Method for producing recombinant proteins

Abstract

This invention provides a novel method for increasing the viable cell density in N-1 large-scale bioreactor cell cultures. [Solution] The method involves culturing host cells expressing the target recombinant polypeptide in a non-perfusion-based culture system, where the live cell density is at least 5 × 10⁶. 6 The number of cells increases up to cells / mL. In one embodiment, the present disclosure provides a novel method for the large-scale production of a recombinant polypeptide of interest, the method comprising the steps of: (1) culturing host cells expressing the recombinant polypeptide of interest in a non-perfusion-based culture system in step N-1, where the viable cell density is at least 5 × 10⁶ 6 (2) Increase up to cells / mL; and (2) N-fed batch-produced cells at least 1.5 × 10 6 Cells are cultured in a concentrated medium with a high seeding density of cells / mL, where N-fed batch-producing cells are inoculated from the N-1 stage in a non-perfusion-based culture system.

Description

Technical Field

[0001] The present invention generally relates to a method for increasing viable cell density in an N-1 culture stage using a non-perfusion strategy for inoculation of an N-producing bioreactor at a high seeding density for cell culture production.

Background Art

[0002] Proteins and polypeptides are becoming increasingly important as therapeutic agents. In most cases, therapeutic proteins and polypeptides are produced in cell culture from cells that have been designed and / or selected to produce the polypeptide of interest at abnormally high levels. Control and optimization of cell culture conditions are extremely important for the successful commercial production of proteins and polypeptides.

[0003] Many proteins or polypeptides produced in cell culture are manufactured by a fed-batch method in which cells are cultured for a certain period of time, the culture is then terminated, and the produced protein or polypeptide is isolated. The final amount and quality of the protein or polypeptide produced can be greatly affected by the seeding density during N-1 seeding culture and N production. Efforts have been made to improve the production of proteins or polypeptides in the fed-batch culture method, but there is a need for further improvement.

[0004] Perfusion cell culture can achieve a much higher viable cell density than conventional fed-batch cell culture systems. Perfusion cell culture provides a continuous supply of fresh medium to the culture system while removing waste products, which provides a rich environment for cells to grow. Compared to conventional low-seeding density fed-batch production cultures, high-seeding density fed-batch production cultures inoculated with N-1 perfusion seeding can achieve a higher final titer in a shorter time. However, perfusion cell culture becomes expensive when used in large-scale culture systems (e.g., bioreactors exceeding 200 L) because it consumes large amounts of cell culture medium. Also, perfusion cell culture can become complex in terms of the cell retention system that prevents cells from being removed from the cell culture system, especially in large-scale manufacturing.

[0005] There is a particular need for the development of improved systems for producing proteins and polypeptides through large-scale cell culture at high seeding density using non-perfusion systems. [Overview of the project]

[0006] This disclosure relates to a method for increasing the viable cell density of N-1 large-scale bioreactor cell cultures, the method comprising culturing host cells expressing a recombinant polypeptide of interest in a non-perfusion-based culture system, wherein the viable cell density is at least 5 × 10⁶. 6 The number of cells / mL increases. In some embodiments, the non-perfusion-based culture system is a batch or fed-batch bioreactor. In some embodiments, the viable cell density at the N-1 stage is at least 5 × 10⁶ 6 , at least 10 × 10 6 , at least 15 × 10 6 , at least 20 × 10 6 , at least 25 × 10 6 , or at least 30 × 10 6 The cell viability is live cells / mL. In some embodiments, the cell viability is at least 80% on the final day of the N-1 phase, at least 85% on the final day of the N-1 phase, or at least 90% on the final day of the N-1 phase.

[0007] In some embodiments of the present invention, host cells are cultured in concentrated medium for N-1 batch culture. In some embodiments, host cells are cultured in seed medium supplemented with feed medium for N-1 fed batch culture.

[0008] In some embodiments of the present invention, the culture medium is concentrated by a feed medium by at least 5% relative to the unconcentrated medium, at least 10% relative to the unconcentrated medium, at least 15% relative to the unconcentrated medium, or at least 20% relative to the unconcentrated medium. In some embodiments, the concentrated medium or feed medium contains an increased amount of a carbon source. In some embodiments, the carbon source is glucose. In some embodiments, the concentrated medium or feed medium contains an increased amount of nutrients. In some embodiments, the nutrients are selected from amino acids, lipids, vitamins, minerals, and polyamines. In some embodiments, the concentrated medium contains an increased amount of both a carbon source and nutrients. In some embodiments, the carbon source is glucose, and the nutrients are selected from amino acids, lipids, vitamins, minerals, and polyamines.

[0009] In some embodiments of the present invention, the host 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 host cell is a CHO cell.

[0010] In some embodiments of the present invention, the polypeptide of interest is a therapeutic polypeptide. In some embodiments, the polypeptide of interest is an antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to an antigen selected from the group consisting of PD-1, PD-L1, LAG-3, TIGIT, GITR, CXCR4, CD73 HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, EGFR, HER3, and HER4.

[0011] In some embodiments of the present invention, the bioreactor has a capacity of at least 50 L, at least 500 L, at least 1,000 L, at least 5,000 L, or at least 10,000 L.

[0012] In some embodiments of the present invention, the method comprises culturing at least 5×10 6 live cells / mL in a batch culture or fed-batch culture concentrated for inoculation at the N production stage in order to produce the recombinant polypeptide of interest. In some embodiments, the method further comprises isolating the polypeptide of interest from the production culture system.

[0013] The present disclosure also relates to a method for the large-scale production of a recombinant polypeptide of interest, comprising: (1) culturing host cells expressing the recombinant polypeptide of interest in a non-perfusion-based culture system at the N-1 stage, where the live cell density increases to at least 5×10 6 cells / mL; and (2) culturing N fed-batch production cells in a basal medium or concentrated basal medium at a high seeding density of at least 1.5×10 6 cells / mL, where the N fed-batch production cells are inoculated from the N-1 stage in a non-perfusion-based culture system. In some embodiments, the N production culture system is a fed-batch bioreactor.

[0014] In some embodiments of the present invention, the concentrated basal medium is concentrated by at least 5%, at least 10%, at least 15%, at least 20% with respect to the non-concentrated medium by the feed medium. In some embodiments, the concentrated medium contains an increased amount of a carbon source. In some embodiments, the carbon source is glucose. In some embodiments, the concentrated medium contains an increased amount of nutrients. In some embodiments, the nutrients are selected from amino acids, lipids, vitamins, minerals and polyamines. In some embodiments, the concentrated medium contains an increased amount of a carbon source and nutrients. In some embodiments, the carbon source is glucose and the nutrients are selected from amino acids, lipids, vitamins, minerals and polyamines.

[0015] In some embodiments of the present invention, the bioreactor has a volume of at least 50 L, at least 500 L, at least 1,000 L, at least 5,000 L, at least 10,000 L, at least 15,000 L, or at least 20,000 L.

[0016] In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a CHO cell.

[0017] In some embodiments of the present invention, the potency of the target polypeptide is at least 100 mg / L, at least 1 g / L, at least 3 g / L, at least 5 g / L, or at least 10 g / L.

[0018] In some embodiments of the present invention, host cells are cultured in a basic medium or concentrated basic medium for an N-fed batch-producing bioreactor, with at least 1.5 × 10⁶ cells. 6 , at least 5 × 10 6 , or at least 10 × 10 6 A live cell density of live cells / mL can be obtained.

[0019] In some embodiments of the present invention, the method further includes the step of isolating the polypeptide of interest. In some embodiments, the polypeptide of interest is a therapeutic polypeptide. In some embodiments, the polypeptide of interest is an antibody or antigen-binding fragment. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1A shows the viable cell density ("VCD") of N-1 cell cultures grown for CHO cell line A in the following cell culture systems: perfusion, fed batch, batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients. Figure 1B shows the cell viability (%) of N-1 cell cultures grown for cell line A in the following cell culture systems: perfusion, fed batch, batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients.

[0021] [Figure 2A] Figure 2A shows the viable cell density of N-producing cultures for polypeptide-1 by cell line A using seeded cultures from the following N-1 cell culture systems: perfusion, fed batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients. [Figure 2B] Figure 2B shows the titer of the target polypeptide grown in production cultures using seeded cultures from the following N-1 cell culture systems: perfusion, fed batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients. [Figure 2C] Figure 2C shows imaging capillary isoelectric focusing ("iCIEF"), size exclusion chromatography ("SEC"), and N-glycan analysis of the target polypeptide grown in the CHO cell line A production culture using seeded cultures from the following N-1 cell culture systems: perfusion, fed batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients.

[0022] [Figure 3] Figure 3A shows the VCD of N-1 cell cultures grown in the following cell culture systems for CHO cell line B: perfusion, fed batch, batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients. Figure 3B shows the cell viability (%) of N-1 cell cultures grown in the following cell culture systems for CHO cell line B: perfusion, fed batch, batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients.

[0023] [Figure 4A] Figure 4A shows the viable cell density of N-producing cultures for polypeptide-2 using CHO cell line B with seeded cultures from the following N-1 cell culture systems: perfusion, fed batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients. [Figure 4B] Figure 4B shows the titer of the target polypeptide grown in N-producing culture for polypeptide-2 using CHO cell line B with seeded cultures from the following N-1 cell culture systems: perfusion, fed batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients. [Figure 4C] Figure 4C shows the iCIEF, SEC, and N-glycan analysis of the target polypeptide grown in N-producing culture of polypeptide-2 using CHO cell line B with seeded cultures from the following N-1 cell culture systems: perfusion, fed batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients.

[0024] [Figure 5] Figure 5A shows the VCD of N-1 cell cultures grown in the following cell culture systems for CHO cell line C: perfusion, fed batch, batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients. Figure 5B shows the cell viability (%) of N-1 cell cultures grown in the following cell culture systems: perfusion, fed batch, batch, batch with concentrated glucose, and batch with concentrated glucose and nutrients.

[0025] [Figure 6A] Figure 6A shows the viable cell density of N-producing cultures for polypeptide-3 by CHO cell line C using seeded cultures from the following N-1 cell culture systems: fed batches, and batches with concentrated glucose and nutrients. [Figure 6B] Figure 6B shows the titer of the target polypeptide grown in production culture for polypeptide-3 using CHO cell line C with seeded cultures from the following N-1 cell culture system: fed batch and batch with concentrated glucose and nutrients. [Figure 6C]Figure 6C shows iCIEF, SEC, and N-glycan analysis of the target polypeptide grown in N-producing culture of polypeptide-3 using CHO cell line C with seeded cultures from the following N-1 cell culture system: fed batch and batch with concentrated glucose and nutrients.

[0026] [Figure 7A] Figure 7A shows the viable cell density of N-producing cultures for polypeptide-3 by CHO cell line C using seeded cultures from the following N-1 cell culture systems: perfusion and fed batch. [Figure 7B] Figure 7B shows the titer of the target polypeptide grown in the production culture for polypeptide-3 using CHO cell line C with seeded cultures from the following N-1 cell culture system: perfusion and fed batch. [Figure 7C] Figure 7C shows the iCIEF, SEC, and N-glycan analysis of the target polypeptide grown in N-producing culture of polypeptide-3 using CHO cell line C with seeded cultures from the following N-1 cell culture system: perfusion and fed batch.

[0027] [Figure 8A] Figure 8A shows the viable cell density of N-producing cultures for polypeptide-1 by CHO cell line A at a 1000L scale (n=3) and 5L satellite (n=2) using seeded cultures from the following N-1 cell culture system: batches with concentrated glucose and nutrients. [Figure 8B] Figure 8B shows the titer of the target polypeptide grown in production cultures of polypeptide-1 using CHO cell line A with seeded cultures from the following N-1 cell culture system, in 1000L scale (n=3) and 5L satellite (n=2) batches with concentrated glucose and nutrients. [Figure 8C]Figure 8C shows iCIEF, SEC, and N-glycan analysis of the target polypeptide grown in N-producing cultures of CHO cell line A for polypeptide-1 using seeded cultures from the following N-1 cell culture system: batches with concentrated glucose and nutrients.

[0028] [Figure 9A] Figure 9A shows the viable cell density of N-producing cultures for polypeptide-2 by CHO cell line B at a 500L scale (n=1) and 5L satellite (n=2) using seeded cultures from the following N-1 cell culture system: batches with concentrated glucose and nutrients. [Figure 9B] Figure 9B shows the titer of the target polypeptide grown in production cultures of polypeptide-2 using CHO cell line B with seeded cultures from the following N-1 cell culture system, in 500L scale (n=1) and 5L satellite (n=2) batches with concentrated glucose and nutrients. [Figure 9C] Figure 9C shows iCIEF, SEC, and N-glycan analysis of the target polypeptide grown in N-producing cultures of CHO cell line B for polypeptide-2 using seeded cultures from the following N-1 cell culture system: batches with concentrated glucose and nutrients.

[0029] [Figure 10A] Figure 10A shows the viable cell density of N-producing cultures for polypeptide-3 by CHO cell line C at a 500L scale (n=1) and 5L satellite (n=2) using seeded cultures from the following N-1 cell culture system: fed batch. [Figure 10B] Figure 10B shows the titer of the target polypeptide grown in production cultures of polypeptide-3 using CHO cell line C with seeded cultures from the following N-1 cell culture system: fed batch. [Figure 10C] Figure 10C shows iCIEF, SEC, and N-glycan analysis of the target polypeptide grown in N-producing cultures of CHO cell line C for polypeptide-3 in 500L scale (n=1) and 5L satellite (n=2) using seeded cultures from the following N-1 cell culture system: fed batch. [Modes for carrying out the invention]

[0030] In one embodiment, the disclosure provides a novel method for increasing the viable cell density of an N-1 large bioreactor cell culture, the method comprising culturing host cells expressing a recombinant polypeptide of interest in a non-perfusion-based culture system, wherein the viable cell density is at least 5 × 10⁶. 6 The number of cells increases up to cells / mL. In one embodiment, the present disclosure provides a novel method for the large-scale production of a recombinant polypeptide of interest, the method comprising the steps of: (1) culturing host cells expressing the recombinant polypeptide of interest in a non-perfusion-based culture system in step N-1, where the viable cell density is at least 5 × 10⁶ 6 (2) Cells inoculated from N-1 cell cultures in a non-perfusion-based culture system increase to at least 1.5 × 10⁶ cells / mL at the N production stage; and (2) Cells inoculated from N-1 cell cultures in a non-perfusion-based culture system increase to at least 1.5 × 10⁶ cells / mL. 6 Culture in concentrated medium at high seeding densities up to cells / mL.

[0031] definition The indefinite article "a" or "an" should be understood to refer to "one or more" of any listed or enumerated components.

[0032] In this specification, the term “about” refers to a value or composition that falls within the tolerance range of a particular value or composition as determined by those skilled in the art, and this depends in part on how the value or composition is measured or determined (i.e., the limits of the measurement system). For example, “about” may, by convention in the art, mean within one standard deviation or within a standard deviation of more than one. Alternatively, “about” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean a value of up to one order of magnitude or up to five times the value. Where a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of “about” should be assumed to be within the tolerance range of that particular value or composition.

[0033] The term "and / or" as used herein should be interpreted as a specific disclosure that includes, or does not include, the other of two identified features or components. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" 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 (alone); B (alone); and C (alone). The use of substitutes (e.g., "or") should be understood to mean either one, both, or any combination thereof of the substitutes.

[0034] In this specification, the term “amino acid” refers in its broadest sense to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N--C(H)(R)--COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. Amino acids (including amino acids at the carboxyl and / or amino terminus of a peptide) may be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the cyclic half-life of the peptide without adversely affecting the peptide's activity. Amino acids may be involved in disulfide bonds. Amino acids may include one or more post-translational modifications (such as bonding with one or more chemical substances (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl components, isoprenoid groups, sulfate groups, polyethylene glycol components, lipid components, glycans, biotin components, etc.)). In some embodiments, the amino acids of the present invention may be provided in a cell culture medium or used for addition to said medium. In some embodiments, the amino acids provided in or used for addition to the cell culture medium may be provided in the form of salts or hydrates.

[0035] As used herein, the term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof) via at least one antigen-recognizing site within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv(scFv) antibodies, multispecific antibodies (such as bispecific antibodies made from at least two intact antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen-recognizing site, insofar as the antibody exhibits the desired biological activity. Antibodies can be one of the following five major classes of immunoglobulins, based on the identity of their heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different known subunit structures and three-dimensional configurations. Antibodies can be naked antibodies or conjugated to other molecules (including, but not limited to, toxins and radioisotopes).

[0036] In this specification, the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" include, for example, (i) Fab fragments (fragments from papain cleavage) or similar monovalent fragments consisting of VL, VH, LC, and CH1 domains; (ii) F(ab')2 fragments (fragments from pepsin cleavage) or similar bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs that may be arbitrarily bound by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together using recombinant methods by a synthetic linker that allows the VL and VH regions to form a single protein chain (known as single-chain Fv (scFv); see, for example, 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 included in the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0037] In this specification, the term “batch culture” refers to a method of culturing cells in which all components ultimately used to culture the cells (including the culture medium (see the definition of “culture medium” below) and the cells themselves) are provided at the beginning of the culture process. Batch cultures are typically stopped at some point, and the cells and / or components in the medium are recovered and optionally purified. The term “fed batch culture” means the addition of a second liquid medium to the first cell culture incrementally or sequentially without substantially or significantly removing the first liquid medium from the cell culture. In some examples, the second liquid medium is the same as the first liquid medium. In other examples, the second liquid medium is a concentrated form of the first liquid medium and / or is added as a dry powder.

[0038] As used herein, the term “bioreactor” refers to any container used for growing mammalian cell cultures. Bioreactors can be of any size as long as they are useful for culturing mammalian cells. Typically, bioreactors are at least 1 liter and can be 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000, 15,000, 20,000 liters or more, or any volume in between. The internal conditions of the bioreactor (including, but not limited to, pH and temperature) are typically controlled during the culture period. Bioreactors may be constructed of any material (including glass, plastic, or metal) suitable for holding mammalian cell cultures suspended in culture medium under the culture conditions of the present invention. As used herein, the term “production bioreactor” refers to the final bioreactor used for the production of the polypeptide or protein of interest. The volume of a large-scale cell culture production bioreactor is typically at least 500 liters, and may be 1,000, 2,500, 5,000, 8,000, 10,000, 12,000, 15,000, 20,000 liters or more, or any volume in between. Those skilled in the art will be able to recognize and select a bioreactor suitable for use in the implementation of the present invention.

[0039] In this specification, the term "live cell density" refers to the number of live cells present in a given volume of culture medium. The term "target cell density" refers to the specific concentration of cells per unit volume of culture medium required to produce recombinant protein during culture. The target cell density may vary depending on the specific mammalian cells being cultured.

[0040] As used herein, the term "cell viability" refers to the ability of cells in culture to survive under given culture conditions or experimental variations. As used herein, this term also refers to the proportion of living cells at a given point in time relative to the total number of living and dead cells in a culture at that time.

[0041] In this specification, the terms “culture,” “cell culture,” and “mammalian cell culture” refer to a population of mammalian cells suspended in a culture medium under conditions suitable for the survival and / or proliferation of the cell population. As will be apparent to those skilled in the art, these terms in this specification may refer to a combination including a population of mammalian cells and the culture medium in which the population is suspended.

[0042] The term "culturing" or "cell culture" refers to the maintenance or proliferation of mammalian cells in a liquid medium under a set of controlled physical conditions.

[0043] In this specification, the terms “culture medium,” “cell culture medium,” and “culture medium” refer to a solution containing nutrients that nourish growing mammalian cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements that cells require for minimal growth and / or survival. The solution may also contain components (including hormones and growth factors) that enhance growth and / or survival in proportions greater than the minimum required. The solution is preferably formulated to the optimal pH and salt concentration for cell survival and growth. The medium may also be a “synthetic medium,” i.e., a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. A limited medium does not contain animal-derived components, and all components have known chemical structures. The term “concentrated medium” or “concentrated synthetic medium” refers to a culture medium that contains additional or increased amounts of carbon sources and / or nutrients compared to a standard culture medium.

[0044] In this specification, the term "N-1 stage" refers to the final seeding and growth stage immediately preceding production inoculation. The N-1 stage is the final cell growth stage before seeding the production bioreactor for polypeptide production. In this specification, the terms "N-2 stage" and "N-3 stage" refer to the cell growth period (typically before inoculation of the N production stage). The N-3 stage is a cell growth stage used to increase the viable cell density for use in the N-2 stage. The N-2 stage is a cell growth stage used to increase the viable cell density for use in the N-1 stage.

[0045] In this specification, the term “perfusion” or “perfusion process” refers to a cell culture method in which an equal volume of culture medium (including nutrient supplements) is simultaneously added to and removed from a bioreactor while cells are retained within the reactor. The corresponding volume of cells and medium is typically removed sequentially or semi-sequentially and optionally purified. Typically, a cell culture process that includes a perfusion process is called “perfusion culture.” In some embodiments, the fresh medium may be identical or similar to the basal medium used in the cell culture process. In some embodiments, the fresh medium may differ from the basal medium and may contain desired nutrient supplements. In some embodiments, the fresh medium is a synthetic medium.

[0046] In this specification, the terms “polynucleotide” or “nucleotide” are intended to encompass single and multiple nucleic acids and refer to isolated nucleic acid molecules or constructs (e.g., messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA)). In some embodiments, polynucleotides include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds found in peptide nucleic acids (PNAs), etc.).

[0047] In this specification, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a product of a specific length. In this specification, the term "protein" is intended to encompass molecules composed of one or more polypeptides (which in some examples may be linked by bonds other than amide bonds). On the other hand, a protein can be a single polypeptide chain. In the latter example, a single polypeptide chain may contain two or more polypeptide subunits fused to form a protein in some examples. The terms "polypeptide" and "protein" also refer to products of post-expression modifications (including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, or modification with amino acids that do not exist in nature). Polypeptides or proteins may originate from natural biological sources or may be produced by recombinant technology, but not necessarily translated from a specified nucleic acid sequence. Polypeptides or proteins may be produced by any method, including chemical synthesis.

[0048] As used herein, the term “polypeptide of interest” is used in the broadest sense to include any protein (whether native or recombinant) present in the mixture to be purified. Such polypeptides of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins.

[0049] The term "production stage" in cell culture refers to the final stage of cell culture. During the production stage, cells first proliferate, and then polypeptides are produced. The production stage is commonly referred to as "N" or the final stage of cell culture production.

[0050] The terms “purify,” “separate,” “isolate,” or “recover” as used interchangeably herein refer to at least partially purifying or isolating recombinant proteins from one or more other components present in a cell medium (e.g., mammalian cells or culture medium proteins) or from one or more other components present in a mammalian cell lysate (e.g., DNA, RNA, or other proteins) (e.g., purity by weight of at least or about 5%, e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least or about 95%). Typically, the degree of purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0051] In this specification, the terms “recombinantly expressed polypeptide” and “recombinant polypeptide” refer to a polypeptide expressed from a mammalian host cell that has been genetically engineered to express that polypeptide. Recombinantly expressed polypeptides may be identical or similar to polypeptides normally expressed in mammalian host cells. Recombinantly expressed polypeptides may also be exogenous to the host cell (i.e., heterogeneous to peptides normally expressed in mammalian host cells). Alternatively, a recombinantly expressed polypeptide may be a chimeric polypeptide in which a portion of the polypeptide contains an amino acid sequence identical or similar to that of polypeptides normally expressed in mammalian host cells, while the other portion is exogenous to the host cell.

[0052] In this specification, the term “seed” refers to the process of providing a cell culture to a bioreactor or another container. The cells may have been previously grown in another bioreactor or container. Alternatively, the cells may be frozen and thawed immediately before being provided to the bioreactor or container. This term refers to any number of cells, including a single cell.

[0053] The term "shaking flask" refers to a container (e.g., a sterile container) capable of holding a volume of liquid culture medium having at least one gas-permeable surface. For example, a shaking flask may be a cell culture flask such as a T-flask, Erlenmeyer flask, or any modified version thereof approved in the art.

[0054] In this specification, the term "titer" refers to the total amount of recombinant expression polypeptide or protein produced by a mammalian cell culture divided by a given volume of culture medium. Titer is usually expressed in milligrams of polypeptide or protein per milliliter of culture medium.

[0055] Various aspects of this disclosure are described in further detail in the following subsections.

[0056] Method of the present invention In one embodiment, the disclosure provides a novel method for increasing the viable cell density of an N-1 large bioreactor cell culture, comprising culturing host cells expressing a recombinant polypeptide of interest in a non-perfusion-based culture system, wherein the viable cell density is at least 5 × 10⁶. 6 This method provides a way to increase the cell count to a maximum of cells / mL.

[0057] In one embodiment, the present disclosure provides a novel method for large-scale production of a recombinant polypeptide of interest, comprising: (1) culturing host cells expressing the recombinant polypeptide of interest in a non-perfusion-based culture system in step N-1, where the viable cell density is at least 5 × 10⁶ 6 (2) Cells inoculated from N-1 cell cultures in a non-perfusion-based culture system increase to at least 1.5 × 10⁶ cells / mL at the N production stage; and (2) Cells inoculated from N-1 cell cultures in a non-perfusion-based culture system increase to at least 1.5 × 10⁶ cells / mL. 6 Culture in concentrated medium at high seeding densities up to cells / mL.

[0058] host cell Any mammalian cell or cell type sensitive to cell culture and polypeptide expression can be used in accordance with the present invention. Some examples of mammalian cells that may be used in accordance with the present invention include: BALB / c mouse myeloma cell line (NSO / 1, ECACC number: 85110503); human retinoblastocytes (PER.C6 (CruCell, Leiden, The Netherlands)); SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for proliferation in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells ± DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251) (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). In some embodiments, the present invention is used for culturing CHO cell lines and for the expression of polypeptides and proteins from CHO cell lines.

[0059] Furthermore, any number of commercially available and uncommercial hybridoma cell lines expressing polypeptides or proteins can be used in accordance with the present invention. Those skilled in the art will understand that hybridoma cell lines may have different nutritional requirements and / or require different culture conditions for optimal growth and polypeptide or protein expression, and these conditions can be modified as needed.

[0060] As mentioned above, in many cases, cells are selected or engineered to produce high levels of protein or polypeptide. Often, cells are genetically engineered to produce high levels of protein (for example, by introducing a gene encoding the protein or polypeptide of interest and / or by introducing a regulatory element that modulates the expression of the gene encoding the polypeptide of interest (either endogenous or introduced)).

[0061] Certain polypeptides may have adverse effects on cell proliferation, cell viability, or other characteristics of cells that ultimately limit the production of the polypeptide or protein of interest in some way. Even among certain types of cell populations engineered to express a particular polypeptide, there is variability within the cell population such that certain individual cells proliferate more and / or produce more of the polypeptide of interest. In one embodiment of the present invention, a cell line is empirically selected by the practitioner for robust proliferation under specific conditions chosen for culturing the cells. In other embodiments, individual cells engineered to express a particular polypeptide are selected for mass production based on cell proliferation, final cell density, % cell viability, titer of the expressed polypeptide, or any combination thereof, or any other condition deemed important by the practitioner.

[0062] Fedobatch cell culture production Typical methods for producing the target polypeptide include batch culture and fed-batch culture production stages for seeding and growth. The batch seeding process traditionally involves inoculating a large-scale production culture with a seeded culture of a specific cell density, growing the cells under conditions that aid cell proliferation and viability, and moving the seeded culture to the next stage when the cells reach a specific cell density. Fed-batch culture methods include further steps of replenishing the batch culture with nutrients and other components consumed during cell growth. Those skilled in the art will recognize that the present invention can be used in any system for culturing cells (including, but not limited to, batch, fed-batch, and perfusion systems). In certain preferred embodiments of the present invention, cells are grown in a batch or fed-batch system.

[0063] Concentrated culture medium The present invention provides concentrated synthetic culture medium formulations that, when used according to other culture steps described herein, increase the viable cell density of host cells in N-1 culture and / or provide more nutrients in production culture at high seeding density compared to host cells cultured in unconcentrated medium. The concentrated culture medium formulations of the present invention, which have been shown to have beneficial effects on cell proliferation or the production of the polypeptide of the desired product, contain i) an increased amount of carbon source, and / or ii) increased nutrients compared to standard culture medium. Furthermore, the carbon source may be: casein, lactate, glucose, fructose, fructan, glucose, sucrose, lactose, maltose, acetate, glycerol, sorbitol, mannitol, saccharose, xylose, molasses, fucose, glucosamine, dextran, fat, oil, glycerol, sodium acetate, arabinose, soy protein, soluble protein, raffinose, amylose, starch, tryptone, yeast extract, and combinations thereof. The nutrients may be amino acids. The concentrated medium is concentrated by 5%, at least 10%, at least 15%, or at least 20% with the feed medium, along with the carbon source and / or nutrients, compared to the unconcentrated medium. Those skilled in the art will understand that the medium formulations of the present invention encompass both limited and unlimited media.

[0064] An unexpected result using concentrated media, as shown in Examples 1-3, is that host cells cultured in batches using concentrated media showed increased viable cell density in the N-1 culture stage compared to host cells cultured in batches using unconcentrated media. Furthermore, host cells cultured in batches using concentrated media showed viable cell density and / or cell viability similar to host cells cultured in a fed-batch system without concentrated media. Therefore, host cells cultured in batches using concentrated media can achieve results similar to those of host cells cultured in a perfusion system or fed-batch system using unconcentrated media.

[0065] Another unexpected result using the concentrated medium shown in Examples 1-3 was that the cultures seeded from cells grown in batch culture using the concentrated medium had similar titers for the target polypeptide as the cultures seeded from cells grown using perfusion or fed-batch methods without the concentrated medium. The above conditions may be used individually or in various combinations.

[0066] Any of these culture medium formulations disclosed in the present invention may be optionally supplemented with hormones and / or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds that are normally present at very low final concentrations), amino acids, lipids, protein hydrolysates, or glucose, or other energy sources, as needed. In some embodiments of the present invention, it may be beneficial to supplement the culture medium with chemical inductants (such as hexamethylenebis(acetamide) ("HMBA") and sodium butyrate ("NaB")). Any of these supplements may be added at the start of the culture, or at a later point in time to replenish depleted nutrients or for other reasons. Those skilled in the art will recognize any desirable or necessary supplements that may be included in the disclosed culture medium formulations.

[0067] Provision of mammalian cell cultures Once cells expressing the target polypeptide or protein are identified, the cells are grown in a culture by one of the various methods well known to those skilled in the art. Cells expressing the target polypeptide or protein are typically grown by growing them in a temperature and medium that aids cell survival, proliferation, and viability. The initial culture volume can be of any size, but is often smaller than the culture volume of the production bioreactor used for the final production of the target polypeptide or protein, and the cells are often passaged several times in a bioreactor with increased volume before seeding into the production bioreactor. Once the cells reach a certain viable cell density, they are grown in a bioreactor to further increase the number of viable cells. These bioreactors are referred to as N-1, N-2, N-3, etc. "N" refers to the bioreactor for the main production culture, "N-1" refers to a bioreactor prior to the main production culture, and so on.

[0068] Cell cultures may be agitated or shaken to increase oxygen supply to the medium and dispersion of nutrients to the cells. Alternatively or additionally, special dispensing devices known in the art may be used to increase and control the oxygen supply to the culture. In accordance with the present invention, those skilled in the art will understand that it may be beneficial to control or regulate certain internal conditions of the bioreactor (including, but not limited to, pH, temperature, and oxygen supply).

[0069] The initial cell density in the N-3 bioreactor can be selected by those skilled in the art. According to the present invention, the initial cell density in the production bioreactor is 2 × 10⁻⁶ 4 It may be as low as live cells / mL. In one embodiment of the present invention, the initial cell density in the N-3 bioreactor is 2 x 10⁻¹⁶ 4 , 2x10 5 , 2x10 6 , 5x10 6 , 10x10 6 Live cells / mL and above. Culture using concentrated medium for N-3 host cells requires at least 5x10 6 Live cells / mL to 5x10 6 , 10x106 , 15x10 6 , 20x10 6 , 25x10 6 or 30x10 6 This can result in live cell densities of up to and above live cells / mL.

[0070] The initial cell density in the N-2 bioreactor can be selected by those skilled in the art. According to the present invention, the initial cell density in the production bioreactor is 2 × 10⁻⁶ 4 The starting cell density may be as low as live cells / mL. In one embodiment of the present invention, the starting cell density in the N-2 bioreactor is approximately 2 x 10⁶ 4 Approximately 2 x 10⁶ cells per mL of live cells 5 , 2x10 6 , 5x10 6 , 10x10 6 Live cells / mL and above. Culture using concentrated medium for N-2 host cells requires at least 5x10⁶ cells. 6 Live cells / mL to 10x10 6 , 15x10 6 , 20x10 6 , 25x10 6 or 30x10 6 This can result in live cell densities of up to and above live cells / mL.

[0071] The starting cell density in the N-1 bioreactor can be selected by those skilled in the art. According to the present invention, the starting cell density in the production bioreactor may be as low as a single cell per culture volume. In one embodiment of the present invention, the starting cell density in the production bioreactor is about 2 x 10⁻¹⁶ 4 Approximately 2 x 10⁶ cells per mL 5 , 2x10 6 , 5x10 6 , 10x10 6 Live cells / mL and above. Culture using concentrated medium for N-1 host cells requires at least 5x10⁶ cells. 6 Approximately 5 x 10 cells per mL of live cells 6 , 10x10 6 , 15x10 6 , 20x10 6, 25x10 6 or 30x10 6 This can result in live cell densities of up to and above live cells / mL.

[0072] The initial cell density in an N-producing bioreactor can be selected by those skilled in the art. According to the present invention, the initial cell density in an N-producing bioreactor is 1 × 10⁻⁶ 6 The starting cell density may be as low as live cells / mL. In one embodiment of the present invention, the starting cell density in the production bioreactor is approximately 1 x 10⁶ 6 Approximately 2 x 10⁶ cells per mL 6 , 5x10 6 , 10x10 6 Live cells / mL may range from and above. Culturing with concentrated host cell medium requires at least 1 x 10⁶ cells. 6 Approximately 2 x 10⁶ cells per mL 6 , 5x10 6 , 10x10 6 , 15x10 6 , 20x10 6 , 25x10 6 or 30x10 6 This can result in live cell densities of up to and above live cells / mL.

[0073] Generally, N-1 cell cultures can be grown to a desired density before seeding in the next production bioreactor. Overall survival or near-overall survival is not required, but it is preferable that the majority of cells remain viable before seeding. In some embodiments of the present invention, cells can be removed from the supernatant (e.g., by slow centrifugation). It may also be desirable to wash the removed cells with culture medium before seeding in the next bioreactor to remove any unwanted metabolic waste or culture medium components. The culture medium may be the medium in which the cells were previously grown, or it may be a different medium or washing solution selected by the practitioner of the present invention.

[0074] Next, the N-1 cells can be diluted to an appropriate density for seeding in a production bioreactor. In one embodiment of the present invention, the cells are diluted in the same medium used in the production bioreactor. Alternatively, the cells may be diluted in a different medium or solution, depending on the needs and requirements of the practitioner of the present invention, or to adapt to the specific requirements of the cells themselves (for example, if the cells need to be stored for a short period before seeding in a production bioreactor).

[0075] According to the present invention, the production bioreactor may be of any volume suitable for large-scale production of polypeptides or proteins. In one embodiment, the volume of the production bioreactor is at least 500 liters. In other embodiments, the volume of the production bioreactor may be 1,000, 2,500, 5,000, 8,000, 10,000, 15,000, 20,000 liters or more, or any volume in between. Those skilled in the art will be able to recognize and select a bioreactor suitable for use in the implementation of the present invention. The production bioreactor may be composed of any substance that does not inhibit the expression or stability of the produced polypeptide or protein and that aids cell proliferation and viability.

[0076] In some embodiments of the present invention, the production step includes a concentrated medium compared to a non-concentrated medium. For example, the medium is concentrated by the feed medium by at least 5%, at least 10%, at least 15%, or at least 20% compared to the non-concentrated medium. In some embodiments, the concentrated medium includes an increased amount of a carbon source (e.g., glucose). In some embodiments, the concentrated medium includes an increased amount of nutrients (e.g., amino acids). In some embodiments, the concentrated medium includes an increased amount of both a carbon source and nutrients.

[0077] The cell culture temperature in the N-1 or production stage is selected primarily based on the temperature range in which the cell culture remains viable. Generally, most mammalian cells grow well in the range of approximately 25°C to 42°C. Preferably, mammalian cells grow well in the range of approximately 35°C to 40°C. Those skilled in the art can select an appropriate temperature for cell growth depending on the cell needs and the practitioner's production requirements. Optionally, the temperature is maintained at a single constant temperature. Optionally, the temperature is maintained within a certain temperature range. For example, the temperature may steadily increase or decrease. Alternatively, the temperature may increase or decrease by discrete amounts over various periods of time. Those skilled in the art can determine whether to use a single temperature or multiple temperatures, and whether to adjust the temperature steadily or by discrete amounts.

[0078] Cells in the N-1 or production stage may be grown for more or less time depending on the practitioner's needs and the requirements of the cells themselves. In some embodiments, cells are grown for a period of time sufficient to achieve a viable cell density which is a predetermined percentage of the maximum viable cell density that can ultimately be achieved if the cells can grow without disturbance. Cells may be grown for a specified period. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, cells may be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or longer. Practitioners of the present invention can select the growth period according to the polypeptide production requirements and the needs of the cells themselves.

[0079] Monitoring of culture conditions In one embodiment of the present invention, specific conditions of a growing cell culture are monitored. Monitoring of cell culture conditions makes it possible to determine whether the cell culture is producing recombinant polypeptides or proteins at suboptimal levels, or whether the culture is about to enter a suboptimal production stage.

[0080] As a non-limiting example, monitoring temperature, pH, cell density, cell viability, integrated viable cell density, lactate levels, ammonium levels, osmolality, or titer of expressed polypeptides or proteins may be beneficial or necessary. Numerous techniques are well known in the art that enable those skilled in the art to measure these conditions. For example, cell density can be measured using a hemocytometer, Coulter counter (Vi-Cell), or cell density test (CEDEX). Viable cell density can be determined by staining the culture sample with trypan blue. Since only dead cells take up trypan blue, viable cell density can be determined by counting the total number of cells, dividing the number of cells that take up the dye by the total number of cells, and taking the reciprocal. HPLC can be used to determine the levels of lactate, ammonium, or expressed polypeptides or proteins. Alternatively, the levels of expressed polypeptides or proteins can be determined by standard molecular biological techniques (such as Coomassi staining of SDS-PAGE gels, Western blotting, Bradford assay, Lowry assay, Biuret assay, and UV absorbance). Furthermore, monitoring post-translational modifications (including phosphorylation and glycosylation) of expressed polypeptides or proteins may be beneficial or necessary.

[0081] Isolation of expressed polypeptides Generally, it is typically desirable to isolate and / or purify the protein or polypeptide expressed according to the present invention. In some embodiments, the expressed polypeptide or protein is secreted into the culture medium, and thus cells and other solids can be removed (e.g., as the first step in the purification process) by centrifugation or filtration. The methods and compositions described herein result in increased cell viability, and therefore these embodiments are particularly useful when used according to the present invention. As a result, fewer cells die during the culture process, and less proteolytic enzymes, which can potentially reduce the yield of the expressed polypeptide or protein, are released into the culture medium.

[0082] Recombinant polypeptides The methods of the present invention can be used for the large-scale production of any recombinant polypeptide of interest (including therapeutic antibodies). Examples of recombinant polypeptides that can be produced by the methods provided herein, but are not limited to, antibodies (including intact immunoglobulins or antibody fragments), enzymes (e.g., galactosidase), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF), or interferon alpha or beta), cell receptors (e.g., EGFR), or immunogenic or antigenic proteins or protein fragments (e.g., proteins for use in vaccines). Antibodies within the scope of this invention include, but are not limited to, the following: anti-HER2 antibodies including trastuzumab (HERCEPTIN®) (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992); anti-HER3 antibodies; anti-HER4 antibodies; U.S. Patent No. 5,725,856); anti-CD20 antibodies such as those described in U.S. Patent No. 5,736,137 (RITUXAN®) (e.g., chimeric anti-CD20 "C2B8"), chimeric or humanized variants of 2H7 antibodies such as those described in U.S. Patent No. 5,721,108B1, or tositumomab (BEXXAR®); anti-IL-8 (St John et al., Chest, 103:932 (1993), and International Publication No. WO 95 / 23865); and humanized anti-VEGF antibodies huA4.6.1 Anti-VEGF antibodies including humanized and / or affinity-mature anti-VEGF antibodies such as AVASTIN® (Kim et al., Growth Factors, 7:53-64 (1992), International Publication No. WO 96 / 30046, and WO 98 / 45331 published on October 15, 1998); anti-PSCA antibodies (WO01 / 40309); anti-CD40 antibodies including S2C6 and its humanized variants (WO00 / 75348); anti-CD11a (U.S. Patent No. 5,622,700, WO 98 / 23761, Steppe et al., Transplant Intl. 4:3-7 (1991), and Hourmant et al.), Transplantation 58:377-380 (1994)); Anti-IgE (Presta et al., J. Immunol. 151:2623-2632 (1993), International Publication No. WO 95 / 19181); Anti-CD18 (as per U.S. Patent No. 5,622,700 issued on April 22, 1997, or WO 97 / 26912 published on July 31, 1997); Anti-IgE (including E25, E26 and E27; as per U.S. Patent No. 5,714,338 issued on February 3, 1998, or U.S. Patent No. 5,091,313 issued on February 25, 1992, WO 95 / 19181 published on March 4, 1993) 93 / 04173, or International Application PCT / US98 / 13410, filed June 30, 1998, U.S. Patent No. 5,714,338); anti-Apo-2 receptor antibody (WO 98 / 51793, published November 19, 1998); anti-TNF-α antibody containing cA2 (REMICADE®), CDP571 and MAK-195 (see U.S. Patent No. 5,672,347, issued September 30, 1997, Lorenz et al., J. Immunol. 156(4):1646-1653 (1996), and Dhainaut et al., Crit. Care Med. 23(9):1461-1469 (1995)); anti-tissue factor (TF) (European Patent No. 0420, granted November 9, 1994) 937 B1); anti-human α4β7 integrin (WO 98 / 06248, published February 19, 1998); anti-EGFR (chimeric or humanized 225 antibodies as described in WO 96 / 40210, published December 19, 1996); anti-CD3 antibodies such as OKT3 (US Patent No. 4,515,893, issued May 7, 1985); anti-CD25 antibodies or anti-tac antibodies such as CHI-621 (SIMULECT®) and (ZENAPAX®) (see US Patent No. 5,693,762, issued December 2, 1997); anti-CD4 antibodies such as cM-7412 antibody (Choy et al., Arthritis Rheum 39(1):52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al.Anti-Fc receptor antibodies such as M22 antibodies against FcγRI, as described in Nature 332:323-337 (1988); Graziano et al., J. Immunol. 155(10):4996-5002 (1995); anti-cancer fetal antigen (CEA) antibodies such as hMN-14 (Sharkey et al., Cancer Res. 55(23 Suppl): 5935s-5945s (1995); antibodies against mammary epithelial cells including huBrE-3, hu-Mc3 and CHL6 (Ceriani et al., Cancer Res. 55(23): 5852s-5856s (1995); and Richman et al., Cancer Res. 55(23 Supp): 5916s-5920s (1995)); antibodies that bind to colon cancer cells such as C242 (Litton et al., Eur J. Immunol. 26(1):1-9 (1996)); anti-CD38 antibodies, e.g., AT13 / 5 (Ellis et al., J. Immunol. 155(2):925-937 (1995)); anti-CD33 antibodies such as Hu M195 (Jurcic et al., Cancer Res 55(23 Suppl): 5908s-5910s (1995)) and CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al.Cancer Res 55(23 Suppl): 5899s-5907s (1995)); anti-EpCAM antibodies such as 17-1A (PANOREX®); anti-GpIIb / IIIa antibodies such as absiximab or c7E3 Fab (REOPRO®); anti-RSV antibodies such as MEDI-493 (SYNAGIS®); anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodies such as PRO542; anti-Hep Anti-hepatitis antibodies such as the B antibody OSTAVIR®; anti-CA125 antibody OvaRex; anti-idiotype GD3 epitope antibody BEC2; anti-αvβ3 antibody VITAXIN®; anti-human renal cell carcinoma antibodies such as ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-human colorectal tumor antibody (A33); anti-human melanoma antibody R24 against GD3 ganglioside; anti-human squamous cell carcinoma antibody (SF-25); anti-human leukocyte antigen (HLA) antibodies such as Smart ID10; anti-PD-1 antibody; anti-PD-L1 antibody; anti-LAG-3 antibody; anti-GITR antibody; anti-TIGIT antibody; anti-CXCR4 antibody; anti-CD73 antibody; and anti-HLA DR antibody Oncolym (Lym-1).

[0083] The above description should be understood as representative only and is not intended to be limiting. Alternative methods and materials for carrying out the present invention, as well as additional applications, will be apparent to those skilled in the art and are intended to be included within the scope of the appended claims. [Examples]

[0084] Cell lines and culture media Three different CHO cell lines, each producing three different monoclonal antibodies or polypeptides, were used in these experiments. The seeding medium, basal medium, and feed medium used were of known composition.

[0085] N-1 seeding culture For batch and fed-batch N-1 cultures, cells were grown in either a 250 ml shaking flask containing an initial volume of 80–100 ml or a 2 L shaking flask containing an initial volume of 1000 ml. A shaking rate of 150 rpm was used in an orbital shaker with an input distance of 25 mm. The incubator was set to a constant temperature of 36.5°C, and CO2 was adjusted to 5%. For batch N-1 cultures, the seeding medium was either unconcentrated, glucose-concentrated, or glucose-concentrated and nutrient-concentrated. No feed was added to the batch N-1 cultures. For fed-batch N-1 cultures, seeding medium was added daily starting from day 3.

[0086] Perfusion N-1 culture involved growing cells in a 10 L cell bag containing an initial volume of 5 L. The agitation speed was adjusted to 28 rpm and the agitation angle was set to 7°. CO2 was adjusted to 4% on days 0-1 and then turned off. A supplemental ATF-2 (Repligen) was connected to the cell bag and the culture was perfused. Old culture medium was continuously removed at the same rate according to the schedule while fresh culture medium (1 × concentrate) was continuously added: increasing to 0.5 VVD D2-4, 1.0 VVD D4-5, and finally increasing to 2.0 VVD D5-6.

[0087] Production culture Fed batch production bioreactors were used in a 5L Sartorius bioreactor with an initial working volume of 3.3L.

[0088] analysis Cell density (VCD) and cell viability were measured offline using a Vi-Cell automated cell counter (Beckman Coulter). Culture samples were also analyzed offline using a Cedex Bio HT (Roche) to monitor glucose, glutamine, glutamic acid, lactate, and ammonium. pH, pCO2, and pO2 were measured offline for bioreactor cultures using a BioProfile pHOX (Nova Biomedical). Protein titers were measured using the Protein A UPLC method and reported as normalized values.

[0089] High molecular weight (HMW) size exclusion chromatography (SEC) is performed on a Waters Alliance HPLC system (Milford, MA) equipped with a temperature-controlled autosampler and a Waters 2996 PDA detector, with a uniform concentration gradient monitored at 280 nm using a Tosoh TSK G3000SW. xl The procedure was performed using a 7.8 x 30 cm, 5 μm column.

[0090] Charge variants were assayed by imaging capillary isoelectric focusing (iCIEF), performed on a Protein Simple iCE3 instrument with an Alcott 720NV autosampler (San Jose, CA). Samples were mixed with appropriate pI markers, amphoteric electrolytes, and urea and injected into fluorocarbon-coated capillary cartridges. High voltage was applied, and the charge variants migrated to their respective pIs. Images were acquired using a UV camera at 280 nM. Major peaks were identified, and peaks that migrated to acidic and basic regions were totaled and quantified, reported as relative percentage areas.

[0091] N-glycan analysis was performed using the commercially available kit GlykoPrep® Rapid N-Glycan Preparation with 2-AB (Hayward, CA) from Prozyme. Free oligosaccharides were profiled using an Acquity UPLC Glycan BEH Amide, 130 Å, 1.7 μm, 2.1 x 10 mm column (Milford, MA) on a Waters Acquity H-Class system (Milford, MA) equipped with a temperature-controlled autosampler and fluorescence detector.

[0092] Example 1 Cell line A:N-1 seeding culture For cell line A, N-1 cultures were grown in batches, batches with glucose concentration, batches with glucose and nutrient concentration, fed batches, or perfusion. Batch N-1 cultures were only 15 × 10⁶. 6 The cell-to-mL (VCD) peak was reached, and high cell viability could not be maintained near the end of the culture period (Figure 1A). In contrast, batch N-1 culture with glucose concentration yielded 17 × 10⁶ cells per mL. 6 VCDs were reached at cells / mL, maintaining a cell viability of >99% (Figure 1A). Similarly, both Fed batch N-1 and batch N-1 with glucose and nutrient concentration achieved >20 × 10⁶ cells on day 6. 6 Cells proliferated to a level of cells / mL, and viability was maintained at >99% (Figures 1A and 1B). Cells in perfused N-1 culture reached 44 × 10⁶ cells on day 6. 6 The cells proliferated to a level of cells / mL, and the survival rate was >99% (Figures 1A and 1B).

[0093] Cell line A for polypeptide-1 production: High-density fed-batch production culture For cell line A, high-density fed-batch production culture was initiated using batches with concentrated glucose, batches with concentrated glucose and nutrients, fed-batch cultures, or seed cultures grown in perfusion culture.

[0094] N-produced culture 5 × 10 6The cells were inoculated at a high seeding density of cells / mL for 14 days. Daily feeding was started on day 2 with a feed volume of 3.5% of the culture volume. Dissolved oxygen (DO) was maintained at 40%, and the pH was adjusted to 6.8–7.6. The temperature was initially maintained at 36.5°C and then reduced to 34°C on day 4.

[0095] Figure 2A shows that all produced cultures maintained a cell viability of >90% throughout the entire culture period. 17 × 10 batch seeding cultures with either concentrated glucose or concentrated glucose and nutrients. 6 Compared to cells / mL, perfusion seeding culture yielded 22 × 10⁶ cells / mL. 6 The maximum viable cell density was observed in cells / mL (Figure 2A). The titer of polypeptide-1 from perfusion-seeded cultures was approximately 9.3 g / L, while that of fed-batch-seeded cultures was approximately 9 g / L (Figure 2B). The titers of the target polypeptide from batch seedings enriched with either glucose or glucose and / or nutrients were approximately 8.5 g / L and 9 g / L, respectively. Figure 2C shows that quality characteristics (iCIEF, SEC, and N-glycan, etc.) were similar under all N-production conditions regardless of the type of N-1 seeding.

[0096] Example 2 Cell line B:N-1 seeding culture For cell line B, N-1 cultures were grown in batches, batches with glucose concentration, batches with glucose and nutrient concentration, fed batches, or perfusion. Each batch of N-1 culture was only 24.5 × 10⁶. 6 The peak VCD of cells / mL was reached, and high viability could not be maintained (Figures 3A and 3B). In contrast, batch N-1 cultures enriched with glucose only, or batch N-1 cultures enriched with both glucose and nutrients, achieved ≥25.5 × 10⁶ viability. 6 Live cells / mL were reached, maintaining a cell viability of ≥99% (Figures 3A and 3B). Similarly, Fed batch N-1 cultures reached ≥30 × 10⁶ cells on day 5. 6Cells proliferated to live cells / mL, and viability was maintained at >99% (Figures 3A and 3B). Cells in perfused N-1 culture reached 41 × 10⁶ cells on day 5. 6 The cells proliferated to a level of 4 / mL, and the survival rate was ≥99% (Figures 3A and 3B).

[0097] Cell line B for polypeptide-2 production: high-density fed-batch production culture For cell line B, we initiated high-density fed-batch production culture using batches, batches with concentrated glucose and nutrients, fed-batch cultures, or seed cultures grown in perfusion culture.

[0098] The produced culture was 3 × 10 6 The cells were inoculated at a high seeding density of cells / mL for 14 days. Daily feeding was started on day 2 with a feed volume of 3.1% of the culture volume. Dissolved oxygen (DO) was maintained at 40%, and the pH was adjusted to 6.7–7.6. The temperature was maintained at 36.5°C.

[0099] Figure 4A shows that all produced cultures maintained a cell viability of >90% throughout the entire culture period. This is only about 24 × 10⁶ of the fed batch seeded cultures compared to batch seeded cultures with either concentrated glucose or concentrated glucose and nutrients. 6 Compared to cells / mL, perfusion seeding culture is approximately 26 × 10⁶ 6 The maximum viable cell density was observed at cells / mL (Figure 4A). The titer of the target polypeptide from perfusion and batch seeded cultures (with concentrated glucose and nutrients) was approximately 3.2 g / L, while the titer from batch and fed-batch seeded cultures was approximately 3 g / L (Figure 4B). Figure 4C shows that quality characteristics (iCIEF, SEC, and N-glycans, etc.) were similar under all N-production conditions regardless of the N-1 seeding.

[0100] Example 3 Cell line C:N-1 seeding culture For cell line C, N-1 cultures were grown in batches, batches with glucose concentration, batches with glucose and nutrient concentration, fed batches, or perfusion. Batch N-1 cultures were only 26 × 10⁶.6 reached the peak VCD of cells / mL and could not maintain a high cell viability (Figures 5A and 5B). In contrast, the batch N-1 culture with only concentrated glucose or the batch N-1 culture with both concentrated glucose and nutrients reached 6 ≧30×10 viable cells / mL and maintained a cell viability of ≧99% (Figures 5A and 5B). Similarly, the fed-batch N-1 culture grew to ≧33×10 6 viable cells / mL on day 5, and the viability was maintained at ≧99% (Figures 5A and 5B). The cells in the perfusion N-1 culture grew to 62×10 6 cells / mL on day 5, and the viability was ≧99% (Figures 5A and 5B).

[0101] Cell line C for polypeptide-3 production (Experiment 1): High-density fed-batch production culture using fed batches or batches with concentrated glucose and nutrient seeds. For cell line C, a high-density fed-batch production culture was initiated using seeds grown in a fed-batch or batch with concentrated glucose and nutrient cultures.

[0102] The production culture was initiated at a high seeding density of 6×10 6 cells / mL for 14 days. The daily feed was started from day 2 at a feed volume of 5% of the culture volume D2-10 and then 3.3% of the starting culture volume D11-13. The feed was performed twice a day at half of the specified amount. Dissolved oxygen (DO) was maintained at 40%, and the pH was adjusted to 6.8 - 7.3. The temperature was initially maintained at 36.5 °C and shifted to 33 °C on day 6.

[0103] Figure 6A shows that all production cultures maintained a cell viability of >80% throughout the entire culture period. The fed-batch seeded culture and the batch seeded culture (with concentrated glucose and nutrients) reached approximately 27×10 6The maximum viable cell density was observed at cells / mL (Figure 6A). The titer of the target polypeptide from fed batches and batch seeded cultures (with concentrated glucose and nutrients) was approximately 4.3 g / L (Figure 6B). Figure 6C shows that quality characteristics (iCIEF, SEC, and N-glycans, etc.) were similar under all N-production conditions regardless of the N-1 seeding.

[0104] Cell line C for polypeptide-3 production (Experiment 2): High-density fed-batch production culture using fed-batch or perfusion seeding For cell line C, we initiated high-density fed-batch production culture using seeds grown in fed-batch or perfusion culture.

[0105] Production culture 6 × 10 6 The culture was initiated for 14 days at a high seeding density of cells / mL. Daily feeding was started from day 1 with a feed volume of 3.7% of the culture volume. Dissolved oxygen (DO) was maintained at 40%, and the pH was adjusted to 6.8–7.3. The temperature was initially maintained at 36.5°C and then reduced to 33°C on day 6.

[0106] Figure 7A shows that all produced cultures maintained a cell viability of >80% throughout the entire culture period. Perfusion seeded cultures were approximately 33 × 10⁶ 6 It has a maximum viable cell density of cells / mL, and a fed batch seeded culture is approximately 30 × 10⁶ 6 The maximum viable cell density was observed at cells / mL (Figure 7A). The titer of the target polypeptide from perfusion and fed-batch seeded cultures was approximately 7 g / L (Figure 7B). Figure 7C shows that quality characteristics (iCIEF, SEC, and N-glycans, etc.) were similar under all N-production conditions, regardless of the N-1 seeding.

[0107] Example 4 Large-scale production processes for the three molecules were carried out in either a 1000L scale (for cell line A) Bristol-Myers Squibb GMP facility or a 500L scale (for cell lines B and C) scale-up facility. N-1 seeding cultures for cell lines A and B utilized glucose and nutrient-enriched batch cultures, while N-1 seeding for cell line C was cultured in a fed-batch manner. All three processes were robust and demonstrated to be scalable up to 1000L or 500L. Cell density, titer, and product quality profiles were consistent with those of satellite cultures in laboratory-scale bioreactors (Figures 8-10). For cell line A, the product culture was harvested on day 10 due to exceptionally high titer exceeding downstream purification capacity. The shortened culture period for cell line A allowed for weekly inoculation of new product cultures (using two production vessels), significantly increasing yield.

Claims

1. A method for increasing the viable cell density of an N-1 large-scale bioreactor cell culture, comprising culturing host cells expressing a target recombinant polypeptide in a non-perfusion-based culture system, wherein the viable cell density is at least 5 × 10⁻⁶. ６ A method to increase the number of cells / mL.

2. The method according to claim 1, wherein the non-perfusion-based culture system is a batch or fed-batch bioreactor.

3. The living cell density at the N-1 stage is at least 5 × 10 ６ , at least 10 x 10 ６ , at least 15 × 10 ６ , at least 20 x 10 ６ , at least 25 × 10 ６ , or at least 30 x 10 ６ The method according to claim 1 or 2, wherein the amount is live cells / mL.

4. The method according to claim 3, wherein the cell viability is at least 80% on the final day of the N-1 stage, at least 85% on the final day of the N-1 stage, or at least 90% on the final day of the N-1 stage.

5. The method according to claim 1 or 2, wherein host cells are cultured in a concentrated medium for N-1 batch culture.

6. The method according to claim 1 or 2, wherein host cells are cultured in seeding medium supplemented with feed medium for N-1 fed batch culture.

7. The method according to claim 5 or 6, wherein the culture medium is concentrated by the feed medium by at least 5%, at least 10%, at least 15%, or at least 20% relative to the unconcentrated medium.

8. The method according to any one of claims 5 to 7, wherein the concentrated medium or feed medium comprises an increased amount of carbon source.

9. The method according to claim 8, wherein the carbon source is glucose.

10. The method according to any one of claims 5 to 7, wherein the concentrated medium or feed medium contains an increased amount of nutrients.

11. The method according to claim 10, wherein the nutrients are selected from amino acids, lipids, vitamins, minerals, and polyamines.

12. The method according to any one of claims 5 to 11, wherein the concentrated medium contains an increased amount of carbon source and nutrients.

13. The method according to claim 12, wherein the carbon source is glucose and the nutrients are selected from amino acids, lipids, vitamins, minerals and polyamines.

14. The method according to any one of claims 1 to 13, wherein the host cell is a mammalian cell.

15. The method according to claim 14, wherein the mammalian cells are selected from the group consisting of CHO, VERO, BHK, HEK, HeLa, COS, MDCK, and hybridoma cells.

16. The method according to claim 14, wherein the host cell is a CHO cell.

17. The method according to any one of claims 1 to 16, wherein the target polypeptide is a therapeutic polypeptide.

18. The method according to any one of claims 1 to 17, wherein the target polypeptide is an antibody or an antigen-binding fragment.

19. The method according to claim 18, wherein the antibody or antigen-binding fragment binds to an antigen selected from the group consisting of PD-1, PD-L1, LAG-3, TIGIT, GITR, CXCR4, CD73 HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, EGFR, HER3, and HER4.

20. The method according to any one of claims 1 to 19, wherein the bioreactor has a volume of at least 50 L, at least 500 L, at least 1,000 L, at least 5,000 L, or at least 10,000 L.

21. In concentrated batch cultures or fed batch cultures used for inoculation at the N production stage to produce the target recombinant polypeptide, at least 5 × 10¹⁶ cells are present at the N-1 stage. ６ The method according to claim 1, further comprising culturing live cells / mL.

22. The method according to claim 21, further comprising the step of isolating the target polypeptide from a production culture system.

23. A method for the large-scale production of a recombinant polypeptide of interest, comprising: (1) culturing a host cell expressing the recombinant polypeptide of interest in a non-perfusion-based culture system in an N-1 stage, where the viable cell density increases to at least 5×10 ６ cells / mL; and (2) culturing N fed-batch production cells in a basal medium or concentrated basal medium at a high seeding density of at least 1.5×10 ６ cells / mL, where the N fed-batch production cells are inoculated from the N-1 stage in a non-perfusion-based culture system.

24. The method according to claim 23, wherein the N-producing culture system is a fed-batch bioreactor.

25. The method according to any one of claims 23 to 25, wherein the concentrated basic medium is concentrated by the feed medium by at least 5%, at least 10%, at least 15%, or at least 20% relative to the unconcentrated medium.

26. The method according to any one of claims 23 to 26, wherein the concentrated culture medium contains an increased amount of carbon source.

27. The method according to claim 27, wherein the carbon source is glucose.

28. The method according to any one of claims 23 to 26, wherein the concentrated culture medium contains an increased amount of nutrients.

29. The method according to claim 29, wherein the nutrients are selected from amino acids, lipids, vitamins, minerals, and polyamines.

30. The method according to any one of claims 23 to 26, wherein the concentrated culture medium contains an increased amount of carbon source and nutrients.

31. The method according to claim 31, wherein the carbon source is glucose and the nutrients are selected from amino acids, lipids, vitamins, minerals and polyamines.

32. The method according to claim 23, wherein the bioreactor has a capacity of at least 50 L, at least 500 L, at least 1,000 L, at least 5,000 L, at least 10,000 L, at least 15,000 L, or at least 20,000 L.

33. The method according to claim 23, wherein the host cell is a mammalian cell.

34. The method according to claim 34, wherein the host cell is a CHO cell.

35. The method according to any one of claims 23 to 35, wherein the potency of the target polypeptide is at least 100 mg / L, at least 1 g / L, at least 3 g / L, at least 5 g / L, or at least 10 g / L.

36. Host cells are cultured in basic medium or concentrated basic medium for N-fed batch-producing bioreactors, with at least 1.5 × 10⁶ cells. ６ , at least 5 × 10 ６ , or at least 10 x 10 ６ The method according to any one of claims 23 to 35, wherein a live cell density of live cells / mL is obtained.

37. The method according to claim 23, further comprising the step of isolating the target polypeptide.

38. The method according to any one of claims 23 to 38, wherein the target polypeptide is a therapeutic polypeptide.

39. The method according to any one of claims 23 to 38, wherein the target polypeptide is an antibody or an antigen-binding fragment.

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