Transient expression of therapeutic proteins

Abstract

The present disclosure provides a novel method for large-scale production of therapeutic proteins, such as recombinant proteins (e.g., antibodies), comprising concentrating eukaryotic cell cultures to high density and transiently transfecting the eukaryotic cells with a polynucleotide encoding the recombinant protein using electroporation (e.g., flow-type electroporation). In some embodiments, the culture is performed under perfusion conditions, e.g., using tangential flow filtration methods, such as alternative tangential flow filtration. The proteins obtained using the disclosed methods are comparable to those produced by stable transfection. The methods disclosed herein can be used, for example, to accelerate therapeutic drug development, reduce host cell toxicity, or for small-scale manufacturing of personalized therapeutic drugs (e.g., drugs for rare or orphan diseases).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 318,370, filed March 9, 2022, and U.S. Provisional Application No. 63 / 322,472, filed March 22, 2022, each of which is incorporated by reference in its entirety herein. [Technical field]

[0002] This application relates to the field of transient gene expression of therapeutic recombinant proteins. [Background technology]

[0003] With the increasing need to shorten timelines and improve throughput in recombinant protein production, there is an urgent need to rapidly produce drug substance material with a product quality profile equivalent to that of material produced in traditional stable cell lines. Transient gene expression (TGE) has the potential to meet this demand by rapidly and stably producing target proteins, typically clonal material. TGE can be produced several weeks faster than clonal material production in a typical stable cell line, accelerating timelines to drug manufacturing by supporting downstream process, formulation and analytical development [Kelley (2020) Nat. Biotechnol. 38(5):540-545;Coffman et al. (2008) Biotechnol. Bioeng. 100(4):605-618;Bolisetty et al. (2020) MAbs 12(1):p176372]. Thus, development programs can be conducted with minimal risk to downstream operational steps before final cell line selection. Furthermore, TGE allows for earlier toxicity testing using material from the same mammalian host system as the subsequent development stages, making evaluation between program stages easier and more accurate. Additional applications of TGE include small-scale manufacturing for rare or orphan diseases, for example, creating small drug batches that allow for the design of targeted medicines for specific patient groups or personalized therapies (Gutierrez-Granados et al. (2018) Crit. Rev. Biotechnol. 38(6):918-940), which supports versatility for products with low development needs, allowing for more efficient production of rare disease drugs, which may lower the overall cost of treatment per patient (Sun et al. J. Med. Genet. A 173(9):2307-2322). However, low productivity (low titer), product quality differences with clonal material, and scalability have limited the use of TGE to non-GLP preclinical studies, preventing the widespread application of transient expression in clinical development.

[0004] Summary of the Invention The present disclosure relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: (i) culturing eukaryotic cells to enrich for high densities of eukaryotic cells; and (ii) transiently transfecting the eukaryotic cell with a polynucleotide encoding the recombinant protein using electroporation; The present invention provides a method for producing recombinant proteins in large volume bioreactors with high yield and / or high quality, comprising:

[0005] In some embodiments, the quality of the recombinant protein is comparable to that obtained using clonal material, i.e., stably transfected cells. In some embodiments, the step of culturing and concentrating eukaryotic cells to a high density comprises maintaining the cells in an optimal growth phase.

[0006] In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is selected from the group consisting of CHO, VERO, BHK, HEK, HeLa, COS, MDCK and hybridoma cells. In some embodiments, the mammalian cell is a CHO cell. In some embodiments, the recombinant protein comprises an antibody or an antigen-binding portion thereof. In some embodiments, the antibody or antigen-binding fragment binds to an antigen selected from the group consisting of PD-1, PD-L1, CVTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, EGFR, HER3 and HER4. In some embodiments, the bioreactor is a high throughput bioreactor. In some embodiments, the high throughput bioreactor is an AMBR® 250 or AMBR® 15 bioreactor. In some embodiments, the bioreactor is a 1 L, 2 L, 5 L, 10 L, 25 L, 50 L, 100 L, 500 L, 1000 L, 2000 L, 5000 L, 10,000 L, or 20,000 L bioreactor. In some embodiments, the bioreactor is a fed-batch production bioreactor. In some embodiments, the bioreactor is a single-use bioreactor. In some embodiments, the bioreactor is a glass tank bioreactor.

[0007] In some embodiments, the culture, e.g., pre-transfection culture, is a perfusion culture. In some embodiments, the perfusion is performed by tangential flow filtration. In some embodiments, the tangential flow filtration is an alternating tangential flow filtration (ATF). In some embodiments, the perfusion is performed using a REPLIGEN® ATF2 system. In some embodiments, the cells are cultured at a density of at least about 40×10 6 , at least about 50x10 6 , at least about 60x10 6 , at least about 70x10 6 , at least about 80x10 6, at least about 90x10 6 , at least about 100x10 6 , at least about 110x10 6 , at least about 120x10 6 , at least about 130x10 6 , at least about 140x10 6 , at least about 150x10 6 , at least about 160x10 6 , at least about 170x10 6 , at least about 180x10 6 , at least about 190x10 6 , or at least about 200x10 6 In one embodiment, the cells are concentrated to a density of about 40x10 6 ~about 200x10 6 , about 60x10 6 ~about 200x10 6 , about 80x10 6 ~about 200x10 6 , about 100x10 6 ~about 200x10 6 , about 120x10 6 ~about 200x10 6 , about 140x10 6 ~about 200x10 6 , about 160x10 6 ~about 200x10 6 , about 180x10 6 ~about 200x10 6 , about 40x10 6 ~Approx. 180x10 6 , about 60x10 6 ~Approx. 180x10 6 , about 80x10 6 ~Approx. 180x10 6 , about 100x10 6 ~Approx. 180x10 6 , about 120x10 6 ~Approx. 180x10 6 , about 140x10 6 ~Approx. 180x10 6 , about 160x10 6 ~Approx. 180x10 6 , about 40x10 6 ~about 160x106 , about 60x10 6 ~about 160x10 6 , about 80x10 6 ~about 160x10 6 , about 100x10 6 ~about 160x10 6 , about 120x10 6 ~about 160x10 6 , about 140x10 6 ~about 160x10 6 , about 40x10 6 ~Approx. 140x10 6 , about 60x10 6 ~Approx. 140x10 6 , about 80x10 6 ~Approx. 140x10 6 , about 100x10 6 ~Approx. 140x10 6 , about 120x10 6 ~Approx. 140x10 6 , about 40x10 6 ~about 120x10 6 , about 60x10 6 ~about 120x10 6 , about 80x10 6 ~about 120x10 6 , about 100x10 6 ~about 120x10 6 , about 40x10 6 ~about 100x10 6 , about 60x10 6 ~about 100x10 6 , about 80x10 6 ~about 100x10 6 In one embodiment, the cells are concentrated to a density of about 100x10 6 , about 110x10 6 , about 120x10 6 , about 130x10 6 , about 140x10 6 , about 150x10 6 , about 160x10 6 , about 170x10 6 , about 180x10 6 , about 190x10 6 , about 200x10 6 , about 210x10 6 , about 220x106 , about 230x10 6 , about 240x10 6 , or about 250x10 6 Concentrated to a density of 100 cells / mL.

[0008] In one embodiment, electroporation is performed at about 100x10 6 , about 110x10 6 , about 120x10 6 , about 130x10 6 , about 140x10 6 , about 150x10 6 , about 160x10 6 , about 170x10 6 , about 180x10 6 , about 190x10 6 , about 200x10 6 , about 210x10 6 , about 220x10 6 , about 230x10 6 , about 240x10 6 or about 250x10 6 In some embodiments, electroporation is performed at a cell density of about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5 μg DNA / 1×10 6 It is carried out in cells.

[0009] In one embodiment, the starting density of the cell culture after transfection is 5x10 6 ~20x10 6cells / mL. In an embodiment, the cell culture after transfection is temperature shifted for a portion of the culture period. In an embodiment, the temperature of the cell culture after transfection is shifted for a portion of the culture period. In an embodiment, the temperature of the cell culture after transfection includes a temperature shift on day 1 or day 2. In an embodiment, the temperature shift is a temperature shift from about 37° C. to about 32° C. In an embodiment, the temperature shift is a temperature shift from about 37° C. to about 34° C. In an embodiment, the cell culture after transfection includes the addition of N,N-dimethylacetamide (DMA). In an embodiment, DMA is added at about 0.125% v / v or about 0.250% v / v. In an embodiment, DMA is added at between about 0.125% v / v and about 0.250% v / v. In an embodiment, the cell culture after transfection includes the addition of sodium butyrate (NaBu). In an embodiment, NaBu is added at a concentration between about 1 mM and about 2 mM.

[0010] In some embodiments, the protein yield of the method is at least about 0.1 g / L, at least about 0.2 g / L, at least about 0.3 g / L, at least about 0.4 g / L, at least about 0.5 g / L, at least about 0.6 g / L, at least about 0.7 g / L, at least about 0.8 g / L, at least about 0.9 g / L, at least about 1 g / L, at least about 2 g / L, at least about 3 g / L, at least about 4 g / L, at least about 5 g / L, or at least about 6 g / L after 7 days of culture. In some embodiments, the protein yield is up to 2 g / L after 14 days of culture.

[0011] The present disclosure relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: (i) culturing eukaryotic cells under continuous ATF perfusion prior to transfection; (ii) Prior to transfection, cells were cultured at 40–200 × 10 cells using continuous ATF perfusion. 6 concentrating to a density of cells / mL; (iii) transiently transfecting eukaryotic cells with a polynucleotide encoding the recombinant protein using flow electroporation; and (iv) culturing the transiently transfected eukaryotic cells after transfection; The present invention relates to a method for producing recombinant proteins in a bioreactor in high yield and / or high quality, comprising:

[0012] In some embodiments, the method produces recombinant protein that exhibits product quality equivalent to that of the clonal material. In some embodiments, the post-transfection culture produces (i) about 15x10 6 (ii) cell culture starting density in cells / mL; (ii) about 0.125% (v / v / ) DMA; (iii) about 1 mM NaBu; and (iv) temperature shift on day 1 from about 36.5° C. to about 32° C., wherein the volume of said bioreactor is between about 250 mL and about 5 L. In an embodiment, the product quality attribute value of the recombinant protein obtained by transiently transfecting eukaryotic cells exhibits ±10% of the product quality attribute value of the recombinant protein obtained by stable transfection. In an embodiment, the quality attribute is: (i) Protein aggregates; (ii) reduced and non-reduced species; (iii) charge variants (charge isomers); (iv) glycosylation profile; and (v) any combination thereof; is selected from the group consisting of:

[0013] In some embodiments, the quality characteristics of the protein aggregates are selected from the group consisting of: (i) percentage of high molecular weight species (HMW%); (ii) percentage of monomeric species; and (iii) any combination thereof. In some embodiments, the quality characteristics of the protein aggregates are determined using HPLC size exclusion chromatography. In some embodiments, the quality characteristics of the reduced and non-reduced species are selected from the group consisting of: (i) percentage of reduced species recombinant protein; (ii) percentage of non-reduced species recombinant protein; and (iii) any combination thereof. In some embodiments, the quality characteristics of the reduced and non-reduced species are determined using capillary electrophoresis under reducing and non-reducing conditions (CE-SDS). In some embodiments, the quality characteristics of the charge variants are selected from the group consisting of: (i) percentage of basic variants; (ii) percentage of acidic variants; (iii) percentage of major species; and (iv) any combination thereof. In some embodiments, the quality characteristics of the charge variants are determined by analyzing the isoelectric distribution by capillary isoelectric focusing (iCIEF). In some embodiments, the glycosylation profile comprises one or more N-linked glycans. In some embodiments, the N-linked glycan comprises (mannose-3-N-acetylglucosamine-4-fucose) (GOF), mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), mannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F), monosialylated mannose-3-N-acetylglucosamine-4-galactose-1-fucose (S1G1F), monosialylated mannose-3-N-acetylglucosamine-4-galactose-2-fucose (S1G2F), monosialylated mannose-3-N-acetylglucosamine-4-galactose-3-fucose (S2G3F), disialylated mannose-3-N-acetylglucosamine-4-galactose-2-fucose (S2G2F), or any combination thereof. In one embodiment, the quality characteristic of the glycosylation profile is selected from the group consisting of: (i) percentage of G1F; (ii) percentage of G0F; (iii) percentage of G2F; (iv) percentage of total non-fucosylated protein; and (v) any combination thereof.In some embodiments, the quality characteristic of the glycosylation profile is determined using an HPLC method, in some embodiments, the HPLC method is ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR).

[0014] The present disclosure also provides a recombinant protein obtained according to any of the methods disclosed above. In some embodiments, the recombinant protein is an antibody or antigen-binding fragment that binds to an antigen selected from the group consisting of PD-1, PD-L1, CVTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, EGFR, HER3, and HER4.

[0015] The present disclosure also provides a pharmaceutical composition comprising a recombinant protein obtained according to any of the methods disclosed above. Also provided is a cell or a plurality of cells obtained according to any of the methods disclosed above. In an embodiment, the cell or a plurality of cells is a Chinese Hamster Ovary (CHO) cell. The present disclosure also provides a bioreactor for producing a recombinant protein produced according to any of the methods disclosed above. Also provided is a bioreactor comprising a cell or a plurality of cells disclosed above. The present disclosure also provides a system for high yield production of recombinant protein by transient transfection, comprising: (i) a bioreactor; (ii) an ATF perfusion system; and (iii) an electroporation transfection system. In an embodiment, the system is used to carry out the methods disclosed above or to obtain a composition (e.g., recombinant protein).

[0016] The present disclosure also provides a method for accelerating or shortening the development timeline of a recombinant protein, comprising: (i) culturing eukaryotic cells in a medium to enrich eukaryotic cells to a high density; and (ii) transiently transfecting the eukaryotic cells with a polynucleotide encoding the recombinant protein using electroporation. Also provided is a method for reducing host cell toxicity of a recombinant protein, comprising: (i) culturing eukaryotic cells in a medium to enrich eukaryotic cells to a high density; and (ii) transiently transfecting the eukaryotic cells with a polynucleotide encoding the recombinant protein using electroporation. The present disclosure also provides a method for producing a recombinant protein for a personalized therapeutic, comprising: (i) culturing eukaryotic cells in a medium to enrich eukaryotic cells to a high density; and (ii) transiently transfecting the eukaryotic cells with a polynucleotide encoding the recombinant protein using electroporation. [Brief description of the drawings]

[0017] [Figure 1] Figures 1A-1E show plots of the main effects of key transient gene expression process parameters on shake flask screening. Figure 1A: Fold change (FC) titer versus starting density. Figure 1B: Time of application of temperature shift at 32 °C. Figure 1C: Addition of N,N-dimethylacetamide (DMA). Figure 1D: Addition of sodium butyrate (NaBu). Figure 1E: DNA:cell ratio (μg DNA / 106 cells / mL). Error bars are represented as 1 standard deviation. [Diagram 2] Figures 2A-2D show bioreactor performance for N-1 seed perfusion and in-process characteristics (N=4). Figure 2A: Viable cell density (VCD, x106 cells / mL) and viability determined by trypan blue dye exclusion using a Vi-CELL instrument. Figure 2B: Glucose (g / L) levels. Figure 2C: Lactate (g / L) levels. Figure 2D: Ammonium (mM) levels. All levels were determined offline by optical measurements using a BioProfile FLEX2® instrument. Error bars are represented as 1 standard deviation. [Diagram 3] Figures 3A-3H show that continuous ATF perfusion enrichment improves cell performance, cell replication and final titer before transfection. Figures 3A and 3E: Glucose concentration after N-1 seed perfusion enrichment. Figures 3B and 3F: G0 / G1, S and G2 / M phase distribution in cells before transfection determined by Guava cell cycle reagent. Figures 3C and 3G: Cell culture titer at day 14. Figures 3D and 3G: Transfection efficiency determined at day 2 by direct staining of surface IgG with AF 488 anti-human IgG (H+L). Continuous perfusion (N=3) and discontinuous perfusion (N=1). Statistical differences were calculated by t-test analysis and error bars are represented as 1 standard deviation. [Figure 4] Figures 4A-4B show the development strategy of a transient gene expression process in an AMBR® 250 bioreactor that resulted in several reactor conditions with production titers >1 g / L. Figure 4A: Main effect plots for each individual process parameter. Figure 4B: Titers of the top production conditions. Common levels across all top conditions are highlighted. [Diagram 5] Figures 5A-5E show successful scale-up of the transient gene expression process in a 5 L reactor. Figure 5A: Final titer in an AMBR® 250 A18 reactor (circles) and scale-up process in a 5 L reactor (squares). Figure 5B: Viable cell density (VCD, x106 cells / mL) and viability measured by trypan blue exclusion using a Vi-CELL instrument. Figure 5C: Lactate levels (g / L). Figure 5D: Ammonium levels (mM). Lactate and ammonium levels determined offline by optical measurements using a BioProfile FLEX2® instrument. Figure 5E: Lactate dehydrogenase (LDH) levels (U / L) determined offline using a CEDEX® Bio HT® analyzer. [Figure 6]Figures 6A-6D show that the transient to stable material in terms of product quality attributes at day 14 harvest for fed-batch cell culture capacity for mAb production using AMBR® 250 (n=1) and 5 L (n=2) bioreactors. Figure 6A: Size Exclusion Chromatography (SEC). Figure 6B: Capillary Electrophoresis under reducing and non-reducing conditions. Figure 6C: Capillary Electrophoresis (iCE). Figure 6D: N-glycosylation profile. [Figure 7] FIG. 7 is a schematic comparing transient gene expression with expression in stable cell lines, demonstrating that transient gene expression allows for rapid production of drug substance. [Figure 8] FIG. 8 is a schematic diagram illustrating the potential use of fugitive agents produced according to the methods of the present disclosure to treat patients with unmet clinical need. [Figure 9] Figure 9 is a schematic diagram of a representative workflow for transient gene expression described herein, which is capable of high protein yields and is feasible on a large scale. Detailed Description of the Invention

[0018] Traditional stable cell line development (CLD) producing a single clone typically requires 3-5 months. Therefore, transient expression of therapeutics is routinely used to rapidly produce small amounts of candidate proteins for preclinical testing, shortening timelines and reducing overall costs. However, issues with low productivity, heterogeneity in the quality of expressed proteins, and scalability have inhibited the use of transient gene expression outside of non-GLP preclinical testing. The present disclosure provides a large-scale transient expression platform for rapid production of proteins (e.g., monoclonal antibodies (mAbs)) using a eukaryotic cell platform (e.g., CHO cells) using large-scale (e.g., up to 5 L or greater scale) flow-type electroporation technology. The platform can produce gram quantities of proteins that exhibit a product quality profile comparable to proteins produced in stable cell lines. The present disclosure also provides a method for perfusion using a tangential flow filtration system, e.g., an alternating tangential flow filtration (ATF) system, followed by concentrating the host cells to high densities (e.g., 40x10 6 ~200x10 6 Density of cells / mL, e.g., approximately 100x10 6 They described that a high-throughput mAb-based recombinant mAb with a densitometry of 1000 ng / mL could be achieved by flow electroporation. Extensive screening studies to optimize cell expansion, transfection, and process conditions were performed to achieve high productivity and the desired product quality profile. With overall process improvements, transient titers of model mAbs reached up to 2 g / L at a scale of up to 5 L, and the quality of the transient recombinant products was comparable to that of recombinant products obtained from stably transfected host cells.

[0019] term In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless expressly indicated otherwise herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout this specification.

[0020] The singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. The term "a" (or "an"), as well as the terms "one or more" and "at least one," can be used interchangeably herein. In certain embodiments, the term "a" or "an" means "one." In other embodiments, the term "a" or "an" includes "two or more" or "plural."

[0021] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two features or components it identifies, regardless of the presence or absence of the other. Thus, when the term "and / or" is used herein in a phrase such as "A and / or B", it is intended to include "A and B", "A or B", "A" (single) and "B" (single). Similarly, when the term "and / or" is used in a phrase such as "A, B and / or C", it is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0022] The term "about" or "essentially comprising" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, the error range depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within 1 or more standard deviations per practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is presented in this application and claims, unless otherwise stated, the meaning of "about" or "essentially comprising" should be considered to be within an acceptable error range for that particular value or composition.

[0023] Where embodiments are described herein with the term "comprising," it is understood that analogous embodiments described in connection with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0024] As used herein, the term "approximately" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than the reference value) of the stated reference value (reference value), unless otherwise stated or clear from the context (except when such value exceeds 100% of possible values).

[0025] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the described range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of an integer), unless otherwise stated.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press are provided to those skilled in the art with many of the general dictionaries of the terms used in this disclosure.

[0027] Units, prefixes, and symbols are expressed in their International System of Units (SI) accepted form. The headings provided herein are not intended to limit the various aspects of this disclosure, which may be understood by reference to the specification as a whole. Thus, defined terms are more fully defined by reference to the specification as a whole.

[0028] Abbreviations used herein are defined throughout this disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0029] The terms "purified," "separated," or "isolated," as used interchangeably herein, refer to increasing the purity of a protein of interest from a composition or sample that contains the protein of interest and one or more impurities. Typically, the purity of a protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0030] As used herein, the terms "culture," "cell culture," and "eukaryotic cell culture" refer to a population of cells attached to a surface or in suspension that is maintained or grown in a medium (see definition of "medium" below) under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, these terms as used herein can refer to a combination that includes the cell population and the medium in which the population is suspended.

[0031] As used herein, the terms "medium(s)", "cell culture medium", "culture medium", "tissue culture medium(s)", "growth medium" refer to a solution containing nutrients that can be used to nourish growing cultured host cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids and trace elements required by cells for minimal growth and / or survival. The solution may contain components that promote growth and / or survival above a minimal rate, such as hormones and growth factors. The solution is adjusted to an optimal pH and salt concentration for cell survival and growth. The medium may also be a "defined medium" or a "chemically defined medium" - a serum-free medium that does not contain proteins, hydrolysates or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. Those skilled in the art will appreciate that defined media may include, but are not limited to, recombinant glycoproteins or proteins, such as hormones, cytokines, interleukins and other signaling molecules. Without limitation, these solutions typically provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all the essential amino acids, usually a basic set of 20 amino acids plus cysteine; (3) vitamins and / or other organic compounds required in low concentrations; (4) free fatty acids or lipids, e.g., linoleic acid; and (5) trace elements, defined as inorganic compounds or naturally occurring elements required in very low concentrations, usually in the micromolar range.The nutrient solution may be selectively supplemented with one or more components from any of the following categories: (1) hormones and other growth factors, such as serum, insulin, transferrin, and epidermal growth factor; (2) salts, such as magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; (5) protein and tissue hydrolysates, such as purified gelatin, peptones or peptone mixtures that may be obtained from plant materials or animal by-products; (6) antibiotics, such as gentamicin; (7) cytoprotectants, such as pluronic polyols; and (8) galactose. For culturing the host cells, commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), Sigma, RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), (Sigma) are suitable. In addition, any of the media described in Ham et al., Meth. Enz. 58:44(1979), Barnes et al., Anal. Biochem.102:255(1980) can be used as a culture medium for the host cells. Other necessary supplementary components can also be included at appropriate concentrations.

[0032] As used herein, "culturing" refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culture conditions that may be defined include temperature, gas mixture, time, and medium composition.

[0033] As used herein, "expanding" refers to culturing one or more cells in vitro for the purpose of obtaining a greater number of cells in culture.

[0034] As used herein, the term "expression" or "expressing" is used to refer to the transcription and translation that occurs in a cell. The expression level of a product gene in a host cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell, or both.

[0035] As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term also refers to the proportion of cells that are viable at a particular time relative to the total number of live and dead cells in the culture at that time.

[0036] In some embodiments, the medium comprises a sufficient concentration of glucose to maintain a high cell density, in some embodiments, the glucose concentration in the medium is at least about 0.1 g / L, at least about 0.5 g / L, at least about 1.0 g / L, at least about 1.5 g / L, at least about 2.0 g / L, at least about 2.5 g / L, at least about 3.0 g / L, at least about 3.5 g / L, at least about 4.0 g / L, at least about 4.5 g / L, at least about 5.0 g / L, at least about 6.0 g / L, at least about 7.0 g / L, at least about 8.0 g / L, at least about 9.0 g / L, or at least about 10.0 g / L.

[0037] In certain embodiments, the glucose concentration in the medium is about 0.1 g / L, about 0.5 g / L, about 1.0 g / L, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, about 3.0 g / L, about 3.5 g / L, about 4.0 g / L, about 4.5 g / L, about 5.0 g / L, about 6.0 g / L, about 7.0 g / L, about 8.0 g / L, about 9.0 g / L, or about 10.0 g / L.

[0038] The term "reference method", "reference process" or "baseline conditions" as used herein refers to a process or method for producing the same recombinant protein that is identical to the disclosed method except for the specific conditions used in the disclosed method to increase protein yield using transient expression. For example, a reference process or method can be a process that does not include (i) transient transfection by electroporation, e.g., flow-type electroporation, (ii) cell concentration using tangential flow filtration (e.g., alternating tangential flow filtration), (iii) perfusion cell culture, e.g., continuous perfusion; or (iv) a combination thereof. A reference method can be used as a baseline ("baseline conditions") to which the disclosed method is compared. In some embodiments, the value of a parameter (e.g., protein yield) measured after applying the disclosed method can be normalized to the baseline conditions.

[0039] The term "antibody" refers, in certain embodiments, to a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies (e.g., naturally occurring IgG antibodies), the heavy chain constant region is composed of a hinge and three domains, namely, CH1, CH2 and CH3. In certain antibodies (e.g., naturally occurring IgG antibodies), each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain (abbreviated herein as CL). The VH and VL regions are further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term "antibody" can include bispecific or multispecific antibodies.

[0040] As used herein, "IgG antibodies", e.g., human IgG1, IgG2, IgG3 and IgG4 antibodies, in certain embodiments have the structure of a naturally occurring IgG antibody, i.e., have the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an IgG1, IgG2, IgG3 or IgG4 antibody may be composed of two heavy chains (HC) and two light chains (LC), the two HCs and LCs being linked by the same number and positions of disulfide bonds as present in naturally occurring IgG1, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been mutated to alter the disulfide bonds).

[0041] Immunoglobulins can be obtained from any of the commonly known isotypes (e.g., IgA, secretory IgA, IgG, and IgM, but are not limited thereto). IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans; and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins (e.g., IgG1) exist in several allotypes, which differ from each other by at most a few amino acids. "Antibody" includes, by way of example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, and totally synthetic antibodies.

[0042] The term "antigen-binding portion" of an antibody as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (papain cleavage fragment) or similar monovalent fragments consisting of the VL, VH, LC and CH1 domains; (ii) a F(ab')2 fragment (pepsin cleavage fragment) or similar bivalent fragments that contain two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR) and (vii) a combination of two or more isolated CDRs that can be optionally linked by a synthetic linker, if desired. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that can be engineered using recombinant methods to produce a single-chain protein in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art and screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0043] The term "recombinant human antibody" as used herein includes all human antibodies produced, expressed, created or isolated by recombinant means, including, for example, (a) antibodies isolated from animals transgenic or transchromosomal animals (e.g., mice) of human immunoglobulin genes or hybridomas produced therefrom, (b) antibodies isolated from host cells transformed to express the antibody (e.g., transfectomas), (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies produced, expressed, created or isolated by other means, including splicing of human immunoglobulin sequences to other DNA sequences.

[0044] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies) that is encoded by heavy chain constant region genes.

[0045] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted one-letter codes.

[0046] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The terms "polypeptide" or "protein" or "product" or "produced protein" or "amino acid residue sequence" are used interchangeably. The term "polypeptide" refers to a chain of two or more amino acids and does not refer to a specific length of the product. As used herein, the term "protein" is intended to encompass molecules composed of one or more polypeptides, which may optionally be linked by bonds other than amide bonds. On the other hand, a protein may also be a single polypeptide chain. In this latter example, a single polypeptide chain may optionally contain two or more polypeptide subunits that are fused together to form a protein. The terms "polypeptide" and "protein" refer to the products of post-expression modifications, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide or protein may be obtained from a natural biological source or produced by recombinant technology.

[0047] The term "polynucleotide" or "nucleotide" as used herein is intended to encompass one nucleic acid and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), complementary DNA (cDNA) or plasmid DNA (pDNA). The term "nucleic acid" refers to one or more nucleic acid segments present in a polynucleotide, e.g., DNA, cDNA or RNA fragments. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA or RNA, that has been removed from its native environment, e.g., a recombinant polynucleotide encoding an antigen-binding protein contained in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides that have been purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the disclosure. Additionally, isolated polynucleotides or nucleic acids according to the disclosure include said molecules that have been produced synthetically. In addition, a polynucleotide or nucleic acid can include regulatory elements, such as a promoter, enhancer, ribosomal binding site, or transcription termination signals.

[0048] I. Methods for large-scale transient expression of therapeutic proteins The present disclosure relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: (i) culturing eukaryotic cells in a medium to enrich for eukaryotic cells at a high density; and (ii) transiently transfecting a eukaryotic cell with a polynucleotide encoding the recombinant protein using electroporation; In one embodiment, the method further comprises (iii) culturing the transiently transfected eukaryotic cells after transfection.

[0049] The present disclosure also provides a method for producing ... semiconductor device comprising the steps of: (i) culturing eukaryotic cells in a medium to enrich for eukaryotic cells at a high density; and (ii) transiently transfecting a eukaryotic cell with a polynucleotide encoding the recombinant protein using electroporation; The present invention provides a method for high quality production of a recombinant protein (e.g., a therapeutic antibody) in a bioreactor (e.g., a high throughput bioreactor or a large volume bioreactor), comprising: (iii) culturing the transiently transfected eukaryotic cells after transfection.

[0050] In addition, the following process: (i) culturing eukaryotic cells in a medium to enrich for eukaryotic cells at a high density; and (ii) transiently transfecting a eukaryotic cell with a polynucleotide encoding the recombinant protein using electroporation; The present invention provides a method for producing a recombinant protein (e.g., a therapeutic antibody) in a bioreactor (e.g., a high-throughput bioreactor or a large-volume bioreactor) in high yield and / or high quality, comprising: In one embodiment, the method further comprises culturing the transiently transfected eukaryotic cells after transfection.

[0051] The methods of transient gene expression disclosed herein can rapidly generate drug substance material (see Figure 7). In stable cell line expression (typically used for cGMP production), the transgene is integrated into the host genome and stably expressed for many generations, whereas in transient gene expression (used primarily for research and development), the transgene is encoded on a plasmid instead of integrated into the genome, and protein synthesis ceases when the vector is degraded or diluted by cell division. The methods disclosed herein allow for the use of transient gene expression beyond research and development, for example, accelerating the timeline for cGMP production. Most importantly, the transient technology disclosed herein can generate material that is representative of that produced by stable cell lines.

[0052] Methods of producing high quality material using transient gene expression can be used, for example, to (i) expedite development timelines, (ii) avoid host cell toxicity, or (iii) develop personalized therapeutics (Figure 8). The potential for expedited development timelines allows for further therapeutic development at a pandemic pace, for example, producing pharmaceuticals to treat AIDS, H1N1 influenza, or COVID19. In some instances, recombinant products can be toxic to host cells. The transient gene methods disclosed herein can provide an alternative platform to constitutive expression of recombinant proteins that are toxic to host cells by reducing the time that cells are exposed to toxic recombinant products. Another application of the transient gene expression disclosed herein is the development of personalized therapeutics. This allows for the production of high quality pharmaceuticals to treat rare and orphan diseases via small-scale manufacturing pipelines.

[0053] A workflow for transient gene expression that can be used for the applications outlined above is shown in Figure 9. The workflow shown in Figure 9 may, for example, include the following: (i) Replacing the perfusion system with a larger system or running multiple equivalent systems in parallel; (ii) replacing the flow-type electroporation system with a larger system or running multiple equivalent systems in parallel; (iii) increasing the scale of the culture vessel after cell harvest; or (iv) combining them; This allows for scale expansion.

[0054] To scale up the culture vessel after cell harvest, shake flasks can be used in combination with high-throughput reactors (e.g., AMBRs). (登録商標) The scale-up of the culture vessel may include substituting a larger capacity production vessel, such as a 10 L, 50 L, 100 L, or larger capacity bioreactor. In some embodiments, the scale-up of the culture vessel may include expanding to a 10 L, 50 L, 100 L, or larger capacity bioreactor. In some embodiments, the workflow can be scaled up by using a larger capacity high throughput bioreactor or multiple high throughput bioreactors in series. Similarly, multiple lower volume bioreactors with equivalent total volume can be used in place of a larger capacity bioreactor (e.g., a 10 L bioreactor) (e.g., two 5 L bioreactors).

[0055] The present disclosure provides a method for accelerating or shortening the development timeline of a recombinant protein (e.g., a therapeutic antibody), comprising the steps of: (i) culturing eukaryotic cells in a medium to enrich for eukaryotic cells at a high density; and (ii) transiently transfecting a eukaryotic cell with a polynucleotide encoding the recombinant protein using electroporation; Includes.

[0056] In some embodiments, the method further comprises culturing the transiently transfected eukaryotic cells after transfection. In some embodiments, development is shortened by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% compared to a normal development timeline.

[0057] The present disclosure provides a method for reducing host cell toxicity of a recombinant protein (e.g., a therapeutic antibody), comprising the steps of: (i) culturing eukaryotic cells in a medium to enrich for a high density of eukaryotic cells; and (ii) transiently transfecting a eukaryotic cell with a polynucleotide encoding the recombinant protein using electroporation; Includes.

[0058] In some embodiments, the method further comprises culturing the transiently transfected eukaryotic cells after transfection, hi some embodiments, the host toxicity of the cells is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the host cell toxicity observed in a stably transfected cell line.

[0059] The present disclosure provides a method for producing a recombinant protein for personalized therapy (e.g., a therapeutic antibody), comprising the steps of: (i) culturing eukaryotic cells in a medium to enrich for eukaryotic cells at a high density; and (ii) transiently transfecting a eukaryotic cell with a polynucleotide encoding the recombinant protein using electroporation; Includes.

[0060] In some embodiments, the method further comprises culturing the transiently transfected eukaryotic cells after transfection. In some embodiments, the recombinant protein for personalized therapy (e.g., therapeutic antibody) is a therapeutic agent for treating rare diseases. In some embodiments, the recombinant protein for personalized therapy (e.g., therapeutic antibody) is a therapeutic agent for treating rare diseases. In some embodiments, the recombinant protein for personalized therapy (e.g., therapeutic antibody) is a therapeutic agent for personalized therapy.

[0061] In one embodiment, cells are concentrated to high density prior to transient transfection while being maintained in optimal growth phase.

[0062] Post-transfection culture of transiently transfected eukaryotic cells according to the present disclosure produces recombinant protein (transient transfectant) with product quality equivalent to that of the clonal material.

[0063] As used herein, the term "transient transfection agent" refers to a recombinant protein or combination thereof obtained by a production process resulting from the use of transiently transfected cells as described in this disclosure.

[0064] As used herein, the term "clonal material" refers to a recombinant protein or combination thereof obtained by a production process that includes the use of stably transfected cells. In some embodiments, the clonal material is produced by a process that includes upstream steps (cell culture conditions, cell culture medium, etc.) and downstream steps (e.g., harvesting, filtration steps, chromatography steps) that are identical or substantially identical to those used to produce the transient material, except that the cells are stably transfected.

[0065] As used herein, the term "product quality equivalent to clone material" refers to a transient transfection material having quality characteristics selected from the group consisting of, for example, (i) protein aggregates; (ii) reduced and non-reduced species; (iii) charge variants; (iv) glycosylation profile; and (v) any combination, which differs by less than about 1%, 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% from the value for the corresponding characteristic or combination thereof determined for the clone material.

[0066] The term "optimal growth phase" as used herein refers to cells in culture growing such that the majority of the cells are in the replicative phase (S and G2 / M). In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% of the cells are in the replicative phase. In some embodiments, about 60% to about 80% of the cells are in the replicative phase. In some embodiments, about 65% to about 75% of the cells are in the replicative phase. In some embodiments, about 50% of the cells are in the replicative phase. In some embodiments, about 60% of the cells are in the replicative phase. In some embodiments, about 70% of the cells are in the replicative phase. In some embodiments, about 75% of the cells are in the replicative phase. In some embodiments, about 80% of the cells are in the replicative phase.

[0067] In some embodiments, the medium in (i) contains a sufficient concentration of glucose to maintain a high cell density. In some embodiments, the glucose concentration in the medium is at least about 0.1 g / L, at least about 0.5 g / L, at least about 1.0 g / L, at least about 1.5 g / L, at least about 2.0 g / L, at least about 2.5 g / L, at least about 3.0 g / L, at least about 3.5 g / L, at least about 4.0 g / L, at least about 4.5 g / L, or at least about 5.0 g / L. In some embodiments, the glucose concentration in the medium is between about 2 g / L and about 6 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L.

[0068] The methods of the present disclosure can be carried out in a variety of vessel types or bioreactors to accommodate a variety of recombinant protein production strategies. In some embodiments, the bioreactor is a high-throughput bioreactor, e.g., an AMBR® 250 bioreactor or an AMBR® 15 bioreactor. In some embodiments, the high-throughput bioreactor is a system for parallel fermentation. In some embodiments, each bioreactor in the high-throughput bioreactor has a volume between 100 mL and 250 mL, e.g., a volume of 100 mL, 150 mL, 200 mL, or 250 mL. In some embodiments, the high-throughput bioreactor includes multiple separate bioreactors, e.g., 12 or 24 bioreactors. In some embodiments, each bioreactor is a single-use bioreactor. In some embodiments, the high-throughput bioreactor is a fully automated system that controls, e.g., medium fill, inoculation, sampling, feed rate, temperature, impeller speed, pH, optical density (OD), or any combination thereof.

[0069] In some embodiments, the bioreactor (e.g., a high-throughput bioreactor such as an AMBR® 250 bioreactor) includes at least one cell culture analyzer. In some embodiments, the cell culture analyzer is a pH measurement module, e.g., an AMBR® Analysis Module. In some embodiments, the cell culture analyzer is an integrated cell counter, e.g., a Beckman Vicell XR cell counter or a Cedex HiRes cell counter. In some embodiments, the cell culture analyzer includes a Nova Biomedical FLEX2 External Sampling Module (ESM) and a FLEX2 Analyzer, or a combination thereof. In some embodiments, automated sample collection, sample transfer and analysis, data transfer, and automated feedback control are performed by the cell culture analyzer (e.g., a FLEX2 analyzer). In some embodiments, control operations such as addition of glucose or feed substances based on glucose levels, cell counts, viable cell counts, or other parameters are performed by the cell culture analyzer (e.g., a FLEX2 analyzer).

[0070] In some embodiments, the bioreactor is a large-volume bioreactor. As used herein, the term "large-volume bioreactor" refers to a bioreactor having a volume of 1 L, 2 L, 5 L, 10 L, or more, such as about 20 L, about 25 L, about 50 L, about 100 L, about 500 L, about 1000 L, about 5000 L, about 10,000 L, or about 20,000 L. In some embodiments, the term "large-volume bioreactor" refers to a volume of at least 1000 L, such as 1000 L to 10,000 L, such as 5000 L. In some embodiments, the term "large-volume bioreactor" refers to a volume of at least 2000 L.

[0071] The methods of the present disclosure may include fed-batch culture. Fed-batch culture is a method of culturing cells in which additional components are supplied to the medium at some point after the start of the culture process. Fed-batch culture may be initiated with a basal medium. The culture medium in which the culture is supplied with additional components at some point after the start of the culture process is a feed medium. Fed-batch culture is usually stopped at some point and the cells and / or components in the medium are harvested and optionally purified. Thus, in some embodiments of the present disclosure, the bioreactor is a fed-batch production bioreactor. In some embodiments, the bioreactor is a single-use bioreactor. In some embodiments, the bioreactor is a glass tank bioreactor.

[0072] The disclosed method can include perfusion culture. Perfusion culture involves flowing a physiological nutrient solution through or continuously over a cell population at a constant rate. Because perfusion systems generally keep the cells in a culture unit, perfusion culture is characterized by a relatively high cell density, but the culture conditions are difficult to maintain and control. In addition, because the cells are kept at high density in the culture unit after proliferation, the proliferation rate generally decreases continuously over time, leading to a late logarithmic or stationary phase of cell growth.

[0073] As used herein, "perfusion" or "perfusion culture" or "perfusion reactor process" refers to flowing a physiological nutrient solution through or over a cell population at a constant rate. Because perfusion systems generally involve the retention of cells in a culture unit, perfusion cultures are characterized by having relatively high cell densities, but the culture conditions are difficult to maintain and control. In addition, because cells are kept at high density in the culture unit after proliferation, the proliferation rate generally decreases continuously over time until the late logarithmic or stationary phase of cell growth. This culture strategy generally involves the culture of mammalian cells, e.g., non-anchorion-dependent cells, expressing a polypeptide and / or virus of interest during the proliferation phase of a continuous cell culture system. In some embodiments, the perfusion may be "continuous perfusion".

[0074] The term "continuous perfusion" refers to a steady-state cell culture system or process in which cells or microorganisms are maintained in culture by the continuous addition of new medium and operate without interruption. The term "continuous" means uninterrupted in time, sequence and / or operation for extended periods of time. When used in reference to the processes of the present disclosure, "continuous" means that the process is physically and logistically integrated and can operate without interruption for extended periods of time. In another embodiment, the perfusion can be "discontinuous perfusion."

[0075] "Non-anchorion dependent cells" refers to cells that grow freely in suspension throughout the bulk of the culture, as opposed to being attached or fixed to a solid substrate during growth. Continuous cell culture systems can include cell retention devices similar to those used in perfusion systems, but a significant portion of the cells can be continuously removed such that a smaller percentage of cells are retained than in perfusion cultures.

[0076] "Cell retention device" refers to any structure capable of retaining cells (especially non-anchorage-dependent cells) in a specific location during cell culture. Non-limiting examples include microcarriers, fine mesh spin filters, hollow fibers, flat membrane filters, sedimentation tubes, ultrasonic cell retention devices, etc., which can retain non-anchorage-dependent cells in a bioreactor. The polypeptide and / or virus of interest (e.g., recombinant polypeptide and / or recombinant virus) can be recovered from the cell culture system, e.g., from the medium removed from the cell culture system.

[0077] In some embodiments, perfusion cell culture is performed using tangential flow filtration. In some embodiments of the present disclosure, tangential flow filtration can also be used to concentrate cells to high density, for example, prior to transfection.

[0078] The term "tangential flow filtration" or "TFF", also known as cross-flow filtration, refers to a specific filtration method in which a solution containing solutes flows tangentially through an ultrafiltration membrane (tangential flow), and the application of pressure causes low molecular weight solutes to pass through the surface of the filter surface (e.g., filtration membrane). Tangential flow filtration is used in concentration processes, for example, to increase cell concentration in media such as cell culture media. As used herein, "tangential flow" refers to flow substantially parallel to the filter surface, e.g., unidirectional tangential flow (TFF) or cross-flow.

[0079] In a particular embodiment, the tangential flow filtration is alternating tangential flow filtration (ATF). The term "alternating tangential flow" as used herein refers to a flow arrangement in which a tangential flow moves back and forth along the membrane surface of a hollow fiber filter, and another flow moves substantially perpendicular to the filter surface. Tangential flow or alternating tangential flow can be performed according to methods known to those skilled in the art. For example, US 6,544,424 describes a method for generating alternating tangential flow in a hollow fiber filter.

[0080] The solution containing high molecular weight solutes that flow tangentially through the ultrafiltration membrane is retained, and is referred to herein as the "retentate." The low molecular weight solutes that pass through the ultrafiltration membrane are referred to herein as the "permeate." Thus, the permeate is concentrated by passing (e.g., tangentially) along the surface of the ultrafiltration membrane under pressure. The ultrafiltration membrane has a pore size with a cutoff value. In some embodiments, the cutoff value is about 50 kDa or less, e.g., 50 kDa, 40 kDa, 30 kDa, 20 kDa, or 10 kDa. In some embodiments, the cutoff value is 30 kDa or less.

[0081] In one embodiment, the system used to carry out the method disclosed herein includes a cell culture device including at least one filter-containing housing, a fluid connector for conducting cell culture fluid from a cell culture vessel (bioreactor) to the cell culture device, at least one pump for pumping fluid in one or alternate directions through the filter-containing housing, and a fluid filtration system including at least one fluid collection port. This system is useful for performing rapid and low shear tangential flow filtration. Such a system can be applied to perfusion cell culture systems or other culture systems that require the retention of viable cells in a bioreactor.

[0082] In some embodiments, perfusion culture and cell concentration are performed using a single-use device, such as, for example, the REPLIGEN® ATF system. In some embodiments, the REPLIGEN® ATF system is the Xcell ATF® 1 system (0.5 L to 2 L suspension culture volume), the Xcell ATF® 2 system (2 L to 10 L suspension culture volume), the Xcell ATF® 4 system (10 L to 50 L suspension culture volume), the Xcell ATF® 6 system (50 L to 200 L suspension culture volume) or the Xcell ATF® 10 system (200 L to 1000 L suspension culture volume). In some embodiments, the REPLIGEN® ATF system is connected to an XCell® Lab controller or an XCell® C410 controller. Similar devices known in the art, such as the EMD Millipore PROSTAK® TFF system, can be used to carry out the methods disclosed herein.

[0083] As used herein, the term "high density" when used in culturing cells according to the present disclosure and in the disclosed methods for the same, such as performing a transfection workflow, generally refers to a cell culture medium having a density of, for example, about 40x10 6 Up to a density of more than 40x10 cells / mL or approximately 6 cells / mL ~ approx. 200x10 6 High density refers to a known cell line or known cell line variant capable of growing or culturing between 200x10 cells / mL. In some specific embodiments, high density refers to a known cell line or known cell line variant capable of growing or culturing between 200x10 cells / mL. 6 Can refer to cells / mL or more.

[0084] In certain embodiments of the methods disclosed herein, the cells are cultured at a concentration of at least about 40×10 6 Cells / mL, at least about 50x10 6 Cells / mL, at least about 60x10 6Cells / mL, at least about 70x10 6 Cells / mL, at least about 80x10 6 Cells / mL, at least about 90x10 6 Cells / mL, at least about 100x10 6 Cells / mL, at least about 110x10 6 Cells / mL, at least about 120x10 6 Cells / mL, at least about 130x10 6 Cells / mL, at least about 140x10 6 Cells / mL, at least about 150x10 6 Cells / mL, at least about 160x10 6 Cells / mL, at least about 170x10 6 Cells / mL, at least about 180x10 6 Cells / mL, at least about 190x10 6 cells / mL or at least about 200x10 6 In some embodiments of the methods disclosed herein, the cells are concentrated to a density of about 40×10 cells / mL using a tangential flow filtration method such as ATF. 6 cells / mL, approximately 50x10 6 cells / mL, approximately 60x10 6 cells / mL, approximately 70x10 6 cells / mL, approximately 80x10 6 cells / mL, approximately 90x10 6 cells / mL, approximately 100x10 6 cells / mL, approximately 110x10 6 cells / mL, approximately 120x10 6 cells / mL, approximately 130x10 6 cells / mL, approximately 140x10 6 cells / mL, approximately 150x10 6 cells / mL, approximately 160x10 6 cells / mL, approximately 170x10 6 cells / mL, approximately 180x10 6 cells / mL, approximately 190x10 6 cells / mL, approximately 200x10 6 cells / mL, approximately 210x10 6 cells / mL, approximately 220x10 6 cells / mL, approximately 230x106 cells / mL, approximately 240x10 6 cells / mL or approximately 250x10 6 In one embodiment, the cells are concentrated to a density of about 40x10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 50x10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 60×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 70×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 80×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 90×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 100x10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 110×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 120×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 130×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 140×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 150×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 160×10 cells / mL using a tangential flow filtration method such as ATF.6 In one embodiment, the cells are concentrated to a density of about 170×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 180×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 190×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 200x10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 210×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 220×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 230×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 240×10 cells / mL using a tangential flow filtration method such as ATF. 6 In one embodiment, the cells are concentrated to a density of about 250×10 cells / mL using a tangential flow filtration method such as ATF. 6 Concentrated to a density of 100 cells / mL.

[0085] In one embodiment, the cells are filtered using a tangential flow filtration method such as ATF at a concentration of about 40×10 6 cells / mL ~ approx. 200x10 6 cells / mL, approximately 60x10 6 cells / mL ~ approx. 200x10 6 cells / mL, approximately 80x10 6 cells / mL ~ approx. 200x10 6 cells / mL, approximately 100x10 6 cells / mL ~ approx. 200x10 6 cells / mL, approximately 120x10 6 cells / mL ~ approx. 200x106 Cells / mL, about 140x10 6 Cells / mL~about 200x10 6 Cells / mL, about 160x10 6 Cells / mL~about 200x10 6 Cells / mL, about 180x10 6 Cells / mL~about 200x10 6 Cells / mL, about 40x10 6 Cells / mL~about 180x10 6 Cells / mL, about 60x10 6 Cells / mL~about 180x10 6 Cells / mL, about 80x10 6 Cells / mL~about 180x10 6 Cells / mL, about 100x10 6 Cells / mL~about 180x10 6 Cells / mL, about 120x10 6 Cells / mL~about 180x10 6 Cells / mL, about 140x10 6 Cells / mL~about 180x10 6 Cells / mL, about 160x10 6 Cells / mL~about 180x10 6 Cells / mL, about 40x10 6 Cells / mL~about 160x10 6 Cells / mL, about 60x10 6 Cells / mL~about 160x10 6 Cells / mL, about 80x10 6 Cells / mL~approximately 160 x10 6 Cells / mL, about 100x10 6 Cells / mL~about 160x10 6 Cells / mL, about 120x10 6 Cells / mL~about 160x10 6 Cells / mL, about 140x10 6 Cells / mL~about 160x10 6 Cells / mL, about 40x10 6 Cells / mL~about 140x10 6 Cells / mL, about 60x10 6 Cells / mL~about 140x10 6 Cells / mL, about 80x10 6 Cells / mL~about 140x10 6cells / mL, approximately 100x10 6 cells / mL ~ approx. 140x10 6 cells / mL, approximately 120x10 6 cells / mL ~ approx. 140x10 6 cells / mL, approximately 40x10 6 cells / mL ~ approx. 120x10 6 cells / mL, approximately 60x10 6 cells / mL ~ approx. 120x10 6 cells / mL, approximately 80x10 6 cells / mL ~ approx. 120x10 6 cells / mL, approximately 100x10 6 cells / mL ~ approx. 120 x10 6 cells / mL, approximately 40x10 6 cells / mL ~ approx. 100x10 6 cells / mL, approximately 60x10 6 cells / mL ~ approx. 100x10 6 cells / mL, approximately 80x10 6 cells / mL ~ approx. 100x10 6 Concentrated to a density of 100 cells / mL.

[0086] As used herein, the term "transfection" refers to the delivery of an exogenous nucleic acid (e.g., a polynucleotide encoding a recombinant protein) to a target or host cell such that the nucleic acid is expressed in the cell. The term "transient transfection" refers to a process in which the nucleic acid introduced into a cell does not require integration into the genome or chromosomal DNA of the cell. In fact, the nucleic acid is largely maintained in the cell as an extrachromosomal element, e.g., an episome. The transcription process of the episomal nucleic acid is not affected, e.g., the protein encoded by the episomal nucleic acid is produced. A "transiently transfected cell" refers to a cell that contains an exogenous nucleic acid that is not stably integrated into the genome / chromosome of the host cell. The exogenous nucleic acid may be transiently heritable or non-heritable. An example of transient transfection is a vector that is transfected into a cell but is not integrated into the chromosome, and may require additional selection pressure to remain in the cell host for a period of time (e.g., 1-5 days, or 2-3 days). The term "transient expression" refers to the expression of an exogenous nucleic acid (eg, a polynucleotide encoding a recombinant protein) to obtain a recombinant protein in a transiently transfected cell of the present disclosure.

[0087] The disclosed method includes using electroporation to facilitate the introduction of one or more nucleic acid molecules into a host cell. As used herein, the term "electroporation" refers to the use of an electric current or electric field on a cell to facilitate the introduction of nucleic acid molecules into the cell. Those skilled in the art will understand that any method and technique of electroporation is contemplated by the present invention. In some embodiments, cells are electroporated using flow-type electroporation. The term "flow-type electroporation" refers to the electroporation of cells in a fluid chamber flow path. This method is particularly effective for large volumes of cells. In contrast, static electroporation involves a certain limited volume of cell electroporation due to the limitations associated with electrical movement across liquid and the distance between opposing electrodes.

[0088] Two major advantages of using flow electroporation are the speed with which large cell populations can be transfected and the ability to overcome thermal degradation of the sample. In one embodiment, electroporation is performed using the MAXCYTE® Transfection System.

[0089] In certain embodiments, flow electroporation can be performed using a MAXCYTE™ STX®, MAXCYTE™ VLS®, or MAXCYTE™ GT® flow electroporation device. In some specific embodiments, the transfection system is a MAXCYTE® STX® transfection system. In some specific embodiments, the transfection system is a MAXCYTE™ VLX® transfection system.

[0090] The method of transfecting cells by electroporation disclosed herein, for example, using flow-type electroporation, can achieve a transfection efficiency of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Transfection efficiency can be measured either by the percentage of cells expressing the gene product or the secretion level of the product expressed by the gene. The cells maintain a high viability during and after the electroporation process. The viability of the electroporated cells can be at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% of the viability of the starting host cell population that is not electroporated or that is transfected with an electroporated control polynucleotide.

[0091] Flow-type electroporation includes, for example, transferring a suspension of cells and carrier molecules to a device that is composed of a fluid chamber or a fluid flow path; the fluid chamber or the fluid flow path includes electrodes arranged along the sides of the fluid chamber or the fluid flow path and arranged to subject the biological particles in the fluid chamber or the fluid flow path to an electric field suitable for electroporation; and transferring the electroporated cell suspension from the device to the outside. In some embodiments, the method disclosed herein includes using a flow-type electroporation device for electrical stimulation of a particle suspension, and includes a flow-type electroporation cell assembly having one or more inlet flow portals, one or more outlet flow portals and one or more flow paths, the flow paths including two or more walls, the flow paths further configured to receive and temporarily hold a continuous flow of particles in suspension from the inlet flow portal; and a pair of electrodes arranged relative to the flow path such that each electrode forms at least one wall of the flow path, and the electrodes are arranged to be electrically connected to an electrical energy source, so that the particle suspension flowing through the flow path can be subjected to an electric field formed between the electrodes.

[0092] The process begins with the installation of a flow cell containing the solution and cell suspension, and containers containing the required liquids and samples. The priming solution (saline) and cell suspension are introduced by entering the necessary commands into the electroporation system, which controls the operation of the pumps and pinch valves. As the cells pass through the flow path between the electrodes, an electric pulse of selected voltage, duration and frequency is applied. The product and waste liquids are collected in designated containers. The user enters the desired voltage and other parameters into the flow-type electroporation system of the present invention. A variety of settings are possible. The computer communicates with the electronics in the tower to charge the capacitor bank to the desired voltage. The voltage is then manipulated by appropriate switches to create an electric field before being delivered to the flow path (the switches provide selective pulse or burst switching to minimize wear on the electrodes caused by prolonged exposure to the electric field). The voltage is delivered according to the duration and frequency parameters set by the operator into the flow-type electroporation system of the present invention.

[0093] The flow-type electroporation process can be initiated, for example, by placing the electroporation chamber in fluid communication with the solution and cell suspension in the container (e.g., via tubing), which can be performed in a sterile or aseptic environment. The cell suspension and / or other reagents can be introduced into the electroporation chamber using one or more pumps, vacuums, valves, other mechanical devices that change the pressure or volume inside the electroporation chamber, and combinations thereof, which allows the cell suspension and / or other reagents to flow through the electroporation chamber for a desired time and at a desired rate. Once a portion of the cell suspension and / or other reagents is placed in the electroporation chamber, an electrical pulse of a desired voltage, duration, and / or interval is applied to the cell suspension and / or other reagents. After electroporation, the cell suspension and / or other reagents that have undergone this process can be removed from the electroporation chamber using one or more pumps, vacuums, valves, other electrical, mechanical, pneumatic, or microfluidic devices, and combinations thereof that change the displacement, pressure, or volume within the electroporation chamber. In certain embodiments, the sample or the sample after the process may be moved into or out of the electroporation chamber using gravity or manual movement. If necessary, new cell suspension and / or other reagents may be introduced into the electroporation chamber. The electroporated sample may be collected separately from the sample that has not yet been electroporated. The preceding series of events may be temporally coordinated, for example, by a computer linked to electronic circuitry (e.g., providing electrical pulses), pumps, vacuums, valves, combinations thereof, and other components that effectively control the flow of the sample into and out of the electroporation chamber.As an example, the electroporation process can be performed by a computer, for example, by an operator through a graphical user interface on a monitor and / or keyboard. Examples of suitable valves include pinch valves, butterfly valves, and / or ball valves. Examples of suitable pumps include centrifugal pumps or positive displacement pumps.

[0094] In one embodiment, electroporation, e.g., flow-type electroporation, is performed at about 1 μg DNA / 1×10 6 Cells ~ approx. 2μg DNA / 1x10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.5 μg DNA / 1×10 6 In some aspects, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1 μg DNA / 1×10 6 Cells, approximately 1.1μg DNA / 1x10 6 Cells, approximately 1.2μg DNA / 1x10 6 Cells, approximately 1.3μg DNA / 1x10 6 Cells, approximately 1.4μg DNA / 1x10 6 Cells, approximately 1.5μg DNA / 1x10 6 Cells, approximately 1.6μg DNA / 1x10 6 Cells, approximately 1.7μg DNA / 1x10 6 Cells, approximately 1.8μg DNA / 1x10 6 Cells, approximately 1.9μg DNA / 1x10 6 Cells or approximately 2μg DNA / 1x10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed on cells at about 1 μg DNA / 1×10 6 In some aspects, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.1 μg DNA / 1×10 6In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.2 μg DNA / 1×10 6 In some aspects, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.3 μg DNA / 1×10 6 In some aspects, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.4 μg DNA / 1×10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.5 μg DNA / 1×10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.6 μg DNA / 1×10 6 In some aspects, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.7 μg DNA / 1×10 6 In some aspects, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.8 μg DNA / 1×10 6 In some aspects, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 1.9 μg DNA / 1×10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a DNA to cell ratio of about 2 μg DNA / 1×10 6 This is done on a cell to cell ratio.

[0095] In one embodiment, electroporation, e.g., flow-type electroporation, is performed at about 40x10 6 cells / mL, approximately 50x10 6 cells / mL, approximately 60x10 6 cells / mL, approximately 70x10 6 cells / mL, approximately 80x106 cells / mL, approximately 90x10 6 cells / mL, approximately 100x10 6 cells / mL, approximately 110x10 6 cells / mL, approximately 120x10 6 cells / mL, approximately 130x10 6 cells / mL, approximately 140x10 6 cells / mL, approximately 150x10 6 cells / mL, approximately 160x10 6 cells / mL, approximately 170x10 6 cells / mL, approximately 180x10 6 cells / mL, approximately 190x10 6 cells / mL, approximately 200x10 6 cells / mL, approximately 210x10 6 cells / mL, approximately 220x10 6 cells / mL, approximately 230x10 6 cells / mL, approximately 240x10 6 cells / mL or approximately 250x10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of at least about 40x10 cells / mL. 6 Cells / mL, at least about 50x10 6 Cells / mL, at least about 60x10 6 Cells / mL, at least about 70x10 6 Cells / mL, at least about 80x10 6 Cells / mL, at least about 90x10 6 Cells / mL, at least about 100x10 6 Cells / mL, at least about 110x10 6 Cells / mL, at least about 120x10 6 Cells / mL, at least about 130x10 6 Cells / mL, at least about 140x10 6 Cells / mL, at least about 150x10 6 Cells / mL, at least about 160x10 6 Cells / mL, at least about 170x10 6 Cells / mL, at least about 180x10 6 Cells / mL, at least about 190x10 6Cells / mL, at least about 200x10 6 cells / mL, approximately 210x10 6 cells / mL, approximately 220x10 6 cells / mL, approximately 230x10 6 cells / mL, approximately 240x10 6 cells / mL or approximately 250x10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 40x10 cells / mL. 6 cells / mL ~ approx. 60x10 6 Between cells / mL, approximately 60x10 6 cells / mL ~ approx. 80x10 6 Between cells / mL, approximately 80x10 6 cells / mL ~ approx. 100x10 6 Between 100x10 cells / mL 6 cells / mL ~ approx. 120x10 6 Between cells / mL, approximately 120x10 6 cells / mL ~ approx. 140x10 6 Between cells / mL, approximately 140x10 6 cells / mL ~ approx. 160x10 6 Between cells / mL, approximately 160x10 6 cells / mL ~ approx. 180x10 6 Between cells / mL, approximately 180x10 6 cells / mL ~ approx. 200x10 6 Between cells / mL, approximately 50x10 6 cells / mL ~ approx. 75x10 6 Between cells / mL, approximately 75x10 6 cells / mL ~ approx. 100x10 6 Between 100x10 cells / mL 6 cells / mL ~ approx. 125x10 6 Between cells / mL, approximately 125x10 6 cells / mL ~ approx. 150x10 6 Between cells / mL, approximately 150x10 6 cells / mL ~ approx. 175x10 6 Between cells / mL, approximately 175x10 6 cells / mL ~ approx. 200x10 6 Between 100x10 cells / mL 6cells / mL ~ approx. 150x10 6 Between cells / mL, approximately 150x10 6 cells / mL ~ approx. 200x10 6 Between 100x10 cells / mL 6 cells / mL ~ approx. 200x10 6 cells / mL or approximately 200x10 6 cells / mL ~ approx. 250x10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of between about 40x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 50x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 60x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 70x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 80x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 90x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 100x10 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 110x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 120x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 130x10 cells / mL. 6In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 140x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 150x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 160x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 170x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 180x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 190x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 200x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 210x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 220x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 230x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 240x10 cells / mL. 6 In one embodiment, electroporation, e.g., flow-type electroporation, is performed at a cell density of about 250x10 cells / mL. 6 Cell densities are performed in cells / mL.

[0096] The eukaryotic cells used in the methods disclosed herein can be transfected with an expression vector or cloning vector to produce recombinant proteins and cultured appropriately. In some embodiments, standard molecular biology techniques can be used to produce recombinant expression vectors, culture cells, and recover products from culture medium. In some embodiments, the cell culture medium described herein can be used as a culture medium for hybridoma cells, monoclonal antibody producing cells, virus producing cells, transfected cells, cancer cells, and / or recombinant peptide producing cells.

[0097] An important aspect of the methods disclosed herein is the tuning of the perfusion reactor after concentration and transfection. In some embodiments, the perfusion system, e.g., the Xcell ATF® 2 system, is adjusted to replenish glucose levels. In some embodiments, the perfusion system, e.g., the Xcell ATF® 2 system, is adjusted to replenish glucose levels to approximately 4 g / L. In some embodiments, the perfusion system, e.g., the Xcell ATF® 2 system, is adjusted to replenish glucose levels to a level sufficient to prevent cells from entering quiescence. In some embodiments, the perfusion system, e.g., the Xcell ATF® 2 system, is adjusted to have at least 60% of the cells in replicative phase (S and G2 / M). In some embodiments, the perfusion system, e.g., the Xcell ATF® 2 system, is adjusted to have at least about 60%, at least about 65%, at least about 70%, or at least about 75% of the cells in replicative phase (S and G2 / M). In some embodiments, the use of continuous ATF perfusion can increase titers by approximately 3-fold compared to batch mode.

[0098] The cells of the present disclosure can be cultured under suitable conditions for a suitable period of time after transfection, and can be cultured under conditions that vary depending on the type of cells being cultured and the product being produced. In some embodiments, the cells are cultured for about 2 to about 14 days after transfection. In some embodiments, the cells are cultured for about 4 to 10 days. In some embodiments, the cells are cultured for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days. In certain embodiments, the cells are cultured for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or about 21 days. In certain embodiments, the cells are cultured for about 2 to about 5 days, about 3 to about 7 days, about 4 to about 10 days, about 5 to about 10 days, about 2 to about 7 days, about 10 to about 15 days, about 15 to about 20 days, about 7 to about 14 days, about 7 to about 21 days, or about 14 to about 21 days.

[0099] In one embodiment, the cell culture starting density after transfection is 5x10 6 ~15x10 6 In one embodiment, the cell culture starting density after transfection is between about 5x10 6 cells / mL, approximately 6x10 6 cells / mL, approximately 7x10 6 cells / mL, approximately 8x10 6 cells / mL, approximately 9x10 6 cells / mL, approximately 10x10 6 cells / mL, approximately 11x10 6 cells / mL, approximately 12x10 6 cells / mL, approximately 13x10 6cells / mL, approximately 14x10 6 cells / mL, approximately 15x10 6 cells / mL, approximately 16x10 6 cells / mL, approximately 17x10 6 cells / mL, approximately 18x10 6 cells / mL, approximately 19x10 6 cells / mL or approximately 20x10 6 In one embodiment, the cell culture starting density after transfection is at least about 5x10 6 Cells / mL, at least about 6x10 6 Cells / mL, at least about 7x10 6 Cells / mL, at least about 8x10 6 Cells / mL, at least about 9x10 6 Cells / mL, at least about 10x10 6 Cells / mL, at least about 11x10 6 Cells / mL, at least about 12x10 6 Cells / mL, at least about 13x10 6 Cells / mL, at least about 14x10 6 Cells / mL, at least about 15x10 6 Cells / mL, at least about 16x10 6 Cells / mL, at least about 17x10 6 Cells / mL, at least about 18x10 6 Cells / mL, at least approximately 19x10 6 cells / mL or at least about 20x10 6 In one embodiment, the cell culture starting density after transfection is about 5x10 6 cells / mL ~ approx. 10x10 6 Between cells / mL, approximately 10x10 6 cells / mL ~ approx. 15x10 6 Between cells / mL, approximately 15x10 6 cells / mL ~ approx. 20x10 6 Between cells / mL, approximately 5x10 6 cells / mL ~ approx. 15x10 6 Between 10x10 cells / mL or approximately 6 cells / mL ~ approx. 20x10 6In one embodiment, the cell culture starting density after transfection is between about 5x10 6 In one embodiment, the cell culture starting density after transfection is about 6x10 6 In one embodiment, the cell culture starting density after transfection is about 7x10 6 In one embodiment, the cell culture starting density after transfection is about 8x10 6 In one embodiment, the cell culture starting density after transfection is about 9x10 6 In one embodiment, the cell culture starting density after transfection is about 10x10 6 In one embodiment, the cell culture starting density after transfection is about 11x10 6 In one embodiment, the cell culture starting density after transfection is about 12x10 6 In one embodiment, the cell culture starting density after transfection is about 13x10 6 In one embodiment, the cell culture starting density after transfection is about 14x10 6 In one embodiment, the cell culture starting density after transfection is about 15x10 6 In one embodiment, the cell culture starting density after transfection is about 16x10 6 In one embodiment, the cell culture starting density after transfection is about 17x10 6 In one embodiment, the cell culture starting density after transfection is about 18x10 6 In one embodiment, the cell culture starting density after transfection is about 19x10 6 In one embodiment, the cell culture starting density after transfection is about 20x10 6 In cells / mL.

[0100] In some embodiments, the cell culture after transfection is temperature shifted for a portion of the culture period. In some embodiments, the cell culture after transfection is temperature shifted for a portion of the culture period. The temperature of a production vessel, such as a bioreactor, can be an important aspect of biological production, as it is involved in cell growth, viable cell density, cell lifespan, and / or glycosylation activity of intracellular glycosylation enzymes. Temperature changes can significantly affect the rate of enzymatic reactions in the cells, denature proteins, and / or cause other effects on the cell culture. After the cells are cultured at a set temperature, such as 37° C., to promote maximum viable cell density, the temperature can be changed to another set temperature (i.e., a second set temperature or a final set temperature) to extend cell lifespan or enhance desired glycosylation activity in the cells. In some embodiments, the methods disclosed herein include one or more temperature adjustments during protein production. In some embodiments, the temperature adjustment can be a decrease in the operating temperature during the manufacturing process.

[0101] In some embodiments, the post-transfection cell culture undergoes a temperature shift on day 1 or 2. In some embodiments, the initial set temperature of the method is about 37° C. and the second set temperature is less than about 36° C., e.g., about 35° C., about 34° C., about 33° C., about 32° C., or about 31° C.

[0102] In some embodiments, the initial set temperature is about 37° C. and the second set temperature is less than about 34° C. In some embodiments, the initial set temperature is about 36° C. and the second set temperature is less than about 35° C., about 34° C. or about 33° C. The initial set temperature is less than about 36.5° C. and the final set temperature is about 31° C. In some embodiments, the initial set temperature is about 36.0° C. and the final set temperature is about 31° C. In some embodiments, the initial set temperature is less than about 35.5° C. and the final set temperature is about 31° C. In some embodiments, the initial set temperature is less than about 35.0° C. and the final set temperature is about 31° C. In some embodiments, the initial set temperature is less than about 36.5° C., the second set temperature is about 33° C. and the final set temperature is less than about 33° C. or 32° C. In one embodiment, the initial setting temperature is about 36.5°C, the second setting temperature is about 33°C, and the final setting temperature is lower than about 33°C or 32°C. In one embodiment, the initial setting temperature is about 36.0°C, the second setting temperature is about 33°C, and the final setting temperature is lower than about 32°C. In one embodiment, the initial setting temperature is about 36.0°C, the second setting temperature is about 33°C, and the final setting temperature is about 31°C. In one embodiment, the temperature shift is about 37°C to about 32°C. In one embodiment, the temperature shift is about 37°C to about 34°C.

[0103] In some embodiments, the second set temperature occurs about 1 day (Day 1), about 2 days (Day 2), about 3 days (Day 3), about 4 days (Day 4), about 5 days (Day 5), about 6 days (Day 6), or about 7 days (Day 7) after the initial set temperature. In some embodiments, the temperature shift is from about 37° C. to about 32° C. on day 1. In some embodiments, the temperature shift is from about 37° C. to about 32° C. on day 2. In other embodiments, the temperature shift is from about 37° C. to about 34° C. on day 1. In some embodiments, the temperature shift is from about 37° C. to about 32° C. on day 2. In another embodiment, the temperature shift is from about 37° C. to about 34° C. on day 2.

[0104] In some embodiments, the cell culture after transfection includes adding N,N-dimethylacetamide (DMA) to the culture medium. In some embodiments, DMA is added to the culture medium at a concentration of about 0.125% v / v to about 0.250% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.125% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.250% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.05% v / v, about 0.1% v / v, about 0.15% v / v, about 0.2% v / v, about 0.25% v / v, about 0.3% v / v, about 0.35% v / v, about 0.4% v / v, about 0.45% v / v, or about 0.5% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.05% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.1% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.15% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.2% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.25% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.3% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.35% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.4% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.45% v / v. In some embodiments, DMA is added to the culture medium at a concentration of about 0.5% v / v. In certain embodiments, DMA is added to the culture medium at a concentration of about 0.05% v / v to about 0.1% v / v, about 0.1% v / v to about 0.15% v / v, about 0.15% v / v to about 0.2% v / v, about 0.2% v / v to about 0.25% v / v, about 0.25% v / v to about 0.3% v / v, about 0.3% v / v to about 0.35% v / v, about 0.35% v / v to about 0.4% v / v, about 0.4% v / v to about 0.45% v / v, or about 0.45% v / v to about 0.5% v / v.

[0105] In some embodiments, the cell culture after transfection includes the addition of sodium butyrate (NaBu.) to the culture medium. In some embodiments, NaBu is added to the culture medium at a concentration of about 1 mM to about 2 mM. In some embodiments, NaBu is added to the culture medium at a concentration of about 1 mM. In some embodiments, NaBu is added to the culture medium at a concentration of about 2 mM. In some embodiments, NaBu is added to the culture medium at a concentration of about 0.1 mM to about 5 mM. In some embodiments, NaBu is added to the culture medium at a concentration of about 0.1 mM, about 0.25 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM. In certain embodiments, NaBu is added to the culture medium at a concentration of about 0.1 mM to about 0.25 mM, about 0.25 mM to about 0.5 mM, about 0.5 mM to about 1 mM, about 1 mM to about 1.5 mM, about 1.5 mM to about 2 mM, about 2 mM to about 2.5 mM, about 2.5 mM to about 3 mM, about 3 mM to about 3.5 mM, about 3.5 mM to about 4 mM, about 4 mM to about 4.5 mM, or about 4.5 mM to about 5 mM.

[0106] The methods disclosed herein can produce recombinant proteins in high yields when using transient transfection. As used herein, the term "high yield" refers to the production of 0.2-1.5 g / L of recombinant protein per liter of cell culture medium. In some embodiments, a high yield is at least 1 g / L. A high yield can also be defined as a yield equivalent to that obtained using clonal cells. In some embodiments, a high yield is a yield that is greater than that obtained using clonal cells, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% or more yield compared to that obtained using stably transfected cells. The methods disclosed herein can also produce high quality recombinant proteins when using transient transfection. As disclosed above, a high quality protein obtained according to the methods disclosed herein is a protein having quality attributes related to, for example, (i) protein aggregates (e.g., percentage of high molecular weight species (HMW%) and / or percentage of monomeric species); (ii) reduced and non-reduced species (e.g., percentage of reduced and / or non-reduced recombinant protein); (iii) charge variants (e.g., percentage of basic variants, percentage of acidic variants, or percentage of major species); (iv) glycosylation profile (e.g., percentage of G1 F, G0 F, G2 F, total non-fucosylated protein, or any combination), which has a product quality equivalent to that of the clonal material (e.g., quality attributes having values ​​of ±5%, 10%, or 20% relative to the corresponding values ​​obtained from the clonal material, or any combination thereof).

[0107] In some aspects, the recombinant protein yield of the methods disclosed herein (e.g., the recombinant protein comprises an antibody or an antigen-binding portion thereof) is at least about 1 g / L, at least about 2 g / L, at least about 3 g / L, at least about 4 g / L, at least about 5 g / L, at least about 6 g / L, at least about 7 g / L, at least about 8 g / L, at least about 9 g / L, or at least about 10 g / L. In some embodiments, the recombinant protein yield of the methods disclosed herein (e.g., the recombinant protein comprises an antibody or an antigen-binding portion thereof) is about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, or about 10 g / L. In some embodiments, the yield of the recombinant mAb produced according to the methods disclosed herein is about 1 g / L. In some embodiments, the yield of the recombinant mAb produced according to the methods disclosed herein is about 2 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 3 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 4 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 5 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 6 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 7 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 8 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 9 g / L. In some embodiments, the yield of recombinant mAb produced according to the methods disclosed herein is about 10 g / L. In some embodiments, these high yields can be achieved after 7 days of culture. In some embodiments, these high yields can be achieved after 14 days of culture. In certain embodiments, the recombinant protein yield of the methods disclosed herein (e.g., wherein the recombinant protein comprises an antibody or an antigen-binding portion thereof) is about 1 g / L after 7 days of culture.In certain embodiments, the recombinant protein yield of the methods disclosed herein (e.g., wherein the recombinant protein comprises an antibody or an antigen-binding portion thereof) is about 2 g / L after 14 days of culture.

[0108] The present disclosure relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: (i) culturing eukaryotic cells (e.g., CHO cells) under continuous ATF perfusion (e.g., using a REPLIGEN® ATF system, such as the Xcell ATF®2 system); (ii) Using continuous ATF perfusion (e.g., a REPLIGEN ATF system such as the Xcell ATF system), cells were perfused at 40–200×10 6 Density of cells / mL (e.g., 160x10 6 cells / mL~200x10 6 concentrating the culture to 1000 cells / mL; (iii) using flow electroporation (e.g., using the MAXCYTE™ STX® transfection system, e.g., at a DNA to cell ratio of about 1.5 μg DNA / 1×10 6 cells), transiently transfecting the eukaryotic cells with a polynucleotide encoding a recombinant protein (e.g., an antibody, such as a mAb, or an antigen-binding portion thereof); (iv) After transfection, culturing the transiently transfected eukaryotic cells in a bioreactor (e.g., a high throughput bioreactor such as an AMBR® 250 bioreactor, or a large volume bioreactor such as a 5 L bioreactor). The present invention provides a method for producing a recombinant protein (e.g., an antibody, such as a mAb, or an antigen-binding portion thereof) in high yield in a bioreactor (e.g., a high throughput bioreactor such as an AMBR® 250 bioreactor or a large volume bioreactor such as a 5 L bioreactor), comprising:

[0109] In one embodiment, the post-transfection culture comprises: (i) Approximately 5x10 6 cells / mL ~ approx. 15x10 6 Cell culture starting density in cells / mL; (ii) optionally, about 0.125% (v / v) DMA; (iii) about 1 mM NaBu; and (iv) a temperature shift from about 36.5° C. to about 32° C. on day 1; The volume of the bioreactor is about 250 mL to about 5 L.

[0110] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) A temperature shift from about 36°C ± 1°C to about 32°C ± 1°C on the first or second day The volume of the bioreactor is about 250 mL to about 5 L.

[0111] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) A temperature shift from about 36° C.±1° C. to about 32° C.±1° C. on day 1 or 2, the volume of the bioreactor being between about 250 mL and about 5 L.

[0112] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) A temperature shift from about 37°C ± 1°C to about 32°C ± 2°C on day 1 and the volume of the bioreactor is approximately 250 mL.

[0113] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) A temperature shift from about 37°C ± 1°C to about 32°C ± 2°C on day 1 and the volume of the bioreactor is about 5 L.

[0114] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) Temperature shift from about 37°C to about 32°C on day 1 The volume of the bioreactor is about 250 mL, The basal medium is formulated for enhanced cell culture production and the culture includes a P / V shift.

[0115] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) Temperature shift from about 37°C to about 32°C on day 1 The volume of the bioreactor is about 250 mL and the culture includes a P / V shift.

[0116] In one embodiment, the post-transfection culture comprises: (i) Approximately 5x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) Temperature shift from about 37°C to about 32°C on day 1 The volume of the bioreactor is about 250 mL, The basal medium is formulated for enhanced cell culture production and the culture is free of P / V shifts.

[0117] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; and (iii) Temperature shift from about 37°C to about 32°C on the second day The volume of the bioreactor is about 250 mL, The basal medium is formulated for enhanced cell culture production and the culture is free of P / V shifts.

[0118] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) about 1 mM ± 10% NaBu; and (iv) Temperature shift from about 37°C to about 34°C on the second day The volume of the bioreactor is about 250 mL, The basal medium is formulated for enhanced cell culture production and the culture contains a P / V shift.

[0119] In one embodiment, the post-transfection culture comprises: (i) Approximately 15x10 6 Cell culture starting density of cells / mL ± 10%; (ii) optionally, about 0.125% (v / v) ± 10% DMA; (iii) Temperature shift from about 37°C to about 32°C on day 1 The volume of the bioreactor is about 250 mL and the culture includes a P / V shift.

[0120] In certain embodiments, the methods disclosed herein increase the yield of recombinant protein by at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 610%, at least about 620%, at least about 630%, at least about 640%, at least about 650%, at least about 660%, at least about 670%, at least about 680%, at least about 690%, at least about 700%, at least about 710%, 20%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390% or at least about 400% improvement.

[0121] In some embodiments, the methods disclosed herein improve protein yield by at least about 100% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 110% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 120% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 130% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 140% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 150% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 160% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 170% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 180% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 190% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 200% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 210% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 220% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 230% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 240% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 250% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 260% compared to the reference method.In some embodiments, the methods disclosed herein improve protein yield by at least about 270% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 280% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 290% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 300% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 310% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 320% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 330% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 340% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 350% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 360% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 370% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 380% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 390% compared to the reference method. In some embodiments, the methods disclosed herein improve protein yield by at least about 400% compared to the reference method.

[0122] In some embodiments, the methods disclosed herein improve protein yield by about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390% or about 400% compared to a reference method that does not use the optimal culture conditions disclosed in the present invention. In some embodiments, the methods disclosed herein improve protein yield by about 100%. In some embodiments, the methods disclosed herein improve protein yield by about 110%. In some embodiments, the methods disclosed herein improve protein yield by about 120%. In some embodiments, the methods disclosed herein improve protein yield by about 130%. In some embodiments, the methods disclosed herein improve protein yield by about 140%. In some embodiments, the methods disclosed herein improve protein yield by about 150%. In some embodiments, the methods disclosed herein improve protein yield by about 160%. In some embodiments, the methods disclosed herein improve protein yield by about 170%. In some embodiments, the methods disclosed herein improve protein yield by about 180%. In some embodiments, the methods disclosed herein improve protein yield by about 190%. In some embodiments, the methods disclosed herein improve protein yield by about 200%. In some embodiments, the methods disclosed herein improve protein yield by about 210%. In some embodiments, the methods disclosed herein improve protein yield by about 220%. In some embodiments, the methods disclosed herein improve protein yield by about 230%. In some embodiments, the methods disclosed herein improve protein yield by about 240%. In some embodiments, the methods disclosed herein improve protein yield by about 250%. In some embodiments, the methods disclosed herein improve protein yield by about 260%. In some embodiments, the methods disclosed herein improve protein yield by about 270%.In some embodiments, the methods disclosed herein improve protein yield by about 280%. In some aspects, the methods disclosed herein improve protein yield by about 290%. In some aspects, the methods disclosed herein improve protein yield by about 300%. In some aspects, the methods disclosed herein improve protein yield by about 310%. In some embodiments, the methods disclosed herein improve protein yield by about 320%. In some embodiments, the methods disclosed herein improve protein yield by about 330%. In some embodiments, the methods disclosed herein improve protein yield by about 340%. In some embodiments, the methods disclosed herein improve protein yield by about 350%. In some embodiments, the methods disclosed herein improve protein yield by about 360%. In some embodiments, the methods disclosed herein improve protein yield by about 370%. In some embodiments, the methods disclosed herein improve protein yield by about 380%. In some embodiments, the methods disclosed herein improve protein yield by about 390%. In some embodiments, the methods disclosed herein improve protein yield by about 400%.

[0123] In some embodiments, the method of the present invention improves protein yield at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold higher than a reference method not using the optimal culture conditions disclosed in the present invention. In some embodiments, the method of the present invention improves protein yield at least about 2-fold. In some embodiments, the method of the present invention improves protein yield at least about 3-fold. In some embodiments, the method of the present invention improves protein yield at least about 4-fold. In some embodiments, the method of the present invention improves protein yield at least about 5-fold. In some embodiments, the method of the present invention improves protein yield at least about 6-fold. In some embodiments, the method of the present invention improves protein yield at least about 7-fold. In some embodiments, the method of the present invention improves protein yield at least about 8-fold. In some embodiments, the method of the present invention improves protein yield at least about 9-fold. In some embodiments, the method of the present invention improves protein yield at least about 10-fold.

[0124] In some embodiments, the method of the present invention improves protein yield by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold higher than a reference method not using the optimal culture conditions disclosed in the present invention. In some embodiments, the method of the present invention improves protein yield by about 2-fold. In some embodiments, the method of the present invention improves protein yield by about 3-fold. In some embodiments, the method of the present invention improves protein yield by about 4-fold. In some embodiments, the method of the present invention improves protein yield by about 5-fold. In some embodiments, the method of the present invention improves protein yield by about 6-fold. In some embodiments, the method of the present invention improves protein yield by about 7-fold. In some embodiments, the method of the present invention improves protein yield by about 8-fold. In some embodiments, the method of the present invention improves protein yield by about 9-fold. In some embodiments, the method of the present invention improves protein yield by about 10-fold. In some embodiments, the method of the present invention improves protein yield by about 2-fold to about 3-fold. In some embodiments, the method of the present invention improves protein yield by about 3-fold to about 4-fold. In some embodiments, the method of the present invention improves protein yield by about 4-fold to about 5-fold. In some embodiments, the method of the present invention improves protein yield by about 5-fold to about 6-fold. In some embodiments, the method of the present invention improves protein yield by about 6-fold to about 7-fold. In some embodiments, the method of the present invention improves protein yield by about 7-fold to about 8-fold. In some embodiments, the method of the present invention improves protein yield by about 8-fold to about 9-fold.

[0125] In some embodiments, the value of a product quality attribute of a recombinant protein obtained by transient transfection of a eukaryotic cell is within ±10% of the value of the product quality attribute of a recombinant protein obtained by stable transfection. In some embodiments, the value of a product quality attribute of a recombinant protein obtained by transient transfection of a eukaryotic cell is within ±15% of the value of the product quality attribute of a recombinant protein obtained by stable transfection. In some embodiments, the value of a product quality attribute of a recombinant protein obtained by transient transfection of a eukaryotic cell is within ±20% of the value of the product quality attribute of a recombinant protein obtained by stable transfection.

[0126] In one embodiment, the product quality attribute is (i) Protein aggregates; (ii) reduced and non-reduced species; (iii) Charge variant; (iv) glycosylation profile; and (v) any combination thereof; is selected from the group consisting of:

[0127] In some embodiments, the quality characteristics of the protein aggregates include: (i) Percentage of high molecular weight species (HMW%); (ii) the percentage of monomeric species; and (iii) any combination thereof; is selected from the group consisting of:

[0128] In some embodiments, the quality characteristics of protein aggregates are determined using high performance liquid size exclusion chromatography (HPLC-SEC). For example, the levels of HMW and monomeric species can be measured by HPLC-SEC using an Alliance 2695 HPLC system equipped with a Model 2487 dual wavelength detector (Waters Corporation, Milford MA USA) and a TSKgel SuperSW3000 main column and guard column (Tosoh Biosciences, King of Prussia, PA, USA). As used herein, "high molecular weight species" or "HMW" refers to aggregates of recombinant proteins composed of two or more monomeric species. For example, HMW species can be dimers, trimers, tetramers, pentamers or hexamers.

[0129] In certain embodiments, the percentage of high molecular weight species (HMW%), percentage of monomeric species, or any combination thereof, of a recombinant protein produced using the disclosed transient expression methods has ±5%, ±10%, or ±20% of the same value as that found when the recombinant protein is produced using a reference method of stable transfection.

[0130] In some embodiments, the quality characteristics of the reduced and non-reduced species are: (i) Proportion of reduced species recombinant protein; (ii) the percentage of non-reduced recombinant protein; and (iii) any combination thereof; is selected from the group consisting of:

[0131] In some embodiments, the quality characteristics of the reduced and non-reduced species are determined using capillary electrophoresis under reducing and non-reducing conditions (CE-SDS). In some embodiments, the percentage of reduced species recombinant protein, the percentage of non-reduced species recombinant protein, or any combination thereof, of the recombinant protein produced using the disclosed transient expression methods has ±5%, ±10%, or ±20% of the same value as that found when the recombinant protein is produced using a reference method of stable transfection.

[0132] In some embodiments, the quality characteristics of the charge variant include: (i) Proportion of basic variants; (ii) the proportion of acidic variants; (iii) the proportion of major species; and (iv) any combination thereof; is selected from the group consisting of:

[0133] As used herein, the term "basic variant" refers to a form of a recombinant protein that has more positive charges than the major species. As used herein, the term "acidic variant" refers to a form of a recombinant protein that has more negative charges than the major species. In some embodiments, the quality characteristics of the charge variants are determined by analyzing the isoelectric focusing of the isoelectric distribution, e.g., imaged capillary isoelectric focusing (iCIEF). iCIEF refers to a method used to separate proteins by isoelectric point (pI). In this method, the sample is adjusted to a final concentration of approximately 1 mg / mL using water, methylcellulose, amphoteric solvents, and pI markers, and then injected into an imaged capillary isoelectric focusing system by an autosampler. Electrophoresis separates the sample based on the charge distribution of isoforms / variants by a pH gradient in a fluorocarbon (FC) coated capillary. The results are compared to a reference separation to compare and characterize the component groups.

[0134] In some embodiments, the quality attributes of the percentage of basic variants, percentage of acidic variants, percentage of major species, or any combination thereof, are determined using isoelectric focusing, e.g., imaging capillary isoelectric focusing (iCIEF). In some embodiments, the percentage of basic variants, percentage of acidic variants, percentage of major species, or any combination thereof, of the recombinant protein produced using the disclosed transient expression methods has ±5%, ±10%, or ±20% of the same value as observed when the recombinant protein is produced using a reference method of stable transfection.

[0135] As used herein, "glycosylation profile" refers to the amount of N-linked or O-linked sugar residues that are covalently attached to a protein molecule, such as a glycoprotein.

[0136] As used herein, the term "glycoprotein" refers to a protein modified by the addition of one or more carbohydrates, e.g., the addition of one or more sugar residues. As used herein, "glycosylation" refers to the addition of complex oligosaccharide structures to a protein at specific sites within the polypeptide chain. Protein glycosylation and subsequent processing of the added sugar chains can affect protein folding and structure, protein stability, including protein half-life, and the functional properties of the protein. Protein glycosylation is divided into two classes, O-linked glycosylation and N-linked glycosylation, depending on the sequence context in which the modification occurs. O-linked polysaccharides are attached to hydroxyl groups, usually those of either serine or threonine residues. O-linked glycans are not attached to all serine and threonine residues. O-linked oligosaccharides are usually monoantennary or biantennary, i.e., they contain one or at most two branches (antennae), each of which has one to four types of sugar residues added. N-linked polysaccharides are linked to the amide nitrogen of asparagine. Only one of the two tripeptide sequences, asparagine-X-serine or asparagine-X-threonine (X is any amino acid except proline), is the target for glycosylation. N-linked oligosaccharides have one to four branches, called mono-, bi-, tri-, or tetraantennary. The structures of the sugar residues present in N-linked and O-linked oligosaccharides are different. Despite the differences, the terminal residue of each branch of both N- and O-linked polysaccharides can be modified with a sialic acid molecule, a modification called sialic acid capping. Sialic acid is the common name for a family of unique nine-carbon monosaccharides that can be linked to other oligosaccharides: N-acetylneuraminic acid, abbreviated as Neu5Ac, NeuAc, or NANA, and N-glycolylneuraminic acid, abbreviated as Neu5Gc or NGNA. The most common form of sialic acid in humans is NANA. N-acetylneuraminic acid (NANA) is the predominant sialic acid present in CTLA4-Ig molecules.However, it should be noted that there are also small but detectable levels of N-glycolylneuraminic acid (NGNA) in CTLA4-Ig molecules. Additionally, the methods described herein can be used to determine the moles of sialic acid for both NANA and NGNA, and therefore the levels of both NANA and NGNA are determined and described for CTLA4-Ig molecules. N-linked and O-linked oligosaccharides have different numbers of branches and therefore different numbers of positions to which sialic acid molecules can be attached. N-linked oligosaccharides provide up to four attachment sites for sialic acid, while O-linked oligosaccharides can provide two attachment sites for sialic acid.

[0137] In certain embodiments, the glycosylation profile comprises one or more N-linked glycans. As used herein, "N-linked glycan" refers to a protein modification in which a glycan is attached to a glycoconjugate via a nitrogen bond. The glycan acceptor is a selected asparagine residue of a polypeptide chain that has entered the periplasm or endoplasmic reticulum lumen, respectively. Oligosaccharyltransferase, a central enzyme in the N-glycosylation pathway, catalyzes the formation of the N-glycosidic bond of an oligosaccharide to the side chain amide of an asparagine residue specified by the consensus sequence NXS / T. All eukaryotic N-glycans share a common core sequence, Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr, and are classified into three types: (1) oligomannose type, in which only Man residues extend the core; (2) complex type, in which an "antenna" initiated by GlcNAc extends the core; and (3) hybrid type, in which Man extends the Manα1-6 arm of the core and one or two GlcNAcs extend the Manα1-3 arm.

[0138] As used herein, "N-linked glycosylation" refers to the attachment of an oligosaccharide to a nitrogen atom, usually the N4 of an asparagine residue. N-glycosylation can occur primarily in eukaryotes and archaea, on secreted or membrane-bound proteins. A detailed review of the biosynthetic pathways and enzymes used to make N-linked glycans (e.g., high mannose oligosaccharides) can be found in Stanley, et al., "N-Glycans" in Essentials of Glycobiology, Ed. Varki, Cummings, and Eskho, Cold Spring Harbor Press, 2009".

[0139] In some embodiments, the N-linked glycan comprises (mannose-3-N-acetylglucosamine-4-fucose) (GOF), mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), mannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F), monosialylated mannose-3-N-acetylglucosamine-4-galactose-1-fucose (S1G1F), monosialylated mannose-3-N-acetylglucosamine-4-galactose-3-fucose (S2G3F), disialylated mannose-3-N-acetylglucosamine-4-galactose-2-fucose (S2G2F), or a combination thereof. In some embodiments, the quality characteristic of the glycosylation profile is selected from the group consisting of: (i) percentage of G1F, (ii) percentage of G0F, (iii) percentage of G2F, (iv) percentage of total non-fucosylated protein, and (v) any combination thereof. In some embodiments, the quality characteristic of the glycosylation profile is determined using an HPLC method, such as ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR).

[0140] In certain embodiments, (i) the percentage of G1F, (ii) the percentage of G0F, (iii) the percentage of G2F, (iv) the percentage of total nonfucosylated protein, and (v) any combination thereof of the recombinant protein produced using the disclosed transient expression method has ±5%, ±10% or ±20% of the same value as that found when the recombinant protein is produced using a reference method of stable transfection.

[0141] The present disclosure also provides recombinant proteins obtained according to the methods disclosed herein. In some embodiments, the recombinant protein is a fusion protein containing, for example, an immunoglobulin component (e.g., Fc component) and a growth factor (e.g., interleukin), an antibody or any antibody-derived molecular form, or an antibody fragment and an antigen-binding fragment. In some embodiments, the recombinant protein comprises a naturally occurring protein. In other embodiments, the recombinant protein may be an antibody, an enzyme, a cytokine, a lymphokine, an adhesion molecule, a receptor, or a derivative or fragment thereof. In some embodiments, the recombinant protein may be an agonist or antagonist, and / or may exhibit therapeutic or diagnostic uses or be used as a research reagent. In some embodiments, the polypeptide is a secreted protein or protein fragment, for example, an antibody or antibody fragment, or an Fc fusion protein.

[0142] In certain embodiments, the recombinant protein comprises an antibody or antigen-binding portion thereof, e.g., a monoclonal antibody (mAb). In certain embodiments, the antibody or antigen-binding fragment binds to an antigen selected from the group consisting of PD-1, PD-L1, CVTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73, HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, EGFR, HER3, and HER4.

[0143] The present disclosure also provides a pharmaceutical composition comprising a recombinant protein or combination thereof obtained according to the method disclosed herein and a pharma- ceutically acceptable carrier. As used herein, "pharma-ceutically acceptable carrier" refers to a vehicle for a pharmacologically active agent, such as a recombinant protein produced according to the method disclosed herein. The carrier facilitates the delivery of the active agent to the target site without stopping the function of the active agent. Non-limiting examples of suitable forms of the carrier include solutions, creams, gels, gel emulsions, jellies, pastes, lotions, ointments, sprays, ointments, powders, solid mixtures, aerosols, emulsions (e.g., water-in-oil or oil-in-water), gel-water solutions, aqueous solutions, suspensions, liniments, tinctures, and patches suitable for topical administration.

[0144] Also provided is a cell or a plurality of cells obtained according to the method of the present disclosure. The cell used in the method disclosed herein is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell, for example, a mammalian cell selected from the group consisting of Chinese Hamster Ovary (CHO), VERO, BHK, HEK, HeLa, COS, MDCK and hybridoma cell. In some specific embodiments, the mammalian cell is a CHO cell.

[0145] Mammalian cells that can be used according to the methods disclosed herein include, for example, DHFR-CHO cells (described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220), NSO myeloma cells, COS cells and SP2 cells, SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney (HEK) lines (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; human hepatocyte cell line cells (Hep G2), NIH 3T3, W138, BT483, Hs578 T, HTB2, BT2O, T47 D, CRL7O3O, COS (e.g., COS1 or COS), PER.C6, HsS78Bst, HEK-293 T, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0146] In some embodiments, the CHO cells are CHO-DG44, CHOZN, CHO / DHFR-, CHOK1 SV GS-KO or CHO-S. In some embodiments, the CHO cells are CHO-DG4. In some embodiments, the CHO cells are CHOZN. Other suitable CHO cell lines disclosed herein include CHO-K (e.g., CHO K1), CHO pro3-, CHO P12, CHO-K1 / SF, DUXB11, CHO DUKX; PA-DUKX; CHO pro5; DUK-BII or derivatives thereof.

[0147] In certain embodiments, the eukaryotic cell is a eukaryotic microorganism, such as a filamentous fungus or a yeast. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species and strains are commonly available and useful herein, for example, Schizosaccharomyces pombe; hosts of the Kluyveromyces genus, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces and filamentous fungi, such as; Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as A. nidulans and A. niger.

[0148] In some embodiments, the eukaryotic cell is from a multicellular organism. In some embodiments, the cell is an invertebrate cell from a plant and an insect cell. Non-limiting examples include cells from Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), silkworm, cottonseed, corn, potato, soybean, petunia, tomato and tobacco.

[0149] The present disclosure also provides a bioreactor for producing recombinant proteins produced according to the methods disclosed herein. Also provided is a bioreactor that includes a cell or a plurality of cells transfected with the cells described herein. Also provided is a bioreactor that cultures the transfected cell or a plurality of transfected cells disclosed herein under the conditions described herein. Particular types of bioreactors that can be used according to the methods disclosed herein, such as high-throughput bioreactors of AMBR® 250 bioreactors or large-volume (e.g., 5 L) bioreactors, are described in detail above.

[0150] In some embodiments, the method of the present disclosure can be used to produce recombinant proteins.In some embodiments, the method of the present disclosure can be used to produce AAV vectors useful for gene therapy.In some embodiments, the method can be used for AAV vector manufacturing lots and mini-batch for personalized biotherapeutics.

[0151] II. Recombinant Proteins The methods disclosed herein can be used to produce recombinant proteins, e.g., therapeutic proteins. In some embodiments, the protein produced by the methods disclosed herein is an antibody. Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies consisting of two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibody described herein refers to a population of polyclonal antibodies. The antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In some embodiments, the antibody described herein is an IgG antibody, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In certain embodiments, the antibody is a humanized monoclonal antibody. In another particular aspect, the antibody is a human monoclonal antibody, preferably an immunoglobulin. In certain embodiments, the antibody described herein is an IgG1 or IgG4 antibody.

[0152] In some embodiments, the proteins described herein are referred to as "antigen-binding domains," "antigen-binding regions," "antigen-binding fragments," and similar terms, which refer to portions of an antibody molecule that contain amino acid residues that confer specificity to the antibody molecule for an antigen (e.g., complementarity determining regions (CDRs)). Antigen-binding regions can be obtained from any animal species, including rodents (e.g., mice, rats, or hamsters) and humans.

[0153] In some embodiments, the protein is an anti-LAG3 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-NKG2a antibody, an anti-ICOS antibody, an anti-CD137 antibody, an anti-KIR antibody, an anti-TGFβ antibody, an anti-IL-10 antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-mesothelin antibody, an anti-CD27 antibody, an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-IL8 antibody, or any combination thereof. In some embodiments, the protein is an abatacept NGP. In other embodiments, the protein is a belatacept NGP. In some embodiments, the protein is an anti-GITR (glucocorticoid-induced tumor necrosis factor receptor family related gene) antibody. In some embodiments, the anti-GITR antibody has the CDR sequences of 6C8, such as a humanized antibody having the CDRs of 6C8 described in WO2006 / 105021, an antibody comprising the CDRs of the anti-GITR antibody described in WO2011 / 028683, an antibody comprising the CDRs of the anti-GITR antibody described in JP2008278814, an antibody comprising the CDRs of the anti-GITR antibody described in WO2015 / 031667, WO2015 / 187835, WO2015 / 184099, WO2016 / 054638, WO2016 / 057841, WO2016 / 057846, WO2018 / 013818, or any other anti-GITR antibody described or referenced herein, all of which are incorporated herein in their entirety. In other embodiments, the protein is an anti-LAG3 antibody. Lymphocyte activation gene 3, also known as LAG-3, is a protein that in humans is encoded by the LAG3 gene. Discovered in the 1990s, LAG3 is a cell surface molecule that exerts a variety of biological effects on T-cell function. It is an immune checkpoint receptor and therefore has been targeted in various drug development programs by pharmaceutical companies seeking to develop new treatments for cancer and autoimmune diseases. In its soluble form, it is also being developed as an anti-cancer drug in its own right.Examples of anti-LAG3 antibodies include, but are not limited to, the antibodies of WO 2017 / 087901 A2, WO 2016 / 028672 A1, WO 2017 / 106129 A1, WO 2017 / 198741 A1, US 2017 / 0097333 A1, US 2017 / 0290914 A1, and US 2017 / 0267759 A1, all of which are incorporated herein.In some embodiments, the protein is an anti-CXCR4 antibody.CXCR4 is a seven-transmembrane protein that is bound to G1. CXCR4 is widely expressed on cells of hematopoietic origin and is the major CD4+ coreceptor for human immunodeficiency virus 1 (HIV-1) (see Feng, Y., Broeder, CC, Kennedy, PE, and Berger, EA (1996) Science 272, 872-877). Examples of anti-CXCR4 antibodies include, but are not limited to, those of WO 2009 / 140124 A1, US 2014 / 0286936 A1, WO 2010 / 125162 A1, WO 2012 / 047339 A2, WO 2013 / 013025 A2, WO 2015 / 069874 A1, WO 2008 / 142303 A2, WO 2011 / 121040 A1, WO 2011 / 154580 A1, WO 2013 / 071068 A2, and WO 2012 / 175576 ​​A1, all of which are incorporated herein. In one embodiment, the protein is an anti-CD73 (ecto-5'-nucleotidase) antibody. In some embodiments, anti-CD73 antibodies inhibit the formation of adenosine. The breakdown of AMP into adenosine creates immunosuppressive and pro-angiogenic niches in the tumor microenvironment that promote the development and progression of cancer. Examples of anti-CD73 antibodies include, but are not limited to, the antibodies in WO 2017 / 100670 A1, WO 2018 / 013611 A1, WO 2017 / 152085 A1 and WO 2016 / 075176 A1, all of which are incorporated herein. In some embodiments, the protein is an anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) antibody.TIGIT is a member of the PVR (poliovirus receptor) family of immunoglobin proteins. TIGIT is expressed in several classes of T cells, including follicular B helper T cells (TFH). The protein has been shown to bind with high affinity to PVR; this binding is believed to assist the interaction between TFH and dendritic cells and regulate T cell-dependent B cell responses. Examples of anti-TIGIT antibodies include, but are not limited to, the antibodies in WO 2016 / 028656 A1, WO 2017 / 030823 A2, WO 2017 / 053748 A2, WO 2018 / 033798 A1, WO 2017 / 059095 A1, and WO 2016 / 011264 A1, all of which are incorporated herein. In one embodiment, the protein is an anti-OX40 (i.e., CD134) antibody. OX40 is a cytokine of the tumor necrosis factor (TNF) ligand family. OX40 functions in the interaction of T cells with antigen-presenting cells (APCs) and mediates adhesion of activated T cells to endothelial cells. Examples of anti-OX40 antibodies include WO 2018 / 031490 A2, WO 2015 / 153513 A1, WO 2017 / 021912 A1, WO 2017 / 050729 A1, WO 2017 / 096182 A1, WO 2017 / 134292 A1, WO 2013 / 038191 A2, WO 2017 / 096281 A1, WO 2013 / 028231 A1, WO 2016 / 057667 A1, WO 2014 / 148895 A1, WO 2016 / 200836 A1, WO 2016 / 100929 A1, WO 2015 / 153514 A1, WO 2016 / 002820 A1, and WO 2016 / 200835 A1, all of which are incorporated herein. In some embodiments, the protein is an anti-IL8 antibody. IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T cells, but not monocytes. IL-8 is also involved in the activation of neutrophils. IL-8 is released from several cell types in response to inflammatory stimuli.

[0154] In some embodiments, the protein produced by the methods of the present disclosure is a fusion protein. A "fusion" or "fusion" protein comprises a first amino acid sequence linked in frame to a first amino acid sequence to which it is not naturally linked. Amino acid sequences that are normally present in separate proteins can be combined in a fusion polypeptide, or amino acid sequences that are normally present in the same protein can be placed in a new arrangement in a fusion polypeptide. A fusion protein is created, for example, by creating and translating a polynucleotide in which peptide regions are encoded in the desired relationship. Once transcribed / translated, a single protein is created. In this manner, multiple proteins or fragments thereof can be incorporated into a single polypeptide.

[0155] In one embodiment, the protein is abatacept (sold as ORENCIA®). Abatacept (also abbreviated herein as Aba) is a drug used to treat autoimmune diseases such as rheumatoid arthritis by inhibiting the immune activity of T cells. Abatacept is a fusion protein that fuses the Fc region of immunoglobulin IgG1 and the extracellular domain of CTLA-4. For T cells to be activated and mount an immune response, antigen-presenting cells must present two signals to T cells. One of these signals is the major histocompatibility complex (MHC) bound to the antigen, and the other signal is the CD80 or CD86 molecule (also known as B7-1 and B7-2).

[0156] In one embodiment, the protein is belatacept (brand name NULOJIX®). Belatacept is a fusion protein combining the Fc fragment of human IgG1 immunoglobulin with the extracellular domain of CTLA-4, a key molecule in controlling T cell costimulation, and selectively inhibits the process of T cell activation. It is intended to prolong graft and post-transplant survival while limiting the toxicity caused by standard immunosuppressive regimens such as calcineurin inhibitors. It differs from abatacept (ORENCIA®) ​​by only two amino acids.

[0157] III. System The present disclosure relates to: (i) Bioreactor; (ii) an ATF perfusion system; and (iii) Electroporation transfection system The present invention provides a system for producing recombinant proteins in high yields by transient transfection, comprising:

[0158] In some embodiments, the bioreactor is a high throughput bioreactor, such as an AMBR® 250 bioreactor. In some embodiments, the bioreactor is a large volume bioreactor (e.g., a 5 L bioreactor).

[0159] In some embodiments, the bioreactor, e.g., a high-throughput bioreactor such as the AMBR® 250 bioreactor, includes at least one integrated cell culture analyzer. In some embodiments, the cell culture analyzer is a pH measurement module, e.g., the AMBR® Analysis module. In some embodiments, the cell culture analyzer is an integrated cell counter, e.g., the Beckman Vicell XR cell counter and the Cedex HiRes cell counter. In some embodiments, the cell culture analyzer includes a Nova Biomedical FLEX2 External Sampling Module (ESM), a FLEX2 Analyzer, or a combination thereof. In some embodiments, automated sample collection, sample transfer and analysis, data transfer, and automated feedback control are performed by the cell culture analyzer (e.g., a FLEX2 analyzer). In some embodiments, control actions such as glucose or feed addition are performed by the cell culture analyzer (e.g., a FLEX2 analyzer) based on glucose levels, cell counts, viable cell counts, or other parameters.

[0160] In some embodiments, the ATF perfusion system is a REPLIGEN® ATF system. In some embodiments, the REPLIGEN® ATF perfusion system is an Xcell ATF® 2 system. In some embodiments, the REPLIGEN® ATF perfusion system is an Xcell ATF® 1 system. In some embodiments, the REPLIGEN® ATF perfusion system is an Xcell ATF® 4 system (10 L to 50 L suspension culture volume). In some embodiments, the REPLIGEN® ATF perfusion system is an Xcell ATF® 10 system (50 L to 200 L suspension culture volume).

[0161] In some embodiments, the REPLIGEN® ATF perfusion system is connected to a REPLIGEN® XCell® Lab Controller. In some embodiments, the REPLIGEN® ATF perfusion system is connected to a REPLIGEN® XCell® C410 Controller.

[0162] In some embodiments, the electroporation transfection system is a flow-type electroporation system. In some embodiments, the flow-type electroporation system is a MAXCYTE® STX® flow transfection system. In some embodiments, the flow-type electroporation system is a MAXCYTE® VLS® flow transfection system. In some embodiments, the flow-type electroporation system is a MAXCYTE® GT® flow transfection system.

[0163] IV. Pharmaceutical Compositions The protein produced by the method of the present disclosure can be further formulated, for example, into a pharmaceutical composition, so that it is suitable for human administration.Compositions acceptable for pharmaceutical administration, the compositions disclosed herein may contain substances that are impurities at levels not exceeding the levels acceptable for pharmaceutical administration (including the absence of impurities), and may contain, in addition to any active agent(s), for example, pharma-ceutically acceptable excipients, vehicles, carriers and other inactive ingredients to formulate the composition for ease of administration.

[0164] Various aspects of the disclosure are described in further detail in the following subsections. The disclosure is further illustrated by the following examples, which should not be construed as further limiting. EXAMPLES

[0165] material and method I. Cell culture The proprietary suspension-adapted BMS-CHO1 cells were cultured in a non-baffled shake flask using the proprietary growth medium supplemented with 8–12 mM L-glutamine (Ajinomoto Co., Inc.) at 37°C, 5% CO2, and 150 rpm. The cells were cultured at 3–4x10 5 The cells were passaged every 2–3 days at a seeding density of 100 cells / mL for a maximum of 7–11 passages.

[0166] For initial small-scale shake flask studies to screen various conditions, cells were cultured at 3–4x10 6 After an initial optimization phase, an alternating tangential flow filtration (ATF) based perfusion system was optimized to achieve the high cell densities required for flow electroporation. To achieve this, cells were cultured in a 5L Applikon Biotechnology (Sartorius) stirred tank bioreactor at 3–3.5x10 5 Cells were inoculated at 1000x10000 cells / mL and controlled using a Finesse G3LAB® Universal TRUEBIO® (Sartorius) control system and software. 24 hours after inoculation, cells were perfused at a cell specific perfusion rate (CSPR) of 0.8 using an XCELL® ATF2® system (Repligen) for the remaining 3 days of culture. At the end of the perfusion phase, cell densities were between 40-60x10 6 cells / mL reached. 200x10 6 To reach a density of 100 x 10 cells / mL, the medium replenishment pump is stopped and the permeation pump is continued for 30-45 minutes, during which the cell culture concentration is 80-100 x 10 6 cells / mL was reached. The cells were then centrifuged as described in the next section.

[0167] II. Transfection On the day of transfection, cells were pelleted by centrifugation at 1,000 rpm for 5-10 min and diluted with 200x106 The cells were suspended in electroporation buffer at 1000 cells / mL (MaxCyte, Gaithersburg, MD). The cell suspension was transfected with plasmid DNA (1.5 μg DNA / 1x10 6 The cells were mixed with 100% ethanol (100% ethanol) and transferred to a CL-1.1 (small-scale electroporation) or CL-2 (large-scale electroporation) processing assembly (MaxCyte, Gaithersburg, MD). The processing assembly was connected to a MaxCyte STX Transfection System and the instrument was set to the "CHO" protocol. Immediately after electroporation, the cells were transferred to a shake flask and incubated at 37°C in a static incubator with 5% CO2 for 30-40 min.

[0168] III. Production run in shake flasks Cells were suspended in proprietary medium containing 8–12 mM L-glutamine and grown in unbaffled shake flasks at 37 °C in a 5% CO2 incubator at 90 rpm (shaking diameter 25 mm). 24 h after electroporation, the shaking speed was increased to 110 rpm. Cells were fed with proprietary feed component A daily until day 4 and then every other day until harvest. Glucose (300 g / L, VWR) was replenished to 4 g / L when glucose levels were below 3 g / L, and anti-aggregation agent (ThermoFisher) was added at a ratio of 1:200 on days 3 or 4. During the optimization process, different initial cell concentrations, medium supplement components and temperature shift strategies were tested, as described in the Results and Discussion section.

[0169] IV. Production Run in AMBR® 250 Reactor The AMBR® 250 (Sartorius, New York) automated bioreactor system was used for inoculation of the transient gene expression (TGE) screening experiments. The AMBR® 250 system is a high-throughput automated bioreactor system for process development with 12 or 24 fully functional single-use 100-250 mL mini-bioreactors.

[0170] Cells were inoculated into 24 separate single-use 250 mL bioreactors according to the conditions outlined in the experimental design. The AMBR® 250 system was supplemented with Vi-Cell (Beckman, California) and Nova Bioprofile Flex2 (Nova Biomedical, Massachusetts) devices to automate feeding and sampling, as well as cell counting and metabolite data collection. Daily supplement feeding was controlled by pump addition. Other initial additions, such as N,N-dimethylacetamide (DMA, Millipore Sigma) and sodium butyrate (NaBu, Millipore Sigma), were done manually by pipetting. The culture period was 14 days, with samples taken periodically and analyzed offline for protein quality and titer.

[0171] V. Production carried out in a 5 L glass tank bioreactor For inoculation of scale-up experiments testing optimal conditions from the TGE screen, a 5 L Applikon (Sartorius) stirred tank bioreactor was utilized in a similar manner to that used previously to prepare for growth with ATF. Cells were inoculated and controlled according to the parameters set in Table 2. pH and DO control was performed using amperometric and optical probes (Mettler Toledo) and connected to control loops supplementing oxygen, carbon dioxide, and sodium carbonate (Jost Chemical). Feeds were automatically controlled by a multi-feed control system (Sartorius). Supplementary feed medium, glucose, and antifoam C were all fed in this manner: (i) feed medium was fed at a constant rate daily, (ii) glucose was added to 3 g / L in a pre-feed, and (iii) antifoam C was added daily to 3 ppm and, if necessary, not to exceed 100 ppm total to control foam. Vessels were manually sampled daily for product quality and to measure cell count and metabolite data.

[0172] VI. Cell counts and metabolites Viable cell density (VCD) and viability were measured offline using a VI-CELL®XR automated cell counter (Beckman Coulter). Whole broth cell culture samples were used for offline gas assays (pH, pCO2, pO2) and offline metabolite assays (glucose, glutamine, glutamate, ammonium and lactate) using a BIOPROFILE®FLEX2® Multitest Cell Culture Analyzer (Nova Biomedical). Clarified supernatants after centrifugation at 1,000 rpm were used for LDH quantification using a CEDEX®BIO HT® Bioprocess Analyzer (Roche).

[0173] VII. Cell cycle and apoptosis assessment Cell cycle analysis was performed using GUAVA® Cell Cycle Reagent (Luminex) according to the manufacturer's instructions. Briefly, cells were cultured at 2x10 5 Cells / samples were diluted with 1X PBS buffer and centrifuged at 500 xg for 5 min, then washed once with 1X PBS. Cells were resuspended in the remaining buffer and 70% cold ethanol (Thermo Fisher) was slowly added. Cells were incubated at -20°C for at least overnight, then washed with 1X PBS. Cells were stained with GUAVA® Cell Cycle Reagent (Luminex) containing Propidium Iodide (PI) and incubated for 30 min at room temperature in the dark. Stained cells were analyzed on a GUAVA® EASYCYTE® Flow Cytometer (Luminex).

[0174] Early and late apoptosis were determined by diluting the cells at 1x10 in 1xPBS buffer. 5 The assay was performed using cells diluted in cells / sample. FBS (Thermo Fisher) was added to 10% (v / v) and stained with the GUAVA NEXIN® Reagent Kit (Luminex) for 20 min according to the manufacturer's instructions. After incubation, cells were analyzed on a GUAVA® EASYCYTE® Flow Cytometer (Luminex).

[0175] VIII. Protein Quality Analysis After completion of the production step, the culture was centrifuged at 1,000xg (Beckman Allegra X-12) for 10 min. The culture was diluted to 2 mg / ml or less and subjected to plate-based purification with MABSELECT® SURE® PCC resin (Cytiva Life Sciences). The plate was then washed three times and eluted with acetate buffer, followed by neutralization with 2 M Tris and measurement of protein concentration at A280 nm. The titer was measured by Protein A UPLC method and the purity of the purified samples was assessed using high performance liquid chromatography size exclusion chromatography (HPLC-SEC) and reduced / non-reduced capillary electrophoresis (CE-SDS) using the technique of Caliper LC90 CE-SDS gel. The quality of the protein was assessed by analysis of the isoelectric distribution by imaging capillary isoelectric focusing (icIEF) and quantification of N-linked glycan species by HPLC method.

[0176] Results and Discussion TGE process screening improved transient gene expression several-fold Several cell culture process parameters were screened, such as inoculation density, sodium butyrate (NaBu) addition, N,N-dimethylacetamide (DMA) supplementation, temperature shift timing and DNA loading. A Box-Behnken design was created with 26 different conditions using 3 levels each of 4 factors (seeding density, NaBu, DMA and temperature shift timing) using the R package "rsm". Fed-batch operation was performed in 250 mL shake flasks with a working volume of 60 mL. Transient gene expression performance was evaluated using baseline conditions (seeding density: 5x10 6 The assay was evaluated based on normalized potency expressed as potency per cell / mL, NaBu: 1 mM, DMA: 0 mM, temperature shift time: 24 h).

[0177] The addition of DMA was found to be statistically significant (p-value <0.01), with a DMA concentration of 0.125% V / V resulting in the highest mean normalized titer (Figure 1A). This process development strategy increased the final titer several-fold, as seen in Figures 1A-1E. Analysis of the individual effects showed that higher seeding densities yielded higher titers (Figure 1B). Similarly, within the time period tested, the slower the temperature shift time, the higher the titer (Figure 1C). The addition of NaBu was detrimental to titers in fed-batch shake flasks (Figure 1D). Furthermore, the increased amount of DNA (10 6 We screened the effect of increasing the DNA to cell ratio (i.e., 10 6 DNA:cell ratios of 1.75 and 2 (as μg DNA per mL of cells) were found to have a substantial undesirable effect on the final titer (Figure IE).

[0178] Continuous ATF perfusion for cell expansion and enrichment improved cell culture characteristics and titers In the above screening study, high seeding density was shown to be favorable for high titer, so N-1 perfusion was introduced into the seeding process to utilize a large amount of host cells to facilitate the development of an enhanced fed-batch process platform for TGE. N-1 seed culture was performed in perfusion mode with a proprietary enrichment medium for 4 days. The bioreactor performance and in-process characteristics of N-1 seed perfusion are shown in Figures 2A-2D.

[0179] For N-1 perfusion culture, the bioreactor will eventually hold approximately 60x10 6 The viable cell density (VCD) was 100 cells / mL with viability >98% over the entire 4-day period (Figure 2A). Measured metabolites, such as glucose (from initial values ​​of approximately 8 g / L to 2 g / L-6 g / L), lactate (approximately <2 g / L, Figure 2C) and ammonia (approximately <5 mM, Figure 2D) as well as process parameters were within the acceptable working ranges.

[0180] On day 4, the cell density was adjusted to 90-100x10 using the ATF2 perfusion system. 6 The concentration of 10 ... 6 We performed a perfusion process in which cells were cultured at low working volumes (1000–15000 cells / mL). Strategies to maintain cells in logarithmic growth phase during transfection to increase product titer have been successfully applied in several studies (Greene et al. (2021) Biotechnol. J. 16(4):e2000251). However, no strategies focusing on tuning after perfusion reactor concentration have been reported. The characteristics and performance during all processes after N-1 seed perfusion concentration are shown in Figures 3A-3D.

[0181] As can be seen in Figure 3A, perfusion after concentration at high density, where glucose levels were replenished to approximately 4 g / L, prevented the cells from entering a quiescent state. In the perfused cell culture, approximately 72% of the cells were in the replicating phase (S and G2 / M phases), whereas in the cell culture without perfusion after concentration (batch mode), only 50% of the cells were in these replicating phases (Figure 3B). This difference in the percentage of replicating cells had a significant impact on the titer in the production culture. As can be seen in Figure 3C, the titer was reduced three-fold in batch mode compared to continuous mode.

[0182] To measure transfection efficiency, direct staining of surface IgG was performed using fluorescent anti-human IgG (H+L) antibody and measured on a GUAVA® EASYCYTE® flow cytometer. However, transfection efficiency in continuous and batch modes was comparable (Figure 3D). This finding indicates that a larger replicating cell population during transfection could contribute to improved transient productivity by other means than more efficient plasmid transfer. Another implication is that glucose depletion could adjust cellular metabolism during the 14-day production run by inducing unfavorable transcriptional and metabolic changes.

[0183] To support these data, N-1 processes with and without perfusion after concentration (discontinuous / batch) were run in parallel and production was performed after electroporation in shake flasks at a higher seeding density (15e6 / mL). N-1 perfusion (ATF2) was performed for 4 days with a seeding density of 3-4e6 / mL and a working volume of 3.5 L. Perfusion started on day 1 when cells reached approximately 8e6 / mL. Perfusion rate was updated daily (VCD) based on cell growth and was performed with a fixed CSPR of 0.04 nL / cell / day during this process. On day 4, when cells reached approximately 60e6 / mL, the perfusion reactor was concentrated to 90-100e6 / mL for 40 minutes at a concentration rate of 24 ml / min. After cell concentration, perfusion was turned on (perfusion) or off (discontinuous / batch). Figures 3E-3F show data for the continuous and discontinuous / batch processes. In the continuous system, glucose levels were higher (Figure 3E) and a higher percentage of cells in G2 / M phase was observed compared to the discontinuous / batch process (Figure 3F).

[0184] Figures 3G and 3H show the performance of transient production in shake flasks using cells from continuous or discontinuous / batch processes. In both processes, cells were inoculated at 15e6 vc / ml and then subjected to the platform's transient production conditions (i.e., temperature shift on day 1 at 32°C, NaBu at 1 mM, and basal medium at 1:1 B6:B9). Although the titer is 20% lower in the discontinuous / batch process, perfusion after cell concentration at the N-1 stage maintains better cell health, indicating improved cell culture performance after electroporation (Figure 3G).

[0185] Process development optimization in an AMBR® 250 bioreactor resulted in titers greater than 1 g / L Following the findings from the shake flask screen, factors such as inoculum seeding density, sodium butyrate (NaBu) addition, temperature shift and shift timing were evaluated in an AMBR® 250 bioreactor system. A custom design of experiment (DoE) available in the JMP v15 software package was set up with three blocks and 24 experiments were performed. [Table 1]

[0186] Two media formulations were evaluated to support enhanced fed-batch transient production: medium A and medium B. Medium B was formulated for enhanced cell culture production and differed from medium A in the concentrations of several individual components, including the addition and removal of some components and the increase or decrease in the concentrations of others.

[0187] In addition to the six process parameters, the agitation speed after transfection was also evaluated in the AMBR® 250 bioreactor. High agitation speeds can have hydrodynamic stress effects (Sieack et al. (2013) J. Biotechnol. 161(1):41-49). To define the optimal operating range, a shift in the power / volume ratio (P / V) (also known as the mean volumetric power or power density) was evaluated 24 hours after transfection to minimize stress due to electroporation-induced cell sensitization (Pakhomova et al. (2011) PLoS One 6(2):e17100). Here, a P / V shift was performed to increase the agitation speed from 350 rpm to 450 rpm. Since three MAXCYTE® CL2® Electroporation Processing Assemblies (PAs) were required to inoculate all 24 AMBR® 250 bioreactors with transfected cells, a block effect was assigned to the electroporated batch of cells. The levels of the six factors that comprise the block effect are shown in Table 1, and the final titers were used to identify optimal conditions.

[0188] Statistical analysis showed that five of the six parameters evaluated were statistically significant (p<0.01), with only the P / V shift having no significant effect on the titer (Table 1). The blocking effect corresponding to the electroporation batch was not significant (p=0.023). Among the four significant process parameters, the seeding density had the greatest effect on the titer, as indicated by the magnitude of the estimate in the statistical test.

[0189] Six reactor conditions (A18, A15, A7, A12, A16, and A19) achieved titers greater than 1 g / L over the 14-day process, with the maximum titer achieved being 1.85 g / L (A18) (Figure 4B). Furthermore, at least two conditions (A18 and A15) achieved titers greater than 1 g / L in just 7 days.

[0190] Figure 4A shows the main effects between the level means of each of the six process parameters. Consistent with the shake flask screening, 6 The seeding density of cells / mL is 5x10 6 The cells / ml of the culture showed higher titers than the cells / ml of the culture medium. The temperature shift on day 1 was more favorable for titer increase than the temperature shift on day 2, and the shift to 32°C was more favorable for monoclonal antibody (mAb) expression than the shift to 34°C. Furthermore, the addition of 1 mM NaBu promoted higher mAb production than cell cultures without NaBu. These results indicated that high seeding density, temperature shift at 32°C on day 1, addition of 1 mM NaBu, and the composition of medium B promoted transient expression of mAbs. Interestingly, reactor A18, which combines this unique optimum level, showed maximum titers on both days 7 and 14 (1.1 g / L and 1.85 g / L, respectively). This result indicated that the selection of these conditions provided a suitable environment for high transient expression of mAbs.

[0191] Moreover, the effect was observed to be close to linear. For example, substituting medium A for base medium B, substituting suboptimal levels, had a detrimental effect on titer, decreasing titer by 18%, from 1.85 g / L to 1.51 g / L (Figure 4B). Similarly, increasing the seeding density from 15x10 6 Cells / mL to 5x10 6 When reduced to cells / mL, the titer was reduced by 36% (1.85 vs. 1.18 g / L).

[0192] During transient gene expression, culturing at temperatures lower than 37°C shifted the cell state from growth to protein production and increased the final titer. In addition, lowering the culture temperature reduced the consumption of certain nutritional elements and the accumulation of secondary metabolites. The timing of the temperature shift significantly affected the expression of the transgene. The time to introduce the temperature shift varies depending on the cell and process, and a complete optimization of the temperature shift strategy is desirable. Our data showed that it was advantageous to introduce the temperature shift on day 1. This strategy allowed for a longer protein production period due to the earlier arrival of the VCD peak, which was useful for intensified cell culture processes.

[0193] The addition of NaBu showed multifunctional effects on transient gene expression, such as suppression of cell proliferation and induction of gene accessibility and expression. The inhibition of cell cycle appears to be a result of gene regulation in response to NaBu. After NaBu treatment, CHO cells are known to have altered expression of several genes related to cell cycle control, including upregulation of three antiproliferative genes, B-cell translocation gene 3 (btg3), tumor susceptibility gene 101 (Tsg101), and cyclin-dependent kinase 3 (Cdkn3). In addition, other important cell cycle regulators such as c-myc and p21 are known to be down- and upregulated, respectively, after NaBu addition in human cell lines. Furthermore, as a histone deacetylase inhibitor, NaBu is known to induce excessive acetylated histones that promote relaxation of chromatin complexes, resulting in more accessible chromatin for transcription factors and improved gene expression.

[0194] Successful scale-up of transfection process in 5L reactor After demonstrating successful transient expression of the model mAb in the AMBR® 250 bioreactor system up to 1.85 g / L, the optimal conditions (reactor A18) were scaled up in a Finesse 5-L bioreactor (Table 2). Agitation was scaled up to match cell growth and protein productivity using impeller tip speed (Table 2).

[0195] Table 2. Comparison of AMBR® 250 and 5 L bioreactor scale transient process platforms [Table 2] The impeller tip speed model for scale-up is often used when limiting cell damage due to shear stress to the cells is more important than altering the mixing and oxygen transfer regimes. The fed-batch production cultures were monitored daily to measure viable cell counts, viability, and metabolite levels (Figures 5A-5E). Overall, cell culture performance was comparable at the two scales, indicating successful scale-up from the AMBR® 250 to a 5 L bioreactor.

[0196] Transfection efficiency was determined based on surface IgG staining 2 days post-transfection with fluorescent anti-human IgG (H+L) measured on a GUAVA® EASYCYTE® flow cytometer. Similar transfection efficiency was achieved at both scales. Furthermore, mAb expression was comparable between the scales, achieving a maximum titer of 1.85 g / L over the 14-day process. Although VCD was higher and peaked later in the 5 L reactor, viability remained above 90% throughout the 14-day period at both scales. The higher VCD indicated a more favorable hydrodynamic environment in the 5 L reactor compared to the AMBR® 250.

[0197] The lactate and ammonia profiles followed the same trend, although lactate was higher in the 5 L reactor. Overall, the metabolic profile showed ideal behavior, peaking in the growth phase followed by lactate and ammonium consumption. Overall, the metabolic profile showed ideal behavior, peaking in the growth phase followed by lactate and ammonium consumption, which is typically seen in productive and stable cell cultures.

[0198] Released LDH levels were also measured as a cell damage marker for 6 days after transfection. Between 24 and 48 h after transfection, the LDH concentration was found to be low, indicating that the transfection conditions were gentle and thus the agitation strategy was successfully implemented in this process of scale-up. At the 5 L scale, the LDH levels were slightly higher, which was attributed to the higher VCD achieved in the 5 L bioreactor.

[0199] Apoptosis analysis revealed that the decline in VCD and the increase in LDH release rate during the production period were due to programmed cell death. As seen in Figures 5A-5E, the early and late apoptotic cell populations increased over time. However, by day 10, the peak of production, at least 50% of the cell population remained healthy.

[0200] The cell cycle was observed to transition from a predominantly dividing population (G2 / S and M determined at the time of transfection was approximately 80%) to a population predominantly in non-dividing G0 / G1 phase (greater than 50%) by day 3. This transition to the protein production phase coincided with the implementation of a temperature shift and addition of NaBu 24 hours after transfection. Importantly, the G0 / G1 population, associated with proliferating cells, remained relatively constant throughout the remaining days of cell culture.

[0201] Transient material was representative of stable clonal material Figures 6A-6D show typical product quality attributes (PQAs) of the purified mAb material. Figures 6A and 6B show typical purity according to size exclusion chromatography and caliper CE-SDS capillary electrophoresis. Material produced at 250 mL (AMBR® 250-A18) and 5 L scales was compared to clone material. Items shown are based on typical acceptance criteria for the model mAb used in this study. HMW and LMW impurities were present at low levels, with over 98% of this material present as intact monomers. Additionally, CE-SDS data showed that the material was intact under reducing conditions, with an expected HC:LC ratio of 2:1. The charge variant profile (Figure 6C) showed the largest deviations in both acidic and basic species, with 10-20% deviation from the profile of the clone material. Overall, the produced fugitive material met the listed specifications. Figure 6D shows an outline of the fucosylated glycoforms and the total nonfucosylated species. The transient material was found to be comparable except for lower nonfucosylated material than the clonal material. ***

[0202] It is understood that it is the Detailed Description section, and not the Abstract and Summary sections, that are intended to be used to interpret the claims. The Abstract and Summary sections may set forth one or more exemplary embodiments of the invention contemplated by the inventor, but cannot set forth all of them, and are therefore not intended to limit the scope of the invention and the appended claims in any way.

[0203] The present invention has been described above with the aid of functional blocks illustrating the implementation of certain functions and their relationships. The boundaries of these functional blocks have been arbitrarily defined herein for the convenience of description. Alternative boundaries may be defined so long as the given functions and their relationships are appropriately implemented.

[0204] The foregoing description of the specific embodiments fully reveals the general nature of the present invention, so that those skilled in the art can easily modify and / or adapt such specific embodiments to various applications without undue experimentation and without departing from the general concept of the present invention. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the terms or phrases in this specification are for the purpose of description and not for the purpose of limitation, as they should be interpreted by those skilled in the art in light of the teaching and guidance.

[0205] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0207] All publications, patent applications, patents and other literature mentioned herein are incorporated by reference in their entirety. Database entries and electronic publications disclosed in this disclosure are incorporated by reference in their entirety. The version of a database entry or electronic publication incorporated by reference in this application is the latest version of the database entry or electronic publication that was publicly available at the time this application was filed. Database entries corresponding to gene or protein identifiers disclosed in this application (e.g., genes or proteins identified by accession numbers or database identifiers in public databases such as Genbank, Refseq or Uniprot) are incorporated by reference in their entirety. The incorporated gene or protein related information is not limited to the sequence data contained in the database entry. The information incorporated by reference includes the entire contents of the database entry in the latest version of the database that was publicly available at the time this application was filed. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Claims

1. (i) A step of culturing eukaryotic cells in a culture medium to concentrate eukaryotic cells to a high density, and (ii) A step of transiently transfecting the eukaryotic cells enriched in (i) with polynucleotides encoding recombinant proteins using electroporation. A method for producing recombinant proteins in high yield and / or high quality in a large-capacity bioreactor, including the following.

2. (a) Eukaryotic cells include mammalian cells, (b) Recombinant protein containing an antibody or its antigen-binding moiety, (c) The large-capacity bioreactors include (i) high-throughput bioreactors, (ii) bioreactors of 1 L, 2 L, 5 L, 10 L, 25 L, 50 L, 100 L, 500 L, 1000 L, 2000 L, 5000 L, 10,000 L or 20,000 L, (iii) bioreactors for fed batch production, or (iv) any combination of (i) through (iii). (d) Electroporation includes (i) flow electroporation, or (ii) being performed using a MAXCYTE® transfection system. (e) The culture medium contains glucose at a concentration sufficient to maintain a high cell density, or (f) Any combination of (a) to (e), The method according to claim 1.

3. The method according to claim 1, wherein the culture includes perfusion culture.

4. The method according to claim 3, wherein the perfusion culture comprises alternative tangential flow filtration (ATF).

5. (a) The eukaryotic cells are concentrated to a density of at least about 40x10 ６ , at least about 50x10 ６ , at least about 60x10 ６ , at least about 70x10 ６ , at least about 80x10 ６ , at least about 90x10 ６ , at least about 100x10 ６ , at least about 110x10 ６ , at least about 120x10 ６ , at least about 130x10 ６ , at least about 140x10 ６ , at least about 150x10 ６ , at least about 160x10 ６ , at least about 170x10 ６ , at least about 180x10 ６ , at least about 190x10 ６ , or at least about 200x10 ６ cells / mL. (b) Electroporation is approximately 100 x 10 ６ , about 110x10 ６ , about 120x10 ６ , about 130x10 ６ , about 140x10 ６ , about 150x10 ６ , about 160x10 ６ , about 170x10 ６ , about 180x10 ６ , about 190x10 ６ , about 200x10 ６ , about 210x10 ６ , about 220x10 ６ , about 230x10 ６ , about 240x10 ６ Or approximately 250 x 10 ６ This is done at a cell density of cells / mL. (c) Electroporation yields approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 ug DNA / 1 x 10 ６ It is done in terms of the ratio of DNA to cells, or (d) Any combination of (a) to (c), The method according to claim 1.

6. The method of claim 1, further comprising culturing transiently transfected eukaryotic cells after transfection, wherein (a) the initial density of transiently transfected eukaryotic cells is 5 x 10 ６ ~20x10 ６ A method in which (a) the temperature is shifted for one or more culture periods during culture after transfection, or (b) both.

7. The method according to claim 6, wherein (a) the temperature is shifted to day 1 or day 2, or (b) the temperature is shifted from about 37°C to about 32°C, or both (a) and (b).

8. The method according to claim 6, further comprising (a) adding N,N-dimethylacetamide (DMA), or (b) adding sodium butyrate (NaBu), or both (a) and (b).

9. The method according to claim 8, wherein (a) DMA is added at about 0.125% v / v or about 0.250% v / v, or (b) NaBu is added at a concentration between about 1 mM and about 2 mM, or both (a) and (b).

10. The method according to claim 1, wherein the product quality characteristic value of the recombinant protein obtained by transiently transfecting eukaryotic cells is within ±10% of the product quality characteristic value of the recombinant protein obtained by stable transfection.

11. The product quality characteristics are, (i) Protein aggregates; (ii) Reduced species and non-reduced species; (iii) Charge variant; (iv) Glycosylation profile; and (v) Any combination of them, The method according to claim 10, selected from the group consisting of the following.

12. Recombinant protein obtained according to the method of claim 1.

13. A pharmaceutical composition comprising the recombinant protein described in claim 12.

14. (i) Bioreactor; (ii) ATF perfusion system; and (iii) Including an electroporation transfection system, A system for producing recombinant proteins in high yield through transient transfection.

15. (i) A step of culturing eukaryotic cells in a culture medium to concentrate eukaryotic cells to a high density; and (ii) A step of transiently transfecting the eukaryotic cells enriched in (i) with polynucleotides encoding recombinant proteins using electroporation. A method for producing recombinant proteins for personalized therapeutics, including, accelerating or shortening the development timeline of recombinant proteins, reducing the host cytotoxicity of recombinant proteins, and / or for personalized therapeutics.

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