield and purity (%) of ceDNA isolated according to several different calcium chloride precipitation methods as described in [link]. Fig. 13c Is Fig. 13a This is a bar graph comparing the ceDNA yield (left bar) and rRNA impurities (right bar) of the four different rRNA removal processes shown in Figure 1. The control in this experiment has no CaCl2 treatment. Fig. 14a This is a chromatography showing the elution peak of the ceDNA product after applying it to a Sartobind® Q column with various concentrations of NaCl added to the loading. Fig. 14b Is Fig. 14a This is an agarose gel electrophoresis image of the elution fraction obtained from the process. Fig. 15 This is a chromatography showing the elution peak of the ceDNA product after application to a Sartobind® Q column. Fig. 16a This is a chromatography showing the stepwise elution profile of the ceDNA product after application to a C4 HLD monolithic column. Fig. 16b Is Fig. 16a This is an agarose gel electrophoresis image of the elution fraction obtained from the process. Fig. 17a This is a chromatography showing the elution profile of the ceDNA product after application to a C4 HLD monolith using a reverse ammonium sulfate step gradient operating as a binding-elution approach. Fig. 17b Is Fig. 17a This is an agarose gel electrophoresis image of the elution fraction obtained from the process. Fig. 18a This is a chromatography showing the elution profile of the ceDNA product after application to a C4 HLD monolith operating in perfusion mode. Fig. 18b Is Fig. 18a This is an agarose gel electrophoresis image of the elution fraction obtained from the process. Fig. 19aThis is a bar graph showing the recovery rate (%) and purity (%) of ceDNA products recovered from several different hydrophobic interaction chromatography (HIC) media. "HIC Loading": qPCR purity %. "Poros Benzyl Ultra": ceSDS purity %. "Poros Ethyl", "Poros Benzyl", and "HIC Monolith": From left to right, the bars represent the recovery rate %, qPCR purity %, and ceSDS purity %, respectively. Fig. 19b Is Fig. 19a This is an agarose gel electrophoresis image of the elution fraction obtained from the process. Fig. 20 This is a diagram showing the study design for using the POROS™ Benzyl Ultra HIC column in binding-elution mode or perfusion mode. Fig. 21a Is Fig. 20 This is a set of chromatography showing the elution profile of the ceDNA product after polishing using a POROS™ Benzyl Ultra HIC column in binding-elution mode or perfusion mode as described in [link]. Fig. 21b Is Fig. 21a This is an agarose gel electrophoresis image of the elution fraction obtained from the process. Fig. 22a This is a chromatography showing the elution profile of the ceDNA product after polishing using Capto™ Core 400 or Capto™ Core 700 core shell resin. Fig. 22b Is Fig. 22a This is an agarose gel electrophoresis image of the filtrate obtained by applying the ceDNA product to the core-shell resin as shown. Fig. 23 This is an agarose gel electrophoresis image of the filtrate obtained by applying purified ceDNA to a Planova™ 35N virus removal filter. Fig. 24 This is an agarose gel electrophoresis image comparing 10 kDa and 30 kDa molecular weight cutoff (MWCO) tangential flow filtration (TFF) cassettes used to concentrate ceDNA material containing approximately 1.5–1.7 M ammonium sulfate. Fig. 25This is a schematic diagram showing the "3-column" ceDNA purification process. Fig. 26 This is a panel of agarose gel electrophoresis images and a table showing the ce-SDS lab chip and agarose gel electrophoresis results of the eluent, product, strip, wash, and regeneration fractions from all column chromatography runs in the 3-column purification approach. The product fraction from each polishing column run is indicated by an asterisk. Fig. 27 This is a schematic diagram showing two polishing strategies with different orderings of Capto™ core-shell-based adsorbents and HIC adsorbents. Fig. 28 This is an agarose gel and table showing the presence and intensity of ceDNA product bands and impurities based on agarose gel concentration measurement or next-generation sequencing short sequence fragment sequencing analysis. Fig. 29 This is an agarose gel and table showing the purity of the ceDNA product using a combination of lab chip, agarose gel concentration measurement, and next-generation sequencing short sequence fragment sequencing analysis. Fig. 30 is an overlaid ion exchange (IEX) chromatogram of four samples ("Loading", "FT", "Wash", and "Elution") taken from the Sartobind® Q ceDNA purification step. rHCP: Residual host cell protein. RFP: Red fluorescent protein (introduced cell marker). rRNA: Residual RNA. FT: Perfusion. Specific details for implementing the invention

[0021] The present disclosure provides a scalable and robust manufacturing process for purifying ceDNA from eukaryotic cells (e.g., insect cells). The ceDNA may contain a sequence of interest (e.g., a sequence encoding a therapeutic protein). Once purified, the ceDNA may be encapsulated in lipid nanoparticles for delivery to a patient. The ceDNA formulation of the present disclosure is expected to have an improved safety profile, such as reduced amounts of nucleic acid impurities like open double-stranded DNA and less anti-drug immune response when delivered to a patient.

[0022] The present disclosure is based on findings related to the purification of ceDNA from eukaryotic cells, such as insect cells. Generally, the purification process comprises: (i) harvesting producer cells and resuspending them to form a cell paste, wherein optionally the cells are harvested by continuous centrifugation; (ii) lysing the cells for a short time in an alkaline buffer containing, for example, sodium hydroxide, without containing detergent (e.g., SDS), and neutralizing the cell lysate with an acidic salt (e.g., potassium acetate), wherein a continuous inline system is used for lysis and neutralization; (iii) chemically pre-clarifying the neutralized cell lysate by adding it to a salt containing, for example, sodium bicarbonate, without containing ammonium salts (e.g., ammonium bicarbonate), and removing aggregates by filtration; (iv) removing RNA by salt precipitation (e.g., calcium chloride precipitation) instead of using RNase; (v) a step of purifying ceDNA from a lysate through anion exchange capture membrane chromatography and more precise polishing chromatography, such as hydrophobic interaction chromatography (e.g., using a resin or monolithic column such as perfusion resin) and / or a mixed mode such as a core-shell resin; and (vi) additional downstream steps such as viral filtration and tangential flow filtration and volumetric filtration. The present method reduces DNA nicking and fragmentation, reduces nucleic acid and host cell protein impurities, and is suitable for environmentally friendly large-scale manufacturing.

[0023] I. ceDNA generation in host cells

[0024] ceDNA may be characterized by having no exposed ends and including loop structures at the ends. In some embodiments, ceDNA has covalently connected ends. That is, the 5' end of the sense strand is covalently connected to the 3' end of the antisense strand, and the 3' end of the sense strand is covalently connected to the 5' end of the antisense strand. In other embodiments, ceDNA includes self-annealed loop structures at both ends of both strands.

[0025] In some embodiments, ceDNA contains a virus-derived inverted terminal repeat (ITR) sequence, and each end of the DNA strand is self-annealed into a hairpin-like structure ( Fig. 1 ). In some embodiments, the ITR sequence is derived from parvoviruses such as adeno-associated virus (AAV) and bocavirus. The ITR sequence may be a wild-type viral sequence or may contain mutations relative to the wild-type viral sequence. In certain embodiments, the ITR sequence may be derived from AAV2. The ITR sequence flanks a target sequence, such as an expression cassette for a transplanted gene expression, e.g., an expression cassette for a therapeutic protein (e.g., enzyme, antibody, cell surface receptor, transcription factor, hormone, or cytokine). The expression cassette may include a promoter (e.g., homeostatic or inducible) and other regulatory elements (e.g., enhancer, insulator, polyadenylation site, etc.) to induce expression of the coding sequence in a host cell. The promoter may be a multifunctional promoter active in multiple tissues or a tissue-specific promoter. For example, promoters can be specific to cells of the liver, lungs, muscles, peripheral or central nervous system, cardiovascular system, ocular system, or immune system.

[0026] ceDNA, such as ceDNA containing viral ITR, can be generated in recombinant eukaryotic host cells. In some embodiments, ceDNA can be generated in mammalian host cells such as HEK293 cells, HeLa cells, and CHO cells. In other embodiments, ceDNA can be generated in non-mammalian host cells such as insect cells. In further embodiments, ceDNA can be generated in insect cells such as Sf21, Sf9, S2, Tni-Hi5, Super9, and ExpresSF+. In specific embodiments, Spodoptera Prugiferda Insect cells such as Sf21 and Sf9 cells derived from are free of rhabdovirus (Sf-rabidovirus-negative).

[0027] In some embodiments, ceDNA may be generated in a stable cell line (e.g., mammalian or insect cell) engineered to contain a copy of a transplanted gene expression cassette flanked by a parvovirus ITR. Rep The ITR-specific replicase protein encoded by the gene recognizes the ITR sequence at the terminal degradation site (TRS) and produces a copy of ceDNA containing the transplanted gene in the production cell. Rep Genes can be stably integrated into the genome of host cells, or, in the case of insect-producing cells, can be transiently expressed from episomal vectors such as baculovirus vectors.

[0028] In some embodiments, the genetically modified host cell lines are derived from insect cells such as Sf21, Sf9, S2, Tni-Hi5, Super9, and ExpresSF+ parvovirus (e.g., AAV). RepGenes can be transiently introduced into insect cells via baculovirus vectors. In a specific embodiment, the template for ceDNA is performed in the same baculovirus vector ("one-bag" system). In another embodiment, the template for ceDNA is performed in separate baculovirus vectors ("two-bag" system). In the "two-bag" system, the two baculovirus vectors can be introduced into insect host cells simultaneously or sequentially. In another system, a production cell line (PCL) is established with a stably integrated copy of the ceDNA template, and Rep A baculovirus vector carrying a gene is transiently introduced into a cell line. These three exemplary systems for producing ceDNA in insect cells are Fig. 2 It is depicted in.

[0029] The ceDNA purification process provided herein can be implemented across various scales, including shaking flasks, mini bioreactors (e.g., 100 to 250 mL), benchtop bioreactors (e.g., 50 L), and large bioreactors (e.g., 500 L, 1000 L, and 10,000 L).

[0030] II. ceDNA purification from host cells

[0031] In the aforementioned ceDNA production system, the host cell genome and baculovirus vector DNA pose significant challenges in the downstream purification of transplant gene-specific full-length ceDNA. This production system also contains intermediate copies of ceDNA that are not full-length and potentially interfere with the purification of transplant gene-specific full-length ceDNA. Furthermore, this process is complicated by the presence of RNA impurities, including those derived from baculoviruses and viruses endemic to the cell line (e.g., rhabdoviruses). The present disclosure provides an improved method for purifying ceDNA from production host cells, which is efficient and scalable for commercial production. The steps of this method are described in detail below.

[0032] A. Cell Harvesting and Re-suspending

[0033] In some embodiments, ceDNA-producing cells are harvested by centrifugation, e.g., continuous flow centrifugation. In continuous flow centrifugation, a large amount of material is centrifuged at a high centrifugal force while the supernatant is extracted through an discharge line. For example, while maintaining a predetermined centrifugation speed, a certain amount of cell-containing culture medium is collected in a vessel, and the supernatant continuously flows out of the vessel and into a collection vessel. Once a certain amount is pumped into the vessel, the supernatant is discarded, and the concentrated cells are distributed through a collection line for further processing. The number of cycles is determined according to a pre-established cell concentration factor required for the process.

[0034] Next, the concentrated cells can be resuspended in a buffer to produce a cell paste. Various resuspension buffers may be used. The pH range of the buffer may be about 6.5 to about 8.5. The buffer may contain sodium salts, potassium salts, and / or buffers.

[0035] In some embodiments, the buffer contains Tris and EDTA, and a polyol (e.g., sucrose) may be used. For example, the buffer contains 100 mM Tris, 10 mM EDTA, and 50 mM sucrose (pH 8).

[0036] In some embodiments, the buffer is phosphate-buffered saline (PBS). For example, the PBS may contain about 100–150 mM NaCl, about 1.5–3.0 mM KCl, and about 10–15 mM phosphate (pH 7.4). In further embodiments, 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).

[0037] B. Dissolution and Neutralization of Cell Paste

[0038] There are various methods for lysing eukaryotic cells. In this process, the cell paste is dissolved in an alkaline buffer that contains no detergent, such as SDS, or contains a very low concentration of detergent (e.g., SDS at a concentration of 0.1% or less). The inventors have discovered that if detergent is present in the lysis buffer, nicking of ceDNA may occur by breaking phosphodiester bonds.

[0039] In some embodiments, the lysis buffer comprises an alkaline agent such as NaOH. In certain embodiments, 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. In some embodiments, the lysis buffer comprises NaOH at a stock concentration of about 300 mM. When the lysis buffer is added to the cells, the effective concentration of NaOH decreases. In some embodiments, the effective NaOH concentration of the lysis buffer in the cell mixture is about 150 mM.

[0040] In some embodiments, the alkaline dissolution buffer has a pH of about 9 to 14. For example, the dissolution buffer has 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. In an additional embodiment, the dissolution buffer contains a pH of about 12.5 or higher.

[0041] A lysis buffer is applied to the cell paste for a predetermined time. This time is also referred to herein as the “lysis retention time.” In some embodiments, the lysis retention time is within 10 minutes, e.g., within 5 minutes. As the lysis retention time increases, the time of exposure to a high pH increases, and consequently, irreversible denaturation of the ceDNA product occurs. In some embodiments, the lysis retention 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. In an additional embodiment, the cells are lysed in an alkaline lysis buffer containing an NaOH concentration of about 300 mM and a pH of about 12.5 or higher, and when the cell solution is mixed with the lysis buffer in equal volume, the effective NaOH concentration is reduced by half to about 150 mM, and the lysis retention time is about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, or about 5 minutes.

[0042] After alkaline dissolution, the solution is neutralized to return the pH of the solution to acidic conditions (e.g., about 5.0 to 6.5, or about 5.5 to 6.0). In some embodiments, the solution is neutralized with an acidic salt such as potassium acetate. In further embodiments, the alkaline solution is neutralized with potassium acetate by mixing it with a potassium acetate solution of about 0.5 M to about 5 M. For example, the acidic salt such as potassium acetate may be provided at concentrations 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. In some embodiments, potassium acetate is provided at a concentration of about 3.0 M (e.g., 3.1 M), and the pH is about 5.2.

[0043] To control the lysis retention time more accurately, cells can be lysed through a continuous inline lysis operation. Fig. 7The figure illustrates this operation. In this operation, concentrated cells (cell paste) and alkaline lysis buffer are fed continuously through the device at a predetermined rate and volume ratio (e.g., 1:1 volume ratio) by controlling the pump speed for each buffer / lysate being processed. The cells pass through a tube where lysis takes place. The lysis holding time is determined by the length of the tube and the feeding rate. At the end of the lysis tube, the mixture is then mixed with a continuous stream of neutralization buffer, and the neutralized cell lysate continues into a collection tank. In some embodiments, continuous cell lysis can be used to process more than 10 L of cell paste with a lysis holding time of about 2.5 minutes (e.g., using a static mixer). A slow mixing rate is preferred to reduce shearing of ceDNA and damage to genomic DNA or viral DNA during the lysis process.

[0044] C. Chemical pre-clarification of neutralized cell lysates

[0045] Once the cell lysate is neutralized, it can be pre-clarified, for example, chemically, to remove impurities such as high molecular weight (HMW) genomic DNA, host cell proteins (HCP), and other cellular components. The inventors have found that chemical pre-clarification yields superior results compared to physical pre-clarification (e.g., by batch or continuous centrifugation).

[0046] In some embodiments, neutralized cell lysates are treated with a pre-clarification salt. Typically, this operation is performed with ammonium bicarbonate. However, ammonium bicarbonate generates harmful ammonia gas and can pose an environmental risk. The inventors have discovered that sodium bicarbonate is not only environmentally friendly but also produces satisfactory pre-clarification results.

[0047] In some embodiments, the pre-clarifying salt is sodium bicarbonate and may be added to a concentration of about 5 to 50 g / L. For example, 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. In a preferred embodiment, the pre-clarifying salt is present at a concentration of 10 g / L.

[0048] In some embodiments, the neutralized cell lysate is treated with a pre-clarifying salt for a predetermined time. In some embodiments, the neutralized cell lysate is treated with a pre-clarifying salt for about 0.5 to 8 hours. For example, the neutralized cell lysate may be treated with a 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. In additional embodiments, the neutralized cell lysate may be treated with a 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 gentle stirring for efficient solubilization.

[0049] D. Clarification of pre-clarified and neutralized cell lysates

[0050] Pre-clarified and neutralized cell lysates can be clarified via deep filtration to remove cell debris and particles from the feed stream. The clarification filter can be selected based on cell density, cell type, and harvest viscosity. Deep filtration can be performed using, for example, polypropylene filters, cellulose filters, silica filters, polyacrylic filters, or mixed material filters. In some embodiments, the filter may have a pore size of 0.6 to 60 μm, for example, 7.5 to 60 μm, or 0.6 to 8.0 μm. Examples of suitable deep filtration systems are Clarisolve® 60HX and D0HC filters (Millipore).

[0051] E. Removal of RNA

[0052] A conventional method for removing RNA impurities from DNA products uses RNase. However, RNase is typically obtained from animals, which not only increases costs but also raises safety concerns regarding therapeutic products. The inventors have discovered that calcium chloride achieves excellent results in removing RNA, as well as other impurities such as genomic DNA fragments, baculovirus DNA, and HCP, from cell lysates.

[0053] In some embodiments, the clarified cell lysate is treated with CaCl2, and the calcium chloride salt is present at a concentration of about 0.5 to 10.0 M. For example, 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. In a preferred embodiment, the salt may be present at a concentration of about 2 M.

[0054] In some embodiments, the clarified cell lysate undergoes ultrafiltration (e.g., TFF) before calcium chloride treatment to concentrate the lysate. For example, the clarified cell lysate is concentrated by about 2 to 10 times, e.g., about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times. After calcium precipitation, the lysate may undergo ultrafiltration and volume filtration in the presence of a buffer suitable for subsequent chromatography. For example, volume filtration may be performed in the presence of a buffer (pH about 8) containing Tris, EDTA, and sodium chloride (e.g., 50 mM Tris, 10 mM EDTA, and 0.3 M NaCl).

[0055] In some embodiments, the clarified cell lysate is treated with calcium chloride to remove RNA before isolating the ceDNA product from the lysate. In some embodiments, the step of removing RNA from the clarified lysate through calcium chloride treatment is performed after ceDNA is isolated from the cell lysate (e.g., via anion exchange) before polishing the ceDNA product. If calcium chloride precipitation is implemented after the capture / isolation step, it may not be necessary to filter the material, and the capture eluent (isolation step product) may undergo volume filtration after calcium chloride precipitation and before polishing.

[0056] Isolation of F. ceDNA products

[0057] The ceDNA product can be isolated from the cell lysate by chromatography. In some embodiments, the method comprises one or more, two or more, or three or more chromatographic steps of the same or different chromatographic modes.

[0058] In some embodiments, the clarified cell lysate is treated with a loading control salt to remove impurities during the chromatography process. In some embodiments, the loading control salt is NaCl. In some embodiments, the loading control salt is present in the cell lysate loading at a concentration of about 50 to 500 mM. For example, the clarified cell lysate loading 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. In additional embodiments, the clarified cell lysate loading may contain NaCl at a concentration of about 300 mM.

[0059] In some embodiments, ceDNA is isolated by anion exchange chromatography. A suitable anion exchange chromatography system is a strongly basic anion ligand exchanger (R-CH2-N) containing a quaternary ammonium. + A weakly basic anion ligand exchange group (R-C2H4-N) comprising (CH3)3) or diethylaminoethyl (DEAE) +It includes H(C2H5)2). For example, the anion exchange chromatography system may be the Sartobind® Q system (Sartorius), Natrix® HD Q (Millipore Sigma), CIMmultus® DEAE (Sartorius), CIMmultus® Q (Sartorius), Mustang™ Q (Pall), ReadytoProcess Adsorber Q (Cytiva), POROS™ 50D (Thermo Fisher), POROS™ 50HQ (Thermo Fisher), Sartorius STIC® PA (Sartorius), or an equivalent system. In some embodiments, the capture chromatography medium is a membrane, monolith, or resin. In some embodiments, the anion exchange chromatography step comprises perfusion of impurities using a loading control of 50 to 500 mM (e.g., 300 mM) of NaCl, a washing step to further remove trace impurities, and subsequent elution of ceDNA using an elution buffer. In some embodiments, the wash buffer contains a salt such as NaCl at a concentration of about 0.15 to 0.8 M. For example, the wash buffer may contain 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. In additional embodiments, the wash buffer contains a salt such as NaCl at a concentration of 0.6 M. In some embodiments, the elution buffer contains a salt such as NaCl at a concentration of 0.5 to 3 M. For example, the elution buffer is approximately 0.5 M, approximately 0.6 M, approximately 0.7 M, approximately 0.8 M, approximately 0.9 M, approximately 1.0 M, approximately 1.1 M, approximately 1.2 M, approximately 1.3 M, approximately 1.4 M, approximately 1.5 M, approximately 1.6 M, approximately 1.7 M, approximately 1.8 M, approximately 1.9 M, approximately 2.0 M, approximately 2.1 M, and approximately 2.It may contain a salt such as NaCl at a concentration of 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. In a further embodiment, the elution buffer contains a salt such as NaCl present at a concentration of about 0.9 M. In some embodiments, the elution buffer contains other salts such as sodium sulfate, sodium acetate, or ammonium acetate.

[0060] Polishing of G. ceDNA products

[0061] In some embodiments, the isolated ceDNA preparation may be further polished by one or more additional chromatographic steps, for example, based on hydrophobic interaction chromatography (HIC). The HIC medium may be a monolithic column or a resin and may include additional operating modes such as multimode core-shell resin-based purification.

[0062] In some embodiments, the ceDNA preparation is treated with an HIC loading control salt before the HIC polishing step. For example, the HIC loading control salt is ammonium sulfate (AS). In some embodiments, the HIC loading control salt is present at a concentration of about 1 to 4 M. For example, the clarified cell lysate may be treated with a 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. In a preferred embodiment, the clarified cell lysate is treated with an HIC loading control salt present at a concentration of about 3.0 M.

[0063] In some embodiments, the HIC step comprises an elution step selected from a linear gradient, or the stepwise gradient may be operated in a combined-elution mode or a perfusion mode. In some embodiments, the linear gradient elution step comprises a reverse salt gradient containing AS with a linear salt concentration gradient of, for example, about 4 to 0 M or about 3 to 0 M.

[0064] In some embodiments, the stepwise gradient elution step includes a stepwise reverse salt gradient. In some embodiments, the stepwise reverse salt gradient includes AS at a concentration of about 10 to 0 M, for example, 2 to 0 M.

[0065] In some embodiments of the binding-elution step, 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 additional embodiments, the binding-elution salt may be present at a concentration of about 1.74 M.

[0066] In some embodiments of the perfusion mode, the AS salt in the loading may be present at a concentration of about 0.75 to 2.5 M. For example, the HIC perfusion purification step may be performed with AS loading concentrations of about 0.75 M, about 1 M, about 1.5 M, about 2 M, or about 2.5 M. In additional embodiments, the AS concentration in the loading is about 1.5 M.

[0067] In some embodiments, the HIC step includes a channel. In some embodiments, the size of the HIC channel is about 0.5 to 10 μm. For example, the HIC channel size may be about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 2.5 μm, about 3.0 μm, about 3.5 μm, about 4.0 μm, about 4.5 μm, about 5.0 μm, about 5.5 μm, about 6.0 μm, about 6.5 μm, about 7.0 μm, about 7.5 μm, about 8.0 μm, about 8.5 μm, about 9.0 μm, about 9.5 μm, or about 10.0 μm. In a preferred embodiment, the size of the HIC channel is about 2.0 μm. In a preferred embodiment, the size of the HIC channel is about 6.0 μm.

[0068] Examples of HIC systems include: C4 HLD monolithic resin (Sartorius), POROS™ ethyl perfusive resin (Thermo Scientific), POROS™ benzyl perfusive resin (Thermo Scientific), or POROS™ benzyl ultra perfusive resin (Thermo Scientific), Capto™ PlasmidSelect (Cytiva), Capto™ phenyl (Cytiva), or Sartobind® phenyl (Sartorius).

[0069] In some embodiments, the polishing step comprises a mixed-mode resin combining size exclusion chromatography and anionic and hydrophobic interaction chromatography properties. In some embodiments, the ceDNA preparation is treated with a loading control salt before application to a size exclusion column. In some embodiments, the loading control salt is NaCl. In some embodiments, the loading control salt is AS. In some embodiments, the loading control salt is present at a concentration of about 0.1 to 4.0 M. For example, the loading control salt may be present at concentrations 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. In additional embodiments, the loading control salt is NaCl present at a concentration of about 0.15 to 0.9 M. In other additional embodiments, the loading control salt is AS present at a concentration of about 1.5 to 2.5 M. Examples of size exclusion chromatography systems include Capto™ Core 700 and Capto™ Core 400 multimode resins (Cytiva) that operate by both size exclusion and HIC. In some embodiments, the size exclusion chromatography has an MWCO of 400 to 900 kDa, e.g., 750 kDa. In some embodiments, Capto™ Core 400 can be used for ceDNA products with a length of about 3 to 5 kb.

[0070] In some embodiments, the polishing step utilizes two different chromatographic methods. For example, ceDNA isolated by Sartobind® may pass through HIC (e.g., C4 HLD HIC monolith in bind-elution mode, C4 HLD HIC monolith in perfusion mode, POROS™ Benzyl Ultra HIC resin in bind-elution mode, or POROS™ Benzyl Ultra HIC resin in perfusion mode) and then through a multimode core-shell resin (e.g., Capto™ Core 400 or 700); or pass through in the reverse order.

[0071] H. Additional filtration step

[0072] In some embodiments, the ceDNA preparation undergoes viral filtration to remove any viral contaminants. In some embodiments, viral filtration is approximately 0.001 to 1.0 m 2 This is performed using a filter with a surface area of ​​. For example, the filter is approximately 0.001 m² 2 , approximately 0.01 m 2 , approximately 0.12 m 2 , about 0.3 m 2 , or about 1.0 m 2 It may exist on the surface of. In a preferred embodiment, the virus filtration has a surface area of ​​about 0.001 m² 2 This is performed using a filter. An example of a virus filtration system includes the Planova™ 35N virus removal filter (Asahi Kasei Bioprocess).

[0073] In some embodiments, the purified ceDNA preparation is concentrated by a tangential flow filtration (TFF) cassette. In some embodiments, the TFF cassette may contain MWCO of about 1 to 100 kDa. For example, the TFF cassette may contain 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. In further embodiments, the TFF contains MWCO of about 10 kDa.

[0074] In some embodiments, the ceDNA product is concentrated by about 2 to 20 times by TFF. For example, the ceDNA product may be concentrated by about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 times by a tangential flow filtration cassette. In a preferred embodiment, the purified ceDNA product is concentrated by about 10 times by a TFF cassette.

[0075] I. Monitoring during the purification process

[0076] The composition of intermediate products of ceDNA production can be monitored. For example, during the chromatographic purification of ceDNA (e.g., purification by Sartobind® Q chromatography), samples taken from different stages of the purification process, such as samples from the loading material, perfusion material, wash product, and elution product, can be analyzed to evaluate the amount of ceDNA and impurities (e.g., host cell proteins and DNA / RNA fragments) present in the samples.

[0077] In some embodiments, analysis can be performed via ion exchange (IEX) ultra-high performance liquid chromatography (UPLC) to separate proteins and nucleic acids according to their charges. The levels of various components in the analyzed sample can be determined by spectrophotometry at wavelengths of 254 nm (for nucleic acids) and 280 nm (for proteins). Non-limiting examples of IEX columns include those comprising non-porous particles (e.g., polymethacrylate particles) coated with a network of ion exchangers (e.g., sulfopropyl, carboxymethyl, and / or quaternary ammonium groups), such as Water™’s Protein-Pak Hi Res Q and Tosoh’s TSKgel DNA-STAT.

[0078] In some embodiments, the ceDNA-producing cell may include an externally introduced expression cassette for expressing a fluorescent protein (e.g., red, blue, yellow, green, or cyan fluorescent protein). In this case, fluorescence spectroscopy may be further used to monitor the amount of the fluorescent protein in the sample as an indicator of the presence of the host cell protein.

[0079] Unless otherwise defined in this specification, scientific and technical terms used in connection with this disclosure have the meanings generally understood by those skilled in the art. While exemplary methods and materials are described below, methods and materials similar or equivalent to those described in this specification may also be used to practice or test the disclosure. In the event of a conflict, this specification shall prevail, including definitions. Additionally, unless otherwise required by the context, singular terms include plural forms and plural terms include singular forms. Throughout this specification and embodiments, variations such as the words “have” and “include” or “having” and “include” are understood to mean the inclusion of the mentioned integer or group of integers, but not the exclusion of any other integer or group of integers. All disclosures and other references mentioned in this specification are incorporated by reference in their entirety, as specifically and individually indicated so that each individual reference is incorporated by reference in its entirety. Although numerous references are cited in this specification, such citations do not constitute an acknowledgment that any of these references form part of the ordinary general knowledge of the art. As used herein, the terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​similar to the mentioned reference value. In certain embodiments, unless otherwise stated or evident from the context, this term refers to a range of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or smaller) of the mentioned reference value.

[0080] Titles in this specification are provided for convenience of organization and are not intended to limit the scope of the claimed invention in any way.

[0081] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0082] Examples

[0083] The following examples describe experiments optimizing the purification procedure of ceDNA from production insect Sf9 cells. These procedures included the resuspension, lysis, neutralization, pre-clarification, clarification, column purification, and analysis of post-harvest production cells. The optimized procedures enable a robust and scalable manufacturing process. In these experiments, ceDNA was generated from Sf9 cells and contained the coding sequence for human factor VIII. The size of the ceDNA was 8.3 kb ( Fig. 1 ).

[0084] Example 1: Evaluation of Resuspended Buffer

[0085] ceDNA-producing Sf9 cells were harvested and centrifuged. The cell pellet was resuspended in a resuspension buffer. Initial process development runs were performed by resuspending cells in 50 mM Tris / 10 mM EDTA buffer (pH 8), similar to the 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 at a final concentration of 50 mM. Data showed that the inclusion of sucrose in the resuspension buffer prevented rapid osmotic shock to Sf9 cells and mitigated cell viability loss during the cell enrichment step using UniFuge®.

[0086] As an alternative to Tris buffer, PBS-based resuspension buffers (with or without sucrose) were examined. It was hypothesized that the presence of sucrose in the resuspension buffer could cause concentrated Sf9 cells to aggregate, thereby hindering the optimal lysis of the concentrated cells. In this study, concentrated Sf9 cells were resuspended in two different resuspension buffer conditions: a buffer containing 100 mM Tris and 10 mM EDTA (pH 8) or 1X PBS (pH 7.4), and then lysed in lysis solutions containing either 66.7 mM NaOH and 0.33% SDS or 150 mM NaOH. For all conditions, the lysates were neutralized, clarified, and finally purified using an ion exchange column (Sartobind® Q). The titer of ceDNA was evaluated using qPCR, purified ceDNA was treated with T5 exonuclease, and the effect of nicking on ceDNA was evaluated by additionally running T5-untreated and treated samples side-by-side on an agarose gel ( Fig. 3 T5 treatment is based on the principle that nicked DNA is completely degraded by T5 exonuclease and will not appear as intact DNA in agarose gel electrophoresis. T5 does not affect unnicked ceDNA. For T5 treatment, DNA samples were incubated with the enzyme at 37°C for 30 minutes.

[0087] Concentration analysis of the agarose gel shows that cells resuspended in Tris buffer and PBS buffer and lysed with 0.15 M NaOH exhibited similar amounts of intact ceDNA ( Fig. 4 However, cells resuspended in PBS under 66.7 mM NaOH + 0.33% SDS lysis conditions exhibited higher ceDNA titers as well as more intact ceDNA than cells suspended in Tris buffer. Based on these results, a PBS-based buffer was selected as the resuspension buffer for further follow-up studies.

[0088] Example 2: Evaluation of Dissolution Buffer

[0089] The plasmid DNA extraction kit sold by Invitrogen uses 0.1 M NaOH + 0.5% SDS as the lysis buffer. A high-throughput study was conducted to determine the lysis conditions suitable for ceDNA-producing Sf9 cells. During this study, various concentrations of NaOH (approx. 0 mM to app. 100 mM) and SDS (approx. 0% to app. 1%) were evaluated. qPCR analysis results showed that 66.7 mM NaOH + 0.33% SDS resulted in higher ceDNA titers and less genomic DNA.

[0090] However, high titers of ceDNA do not necessarily imply high-quality ceDNA. Subsequent results showed that when ceDNA was purified using 66.7 mM NaOH + 0.33% SDS lysis buffer, significant product degradation occurred, suggesting that the degradation of ceDNA product quality may be caused by a combination of nuclease activity and / or specific lysis conditions.

[0091] We included an investigation into dissolution conditions to identify the specific root causes leading to ceDNA nicking and to find optimal dissolution conditions that mitigate ceDNA product quality fragmentation. Different dissolution strategies were tested as described below.

[0092] A. Detergent-based dissolution

[0093] Experimental results using SDS-containing lysis buffers showed that when lysis was performed using SDS alone (0.5% SDS and 1% SDS), complete nicking of ceDNA occurred, as indicated by agarose gel electrophoresis after T5 treatment. An alternative detergent called RIPA buffer, which is commonly used for protein extraction, was also evaluated, but ceDNA degradation was similarly observed. Next, the inventors evaluated a lysis buffer containing both NaOH and SDS (150 mM NaOH + 0.05% SDS, or 150 mM NaOH + 1% SDS). Again, nicking was observed.

[0094] When SDS was removed from the 150 mM NaOH lysis buffer, no ceDNA nicking was observed. Based on these encouraging results, it was decided to evaluate lysis conditions using only NaOH without the presence of any detergent components.

[0095] B. pH-based dissolution (using NaOH only)

[0096] Dissolution studies were performed by varying 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. According to the data, completely intact and unnicked ceDNA was produced under all dissolution conditions within the 50 mM to 200 mM NaOH range ( Fig. 5a In addition, when using a lysis buffer containing only NaOH, the ceDNA yield was higher than when using a lysis buffer containing detergent ( Fig. 5bBased on this study, highly alkaline 150 mM NaOH was selected as the final lysis buffer condition for further follow-up studies. The 150 mM NaOH lysis condition resulted in consistent and reproducible intact ceDNA across multiple harvests / runs. The inventors hypothesized that in the absence of SDS, nuclease activity may be attenuated, and that the presence of highly alkaline conditions (pH 10–12.6) may be favorable for the denaturation of nucleases present in cell lysates.

[0097] Example 3: Effect of dissolution retention time

[0098] For the above experiments, the lysis holding time, i.e., the time of incubating cells with the lysis buffer, was maintained constant at 5 minutes. Because there is a possibility that DNA structure may be irreversibly denatured under highly alkaline conditions, the inventors investigated the effect of various lysis holding times on the homogeneity of the ceDNA product for lysis using 150 mM NaOH. T5 digestion was used to indicate the presence of nicks within the ceDNA. The experiment was designed to evaluate various lysis times of 2.5, 5, 7.5, or 12.5 minutes in batch mode, followed by the purification of the ceDNA using a high-throughput form of Sartobind® Q. Based on agarose gel electrophoresis, the ceDNA bands showed a decrease in intensity and homogeneity over time. The results of this study showed that when the lysis holding time was between 2.5 and 5 minutes, impurities such as baculovirus and insect cell DNA were reduced ( Fig. 6b It was found that intact ceDNA with optimal yield was generated. Fig. 6a ).

[0099] Next, the inventors investigated various mixing methods at the laboratory scale to determine how the mixing method might affect product quality at a commercial scale. The modes were stirring rods, overhead mixers, and combinations of both stirring rods and overhead mixers. Since ceDNA is highly sensitive to shear forces, slow mixing using stirring rods provided the best results at the laboratory scale. However, when scaling up to 500 L, which is the minimum volume generally required for mixing in production tanks, there are limitations such as the large pumps required to add lysis / neutralization buffers and the duration of buffer addition. Therefore, to better control process parameters and achieve efficient lysis, continuous inline cell lysis was used instead of batch lysis. Fig. 7 Flow rates 1, 2, and 3 were identical, and two flow rates of 15 mL / min and 27 mL / min were tested according to shear rates (165 / sec and 308 / sec). Several parameters were studied in the continuous inline cell lysis system. The retention times of the tested lysis durations were 3.7 min, 2.5 min, and 1.5 min. Various neutralization mechanisms, such as a static mixer, a T-mixer, and batch mode (in-bottle), were also tested. According to the data, continuous cell lysis was successfully scaled up to a 10 L scale using a static mixer with a lysis duration of 2.5 min and a neutralization step using a T-mixer. Fig. 8 ).

[0100] Example 4: Pre-clarification

[0101] The inventors confirmed that when cell lysates are neutralized using 3.1 M potassium acetate, HMW genomic DNA, host cell proteins, and other cellular components precipitate, resulting in the formation of large aggregates. Therefore, the inventors included an intermediate pre-clarification / pretreatment step before the clarification step to remove large aggregates and increase the throughput of the clarification device.

[0102] Early pre-clarification approaches focused on removing aggregates using UniFuge® / minifuge-based centrifugation methods (Carr Biosystems). However, this approach resulted in the shearing of the ceDNA product. This approach was also ineffective in separating aggregates; rather, it resulted in the generation of much smaller aggregates, leading to a clarification throughput of 15 L / m². 2 It was reduced to less than 70–100 L / m² through a batch centrifuge-based pre-clarification step. 2 Although intact ceDNA was produced with a higher clarification throughput, it will be difficult to scale this method up to the manufacturing level.

[0103] Subsequently, a salt-based pre-clarification approach was developed for the efficient removal of aggregates and enhanced filtration capacity. Initial attempts in this approach were made using ammonium bicarbonate (i.e., ammonium bicarbonate (NH4HCO3); "AHC"). The use of this salt was based on a mechanism in which aggregates float to the surface of the solution due to the release of carbon dioxide and ammonia following the addition of ammonium bicarbonate to the neutralized solution. AHC concentrations ranging from 2.5 to 25 g / L were evaluated for optimal phase separation. E. coli Compared to the 5 g / L salt concentration emphasized in the public literature regarding plasmid generation in cells (Blom et al., Vaccine (2010) 29(1):6-10), 25 g / L of AHC was found to be the optimal salt concentration for efficient phase separation in the ceDNA pre-clarification process. The AHC-based pre-clarification process also prevented fragmentation of the ceDNA product.

[0104] Although efficient phase separation was observed using AHC, this process poses a risk of generating dangerous ammonia gas in large-scale manufacturing. To prevent the generation of harmful ammonia gas, sodium bicarbonate (i.e., sodium bicarbonate (NaHCO3); "NaHC"), an alternative salt, was evaluated as a possible substitute for AHC. NaHC reacts with water to produce sodium chloride, carbon dioxide, and water, while CO2 gas aids in the phase separation of aggregates. A comparative study was conducted to compare the effects of cell debris separation using AHC and NaHC. The following four groups were designed: 15 g / L AHC, 20 g / L AHC, 15 g / L NaHC, and 20 g / L NaHC. 900 mL of neutralized solution was added to 2 L bags containing 15 or 20 g / L AHC or NaHC (i.e., having approximately 50% empty space in the bag). The inventors observed that NaHC induces less swelling compared to AHC while maintaining the separation of cell debris from the supernatant. In addition, a comparison of ceDNA product quality through T5 exonuclease treatment showed that full-length ceDNA band integrity was maintained when NaHC was used as a pre-clarification salt. Fig. 9 ).

[0105] Subsequently, the inventors evaluated whether they could change the method of salt addition and reduce the amount of salt and duration required for small-scale separation. According to the results, adding 5 g / L or 10 g / L of NaHC was sufficient to allow the aggregate layer to rise to the surface, and it was found that cell debris was effectively separated from the supernatant containing the ceDNA product ( Figures 10a and 10b ).

[0106] The salt-mediated phase separation approach for pre-clarification could be implemented on a 500 L scale without product loss. Approximately 90% (w / w) of the pre-clarified supernatant could be loaded into a deep filter. This approach allows for a deep filter loading capacity of 15–20 L / m² 2 At approximately 70~100 L / m 2 It improved the turbidity of the pre-clarified sample from about 6,000 nephelometric turbidity units (NTU) to about 50 NTU for deep filter loading, and did not affect the quality of the product.

[0107] Example 5: Clarification

[0108] After separating the aggregated and liquid phases of the cell lysate, clarification was performed to remove cell debris and particles from the feed stream. Clarification filters were selected primarily based on cell density, viability, cell type, and harvest viscosity. The objective of this study was to identify filters for robust and scalable ceDNA purification. Performance indicators included filter throughput and turbidity, as well as qPCR. Table 1 below shows the list of filters tested.

[0109]

[0110] Comparative study results showed that the Clarisolve 60HX filter (Millipore) and D0HC filter (Millipore) provide satisfactory yield, throughput, and turbidity. Fig. 11 Pre-clarification using NaHC was shown to increase the throughput of the clarification filter. In addition, two feed rates of 75 LMH and 150 LMH were tested, with a maximum tested feed pressure of 15 psi. The maximum throughput obtained was 66 L / m³ for 75 LMH. 2 , in the case of 150 LMH, 48 L / m 2 was Fig. 12 ).

[0111] Example 6: rRNA removal

[0112] It was found that a significant amount of residual RNA (rRNA) was released after lysis due to changes in lysis buffer conditions. Due to concerns regarding the use of animal-derived (primarily bovine pancreas-derived) RNases in the ceDNA purification process, the inventors investigated the use of CaCl2 for rRNA removal. CaCl2 was added to the samples to achieve final concentrations of 0.5, 1, 1.5, or 2 M. The inventors used purified material (Q eluent) under the following two buffer conditions: (i) 1 M potassium acetate (pH 5.5); and (ii) 25 mM Tris (pH 8.0) + 10 mM EDTA + 0.9 M NaCl. Two total DNA concentrations of 75 and 250 μg / ml were tested based on Nanodrop. According to the data, regardless of DNA concentration and buffer conditions, the amount of RNA in the samples decreased as the amount of CaCl2 increased. For subsequent studies, 2 M CaCl2 was selected.

[0113] To ensure efficient binding to the anion exchange resin (Sartobind® Q), CaCl2-treated cell lysates were concentrated and buffer-exchanged. To this end, the inventors investigated different tangential flow filtration (TFF) cassette screens (T-screen and J-screen) and molecular weight cutoffs (MWCO; 10 kDa and 100 kDa), but low permeate flow rates occurred due to viscosity issues. Therefore, the inventors divided the process into two stages: concentration only (ultrafiltration or UF), and treatment with CaCl2 followed by buffer exchange / concentration (UF / DF). An experimental design with four different processes Fig. 13a It is illustrated in [figure]. Treatment with CaCl2 showed higher ceDNA purity (%) compared to samples not treated with CaCl2 ( Fig. 13bIn addition, ceDNA band intensity was similar across the four processes (left bar of each paired group); however, significant removal was observed in the sample (condition 3) that underwent UF / DF followed by CaCl2 precipitation after UF treatment ( Fig. 13c ).

[0114] The inventors further investigated whether CaCl2 treatment helps remove impurities other than rRNA. This study compared CaCl2-treated samples with and without the addition of RNase A. The precipitates from the samples were dissolved and processed on an agarose gel. According to the data, the addition of calcium chloride resulted in a reduction of approximately 70–90% in impurities, including DNA from the baculovirus vector and Sf9 genome, as well as fragmented ceDNA (less than 5 kb) and rRNA.

[0115] Example 7: Evaluation of capture chromatography medium

[0116] In previous experiments, ceDNA products were recovered using a commercial Gigaprep plasmid DNA purification kit. However, this process lacked selectivity in distinguishing between ceDNA products and other nucleic acid impurities. To optimize ceDNA capture, the inventors screened various chromatographic media ranging from membranes, monoliths, and resins.

[0117] Regarding chromatography membranes, Natrix® HDQ (Millipore Sigma), Mustang™ Q (Cytiva), and Sartobind® Q (Sartorius) membranes were evaluated. Both Natrix® HDQ and Sartobind® Q were found to be capable of handling high hard loadings of 200 mL / MV and 178 mL / MV, respectively. However, Mustang™ Q became contaminated after loading 18 mL / MV of clarified ceDNA cell culture lysate. Both Natrix® HDQ and Sartobind® Q exhibited ceDNA eluent peaks, which were further confirmed by agarose gel electrophoresis.

[0118] Regarding chromatography monoliths, the CIM® DEAE (2 μm channel) anion-exchange monolith (BIA Separations) also faced pressure issues similar to Mustang™ Q. However, the loading capacity was relatively higher at 64 mL / MV.

[0119] Regarding the resin, POROS™ 50D anion exchange resin (Thermo Scientific) did not exhibit contamination issues, but no ceDNA peak was observed in the eluent. Similar behavior was observed in the case of HyperCel™ STAR AX resin (Sartorius).

[0120] Sartobind® Q membranes were selected for further study. After evaluating the elution profiles and adding different concentrations of NaCl to the loading material, the inventors confirmed that 150 mM NaCl and 300 mM NaCl in the loading shifted the impurity peak (composed mainly of low molecular weight species) toward the perfusion fraction. Figures 14a and 14b As a result of droplet digital PCR and qPCR titer analysis, it was confirmed that the addition of NaCl to the loading sample did not affect the titer of ceDNA in the eluent fraction ( Table 2Optimizing loading conductivity allows low molecular weight impurities to be directed toward the perfusion fraction or wash fraction. This strategy potentially increases the binding capacity for target molecules by reducing impurity competition opportunities or available binding sites on the membrane ( Fig. 15 ).

[0121]

[0122] The ceDNA capture process using Sartobind® Q membranes was evaluated using the following two different upstream processes: the one-bag system (using production cell lines (PCL) that stably expressed the Factor VIII transgene and were transiently transfected with a baculovirus vector to express the AAV Rep protein); and the two-bag system (Sf9 cells transiently transfected with two baculovirus vectors encoding the AAV Rep protein and Factor VIII, respectively).

[0123] For the two-back system, the inventors tested various MOIs (0.01 / 0.1, 0.05 / 0.5, 0.2 / 2.0, or 0.3 / 3.0) of the Rep / FVIII transplant gene. The inventors confirmed that the two-back system (0.2 / 2.0 MOI) produced ceDNA titers 4 to 5 times higher than the one-back system. The inventors also observed a reduction in host cell protein (HCP) with a log-lowering value (LRV) of approximately 3. Due to high conductivity loading (approx. 62–70 mS / cm) caused by the presence of 0.75–1.0 M potassium acetate and 300 mM NaCl, most of the HCP could be perfused during the capture step. The final elution of ceDNA was performed with 900 mM NaCl.

[0124] Example 8: Evaluation of the ceDNA polishing method

[0125] Capture and purification using the Sartobind® Q membrane provided good separation of HCP and RNA impurities in the perfusion and wash fractions. Additionally, Sartobind® Q showed a reduction of 20–30% in Sf9 DNA and baculovirus DNA. However, complete removal of nucleic acid impurities was not observed throughout the capture step, and some RNA, Sf9 DNA, and baculovirus DNA eluted along with the ceDNA product. The purified ceDNA from the capture step also consisted of ceDNA fragments (size < 4.5 kb).

[0126] To polish the Sartobind® capture product, the inventors investigated the use of a 1-column or 2-column approach to further separate the ceDNA product from product-related impurities (ceDNA low molecular weight fragments) and process-related impurities (Sf9 DNA, baculovirus DNA, and RNA).

[0127] Several polishing adsorbent candidates were reviewed ( Table 3 Hydrophobic Interaction Chromatography (HIC) mode was selected for further research due to its ability to separate different DNA isoforms and to separate DNA isoforms from more hydrophobic nucleic acid components such as RNA, genomes, and denatured DNA.

[0128]

[0129] A. Evaluation of C4 HLD Monolith Column

[0130] C4 HLD monolithic columns (Sartorius) are butyl-modified with high ligand density. They were evaluated as potential polishing media for ceDNA purification. A large amount of ammonium sulfate (AS) is required to bind DNA to C4 HLD HIC monolithic columns. Therefore, hydrophobic interactions were induced by adjusting the HIC loading to 3 M AS. Subsequently, elution was achieved by performing an ammonium sulfate salt gradient that decreased linearly from 3 M to 0 M over 60 CV. Under these conditions, the ceDNA product was observed to exhibit typical 4-peak elution behavior and elute over a wide elution range (2.25 M to 0 M AS). Size-based selectivity was observed between the full-length ceDNA and low molecular weight (LMW) fragments containing more full-length ceDNA observed in the early elution fraction (2.25 M to 1.5 M AS) and the LMW ceDNA fragments eluted in the later fraction (1.5 M to 0 M AS). The inventors observed that ceDNA fragments, baculovirus DNA, Sf9 DNA, and RNA bind more strongly to the column and can be efficiently separated from the ceDNA product. The high binding strength of these species may be due to stronger interactions between the LMW nucleic acid fragments and the C4 butyl ligand.

[0131] Based on the linear gradient results, a stepwise elution process was implemented for the ceDNA polishing process. Since it was observed that full-length ceDNA was eluted at 2.25 M to 1.5 M AS, stepwise elution was performed at a concentration of 1.74 M AS to recover pure and concentrated full-length ceDNA. The stepwise gradient also included steps of 0.9 M, 0.45 M, 0.24 M, and 0 M AS to capture ceDNA and other nucleic acid fragments. When stepwise elution was implemented at 1.74 M AS, it was observed that approximately 77% of the ceDNA product was recovered ( Figures 16a and 16b and Table 4 ).

[0132]

[0133] C4 HLD monolith polishing purification can be performed in both the binding-elution approach and the perfusion approach for ceDNA purification through the weaker binding of full-length ceDNA and C4 aliphatic ligands. The inventors performed a head-to-head comparison of the two approaches.

[0134] For the binding-elution approach, ceDNA elution was performed in 1.74 M AS, and LMW fragments were removed from the subsequent strip fraction. This approach resulted in a 66% recovery rate of the ceDNA product, an 84% reduction (0.8 LRV) in residual Sf9 DNA, and an 81% reduction (0.7 LRV) in residual baculovirus DNA. Figures 17a and 17b and Table 5 ).

[0135]

[0136] Because the full-length ceDNA product eluted earlier, the inventors investigated a perfusion approach to isolate ceDNA containing nucleic acid impurities (e.g., ceDNA fragments, baculovirus DNA, Sf9 DNA, and RNA). One advantage of the perfusion approach over the binding-elution approach is that more material can be loaded onto the monolith at once without significantly affecting impurity removal. To optimize the perfusion approach, Sartobind® Q eluent was adjusted to a 1.5 M AS concentration and loaded onto the monolith. Analysis of the column fractions by agarose gel electrophoresis revealed that the perfusion fraction contained purified ceDNA product, while the strip fraction showed that LMW species had been removed. This approach yielded a 64% recovery rate of the ceDNA product, a 96% reduction in residual Sf9 DNA (1.4 LRV), and a 97% reduction in residual baculovirus DNA (1.5 LRV). Figures 18a and 18b and Table 6 ).

[0137]

[0138] This study shows that the ceDNA purification process is suitable for both binding-elution and perfusion modes of chromatography using a C4 HLD monolithic column.

[0139] B. Evaluation of Perfusive HIC Resin

[0140] As an alternative to C4 HLD monoliths for ceDNA polishing, POROS™ resins were evaluated for product separation efficiency. POROS™ resins have a wide pore size range of 100–400 nm, making them ideal resin candidates for large-scale biomolecular purification without significantly affecting binding capacity. Three POROS™ HIC candidates were tested to evaluate whether the properties of HIC ligands (ethyl or benzyl) combined with perfusive flow characteristics would provide improved resolution between linear ceDNA products and process- and product-related impurities.

[0141] Experiments were performed by loading Sartobind® Q eluent adjusted to a 3 M AS concentration under a difficult loading of 2 mg / mL resin, and the retention behavior of linear ceDNA products and nucleic acid impurities was mapped by performing a reverse linear gradient of 3–0 M AS over 60 CV. For comparison, C4 HLD was evaluated as a reference candidate using the same chromatographic experiment protocol.

[0142] After superimposing the chromatographic elution profiles of POROS™ Ethyl, POROS™ Benzyl, POROS™ Benzyl Ultra, and C4 HLD Monolith, three distinct elution peaks were observed. Analysis of the elution fractions (E1, E2, and E3) from the four HIC adsorbent candidates on an agarose gel revealed that the E1 elution profile consisted of intact ceDNA of the highest purity, while the E2 and E3 fractions were observed to consist mainly of cleaved ceDNA fragments, RNA impurities, and Sf9 and baculovirus DNA fragments. qPCR analysis showed that all four HIC candidates (POROS™ Ethyl, POROS™ Benzyl, POROS™ Benzyl Ultra, and C4 HLD Monolith) improved ceDNA process-related purity from 48% to over 80%. However, ceSDS lab chip analysis results showed that monomeric ceDNA purity was 25–35% when using POROS™ benzyl and C4 HLD monoliths ( Figures 19a and 19b According to the data, all three resin candidates exhibited sufficient resolution to separate a pure, intact ceDNA product from the first eluent fraction; thus, it was found that all tested POROS™ resin candidates can be used as alternatives to the C4 HLD monolith.

[0143] Although all three POROS™ HIC resins exhibited similar behavior, POROS™ ethyl showed the highest binding capacity compared to benzyl or benzyl ultra and was able to accommodate a hard loading of 2.5 mg / mL resin. The POROS™ HIC adsorbents demonstrated an inverse relationship between the binding capacity to ceDNA material and the hydrophobic strength of the HIC ligands (ethyl, benzyl, and benzyl ultra). Generally, stronger HIC ligands are expected to exhibit a higher binding capacity to biomolecules compared to ligands with lower hydrophobic strength. However, it was interesting to observe that POROS™ ethyl resin exhibited a higher binding capacity to ceDNA compared to POROS™ benzyl and POROS™ benzyl ultra, even though benzyl ultra showed the highest hydrophobic strength among the tested ligands, followed by benzyl, and ethyl showed the lowest hydrophobicity.

[0144] These differences can be attributed to the grafting process used to attach ligands to the resin matrix, which significantly affects pore structure and accessibility. In this regard, due to the grafting properties of hydrophobic interaction chromatographic ligands, the effective pore size of POROS™ Benzyl Ultra is smaller compared to POROS™ Ethyl. The narrow pore size of POROS™ Benzyl Ultra induces steric hindrance for large DNA biomolecules and introduces additional restrictions on diffusion mass transfer, thereby reducing the surface area available for binding and ultimately limiting binding capacity. Conversely, the more open pore structure of POROS™ Ethyl facilitates the diffusion of DNA biomolecules into the pores, enabling access to the internal surface area for binding and thereby enhancing binding capacity. The DNA binding capacities of the HIC adsorbents evaluated for polishing purification in both binding-elution and perfusion modes are shown in Table 7.

[0145]

[0146] To further optimize the use of POROS™ HIC columns for polishing ceDNA elution products, comparative experiments were performed to compare the use of POROS™ Benzyl Ultra HIC resin in the binding-elution approach and the perfusion approach ( Fig. 20 ). For the binding-elution approach, the loading was first adjusted to a final concentration of 3 M AS. Elution from the POROS™ Benzyl Ultra HIC column was performed using a reverse gradient approach by decreasing the AS concentration from 3 M to 0 M over 60 CVs. The POROS™ Benzyl Ultra HIC column was observed to have a binding capacity where breakthroughs close to a difficult 0.7 mg / mL resin loading were observed. In the binding-elution approach, reductions in baculovirus and Sf9 DNA were shown to exceed 90%, and the ceDNA product recovery rate was 42% due to the loss of ceDNA in perfusion as a result of column breakthrough ( Figures 21a and 21b ).

[0147] Compared to binding-elution column operation, the efficiency of perfusion operation of the POROS™ Benzyl Ultra HIC column was also evaluated. In the aforementioned experiments, ceDNA was observed to elute between 2 M and 1.5 M while running a linear gradient on the POROS™ Benzyl Ultra HIC column. For perfusion operation, the loading was first adjusted to a final AS concentration of 1.8 M and loaded onto the POROS™ Benzyl Ultra HIC column. Under an AS concentration of 1.8 M, the ceDNA product was separated in the perfusion section, and only nucleic acid impurities such as Sf9, baculovirus DNA, and RNA were bound to the column and removed in a stripping step performed using a salt-free buffer (pH 8) containing 50 mM Tris and 10 mM EDTA. In the perfusion approach, the ceDNA product recovery rate was found to be 92% and the reduction of process-related impurities was 62%. Evaluation of ceDNA purity through an agarose gel showed that the purity was similar for both binding-elution and perfusion mode operations.

[0148] C. Evaluation of Capto™ Core Resin for Polishing Purified ceDNA

[0149] The inventors also evaluated Cytiva’s core-shell resins (Capto™ Core 400 and Capto™ Core 700) for use in the polishing step. Capto™ Core resins are based on a core-shell or core bead concept in which each resin bead has a ligand-activated core and an outer inactive layer without ligands. The outer layer has size exclusion properties that prevent large targets from entering the bead, whereas smaller protein and DNA impurities can enter the core and bind to hydrophobic and positively charged octylamine ligands. Capto™ Core resins have not previously been used as a polishing approach in DNA purification processes. Capto™ Core 700 resin has a larger pore structure and a larger MWCO than Capto™ Core 400 resin (750 kDa vs. 400 kDa).

[0150] According to the inventors' research, Capto™ Core 700 resin was found to be more effective (greater than 70% reduction) in reducing Sf9 and baculovirus DNA impurities compared to Capto™ Core 400 resin (less than 50% reduction). Table 8 ).

[0151]

[0152] As a result of chromatographic superposition of the perfusion block, the peak height of Capto™ Core 400 was found to be higher than that of 700, indicating a lower removal rate of impurities in the perfusion product portion ( Fig. 22a Agarose gel analysis of the perfusion fractions of these columns revealed that the ceDNA products produced using Capto™ Core 700 resin showed less spreading compared to Capto™ Core 400 resin ( Fig. 22b Overall, Capto™ Core 700 resin demonstrated excellent performance in the polishing step of ceDNA purification.

[0153] Example 9: Feasibility of Virus Filtration

[0154] Baculovirus is inherently present in Sf9 cells due to the use of baculovirus viruses for transduction and protein expression. Additionally, the original Sf9 cell line is known to be contaminated with rhabdovirus. Therefore, the removal of viral contaminants is necessary in this ceDNA purification scheme. A difficulty in using viral filtration for DNA purification is the size of the product. The swivel radius of ceDNA monomers (approximately 150 nm) makes it difficult to distinguish ceDNA from baculovirus DNA through size-exclusion separation due to size limitations. To evaluate the feasibility of viral removal in ceDNA purification, the inventors used a Planova™ 35N size-based viral removal filter.

[0155] To determine the feasibility of implementing viral filtration in the ceDNA platform process, Planova™ 35N 0.001 m was used with the PendoTECH Vertical Flow Filtration (NFF) filter screening system. 2 Three test groups were tested using a viral filter. The experimental groups contained the following three different feed streams: Sartobind® Q eluent (50 mM Tris pH 8.0, 10 mM EDTA, 0.9 M NaCl), C4 HLD loading (50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M AS), and C4 HLD product (perfusion and wash volume) (50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, 1.5 M AS). Pressure was controlled at 12 psi, and the tested throughput was 50 L / m² 2 According to the data, the ceDNA recovery rates of the Sartobind® Q test group and the C4HLD product test group were found to exceed 50% based on A260 absorbance, respectively. Table 9In addition, analysis of ceDNA product purity by agarose gel electrophoresis showed a reduction in contaminant species in the C4 HLD product test group, indicating that the Planova™ 35N virus filter is a feasible size-based virus removal step in ceDNA purification ( Fig. 23 ).

[0156]

[0157] Example 10: Evaluation of Final Tangential Flow Filtration Parameters

[0158] It was found that the presence of 1.5 M AS in the final tangential flow filtration (TFF) loading significantly altered the DNA conformation. To address this issue, the inventors evaluated the efficiency of different TFF cassette screens (L screen, J screen, Repligen) and molecular weight thresholds (10 kDa and 30 kDa) for use in the final TFF step. A C4 HLD column was used in perfusion mode and concentrated 10-fold. The buffer was replaced with 1X TE buffer (approx. 6–8 DV). Subsequently, the residue after UF / DF TFF was run on an agarose gel to determine the quality of the ceDNA product through band intensity. In the case of the 30 kDa MWCO TFF membrane, ceDNA loss in the permeate was observed. However, in the case of the 10 kDa MWCO TFF membrane, no ceDNA product was lost in the permeate, and a 100% recovery rate was obtained based on qPCR and band intensity on the agarose gel ( Fig. 24 Therefore, if the polished ceDNA product contains a high molar concentration of ammonium sulfate, a 10 kDa MWCO TFF membrane can be used as the final purification step.

[0159] Example 11: Development of a 3-Column Purification Process

[0160] A 3-column purification approach was tested for ceDNA purification ( Fig. 25In this approach, ceDNA was purified using a Sartobind® Q capture column. The Sartobind® Q eluent was further polished using four different polishing approaches, including a C4 HLD HIC monolith in binding-elution mode, a C4 HLD HIC monolith in perfusion mode, POROS™ Benzyl Ultra HIC resin in binding-elution mode, and POROS™ Benzyl Ultra HIC resin in perfusion mode. Finally, the eluents from the binding-elution secondary column approach were combined and further purified using Capto™ Core multimode core-shell resin. The quality characteristics of the ceDNA products for all these runs were analyzed using agarose gel electrophoresis with a ce-SDS lab chip.

[0161] The agarose gel and lab chip results are Fig. 26 It is illustrated in [figure]. According to the data, the range of intact purity of ceDNA from the initial purification process (Sartobind® Q - HIC polishing) was found to be 27–34%. After incorporating a 2-column polishing process including CaCl2 precipitation and HIC and Capto™ Core polishing purification steps, the ce-SDS purity of intact ceDNA increased to over 80%.

[0162] Both hydrophobic interaction chromatography adsorbents and Capto™ core-shell resins have demonstrated significant potential in removing process- and product-related impurities. However, to achieve enhanced purity, a 2-column polishing strategy was determined to be essential. This approach combines the ability of HIC adsorbents to remove primarily low molecular weight nucleic acid impurities (0.1 kb to 4 kb) with the ability of Capto™ core resins to remove medium-sized nucleic acid impurities (0.1 kb to 6 kb).

[0163] To improve the quality of intact closed DNA (ceDNA), the following two polishing strategies were evaluated by changing the order of the polishing columns ( Fig. 27 ):

[0164] Polishing Strategy 1: HIC adsorbent following Capto™ Core;

[0165] Polishing Strategy 2: Capto™ Core Resin Following HIC Adsorbent

[0166] In addition, the efficacy of calcium chloride (CaCl2) precipitation of clarified feedstock in improving the quality of the ceDNA product was evaluated. This was achieved by conducting a comparative purification study including capture, intermediate, and final polishing steps using both CaCl2-untreated and CaCl2-treated clarified feedstocks. For the CaCl2-untreated feedstocks, both polishing strategies were evaluated and designated as Test Group 1 and Test Group 2. For the CaCl2-treated feedstocks, only the Capto™ Core sequence following HIC was evaluated and designated as Test Group 3.

[0167] Process conditions were optimized to facilitate direct loading of the eluent from the capture column to the intermediate polishing column and then to the final polishing column with minimal adjustment. This enables a fully integrated continuous approach for the capture, intermediate polishing, and final polishing steps.

[0168] For Test Group 1, in which the polishing process includes a HIC adsorbent following the Capto™ Core resin, the Sartobind® Q eluent in pH 8.5, 50 mM Tris, 10 mM EDTA, and 0.9 M NaCl can be loaded directly onto the Capto™ Core column. Subsequently, the product of the Capto™ Core intermediate polishing step in the same buffer matrix can be loaded onto the final HIC polishing adsorbent after being adjusted to 3 M or 1.5–2 M ammonium sulfate (AS) in pH 8, 50 mM Tris, 10 mM EDTA, and 0.45 M NaCl, depending on whether the HIC step is operated in binding-elution or perfusion mode. The final purified ceDNA material is recovered in an eluent of 1.5–2 M AS in pH 8, 50 mM Tris, 10 mM EDTA, and 0.45 M NaCl (for binding-elution mode) or in a perfusion and wash fraction (perfusion mode).

[0169] After the HIC adsorbent is used as an intermediate polishing step, for Test Groups 2 and 3 using Capto™ Core resin, the Sartobind® Q capture eluent in pH 8.5, 50 mM Tris, 10 mM EDTA, and 0.9 M NaCl is adjusted to 3 M or 1.5 M AS in pH 8, 50 mM Tris, 10 mM EDTA, and 0.45 M NaCl, and loaded onto the HIC adsorbent operating in binding-elution or perfusion mode. After the HIC polishing step, the ceDNA product in the range of 1.5–2 M AS is loaded onto the Capto™ Core column. The final purified ceDNA product is obtained from the perfusion and wash fractions in the same buffer matrix as the Capto™ Core loading conditions.

[0170] The recovery results of this study are presented in Table 10, providing a comparative overview of stepwise ceDNA recovery rates at various stages. Additionally, the product quality characteristics of ceDNA were investigated using various orthogonal analyses, including qPCR, gel concentration (agarose gel), capillary gel electrophoresis (LabChip), and next-generation sequencing (NGS) short sequence fragment sequencing. Product-related purity, representing the percentage of intact full-length ceDNA, was evaluated by gel concentration and capillary gel electrophoresis. Process-related purity, representing the percentage of ceDNA-transplanted genes, was determined more accurately by NGS short sequence fragment sequencing and qPCR analysis. Product quality indicators for the two polishing approaches using CaCl2-untreated and CaCl2-treated feedstocks were further Fig. 28 and Fig. 29 It is presented in.

[0171]

[0172] Example 12: Exemplary purification process

[0173] An exemplary process using the previously mentioned tested steps is further described below.

[0174] A. Cell harvesting and re-table

[0175] Sf9 insect producer cells previously infected with two baculovirus expression vectors were concentrated 4 to 6 times using a 2000 xg continuous centrifugation system at a flow rate of 3 L / min (Minifuge / UniFuge®, CARR Biosystems UniFuge®). The concentrated Sf9 cells were resuspended in 1X PBS (2.7 mM KCl, 11 mM phosphate, 135 mM NaCl) washing buffer at pH 7.4 to produce a cell paste.

[0176] B. Cell lysis and neutralization

[0177] The resuspended cell paste was immediately dissolved by alkaline dissolution by adding 0.3 M NaOH to the suspension to achieve a final concentration of 150 mM NaOH. The cell paste was dissolved by continuous inline dissolution at a flow rate of 15 mL / min. The cell paste was dissolved under alkaline conditions within 5 minutes, preferably within 2.5 minutes.

[0178] As an extension of the continuous inline system, highly alkaline cell lysates (pH >12.5) were immediately neutralized by adding 3.1 M potassium acetate (pH 5.2) until the lysate pH reached pH 5.5–5.6. Neutralizing the cells with potassium acetate caused HMW genomic DNA, HCP, and other cellular components to precipitate, resulting in the formation of large aggregates in the cell lysates.

[0179] C. Pre-clarification of cell lysates

[0180] As a pre-clarification step prior to clarification and filtration, the aggregated cell lysate was treated with 10 g / L sodium bicarbonate and stirred for approximately 30 seconds. Subsequently, the reaction was carried out for approximately 2 hours. Due to this pre-clarification treatment, phase separation occurred between the aggregate and the clarified cell lysate.

[0181] D. Clarification and Concentration of Cell Lysates

[0182] To remove cell debris and particles generated during the neutralization and pre-clarification steps, neutralized cell lysate at 50 L / m³ 2 23 cm at a flow rate of / hr 2 Clarisolve 60HX (Millipore) or 23 cm 2It was passed through a D0HC (Millipore) deep filter. To reduce volume, the clarified cell lysate was treated with a TFF system to concentrate the cell lysate 6 to 10 times. A tangential flow filtration step was performed in 50 mM Tris, pH 8.0, 10 mM EDTA, and 0.3 M NaCl.

[0183] E. Precipitation of impurities

[0184] To precipitate contaminating RNA and DNA impurities from the clarified cell lysate, the clarified cell lysate was supplemented with 5 M CaCl2 to achieve a final concentration of 2 M CaCl2 in solution. The precipitation reaction was carried out at room temperature for 30 minutes. The clarified cell lysate was passed through a second tangential flow filtration system, and the buffer was exchanged with 50 mM Tris, pH 8.0, 10 mM EDTA, and 0.3 M NaCl. The buffer was exchanged 6 times.

[0185] F. Isolation of ceDNA products

[0186] To purify the target ceDNA, the clarified cell lysate was passed through a Sartobind® Q anion exchange chromatography column. After binding the ceDNA to the column, the column was washed with 50 mM Tris, pH 8.0, and 10 mM EDTA. The column was washed again with 50 mM Tris, pH 8.0, 10 mM EDTA, and 0.6 M NaCl. After the washing step, the ceDNA was eluted from the Sartobind® Q column using 50 mM Tris, pH 8.0, 10 mM EDTA, and 0.9 M NaCl. The anion exchange chromatography step was performed at a flow rate of 3 matrix volumes / min.

[0187] G. Polishing

[0188] To polish the Sartobind® Q-eluted ceDNA, the eluent was applied to an HIC chromatography column. The HIC columns were C4 HLD monolithic 2 μm channel columns (Sartorius) or POROS™ perfusive resin (Thermo Scientific). Using the C4 HLD monolithic column, the ceDNA product was isolated via binding-elution or perfusion methods. In the binding-elution method, the Sartobind® Q-eluted ceDNA was first replenished with 3 M ammonium sulfate to induce hydrophobic interactions. After binding of the ceDNA, the C4 HLD monolith was washed with 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, and 1.5 M ammonium sulfate. After the washing step, the bound ceDNA was eluted from the column via a stepwise 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 and 10 mM EDTA. In the perfusion method, Sartobind® Q-eluted ceDNA was first replenished with 1.5 M ammonium sulfate. The unbound ceDNA product was recovered from the column in the perfusion pool using 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, and 1.5 M ammonium sulfate. Some additional ceDNA product was removed during the washing step using 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, and 1.5 M ammonium sulfate.

[0189] Alternatively, in the binding-elution method using POROS™ Perfusive HIC resin, Sartobind® Q-eluted ceDNA was first replenished with 3 M ammonium sulfate. After sample loading, the POROS™ HIC resin was washed with 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, and 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 and 10 mM EDTA. In the perfusion method using POROS™ Perfusive HIC resin, Sartobind® Q-eluted ceDNA was first replenished with 1.8 M AS. Unbound ceDNA was recovered from the column of the perfusion pool using 50 mM Tris pH 8.0, 10 mM EDTA, 0.9 M NaCl, and 1.8 M ammonium sulfate. Some additional ceDNA product was recovered during the washing step using 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, and 1.5 M ammonium sulfate.

[0190] To further polish the eluted ceDNA product, trace nucleic acid fragments were removed from the ceDNA product by applying a Capto™ Core-based shell multimode resin-based purification. Capto™ Core can be used as a secondary polishing chromatography column after the Sartobind® Q capture step, or alternatively, as a tertiary polishing column after a second column polishing step involving hydrophobic interaction chromatography (HIC). The Capto™ Core polishing purification process was optimized to facilitate direct loading of the eluent from the previous column purification step onto the Capto™ Core resin without requiring any supplementation or adjustment to the loading conditions.

[0191] For example, in a scenario where the Capto™ Core functions as a secondary column after the capture step, an eluent from Sartobind® Q containing 50 mM Tris (pH 8.0), 10 mM EDTA, and 0.9 M NaCl was loaded directly onto the Capto™ Core resin. Subsequently, the ceDNA product was removed from the perfusion and wash fractions using the same buffer conditions of 50 mM Tris (pH 8.0), 10 mM EDTA, and 0.9 M NaCl.

[0192] When using Capto™ Core as a tertiary column after the second polishing step of HIC, the ceDNA product obtained from the HIC step was directly loaded onto the Capto™ Core resin under conditions characterized by 50 mM Tris (pH 8.0) and ammonium sulfate concentrations ranging from 1.7 M to 2 M in 10 mM EDTA. Subsequently, the resin was subjected to a washing step using conditions identical to the loading conditions, namely 50 mM Tris (pH 8.0) and 1.7 to 2 M ammonium sulfate in 10 mM EDTA.

[0193] H. Virus Filtering

[0194] To remove contaminating viral DNA from the purified ceDNA composition, the Sartobind® Q eluent in a composition of 50 mM Tris pH 8.0, 10 mM EDTA, and 0.9 M NaCl, or the HIC polishing product in a composition of 50 mM Tris pH 8.0, 10 mM EDTA, 0.45 M NaCl, and 1.5 M ammonium sulfate was 0.001 m in dead-end filtration mode. 2 Filtered through a Planova® 35N virus filter (Asahi Kasei Bioprocess). 50 L / m² 2Filtration was performed under a constant operating pressure of 12 psi at a target throughput. After filtration, 10 L / m² of 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) was used. 2 Flushing was performed after recovery.

[0195] I. TFF

[0196] To prevent conformational changes of the isolated ceDNA product and to concentrate the ceDNA product, the purified ceDNA composition was passed through a two-stage TFF with an MWCO of 10 kDa. Both TFF stages were operated at a constant TMP of 8 psi and an orthocurrent flux of 240 LMH; the throughput of stage 1 was 20–30 L / m² 2 , the throughput of Stage 2 is 40 L / m 2 In Step 1, the ceDNA product was concentrated 10 to 20 times through an ultrafiltration step, and then the buffer was exchanged 10 times through a volume filtration step. In this tangential flow filtration step, the ceDNA product buffer was exchanged with 10 mM Tris (pH 8.0) and 1 mM EDTA. In Step 2, the ceDNA product was further concentrated 10 times to a desired concentration of 2 mg / mL through only an ultrafiltration step.

[0197] Example 13: In-process monitoring of ceDNA quality and quantity

[0198] Considering the complexity of ceDNA generation, the inventors have developed an ion exchange (IEX) ultra-high performance liquid chromatography (UPLC) method capable of separating all process impurities from ceDNA and quantifying impurities and ceDNA at any given step of the entire purification process.

[0199] The performance of the IEX method was evaluated using four samples taken from different stages of the Sartobind® Q purification step. This method separates proteins and nucleic acids using a linear salt gradient. ceDNA samples were either injected directly or diluted with water and then injected into the IEX column. Sartobind® Q was the first purification step specialized for ceDNA in the ceDNA preparation process. The loading of the Sartobind® Q step included ceDNA and many process impurities, such as host cell proteins (including red fluorescent protein, an introduced cell marker), DNA and RNA fragments. In this experiment, the inventors selected a column (Protein-Pak Hi Res Q (Waters™, Catalog No. 186004931)) functionalized with quaternary ammonium anion exchangers on 5 μm diameter particles.

[0200] The inventors loaded four samples, taken from the loading, perfusion, wash products, and eluent, respectively, of the Sartobind® Q purification step, onto an IEX column. Subsequently, the IEX column was run with a linear gradient of sodium chloride ranging from 0.5 M to 2.8 M. The chromatogram records for the four samples are Fig. 30 It is superimposed on [the image]. As shown in the figure, the "loading" and "perfusion" samples contained large peaks for host cell proteins, red fluorescent protein, and nuclear fragments, and the "perfusion" sample contained very small amounts of ceDNA. The "wash" sample contained only impurities (mainly nucleic acid fragments and residual RNA) and did not contain any detectable amounts of ceDNA. The "elution" sample contained mainly ceDNA, and red fluorescent protein, a marker protein expressed by ceDNA-producing cells, was reduced by approximately 2500-fold.

[0201] The principle of IEX UPLC is separation based on differences in the charge characteristics of molecules. In this context, IEX analysis combines different detector wavelengths, namely UV and fluorescence. The results above demonstrate that IEX chromatography is a sensitive and convenient in-process method for monitoring the content of impurities and ceDNA during the ceDNA purification process.

Claims

Claim 1 A method for obtaining a purified preparation of ceDNA from closed DNA (ceDNA)-producing cells, comprising the steps of: incubating the cells in an alkaline buffer to lyse the cells and produce a cell lysate, wherein the alkaline buffer does not contain detergent and has a pH of 10 or higher; and isolating ceDNA from the lysate. Claim 2 A method according to claim 1, further comprising, prior to the isolation step, a step of neutralizing the cell lysate with an acidic salt. Claim 3 A method according to claim 2, further comprising the step of pre-clarifying the neutralized cell lysate by adding sodium bicarbonate and separating the resulting aggregate from the cell lysate prior to the isolation step to produce a pre-clarified cell lysate. Claim 4 A method for obtaining a purified preparation of ceDNA from closed DNA (ceDNA)-producing cells, comprising the steps of: obtaining a lysate of said cells; pre-clarifying said cell lysate by adding sodium bicarbonate and separating the resulting aggregate from said cell lysate to produce a pre-clarified cell lysate; and isolating ceDNA from said lysate. Claim 5 In claim 4, the lysate is obtained by incubating the cells in an alkaline buffer to lyse the cells, wherein the alkaline buffer does not contain detergent and has a pH of 10 or higher. Claim 6 A method according to claim 5, further comprising a step of neutralizing the cell lysate with an acidic salt prior to the pre-clarification step. Claim 7 A method according to any one of claims 1 to 6, wherein the isolation step is performed by anion exchange chromatography. Claim 8 A method according to any one of claims 3 to 7, wherein prior to the isolation step, the method further comprises a step of removing RNA by calcium chloride precipitation, and optionally, the removal of RNA is performed by: a step of filtering the pre-clarified cell lysate to produce a clarified cell lysate, and a step of removing RNA by treating the clarified cell lysate with calcium chloride precipitation. Claim 9 A method according to claim 8, wherein the clarified solution is subjected to ultrafiltration before the addition of calcium chloride, or the solution is subjected to ultrafiltration and volume filtration after calcium chloride precipitation to reduce the calcium chloride concentration. Claim 10 A method according to any one of claims 3 to 7, further comprising, after the isolation step, a step of removing RNA through calcium chloride precipitation. Claim 11 A method according to claim 10, further comprising the step of treating the product of the isolation step with a positive volume filtration after calcium chloride precipitation to reduce the calcium chloride concentration. Claim 12 A method according to any one of paragraphs 8 to 11, wherein the calcium chloride is about 1 to 3 M, optionally about 2 M. Claim 13 A method according to any one of claims 1 to 12, further comprising the step of polishing the isolated ceDNA. Claim 14 A method according to claim 13, wherein the polishing step is performed using one or both of (i) hydrophobic interaction chromatography (HIC) (optional, the HIC is performed with a monolithic or perfusive resin); or (ii) a multimode core-shell resin (optional, the multimode core-shell resin comprises resin beads having a size-exclusion shell, and optionally, the size-exclusion shell has a molecular weight cutoff (MWCO) of 400 kDa or 700 kDa). Claim 15 A method according to any one of claims 1 to 14, further comprising a virus filtration step, optionally performed using a 35 nm filter after the isolation step. Claim 16 A method according to any one of claims 1 to 3 and 5 to 15, wherein the alkaline buffer contains sodium hydroxide at a final concentration of about 100 to 300 mM and optionally about 150 mM after being added to the cell. Claim 17 In paragraph 16, the incubation step does not last for more than 5 minutes, optionally the incubation step lasts for about 2.5 minutes or about 3.5 minutes, and optionally the incubation step is performed in a continuous inline system. Claim 18 A method according to any one of claims 2, 3, and 6 to 17, wherein the acid salt is optionally potassium acetate of about 2.5 to 3.5 M and optionally about 3.1 M. Claim 19 A method according to any one of claims 3 to 18, wherein sodium bicarbonate is added at a concentration of about 5 to about 50 g / L, optionally about 10 g / L, and / or said aggregate is removed by a filter having a pore size of about 7.5 to 60 μm, and optionally the incubation time is about 2 hours. Claim 20 A method according to any one of claims 1 to 19, wherein the isolated ceDNA undergoes tangential flow filtration, optionally having an MWCO of 10 and / or 100 kDa. Claim 21 A method according to any one of claims 1 to 20, wherein the ceDNA-producing cell is an insect cell infected with a recombinant baculovirus expression vector. Claim 22 In claim 21, the recombinant baculovirus expression vector comprises a heterogeneous nucleic acid sequence containing a transplant gene flanked by an inverted terminal repeat (ITR), wherein optionally the ITR is a parvovirus ITR or an AAV ITR, optionally an AAV2 ITR. Claim 23 In paragraph 22, the above heterogeneous nucleic acid encodes a therapeutic protein, a method. Claim 24 A method according to any one of claims 1 to 23, wherein the ceDNA-producing cell is a genetically modified insect cell containing a sequence encoding the ceDNA in its genome. Claim 25 A method according to any one of claims 1 to 24, further comprising the step of monitoring ceDNA purity during the process by measuring the levels of ceDNA and impurities through ion exchange ultra-high performance liquid chromatography in a sample taken before, during, or after the isolation step.

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