Methods for producing therapeutic proteins

Abstract

The present invention relates generally to a method for producing a therapeutic protein of interest that is secreted from host cells cultured in a bioreactor by adding a carboxypeptidase to the cell culture.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 081380, filed September 22, 2020, and U.S. Provisional Application No. 63 / 225832, filed July 26, 2021, each of which is hereby incorporated by reference in its entirety as part of this specification.

[0002] Technical field of the invention The present invention relates generally to methods for producing a therapeutic protein of interest that is secreted from a host cell. [Background technology]

[0003] Background of the Invention Therapeutic proteins are becoming more common in the treatment of many human diseases. Due to various post-translational modifications, they are known to exhibit a certain degree of heterogeneity during manufacturing processes. Among them, charge variant species (acidic species, major peak, and acidic species) are very commonly observed, for example, in monoclonal antibodies (mAbs) (Hintersteiner et al., 2016). From a regulatory perspective, highly basic species derived from C-terminal lysines are of less concern because they are rapidly cleaved by endogenous serum carboxypeptidase B (CpB) after intravenous injection into patients, with an estimated half-life of approximately 1 hour (Brorson & Jia, 2014). However, charge variants must be monitored to ensure manufacturing consistency and product comparability (Chung et al., 2019). The major peak must be controlled to the release specification for mAb products. Successful control of the manufacturing process is crucial to ensure the quality, safety, and lot-to-lot consistency of therapeutic protein products.

[0004] C-terminal lysine variants in mAb heavy chains, with zero, one, or two lysines remaining during mAb production, were thought to result from inefficient cleavage of C-terminal lysine residues by endogenous carboxypeptidases (Zhang et al., 2015). Because lysines are positively charged and lower the PI of mAb molecules, increasing the amount of remaining lysines increases the proportion of basic species in the charge variant profile (Brorson et al., 2014). To reduce the heterogeneity of mAb C-terminal lysines during production, one solution is to shorten the incubation period of host cells during the production phase. However, this approach significantly reduces mAb productivity.

[0005] Therefore, there is a need to develop improved cell culture methods to increase the productivity of therapeutic proteins with high titers and low heterogeneity. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention In one embodiment of the present invention, we developed a cost-effective enzyme treatment strategy by directly administering CpB treatment to the bioreactor at the end of the upstream cell culture process. This strategy was selected for a number of advantages. First, cell culture conditions typically correspond to enzyme reaction conditions in terms of temperature, pH, mixing, etc., so no additional condition adjustments are required after CpB addition. CpB is introduced prior to the downstream purification sequence, and it can be easily removed during the subsequent purification process. We have shown that the Protein A step was able to remove introduced CpB to below the detection limit by ELISA assay, even when 50-fold the working concentration of enzyme was added. This significantly simplifies the overall process while achieving the goal of producing a qualified therapeutic protein product with improved titer.

[0007] In one embodiment of the present invention, the present disclosure provides a method for producing a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase (CP) to the cultured cells; c) recovering a clarified bulk (CB); and d) subjecting the clarified bulk to a purification process.

[0008] In one embodiment of the present invention, the present disclosure provides a method for improving the productivity of a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase (CP) to the cultured cells; c) recovering a clarified bulk (CB); and d) subjecting the clarified bulk to a purification process.

[0009] In one embodiment of the invention, the bioreactor is a production bioreactor.

[0010] In one embodiment of the present invention, the protein is a protein having a lysine or arginine residue at the C-terminus.

[0011] In some embodiments of the invention, the protein is an Fc-fusion protein or an antibody.

[0012] In one embodiment of the invention, the antibody is a mAb.

[0013] In certain aspects of the invention, the carboxypeptidase is selected from carboxypeptidase A (CPA), carboxypeptidase B (CpB), carboxypeptidase D (CPD), carboxypeptidase H (CPH), carboxypeptidase E (CPE), carboxypeptidase M (CPM), carboxypeptidase N (CPN), carboxypeptidase T (CPT), carboxypeptidase Y (CPY), carboxypeptidase M32, glutamate carboxypeptidase (GCP), prolyl carboxypeptidase (PCP), D-alanyl-D-alanine carboxypeptidase (AACP), or procarboxypeptidase.

[0014] In one embodiment of the invention, the carboxypeptidase is carboxypeptidase B.

[0015] In one embodiment of the invention, the cells are cultured for about 14 days.

[0016] In one embodiment of the invention, the carboxypeptidase is added during the production stage of the cultured cells.

[0017] In one embodiment of the invention, carboxypeptidase is added to cultured cells 0 to 24 hours before harvesting.

[0018] In one embodiment of the invention, carboxypeptidase is added to the cultured cells about 2 hours before harvesting.

[0019] In one embodiment of the invention, carboxypeptidase is added to cultured cells at the time of harvest.

[0020] In some aspects of the invention, the cells are in perfusion, batch or fed-batch cell culture.

[0021] In one embodiment of the invention, the carboxypeptidase is added in an amount sufficient to reduce basic species of the protein.

[0022] In one embodiment of the invention, carboxypeptidase is added at a Cp:protein of interest ratio between 0.0001% and 1% w / w.

[0023] In one embodiment of the invention, the carboxypeptidase is added to the production bioreactor one or more times.

[0024] In one embodiment of the invention, the host cell is a CHO cell.

[0025] In certain aspects of the invention, the antibody binds to an antigen selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73 HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, IL-13, SARS-CoV-2 spike protein, EGFR, HER3, and HER4.

[0026] In one embodiment of the invention, the mAb is Nivolumab. [Brief explanation of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figures 1A, 1B, and 1C show the production performance of CHO cell cultures for mAb-1 in laboratory bioreactors; the normalized space-time yield (solid line, normalized to the average day 10 titer of process B as 100%) and the normalized space-time yield (dashed line, calculated based on the normalized titer over time) are shown for (Figure 1A) process B (enhanced fed-batch production using enriched N-1 seeds) (n = 4), (Figure 1B) process C (enhanced fed-batch production using perfused N-1 seeds) (n = 4), and (Figure 1C) process D (enhanced perfusion production) (n = 2, batch 1 and batch 2 separately), respectively. Because the standard deviation is very small, no error bars are observed at some time points.

[0028] [Figure 2]Figures 2A, 2B, and 2C show normalized main peak levels (normalized to the release specification (spec) for the main peak as 100%) for mAb-1 with CpB treatment (dashed line) and untreated control (solid line) in a lab bioreactor (similar to Figures 1A–C): (Figure 2A) Process B (n = 4), (Figure 2B) Process C (n = 4), and (Figure 2C) Process D (n = 2, for batch 1 and batch 2 separately), respectively. Because the standard deviation is very small, no error bars are observed at some time points.

[0029] [Figure 3] Figures 3A, 3B, and 3C show normalized basic species levels (normalized to the release standard for the major peak as 100%) for mAb-1 with CpB treatment (dashed line) and untreated control (solid line) in a lab bioreactor (similar to Figures 1A–C): (Figure 3A) Process B (n = 4), (Figure 3B) Process C (n = 4), and (Figure 3C) Process D (n = 2, for batches 1 and 2 separately), respectively. Because the standard deviation is very small, no error bars are observed at some time points.

[0030] [Figure 4] Figure 4 shows a comparison of iCE chromatograms of mAb-1 with CpB treatment (solid line) and untreated control (dashed line). The main peak and basic species levels were normalized to the mAb-1 release standard for the main peak, which was set at 100%.

[0031] [Figure 5] Figure 5 shows the mAb-1 normalized major peak and basic species levels after CpB treatment at different enzyme concentrations for 2 h at 34 °C in different matrices: CpB-untreated samples were used as controls (n = 3), and whole cell culture at 1%, 0.05%, and 0.01% (w / w) concentrations, respectively; cell culture supernatant at 0.01% and 0.001% (w / w) concentrations; and 5 mg / mL mAb-1 in Tris buffer, pH 7.6, at 0.01% and 0.001% (w / w) concentrations.

[0032] [Figure 6] Figure 6 shows the mAb-1 normalized main peak and basic species levels after CpB treatment at 0.01% (w / w) CpB concentration for 2 hours at different temperatures (e.g., room temperature (RT), 32°C, 34°C, and 36°C) in cell culture supernatants produced from process B, using CpB-untreated samples as controls (n=3).

[0033] [Figure 7] Figure 7 shows the mAb-1 normalized main peak and basic species levels after CpB treatment with purified mAb-1 at 0.01% (w / w) CpB concentration for 2 hours at 34°C using a CpB-untreated sample as a control (n=3) and at different pH values, e.g., pH 5.0 (25 mM sodium acetate, 12 mM acetic acid); pH 6.1 (25 mM sodium acetate, 12 mM acetic acid); pH 7.0 (25 mM Tris-HCl, 100 mM NaCl); pH 8.0 (25 mM Tris-HCl, 100 mM NaCl); and pH 8.9 (25 mM Tris-HCl, 100 mM NaCl).

[0034] [Figure 8] FIG. 8 shows a flow chart of the mAb-1 manufacturing process.

[0035] [Figure 9] Figure 9 shows the performance profile (e.g., VCD, viability, and normalized titer) of mAb-1 cell cultures in 5 L bioreactors used for Process B (n=2) followed by CpB treatment at 0.05% (w / w) concentration at 34°C for 2 hours, after which the entire downstream process was carried out for both CpB-treated and untreated conditions (Table 1).

[0036] [Figure 10] FIG. 10 shows a schematic diagram of the in vitro CpB treatment protocol.

[0037] [Figure 11] Figures 11A and 11B show the performance profile of fed-batch cell cultures for VCD, viability, and normalized titer (Figure 11A) and metabolite profiles for glucose, lactate, and ammonium levels (Figure 11B) for mAb-8 production in 250 mL shake flasks.

[0038] [Figure 12] Figures 12A, 12B, and 12C show in-process quality attributes (normalized basic charge variant species levels and normalized major peak levels, Figure 12A; N-glycans including G0, G0F, G1F, G2F, and Man5, Figure 12B; SEC impurities including high molecular weight (HMW), low molecular weight (LMW), and monomeric impurities, Figure 12C) for control and CpB-treated conditions of whole cell culture (CpB:mAb-8 ratio 1:10,000 (w / w)) for mAb-8 production in 250 mL shake flasks.

[0039] [Figure 13] Figures 13A and 13B show the performance profile of fed-batch cell cultures, e.g., VCD, viability, and normalized titer (Figure 11A) and metabolite profiles of glucose, lactate, and ammonium levels (Figure 11B), during mAb-3 production in 250 mL shake flasks.

[0040] [Figure 14] Figures 14A, 14B, and 14C show in-process quality attributes (normalized basic charge variant species levels and normalized major peak levels, Figure 14A; N-glycans including G0, G0F, G1F, G2F, and Man5, Figure 12B; SEC impurities including high molecular weight (HMW), low molecular weight (LMW), and monomeric, Figure 12C) for control and CpB-treated conditions of whole cell culture (CpB:mAb-3 ratio 1:10,000 (w / w)) for mAb-3 production in 250 mL shake flasks. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description of the Invention The present invention provides a method for producing a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase (CP) to the cultured cells; c) recovering the clarified bulk; and d) subjecting the clarified bulk to a purification process.

[0042] In one embodiment of the present invention, the present disclosure provides a method for improving the productivity of a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase (CP) to the cultured cells; c) recovering the clarified bulk; and d) subjecting the clarified bulk to a purification process.

[0043] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0044] The indefinite article "a" or "an" should be understood to refer to "one or more" of any described or listed components.

[0045] As used herein, the term "about" 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, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean a range of plus or minus 50% of the stated reference value, preferably a range of plus or minus 25%, or more preferably a range of plus or minus 10%. When a particular value or composition is provided in the present application and claims, unless otherwise specified, the meaning of "about" should be assumed to be within an acceptable error range for the particular value or composition.

[0046] The term "and / or" as used herein is to be understood as a specific disclosure of each of two specified features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (only), and "B" (only). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to encompass 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 (only); B (only); and C (only). The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives.

[0047] As used herein, the term "protein" refers to a peptide-linked amino acid chain, regardless of length. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. The term "protein" is used interchangeably with "polypeptide" herein.

[0048] As used herein, the term "protein of interest" is used in the broadest sense and includes any protein (either natural or recombinant) present in a mixture from which purification is desired. Such proteins of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins.

[0049] As used herein, the term "therapeutic protein" refers to any protein known to be useful in the prevention, treatment, or amelioration of a disease or disorder, such as an antibody, growth factor, cell surface receptor, cytokine, hormone, toxin, or fragment and / or fusion protein of any of the foregoing.

[0050] As used herein, the term "Fc-fusion protein" is meant to encompass a therapeutic protein that includes an immunoglobulin-derived portion (i.e., an Fc portion) and a portion derived from a second, non-immunoglobulin protein.

[0051] "Antibody" includes, but is not limited to, immunoglobulin (Ig), a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, which specifically binds to an antigen, or an antigen-binding portion thereof.

[0052] As used herein, "isolated" or "purified" Ab refers to an Ab that is substantially free of other Abs having different antigen specificities. Furthermore, isolated Ab can also refer to an Ab that has been purified to be substantially free of other cellular material and / or chemicals.

[0053] As used herein, the term "monoclonal antibody" (mAb) refers to an Ab molecule of single molecular composition, i.e., a non-naturally occurring preparation of multiple Ab molecules that are substantially identical in their primary sequence and display a single binding specificity and affinity for a particular epitope. An mAB is an example of an isolated Ab. mAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0054] As used herein, the term "mAb of interest" refers to a mAb present in a mixture for which purification is desired.

[0055] As used herein, a "chimeric" Ab refers to an Ab whose variable region is derived from one species and whose constant region is derived from another species, e.g., the variable region is derived from a murine Ab and the constant region is derived from a human Ab.

[0056] As used herein, a "human" mAb (HuMAb) refers to a mAb having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if an Ab contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human Abs of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human" Ab is not intended to include Abs in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" Ab and "fully human" Ab are used interchangeably.

[0057] As used herein, a "humanized" mAb refers to a mAb in which some, most, or all of the amino acids outside the CDR domains of a non-human mAb have been replaced with corresponding amino acids derived from a human immunoglobulin. In one embodiment of a humanized form of an Ab, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids derived from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not impair the Ab's ability to bind to a specific antigen. A "humanized" Ab retains the same antigen specificity as the original Ab.

[0058] As used herein, an "anti-antigen" Ab refers to an Ab that specifically binds to an antigen. For example, an anti-PD-1 Ab is an Ab that specifically binds to PD-1.

[0059] As used herein, the terms "culture," "cell culture," and "mammalian cell culture" refer to a population of mammalian cells suspended in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, as used herein, these terms can refer to a combination comprising a mammalian cell population and the medium in which the population is suspended.

[0060] The terms "culture" or "cell culture" refer to the maintenance or growth of mammalian cells in a liquid culture medium under controlled set of physical conditions.

[0061] As used herein, the terms "seeding," "seeded," "inoculation," "inoculating," and "inoculated" refer to the process of providing a cell culture to a bioreactor or another vessel. The cells may have been previously grown in another bioreactor or vessel. Alternatively, the cells may be frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term refers to any number of cells, including one cell.

[0062] As used herein, the term "basal medium" refers to a solution containing nutrients for supporting growing mammalian cells. Typically, this solution provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal cell growth and / or survival. The solution may also contain components that promote growth and / or survival beyond the above-mentioned minimum rate, including hormones and growth factors. The solution is preferably adjusted to an optimal pH and salt concentration for cell survival and growth. Various components may be added to the basal medium to support cell growth. The medium may also be a "chemically defined medium" that does not contain serum, hydrolysates, or components of unknown composition. Defined media are animal-derived free, and all components have known chemical structures.

[0063] As used herein, the term "batch" or "batch culture" refers to a cell culture method in which all components that will ultimately be used in the cell culture, including the cells themselves and the basal medium, 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 harvested and optionally purified.

[0064] The term "fed-batch" or "fed-batch cultivation" refers to the incremental or continuous addition of a feed medium to an initial cell culture without substantially or significantly removing the initial basal medium from the cell culture. In some cases, the feed medium is the same as the initial basal medium. In some cases, the feed medium is a concentrated form of the basal medium. In some cases, the feed medium may differ from the basal medium and include desired nutritional supplements.

[0065] As used herein, the term "perfusion" or "perfusion process" refers to a cell culture method in which equal volumes of medium (including nutritional supplements) are simultaneously added and removed from a bioreactor while the cells remain in the reactor. An amount of cells and medium equivalent to the replenishment medium is typically removed continuously or semi-continuously and optionally purified. Cell culture processes that include a perfusion process are typically referred to as "perfusion culture." The fresh medium can be the same as, similar to, or different from the basal medium in the cell culture process.

[0066] As used herein, the term "N-1 stage" refers to the final seed growth stage immediately prior to production inoculation. The N-1 stage is the final cell growth stage before inoculating a production bioreactor for production of a protein of interest. As used herein, the terms "N-2 stage" and "N-3 stage" refer to the period during cell growth and propagation, typically prior to inoculation of the N production stage. The N-3 stage is a cell growth stage used to increase viable cell density for use in the N-2 stage. The N-2 stage is a cell growth stage that can be used to increase viable cell density for use in the N-1 stage.

[0067] The term "production phase" or "N production phase" of a cell culture refers to the final stage of cell culture. In the production phase, cells are cultured with the primary focus on producing a protein of interest, e.g., a mAb. The production phase is commonly referred to as the "N" or final stage of cell culture manufacturing.

[0068] As used herein, the term "bioreactor" refers to any vessel used for the growth of mammalian cell cultures. Bioreactors can be of any size, so long as they are useful for culturing mammalian cells. Typically, bioreactors are at least 1 liter and can be 2, 5, 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000, 15,000, 20,000 liters or more, or any volume in between. Internal conditions of the bioreactor include, but are not limited to, pH and temperature, and are typically controlled during the culture period. Bioreactors can be constructed of any material suitable for holding a mammalian cell culture suspended in a medium under the culture conditions of the present invention, including glass, plastic, or metal.

[0069] As used herein, "production bioreactor" refers to the final bioreactor used in the production stage of a cell culture for the production of a protein of interest (e.g., a mAB). Large-scale cell culture production bioreactor volumes are typically at least 500 liters, and can be 1000, 2500, 5000, 8000, 10,000, 12,000, 15,000, 20,000 liters or more, or any volume in between. One of skill in the art would be able to recognize and select a suitable bioreactor for use in the practice of the present invention.

[0070] As used herein, the term "titer" refers to the total amount of recombinantly expressed protein of interest, such as a mAb, produced by a mammalian cell culture divided by a given volume of culture medium. Titer is typically expressed in grams of polypeptide or protein per liter of culture medium.

[0071] The term "harvesting" refers to the procedure of removing cells and cell debris to obtain a clarified bulk (CB) suitable for protein purification, which is generally accomplished by any suitable technique, including but not limited to, centrifugation, depth filtration, or sterile filtration, depending on scale and equipment capabilities.

[0072] As used herein, "clarified bulk" and "CB" are used interchangeably and refer to the solution recovered after a primary recovery step, such as centrifugation, depth filtration, or flocculation, used to clarify a cell culture medium.

[0073] The term "purified" refers to a procedure in which a protein of interest is at least partially purified or isolated (e.g., at least or about 5% pure by weight, 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% pure) from one or more other components present in a cell culture medium (e.g., mammalian cell proteins or culture medium proteins) or from one or more other components present in a mammalian cell lysate (e.g., DNA, RNA, or other proteins). Typically, the purity of the protein of interest is increased by removing (completely or partially) at least one impurity from the composition.

[0074] The term "carboxypeptidase" refers to a multifunctional enzyme that cleaves proteins from the C-terminus. Many different types of carboxypeptidases exist, including carboxypeptidase A (CpA), carboxypeptidase B (CpB), carboxypeptidase D (CpD), carboxypeptidase E (CpE), carboxypeptidase M (CpM), carboxypeptidase N (CpN), carboxypeptidase T (CpT), carboxypeptidase Y (CpY), carboxypeptidase M32, glutamate carboxypeptidase (GCP), prolyl carboxypeptidase (PCP), D-alanyl-D-alanine carboxypeptidase (AACP), procarboxypeptidase, and others.

[0075] In one embodiment of the present invention, the present disclosure provides a method for producing a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase to the cultured cells; c) harvesting and collecting the clarified bulk (CB); and d) subjecting the clarified bulk to a purification process.

[0076] In one embodiment of the present invention, the present disclosure provides a method for improving the productivity of a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase to the cultured cells; c) harvesting and collecting the clarified bulk (CB); and d) subjecting the clarified bulk to a purification process.

[0077] The term "carboxypeptidase B" refers to a carboxypeptidase that sequentially cleaves basic amino acids such as arginine and lysine from the C-terminus of proteins.

[0078] In one embodiment of the invention, the carboxypeptidase is carboxypeptidase B (CpB).

[0079] In one embodiment of the present invention, the present disclosure provides a method for producing a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase B to the cultured cells; c) harvesting and collecting the clarified bulk (CB); and d) subjecting the clarified bulk to a purification process.

[0080] In one embodiment of the present invention, the present disclosure provides a method for improving the productivity of a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase B to the cultured cells; c) harvesting and collecting the clarified bulk (CB); and d) subjecting the clarified bulk to a purification process.

[0081] Various aspects of the disclosure are described in further detail in the following subsections.

[0082] In certain embodiments of the present invention, the present disclosure provides a method for improving productivity of a monoclonal antibody (mAb) of interest, comprising the steps of: a) culturing host cells expressing the mAb in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase (CP) to the cultured cells; c) recovering a clarified bulk; and d) subjecting the clarified bulk to a purification process.

[0083] In some embodiments of the invention, the protein has a C-terminal lysine or arginine.

[0084] In some embodiments of the invention, the protein is an Fc-fusion protein or an antibody.

[0085] In one embodiment of the invention, the antibody is a mAb.

[0086] In one embodiment of the invention, the bioreactor is a production bioreactor.

[0087] In one embodiment of the invention, carboxypeptidase is added to a batch, fed-batch, or perfusion cell culture prior to harvest.

[0088] In certain aspects of the invention, the carboxypeptidase is selected from carboxypeptidase A (CPA), carboxypeptidase B (CpB), carboxypeptidase D (CPD), carboxypeptidase H (CPH), carboxypeptidase E (CPE), carboxypeptidase M (CPM), carboxypeptidase N (CPN), carboxypeptidase T (CPT), carboxypeptidase Y (CPY), carboxypeptidase M32, glutamate carboxypeptidase (GCP), prolyl carboxypeptidase (PCP), D-alanyl-D-alanine carboxypeptidase (AACP), or procarboxypeptidase.

[0089] In some embodiments of the invention, the cells are cultured in the production bioreactor for about 14 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 15 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 16 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 17 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 18 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 19 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 20 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 21 days. In some embodiments of the invention, the cells are cultured in the production bioreactor for about 22 days.

[0090] In one embodiment of the invention, the carboxypeptidase is added during the production stage of the cultured cells.

[0091] In one embodiment of the invention, carboxypeptidase is added to cultured cells 0 to 24 hours before harvesting.

[0092] In certain embodiments of the invention, carboxypeptidase is added to cultured cells about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or 24 hours before harvesting.

[0093] In one embodiment of the invention, carboxypeptidase is added to the cultured cells about 2 hours before harvesting.

[0094] In one embodiment of the invention, carboxypeptidase B is added to the cultured cells about 2 hours before harvesting.

[0095] In one embodiment of the invention, carboxypeptidase is added to cultured cells at the time of harvest.

[0096] In some aspects of the invention, the cells are in perfusion, batch or fed-batch cell culture.

[0097] In one aspect of the invention, the carboxypeptidase is added in an amount sufficient to reduce basic species of the protein of interest.

[0098] In some embodiments of the invention, the carboxypeptidase is added at a CP:protein of interest ratio between 0.0001% and 1% w / w, hi some embodiments of the invention, the carboxypeptidase is added at a CP:protein of interest ratio of about 0.001%, about 0.002%, about 0.005%, about 0.01%, about 0.02%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, or about 1% w / w.

[0099] In one embodiment of the invention, the carboxypeptidase is added in an amount sufficient to reduce basic species of the mAb of interest.

[0100] In one embodiment of the invention, carboxypeptidase B is added at a CP:mAb of interest ratio between 0.0001% and 1% w / w.

[0101] In certain embodiments of the invention, carboxypeptidase B is added at a CP:mAb of interest ratio of about 0.001%, about 0.002%, about 0.005%, about 0.01%, about 0.02%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, or about 1% w / w.

[0102] In one embodiment of the invention, carboxypeptidase B is added at about 0.01%, w / w CP:mAb of interest.

[0103] In some embodiments of the invention, the carboxypeptidase is added multiple times to the production bioreactor.

[0104] In one embodiment of the invention, the host cell is a CHO cell.

[0105] In certain aspects of the invention, the antibody binds to an antigen selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73 HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, IL-13, SARS-CoV-2 spike protein, EGFR, HER3, and HER4.

[0106] In one embodiment, the mAb is nivolumab.

[0107] mAb charge variants Monoclonal antibodies (mAbs) are heterogeneous in their biochemical and biophysical properties due to multiple post-translational modifications and degradation events. The charge heterogeneity of mAbs can be affected by changes in the net charge or local charge distribution caused by these modifications. Charge variants of mAbs are identified as acidic, basic, and major species. As used herein, the terms "major species," "major peak," or "major variant" of a mAb refer to a mAb that elutes as a major peak with a neutral isoelectric point (PI). As used herein, the terms "acidic species" or "acidic variant" of a mAb refer to a variant with a lower pI than the major species. As used herein, the terms "basic species" or "basic variant" of a mAb refer to a variant with a higher pI than the major species. The C-terminal lysine residue of a mAb adds a positive charge, resulting in an increase in the basic species of the mAb. Inefficient cleavage of C-terminal lysine residues by endogenous carboxypeptidases in antibody production is one of the main reasons resulting in mAbs with zero, one, or two C-terminal lysines ( Zhang et al., 2015 ).

[0108] Charge variants can be separated using charge-based separation techniques, such as isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX). Herein, charge variant species were analyzed by imaging capillary isoelectric focusing (iCIEF) using a Protein Simple iCE3 instrument with an Alcott 720NV autosampler. Samples were mixed with appropriate pI markers, ampholytes, and urea and injected into a fluorocarbon-coated capillary cartridge. A high voltage was applied, creating a pH gradient within the column. Charge variants migrated to their respective pIs. Images were taken at 280 nM with a UV camera. The major peaks were identified, and peaks migrating into the acidic and basic regions were summed, quantified, and reported as relative percent areas.

[0109] Several methods for controlling charge variant profiles include using cell culture media with low copper concentrations (Kim et al., 2016), adjusting the lysine concentration in the medium, temperature shifts (Kim et al., 2016), extending the culture period, and constructing mAbs with C-terminal deletions of lysines (Luo et al., 2012; Jiang et al., 2016). These strategies occur during or before the cell culture stage and thus can collectively affect multiple pathways and lead to unpredictable overall cell culture characteristics, titer profiles, or product quality. U.S. Patent No. 5,672,347 discloses a method of adding carboxypeptidase to the clarified bulk, which involves extra steps and incubation times.

[0110] In contrast, the method of the present invention uses carboxypeptidase near the end of the cell culture production stage to avoid these variables that affect cell culture. Because the added carboxypeptidase is removed via the usual downstream purification processes, no additional steps are required to remove this new entity without any changes to the original operational design. This greatly simplifies the overall procedure.

[0111] Charge variant profiles are mAb-dependent, and some mAbs (e.g., mAb-1) have a high proportion of basic species and a low proportion of major species when secreted from host cells during cell culture. For example, the proportion of basic species for mAb-1 is greater than 8 percent after 10 days of culture in a production bioreactor. The proportion of mAb basic variants continues to increase with increasing production time. Therefore, for these mAbs, it may not be possible to achieve a longer production period with high titer but a low proportion of mAb basic variants. In the present invention, for these mAbs that suffer from a high proportion of basic variants, the cell culture period in a production bioreactor can be extended from 10 days to 14 days. This method may be applicable to other biologics with similar charge variant profile problems.

[0112] In this study, we developed a simple, robust, and cost-effective CpB treatment method to improve the quality and productivity of intensified cell culture processes for mAb-1 production using industrial CHO cell lines. The intensified processes, such as Process B intensified with non-perfused N-1, Process C intensified with perfused N-1, and Process D intensified with perfusion during production, achieved significantly higher productivity than the conventional fed-batch manufacturing process A. However, intensified fed-batch processes B and C required much earlier harvesting, and perfusion process D was not viable due to a low main peak that did not meet release specifications. The low main peak was primarily due to the higher abundance of basic species resulting from the higher abundance of C-terminal lysine variants in the intensified processes. After CpB treatment, the C-terminal lysine was effectively removed, resulting in a significant reduction in basic species and an increase in main peak levels for all intensified processes, meeting release specifications throughout the entire culture period. In the longer cell culture production window after CpB treatment, fed-batch processes B and C achieved higher final titers. Additionally, clearance of CpB during downstream processing was demonstrated by similar downstream yields and in-process quality attribute profiles with and without CpB treatment.

[0113] In one embodiment of the invention, carboxypeptidase is added to cultured cells in an amount sufficient to reduce basic species of the mAb of interest.

[0114] In one embodiment of the invention, carboxypeptidase is added to cultured cells in an amount sufficient to enrich for the predominant species of the mAb of interest.

[0115] host cell Any mammalian cell or cell type amenable to cell culture and protein expression can be utilized in accordance with the present invention. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include the BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblastoma cells (PER.C6 (CruCell, Leiden, The Netherlands)); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth 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 carcinoma cells (HeLa, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, 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 (Mather et al., Annals NY Acad. Sci., 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma-derived cell line (Hep G2). In one embodiment of the present invention, the host cell is a CHO cell line.

[0116] Mammalian cell culture offerings Once cells expressing a protein of interest are identified, the cells are expanded in culture by any of a variety of methods known to those skilled in the art. Cells expressing the protein of interest are typically expanded by growing at a temperature and in a medium conducive to cell survival, proliferation, and viability. The initial culture volume can be of any scale but is usually smaller than the culture volume of the production bioreactor used in the ultimate production of the protein of interest, and the cells are often passaged several times in bioreactors of increasing volume before seeding the production bioreactor. Once the cells reach a certain viable cell density, they are grown in bioreactors to further increase the viable cell number. These bioreactors are designated N-1, N-2, N-3, etc. For example, "N" refers to the production culture bioreactor, "N-1" refers to the bioreactor prior to the production culture, etc.

[0117] Generally, the N-1 cell culture can be grown to a desired density before inoculating the next production bioreactor. Prior to inoculation, it is preferred that the majority of cells remain viable, although complete or near complete viability is not required.

[0118] Production of expressed proteins In the production phase, cells are cultured with the primary goal of producing a protein of interest, e.g., a mAb of interest. The production phase is commonly referred to as the "N" or final phase of cell culture manufacturing. The term "production bioreactor" generally refers to the final bioreactor used in the production phase of a cell culture for the production of a protein of interest (e.g., a mAb). The volume of a large-scale cell culture production bioreactor is typically at least 500 liters, and can be 1000, 2500, 5000, 8000, 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 suitable bioreactors for use in practicing the present invention.

[0119] In certain embodiments of the present invention, the cell culture is a perfusion, batch, or fed-batch cell culture. In certain preferred embodiments, the cell culture in the production bioreactor is a fed-batch cell culture.

[0120] Extending the cell culture production window through cell line engineering, medium development, and bioreactor optimization has been one of the major strategies for improving titer in fed-batch processes in recent decades (Druz, Son, Betenbaugh, & Shiloach, 2013; Fan et al., 2015; Powers et al., 2019; Wurm, 2004). Regardless of the cell culture production window, a single production batch requires similar cell growth time to peak VCD, batch-to-batch turnaround time, and batch setup costs, including seed growth, medium preparation, and bioreactor operation (Chen, et al., 2018; Xu, et al., 2020). Therefore, increasing titer by extending the cell culture production window during stationary phase can improve manufacturing productivity and reduce production costs (Wurm, 2004; Xu et al., 2012; Xu, et al., 2020). However, fed-batch cell cultures for periods longer than two weeks can eventually enter a death phase with increased toxic metabolites, decreased VCD, and decreased cell viability, resulting in reduced product quality (Arden & Betenbaugh, 2004; Henry et al., 2020). In some cases, fed-batch cell cultures may be required to be harvested earlier in the stationary phase to meet quality requirements and release specifications.

[0121] Here, fortification processes B and C had to be terminated early because the main peak did not meet specifications on day 14 (Figure 1A–C). However, after CpB treatment, the main peak met specifications throughout the entire fortification process (Figure 1A–C). Process B was extended from 10 to 14 days, and CpB treatment improved the space-time yield by 38% and reduced the upstream consumable cost per gram of final drug substance by 22%. Similarly, process C was extended from 6 to 14 days, improved the space-time yield by 108%, and reduced the upstream consumable cost by 46% (Figure 1A–C and Table 2). Compared to the conventional process A, the addition of this CpB treatment step and the previously described process fortification strategy significantly improved the space-time yield by 3.7-fold, 5.4-fold, and 11-fold for processes B, C, and D, respectively (Figure 1A–C).

[0122] Other mitigation strategies affecting C-terminal lysine levels, such as cell line engineering and optimization of cell culture media and processes, have been reported in the literature. Hu et al. (2017) reported that CHO cells with a C-terminal lysine-deleted DNA sequence indeed produced fewer C-terminal lysine-related basic variants, but exhibited more proline amidation than wild-type CHO cells, resulting in lower titers and an increase in the overall amount of basic species. Furthermore, CHO cells lacking C-terminal lysines and glycines were found to produce fewer basic species and comparable titers than wild-type CHO cells. However, the safety of lysine- and glycine-deleted mAbs still needs to be tested in patients before commercial use (Hu et al., 2017). Furthermore, after knocking out the carboxypeptidase D gene, CHO cells produced 100% C-terminal lysines at the termini of mAb heavy chains (Hu et al., 2016). While this provides another solution to eliminate C-terminal lysine heterogeneity, it is contrary to our goal of minimizing C-terminal lysine, so changing the cell line to produce a more uniform product is not an option. Generally, changing the cell line would require a long time due to the time required for cell line generation, lead clone screening, and new process development. Most importantly, depending on the stage of development, changing the cell line is a major process change that may require strict regulatory scrutiny. Therefore, changing the cell line may be more difficult to implement than adopting the enzymatic treatment we developed.

[0123] Modulation of C-terminal lysine has also been approached using cell culture media. Lowering copper and increasing zinc concentrations (Luo et al., 2012; Yuk et al., 2015) and adjusting lysine concentration in the media (Zhang et al., 2015) can be used to reduce C-terminal levels. Regarding process control strategies, a well-studied study demonstrated that a temperature shift to lower temperatures increases the main peak level in cell cultures (Kishishita et al., 2015; McHugh, Xu, Aron, Borys, & Li, 2020). However, these media and temperature shift strategies are predictably cell line and process dependent.

[0124] In one embodiment of the present invention, cells are grown in a bioreactor for a specified period of time to produce a protein of interest. For example, depending on the starting density of the cell culture, the cell growth temperature, and the intrinsic growth rate of the cells, cells may be grown in the bioreactor for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days. The practitioner of the present invention can select the growth period depending on the production requirements of the polypeptide and the needs of the cells themselves. In one embodiment of the present invention, carboxypeptidase is added after growing cells in the production bioreactor for at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, or at least 22 days.

[0125] At the end of the production bioreactor run, the cell culture broth is removed and harvested to remove cells and cell debris to provide a clarified bulk suitable for protein production, which is generally accomplished by any suitable technique, including but not limited to, centrifugation, depth filtration, and sterile filtration, depending on the scale and facility capacity.

[0126] In some embodiments of the invention, the carboxypeptidase is added at any time during the production phase of the cell culture. In some embodiments of the invention, the carboxypeptidase is added to cells growing in a production bioreactor 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours or more before harvest, or any time in between, or during harvest. In some preferred embodiments of the invention, the carboxypeptidase is added about 2 hours before harvest.

[0127] In certain aspects of the invention, after adding carboxypeptidase to the production bioreactor, the culture temperature is maintained at the same temperature used for the previous culture conditions, or the temperature is shifted. Those skilled in the art will recognize and be able to select suitable temperatures for use in the practice of the present invention.

[0128] In some embodiments of the invention, after adding carboxypeptidase to the production bioreactor, the culture pH is maintained at the same pH used for the previous culture conditions, or the pH is shifted. In some embodiments, after adding carboxypeptidase, the pH is greater than 7.0. Those skilled in the art will recognize and be able to select a suitable pH for use in the practice of the present invention.

[0129] The CpB treatment developed in this invention provides a simple, robust, flexible, and cost-effective method for efficient removal of C-terminal lysines, which is expected to have minimal impact on cell culture production. Due to the nature of the underlying mechanism, this enzymatic treatment strategy may also be applied to other processes with similar quality issues.

[0130] Purification of expressed proteins Generally, it is desirable to isolate and / or purify a protein of interest (e.g., a mAb) from the clarified bulk, which typically undergoes a series of purification steps, including but not limited to affinity chromatography (e.g., Protein A chromatography), viral inactivation & depth filtration, hydrophobic interaction chromatography, ion exchange chromatography, viral filtration, concentration, and diafiltration.

[0131] In one embodiment of the invention, carboxypeptidase is added during any of these purification steps.

[0132] In one embodiment of the present invention, the present disclosure provides a method for improving the productivity of a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) recovering a clarified bulk; c) subjecting the clarified bulk to a purification process; and d) adding a carboxypeptidase to the purification process.

[0133] In one embodiment of the present invention, the present disclosure provides a method for producing a therapeutic protein of interest, comprising the steps of: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) recovering a clarified bulk; c) subjecting the clarified bulk to a purification process; and d) adding a carboxypeptidase to the purification process.

[0134] Recombinant antibodies The methods of the present invention can be used for the large-scale production of any antibody. Antibodies within the scope of the present invention include, but are not limited to: Trastuzumab (HERCEPTIN (登録商標)anti-HER2 antibodies, including (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, e.g., chimeric anti-CD20 "C2B8" in U.S. Patent No. 5,736,137; RITUXAN (登録商標) , chimeric or humanized variants of the 2H7 antibody in U.S. Pat. No. 5,721,108, or Tositumomab (BEXXAR (登録商標) anti-IL-8 (St John et al., Chest, 103:932 (1993), and WO 95 / 23865); humanized and / or affinity matured anti-VEGF antibodies, such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN (登録商標)Anti-VEGF antibodies, including (Kim et al., Growth Factors, 7:53-64 (1992), WO 96 / 30046, and WO 98 / 45331 published October 15, 1998); anti-PSCA antibodies (WO 01 / 40309); anti-CD40 antibodies, including S2C6 and its humanized variants (WO 00 / 75348); anti-CD11a (U.S. Pat. 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. al., J. Immunol. 151:2623-2632 (1993), and International Publication No. WO 95 / 19181; anti-CD18 (U.S. Pat. No. 5,622,700, issued April 22, 1997, or U.S. Pat. No. WO 97 / 26912, published July 31, 1997); anti-IgE (including E25, E26, and E27; U.S. Pat. No. 5,714,338, issued February 3, 1998); No. 5,091,313, issued February 25, 1992, International Publication No. WO 93 / 04173, published March 4, 1993, or International Application No. PCT / US98 / 13410, filed June 30, 1998, U.S. Patent No. 5,714,338; anti-Apo-2 receptor antibodies (WO 98 / 51793, published November 19, 1998); cA2 (REMICADE (登録商標)), anti-TNF-α antibodies including CDP571 and MAK-195 (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. 0420937, granted November 9, 1994); anti-human α4β7 integrin (International Publication No. WO 98 / 06248, published February 19, 1998); anti-EGFR (chimeric or humanized 225 antibody in International Publication No. WO 96 / 40210, published December 19, 1996); anti-CD3 antibodies, e.g., OKT3 (U.S. Patent No. 4,515,893, issued May 7, 1985); anti-CD25 or anti-tac antibodies, e.g., CHI-621 (SIMULECT (登録商標) ) and (ZENAPAX (登録商標)) (see U.S. Patent No. 5,693,762 issued December 2, 1997); anti-CD4 antibodies, such as the cM-7412 antibody (Choy et al., Arthritis Rheum 39(1):52-56 (1996)); anti-CD52 antibodies, such as CAMPATH-1H (Riechmann et al., Nature 332:323-337 (1988)); anti-Fc receptor antibodies, such as the M22 antibody directed against FcγRI in Graziano et al., J. Immunol. 155(10):4996-5002 (1995); anti-carcinoembryonic antigen (CEA) antibodies, such as hMN-14 (Sharkey et al., Cancer Res. 55(23Suppl):5935s-5945s (1995); antibodies directed against breast 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(23Supp):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, such as AT 13 / 5 (Ellis et al., J. Immunol. 155(2):925-937 (1995)); anti-CD33 antibodies, such as Hu M195 (Jurcic et al., Cancer Res. 55(23Suppl):5908s-5910s(1995) and CMA-676 or CDP771; anti-CD22 antibodies, such as LL2 or LymphoCide (Juweid et al., Cancer Res 55(23Suppl):5899s-5907s(1995)); anti-EpCAM antibodies, such as 17-1A (PANOREX (登録商標) ); anti-GpIIb / IIIa antibodies, such as abciximab or c7E3 Fab (REOPRO (登録商標) ); anti-RSV antibodies, e.g., MEDI-493 (SYNAGIS (登録商標) ); anti-CMV antibodies, e.g. PROTOVIR (登録商標)anti-HIV antibodies, such as PRO542; anti-hepatitis antibodies, such as the anti-HepB antibody OSTAVIR (登録商標) ; anti-CA125 antibody OvaRex; anti-idiotype GD3 epitope antibody BEC2; anti-αvβ3 antibody VITAXIN (登録商標) ; anti-human renal cell carcinoma antibodies, such as ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-human colorectal cancer antibody (A33); anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous cell carcinoma (SF-25); anti-human leukocyte antigen (HLA) antibodies, such as Smart ID10; anti-PD-1 antibodies; anti-PD-L1 antibodies; anti-LAG-3 antibodies; anti-GITR antibodies; anti-TIGIT antibodies; anti-CXCR4 antibodies; anti-CD73 antibodies; anti-IL-13 antibodies, anti-SARS-CoV-2 spike protein antibodies and anti-HLA DR antibody Oncolym (Lym-1).

[0135] In one embodiment of the invention, the mAb is nivolumab.

[0136] In one aspect of the present invention, the disclosure provides a method for reducing heterogeneity of a monoclonal antibody (mAb) of interest in a cell culture, the method comprising the steps of: a) culturing host cells expressing the mAb in a production bioreactor under optimized culture conditions for at least 6 days, wherein the proportion of mAb basic species is greater than 8% of the total mAb; b) adding carboxypeptidase to the cultured cells; c) recovering the clarified bulk; and e) subjecting to a purification process.

[0137] In one aspect of the present invention, the disclosure provides a method for reducing heterogeneity of a monoclonal antibody (mAb) of interest in a cell culture, the method comprising the steps of: a) culturing host cells expressing the mAb in a production bioreactor under optimized culture conditions for at least 10 days, wherein the proportion of mAb basic species is greater than 10% of the total mAb; b) adding carboxypeptidase to the cultured cells; c) recovering the clarified bulk; and e) subjecting to a purification process.

[0138] In certain embodiments of the invention, carboxypeptidase is added to the production bioreactor at a CP:mAb ratio sufficient to reduce mAb basic species, such that the percentage of mAb basic species in the CP-treated clarified bulk is reduced to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1% or less of the total mAb.

[0139] In one embodiment of the invention, carboxypeptidase is added to the production bioreactor at a CP:mAb ratio sufficient to reduce mAb basic species, where the proportion of mAb basic species in the CP-treated clarified bulk falls to less than 8% of the total mAb.

[0140] In certain preferred embodiments of the present invention, the proportion of mAb basic species in the CP-treated clarified bulk is reduced to less than 5% of the total Ab.

[0141] The foregoing description should be understood as merely representative and is not intended to be limiting. Alternative methods and materials for practicing the invention, as well as additional uses, will be apparent to those skilled in the art and are intended to be encompassed by the appended claims. For example, the present disclosure provides: [Section 1] 1. A method for producing a therapeutic protein of interest, comprising: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase (CP) to the cultured cells; c) recovering the clarified bulk; and d) subjecting the clarified bulk to a purification process; A method comprising: [Section 2] 1. A method for improving productivity of a therapeutic protein of interest, comprising: a) culturing host cells expressing the protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase (CP) to the cultured cells; c) recovering the clarified bulk; and d) subjecting the clarified bulk to a purification process; A method comprising: [Section 3] The method according to items 1 to 2, wherein the carboxypeptidase is selected from carboxypeptidase A (CPA), carboxypeptidase B (CPB), carboxypeptidase D (CPD), carboxypeptidase H (CPH), carboxypeptidase E (CPE), carboxypeptidase M (CPM), carboxypeptidase N (CPN), carboxypeptidase T (CPT), carboxypeptidase Y (CPY), carboxypeptidase M32, glutamate carboxypeptidase (GCP), prolyl carboxypeptidase (PCP), D-alanyl-D-alanine carboxypeptidase (AACP), or procarboxypeptidase. [Section 4] Item 4. The method according to Item 3, wherein the carboxypeptidase is carboxypeptidase B (CpB). [Section 5] The method of any one of the preceding clauses, wherein the bioreactor is a production bioreactor. [Section 6] The method of any one of the preceding clauses, wherein the cells are cultured for about 14 days. [Section 7] Item 10. The method of any one of the preceding paragraphs, wherein the carboxypeptidase is added to the cultured cells 0 to 24 hours before harvesting. [Section 8] Item 11. The method of any one of the preceding paragraphs, wherein the carboxypeptidase is added to the cultured cells about 2 hours before harvesting. [Section 9] The method of any one of the preceding clauses, wherein the cells are in perfusion, batch, or fed-batch cell culture. [Section 10] Item 10. The method of any one of the preceding paragraphs, wherein the carboxypeptidase is added at a CP:protein of interest ratio of between 0.0001% and 1% w / w. [Section 11] The method of any one of the preceding clauses, wherein the protein has a lysine or arginine residue at the C-terminus. [Section 12] The method of any one of the preceding clauses, wherein the protein is an Fc-fusion protein or an antibody. [Section 13] Item 13. The method of item 12, wherein the antibody is a monoclonal antibody (mAb). [Section 14] 14. The method of paragraph 13, wherein the carboxypeptidase is added in an amount sufficient to reduce basic species of the mAb of interest. [Section 15] 14. The method of paragraph 13, wherein the carboxypeptidase is added in an amount sufficient to enrich for the predominant species of the mAb of interest. [Section 16] The method of any one of the preceding clauses, wherein the host cell is a CHO cell. [Section 17] 13. The method of clause 12, wherein the antibody binds to an antigen selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73 HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, IL-13, EGFR, SARS-CoV-2 spike protein, HER3, and HER4. [Example]

[0142] Example 1 material and method Cell lines, media and upstream cell culture processes for mAb-1 Four different cell culture processes were used for mAb-1 (IgG4) production in this study: Processes A, B, C, and D. A recombinant CHO cell line harboring an endogenous glutamine synthetase (GS) gene and engineered using GS as a selection marker was used in Process A using a proprietary serum-free medium. For Processes B, C, and D, a new CHO cell line in which endogenous GS was knocked out (GS- / -) was used in a different proprietary chemically defined medium.

[0143] Process A is used for commercial production at 5000 L and 15,000 L bioreactor scale with a temperature shift from 36.5 °C to 34 °C on day 5, with an inoculum cell density of 0.3 × 10 6 Process B was a conventional 14-day fed-batch process with enriched N-1 seed culture (Yongky et al., 2019), with a target inoculum cell density of 3 × 10 cells / mL in a 5-L bioreactor (Sartorius, Germany) starting from an initial working volume of 3 L, with a temperature shift from 36.5 °C to 34 °C on day 6 of the 14-day process. 6 Process C was developed as an intensified fed-batch process with a perfused N-1 seed culture (J. Xu, Rehmann, Xu, et al., 2020), starting from an initial working volume of 3 L to a target inoculum cell density of 15 x 10 in a 5 L bioreactor (Sartorius, Germany), with a temperature shift from 36.5 °C to 34 °C on day 4 of the 14-day process. 6 Process D was developed starting from a 1 L working volume with a target inoculation cell density of 10 × 10 cells / mL in a 3 L bioreactor (Applikon, USA). 6 This is a perfusion production process in which the cells are cultured at 36.5°C for the entire period. From day 0, medium changes were performed using the ATF1 system (Repligen, USA), and 50 × 10 cells / mL were cultured. 6The volume of media per bioreactor volume per day (VVD) was slowly increased from 2 to 4 until the target VCD in cells / mL was reached. Batch 1 of Process D reached the target VCD on day 5. A "Push-to-Low" (Konstantinov et al., 2006) strategy was then used for medium exchanges for Batch 1, with 4 VVDs on days 5 to 11, 3 VVDs on days 11 to 18, 2 VVDs on days 18 to 25, and 1 VVD on days 26 to 31. Batch 2 of Process D reached the target VCD on day 6, after which the medium exchanges were changed to 2 VVDs and maintained for the 21-day run.

[0144] Upstream cell culture titer, productivity and other in-process assays for mAb-1 Product concentration or titer (g / L) was measured using Protein A UPLC. To compare the productivity of the fed-batch and perfusion methods, this study used space-time yield or overall volumetric productivity (g / L / day) and space yield (g / L). Space-time yield is defined as the total mAb-1 produced per L of culture volume over the culture period from day 0 to the day the mAb-1 concentration was measured (Bausch et al. 2019). Space yield is a new cell culture term introduced in this study and is defined as the total mAb-1 produced per L of culture volume. The space yield for the fed-batch process is equal to the bioreactor titer, while the space yield for the perfusion process is significantly greater than the bioreactor titer because the product-containing cell culture broth is removed from the bioreactor while fresh medium is continuously fed into the bioreactor. Normalized space yield or titer (normalized weight / L) was further normalized relative to the average titer at day 10 of Process B, which was set at 100%. The normalized space-time yield (normalized weight / L / day) is equal to the normalized space yield (relative to the mean titer on day 10 of process B) divided by the culture period.

[0145] Other in-process cell culture assays were performed as follows: offline pH, pCO2, and pO2 were detected using a Bioprofile pHOx analyzer (Nova Biomedical, USA); VCD and cell viability were measured offline using a Vi-CELL XR automatic cell counter (Beckman Coulter, USA); glucose, glutamine, glutamate, lactate, and ammonia were measured using a CEDEX Bio HT analyzer (Roche, USA).

[0146] CpB treatment method The CpB treatment protocol for complete C-terminal lysine cleavage was as follows: CpB enzyme concentration was 1% (w / w) in Tris buffer (pH 7.6) containing 25 mM Tris-HCl and 100 mM NaCl, and the mixture was incubated overnight at 37 °C. This method was also applied to various matrices, including whole cell cultures or supernatants. For mAb-1, a very low CpB concentration of 0.01% (w / w) was used for 2 hours at 34 °C, which also achieved complete C-terminal lysine cleavage. Because the pH values ​​of the whole cell cultures and supernatants for mAb-1 were within the optimal range of pH 7.5–8.0, no pH adjustment was performed on the whole cell cultures or supernatants. The supernatant was obtained by centrifuging the bioreactor samples at 1000 g for 10 minutes. Supernatants used for CpB treatment were either freshly prepared or thawed from frozen samples stored at -80° C. Methods for studies at different pH and temperatures are described in the Results section.

[0147] Three different CpB products derived from different sources were used in this study: recombinant CpB produced from Escherichia coli (E. coli) (ProSpec-Tany TechnoGene Ltd., Israel), GMP-grade recombinant CpB produced from P. pastoris (Roche, USA), and porcine CpB extracted from porcine pancreas (Sigma-Aldrich Corporation, USA).

[0148] Study of downstream processes and CpB clearance Two 5L Process B bioreactors were used to generate cell cultures for downstream processing and to evaluate CpB clearance, as described in the Results section. At harvest on day 14, one 5L whole cell culture was treated with 0.05% (w / w) CpB for 2 hours at 34°C before harvest, while the other 5L bioreactor served as a control and was maintained at 34°C for 2 hours without CpB treatment. Both the treated and untreated 5L whole cell cultures were then processed through the same downstream steps. After centrifugation at 1000g for 30 minutes, the supernatant was filtered through 0.2 μm sterile filtration. The clarified bulk was then processed through the following downstream steps: purification by protein A affinity chromatography, low-pH viral inactivation and depth filtration, hydrophobic interaction chromatography (HIC), cation exchange chromatography (CEX), and concentration by ultrafiltration and diafiltration (UF / DF) to produce the final formulated DS.

[0149] Product quality characteristics For the assessment of quality attributes of whole cell culture and supernatant samples, purification by Protein A chromatography is required before subjecting them to quality attribute assays. Samples in downstream processes after the Protein A step (such as Protein A elution, HIC and CEX pools) are directly subjected to quality attribute assays.

[0150] Charge variant species (major, acidic, and basic peaks) were analyzed by imaging capillary isoelectric focusing (iCIEF) using a Protein Simple iCE3 instrument with an Alcott 720NV autosampler (San Jose, CA). Samples were mixed with appropriate pI markers, ampholytes, and urea and injected into a fluorocarbon-coated capillary cartridge. Upon application of high voltage, the charge variants migrated according to their respective pIs. Images were taken at 280 nM with a UV camera. The major peak was identified, and peaks migrating in the acidic and basic regions were summed, quantified, and reported as relative percent area. The mAb-1 specification for the major peak was normalized to 100%. If the normalized major peak level was 100% or greater, the mAb-1 drug substance met the specification. If the normalized major peak level was less than 100%, the mAb-1 drug substance did not meet the specification. All major peak and basic species levels were normalized to the major peak value in this study, using this DS standard as 100%. Acidic species levels were not reported because they were well within specification for all processes regardless of CpB treatment.

[0151] N-glycan analysis was performed using the GlycoWorks RapiFluor-MS kit from Waters (Milford, MA). Free oligosaccharides were analyzed by gradient elution using an Acquity UPLC Glycan BEH Amide, 130 Å, 1.7 μm, 2.1 × 10 mm column (Milford, MA) on a Waters Acquity H-Class system (Milford, MA) equipped with a temperature-controlled autosampler and a fluorescence detector.

[0152] Size-exclusion chromatography (SEC) of impurities was performed on a Waters Acquity BEH200, 4.6 mm × 30 mm, 1.7 μm, coupled to a Waters Acquity guard column on a Waters Acquity UPLC system (Milford, MA) equipped with a temperature-controlled autosampler and a Waters 2996 PDA detector, using an isocratic gradient monitored at 280 nm.

[0153] Samples were prepared for protein purity by CE-SDS under both reducing and non-reducing conditions. Samples were then injected onto a PA800 instrument equipped with a UV detector (AB Sciex, Framingham, MA, USA) and 50 μm internal diameter pre-cut capillaries using Karat software. Separation was performed using a constant pressure of 15 kV. Data were exported and analyzed using Empower 3 software from Waters (Milford, MA, USA).

[0154] HCP was quantified using a process-specific sandwich enzyme-linked immunosorbent assay (ELISA). Samples were loaded onto coated assay plates from VWR (Radnor, PA, USA), and absorbance was measured using a Magellan Infinite M1000 Pro plate reader from Tecan Inc. (Morrisville, NC, USA). Concentrations were back-calculated using a 4-parameter logistic curve fitting algorithm.

[0155] Residual host cell DNA was assayed by qPCR using forward and reverse primers from Integrated DNA Technologies (Skokie, IL, USA) and the TaqMan and extraction kit from Applied Biosystems (Foster City, CA, USA). Samples were analyzed using the 7900 real-time PCR system from Applied Biosystems (Foster City, CA, USA), and data were processed using a standard curve.

[0156] Residual protein A was assayed using a commercial ELISA kit from Repligen (Waltham, MA, USA), and absorbance was measured using a Magellan Infinite M1000Pro plate reader from Tecan Inc. (Morrisville, NC, USA). Concentrations were back-calculated using a 4-parameter logistic curve fitting algorithm.

[0157] Throughout the purification process, residual CpB was measured by ELISA. Anti-CpB antibodies were generated in-house, while horseradish peroxidase-conjugated secondary antibodies were purchased from Thermo Fisher Scientific Inc., USA.

[0158] Upstream Cost Analysis Upstream consumable costs were calculated as dollars per gram of final formulated DS, as described in detail in our previous publication (J. Xu, Xu, et al., 2020). In this study, all upstream costs were normalized to 100% for Process B, harvested on Day 10. Briefly, upstream consumable costs focused on media, filters, probes, and bags, and did not include downstream processing (including harvesting) costs. The intensified lab process described in this study was assumed to be scalable to a 2000-L disposable bioreactor with the same titer or space yield and raw material consumption rate as a 5-L bioreactor. Because CpB treatment is required to increase the main peak to meet DS release specifications, an additional CpB cost of 0.01% (w / w) was assumed for Process B and Process C on Day 14. To calculate upstream costs per gram of DS, a 70% overall downstream yield was assumed based on lab-scale performance.

[0159] result Improvement of the productivity and quality (main peak) of mAb-1 during the enrichment process by CpB treatment Three intensification processes were developed in this report to improve the productivity of mAb-1 from conventional manufacturing process A. As described in the Methods section, space yield (a new term, defined in this study as the total grams of protein produced per L of bioreactor culture) and space-time yield were used to compare the productivity of different fed-batch and perfusion processes. The space-time yield is equal to the bioreactor titer in fed-batch processes, but is significantly greater than the bioreactor titer for continuous processes due to the removal of product from the bioreactor by perfusion. The target inoculum cell density was 0.3 x 10 6 Process A, with a target inoculum cell density of 3 x 10 cells / mL, achieved a normalized space yield (or titer) of 37.0 ± 2.4% (normalized wt / L) and a normalized space-time yield (or overall volumetric productivity) of 2.6 ± 0.2% (normalized wt / L / day) at day 14 (n = 29). 6Fed-batch process B, enhanced by using enriched medium in the N-1 seed, achieved a normalized spatial yield of 138 ± 5% (normalized wt / L) and a normalized space-time yield of 9.9 ± 0.9% (normalized wt / L / day) (n = 4) at day 14 (Figure 1A), while achieving a target inoculum cell density of 15 × 10 6 Fed-batch process C, where cells / mL were enhanced by perfusion of an N-1 seed culture, achieved a normalized spatial yield of 198 ± 2% (normalized wt / L) (n = 4) and a normalized space-time yield of 14.1 ± 0.2% (normalized wt / L / day) (n = 4) at day 14 (Figure 1B). Perfusion process D was enhanced by perfusion during the production run. Process D batch 1 with variable medium exchange rates using a "Push-to-Low" strategy, as described in the Methods section, achieved a normalized spatial yield of 994% (normalized wt / L) at day 31 (Figure 1C). Process D batch 1 achieved a normalized space-time yield of 30% (normalized wt / L / day) at day 17, and the normalized space-time yield of batch 1 was maintained between 30 and 33% (normalized wt / L / day) from days 17 to 31 (Figure 1C). Batch 2 of Process D, with a fixed medium exchange rate of 2VVD, achieved a normalized space-time yield of 599% (normalized wt / L) on day 21 (Figure 1C). Batch 2 of Process D achieved a normalized space-time yield of 28% (normalized wt / L / day) on day 18, and thereafter, the normalized space-time yield of Batch 2 was maintained between 28 and 29% (normalized wt / L / day) from days 18 to 21 (Figure 1C).

[0160] As described above, the space-time and space-yields of the fortified processes increased significantly from processes B to C and D, each substantially improving compared to process A. However, in both fed-batch processes B (Figure 2A) and C (Figure 2B), the levels of the major peak decreased significantly with increasing culture duration. For this particular mAb-1, the DS specification limit for the major peak was 100% (normalized value), which was set based on process A in commercial production. While improving cell culture productivity is a significant advantage, protein quality attributes must meet release specifications. Therefore, although the space-time yield increased throughout the 14-day period, harvesting was required on day 10 for process B (Figure 1A) and day 6 for process C (Figure 1B). Perfusion process D yielded the highest space-time and space-yields of all processes, but the major peak levels in process D remained below the specification limits throughout the culture period (Figure 2C), so process D could not be implemented in production. It should be noted that the charge variant profile did not change between downstream processes. Other quality attributes, such as SEC impurities and N-glycans, were not challenged in this study for enrichment steps B, C, and D, regardless of cell culture duration (data not shown).

[0161] Normalized basic species levels in the fortification processes, ranging from 25 to 65% (Figure 3), were significantly higher than those in Process A, ranging from 5 to 18% (n = 164). As shown in Figure 4, an example of the fortification process, the basic species were primarily derived from C-terminal lysines, as evidenced by the substantial decrease in values ​​after standard CpB treatment assays. After CpB treatment, the majority of the basic species were converted to the major peak, which increased from 91.8% to 107.7%. While the 91.8% major peak did not meet the specification (i.e., the major peak was ≥100%), the 107.7% major peak after CpB treatment met the specification (Figure 4). It should be noted that acidic species levels met the specification for all samples from Processes B, C, and D, regardless of CpB treatment (data not shown). Therefore, changes in acidic species after CpB treatment were not considered problematic, and only the major peak and basic species are reported in this study.

[0162] CpB treatment was performed on all in-process samples generated from processes B, C, and D at multiple time points to evaluate its effect on the main peak level. As shown in Figure 2, after CpB treatment (dashed line), the main peak level increased substantially for all fortified processes, accompanied by a decrease in basic species (Figure 3). For all fortified processes, the normalized main peak level at different time points exceeded 100% throughout the test period (Figure 2). Therefore, for process B, the cell culture period could be extended from 10 to 14 days, resulting in an average increase of 38% in the final harvest titer (compared to the titer on day 10, Figure 1A). For process C, the cell culture period could be extended from 6 to 14 days, resulting in an average increase of 108% in the final harvest titer (compared to the titer on day 6, Figure 1B). For process D, CpB treatment (Figures 2C and 3C) had the most significant effect, enabling this process to be a viable option for manufacturing.

[0163] Optimization of CpB treatment for mAb-1 After preliminary laboratory testing (data not shown), we selected a 2-hour CpB treatment because a 2-hour reaction time is sufficient for the removal of C-terminal lysines and is easy to implement in biological manufacturing operations. The CpB treatment method was further optimized based on the initial enzyme:mAb ratio of 1% (w / w). First, enzyme concentrations in different matrices were evaluated for CpB treatment efficiency. In whole cell culture broth, the standard CpB treatment method (e.g., 1% (w / w) enzyme concentration) converted the majority of basic species to a major peak, as expected (Figure 5). A 100-fold reduction in the CpB concentration to 0.01% was observed to yield an efficiency comparable to that of the standard treatment with 1% (w / w) CpB (Figure 5). In addition, the 0.01% (w / w) CpB concentration also performed well in cell culture supernatant and Tris buffer. However, as shown in Figure 5, when the CpB concentration was reduced to 0.001% (w / w), the main peak level was significantly reduced compared to 0.01% (w / w) treatment in both cell culture supernatant (t-test, p<0.0001) and Tris buffer (t-test, p<0.01). To ensure effective excision of the C-terminal lysine, a concentration of 0.01% (w / w) was selected and used in all subsequent studies.

[0164] Second, the temperature of CpB treatment was studied. Fed-batch cell culture was initiated at 36.5°C and shifted to a lower temperature of 34°C, while harvesting and downstream purification steps were performed at room temperature. Therefore, different temperatures ranging from room temperature to 32, 35, and 36.5°C were studied using 0.01% (w / w) CpB for 2 hours (Figure 6). Similar high CpB treatment efficiencies were observed between room temperature and 36.5°C (Figure 6).

[0165] Third, the effect of pH was studied. While fed-batch processes can be operated at pH 6.5–8.0, downstream processes are typically operated over a wider pH range. Therefore, the effect of pH was evaluated over a pH range of 5–9 using 0.01% (w / w) CpB at 34°C for 2 h. The temperature of 34°C was chosen because the final CpB treatment step is preferably performed at the end of the cell culture process for ease of operation, as described in detail later in this study. Similar high efficiency of conversion of basic species to the main peak was observed at pH 7.2–8.9, but approximately 4% lower efficiency was observed at pH 6.0–7.0 (Figure 7). Much lower efficiency was observed at pH 5.0 (Figure 7).

[0166] In conclusion, the CpB treatment appears to be effective over a wide range of pH and temperature in different matrices, including whole cell culture, cell culture supernatant (or clarified bulk at harvest), and Tris buffer (Figures 5–7). The CpB treatment was also found to be effective in other buffers, such as citrate and phosphate (data not shown). Theoretically, these results suggest that the CpB treatment could be introduced at any of several steps in a biological manufacturing process, from bioreactor operation to harvest and different downstream purification steps (Figure 8). We decided to introduce a 2-hour CpB treatment at the end of the cell culture portion of the fed-batch process. The main reasons for this are: The bioreactor is cooled from 34°C to room temperature before the start of harvest by filtration, which takes longer than 2 hours in commercial biological manufacturing. The temperature range and cooling time from 34°C to room temperature are ideal for effective CpB treatment (Figure 6). Although aseptic technique is required for cell culture, the short cooling phase allowed for the addition of CpB to the production bioreactor without aseptic technique. This facilitated CpB treatment while minimizing impact on existing manufacturing processes. The pH of the whole cell culture was between 7.5 and 8.0, which was optimal for treatment (Figure 7). While the clearance of CpB (a novel raw material) in the final drug substance needs to be fully demonstrated, introducing CpB at the completion of the production bioreactor process allows for its effective removal during harvest and downstream purification steps. For perfusion step D, the CpB treatment was introduced into the surge vessel at room temperature for 2 hours prior to 0.2 μm sterile filtration and Protein A purification steps.

[0167] Demonstration of CpB implementation process at 5L scale Because clearance of all raw materials used in biological manufacturing must be demonstrated in the final DS (FDA, 2016; J. Xu, Rehmann, Tian, ​​et al., 2020), two 5L fed-batch bioreactors were run using process B as an example. One was treated with CpB and the other was not, followed by harvest and downstream processing. To ensure sufficient removal of CpB in the final DS, a five-fold CpB level of 0.05% (w / w) was used. Cell culture performance parameters, such as VCD, cell viability, and normalized titer, were similar to those of the untreated control process B. The protein A process alone demonstrated a greater than 2-log reduction in CpB (Figure 9). CpB was below the detection limit (<50 μg / L) during the protein A elution and remained undetectable throughout the remainder of the downstream processing (Table 1). Regarding the charge variant profile, the major peak level in the CpB-treated sample was higher than that in the untreated sample and maintained similar levels for each arm throughout downstream processing (Table 1). All other quality attributes, such as process-related impurities and product-related impurities, were comparable between the two conditions, achieving similar process and overall downstream yields (Table 1).

[0168] CpB Treatment Cost Analysis for mAb-1 Extending the cell culture period by implementing CpB treatment improved the average final titer by 38% for process B and 108% for process C, respectively (Figure 1 and Table 2). Although the addition of 0.01% (w / w) CpB slightly increased the total upstream consumable costs per batch, by 5% for process B and 6% for process C, significant titer improvements were achieved by extending the culture period to 14 days, resulting in a reduction in the upstream consumable costs per gram of DS of 22% for process B and 46% for process C, respectively (Table 2). [Table 1] [Table 2]

[0169] Potential applications of CpB treatment to other mAb products In addition to mAb-1, six other mAb products were tested using the standard CpB treatment method with a 1% (w / w) CpB concentration. For mAb-2, mAb-3, and mAb-4, CpB treatment substantially reduced basic species and increased the main peak (Table 3). For mAb-5, the effect on basic species and the main peak was also evident, but to a lesser extent. However, no changes were observed for mAb-6 and mAb-7 (Table 3). The difference in the effectiveness of CpB in reducing basic species was attributed to the difference in the abundance of C-terminal lysine in the cell cultures of these mAbs. CpB treatment could only convert basic species due to C-terminal lysine, but not basic species resulting from other molecular modifications such as N-terminal pyroglutamic acid and amidation. These results suggest that the CpB treatment strategy for improving protein homogeneity by increasing the main peak can be applied to mAb products containing C-terminal lysine species. [Table 3]

[0170] Example 2 material and method Cell lines, media and cell culture processes The CHO GS cell line was used for the expression of proprietary human mAb-8, while the CHO DG44 cell line was used for the expression of proprietary human mAb-3. Proprietary chemically defined seed, basal, and feed media were used. Vial thawing and seed propagation steps were performed using shake flasks (Corning Life Sciences) containing seed media containing methionine sulfoximine as the selective agent for the CHO GS cell line and another seed medium containing methotrexate as the selective agent for the CHO DG44 cell line. Cells were passaged every 3–4 days for at least 2 weeks prior to inoculation of the production culture. Fed-batch production cell culture runs were performed using an initial volume of 80 mL of basal medium and 1.5 × 10 6 The study was performed using 250 mL shake flasks (n=3) containing seed cultures with a target inoculum viable cell density (VCD) of 1000 cells / mL. A proprietary feed medium and a 300 g / L glucose solution were used to maintain a good nutrient supply to the fed-batch production cultures, which were harvested on day 12. All seed and production cultures were grown in a humidified incubator (Climo-Shaker, Kuhner) using standard conditions of 36.5 °C, 5% CO2, and 150 rpm for the first 5 days. The temperature was then changed to 34 °C on day 5 for all production cell cultures, and they were grown until harvest on day 12.

[0171] In-vitro CpB treatment protocol Prior to use in this experiment, 2.6 mg / mL of GMP-grade recombinant CpB produced from P. pastoris (Roche) was frozen and stored at -20°C. The in vitro CpB treatment protocol for cleaving the C-terminal lysine residue is shown in Figure 10. Immediately before use, a 1 mL aliquot of frozen CpB was thawed and diluted 50-fold with Dulbecco's phosphate-buffered saline (Gibco). At the end of fed-batch production, 20 mL of total cell culture from each shake flask was aliquoted into two 50 mL Falcon tubes. One set of Falcon tubes (n = 3) was kept as a control without CpB. To the other set of Falcon tubes (n = 3), 50-fold diluted CpB was added at a CpB:mAb ratio of 1:10,000 (w / w). Subsequently, all Falcon tubes were returned to a humidified incubator with the same production cell culture conditions (150 rpm, 34 °C, and 5% CO). After 2 h of incubation, the cultures were centrifuged at 1,000 × g for 10 min, and the supernatants were collected and stored at 2–8 °C prior to quality attribute assays.

[0172] In-process cell culture and quality attribute assays Cell culture broth was sampled from each shake flask and directly analyzed for cell number, nutrients, and metabolites. VCD and cell viability were quantified offline using a Vi-CELL XR automatic cell counter (Beckman Coulter). Glucose, lactate, and ammonia were quantified using a CEDEX Bio HT analyzer (Roche).

[0173] For titer determination, the cell culture broth was centrifuged at 1000 × g for 10 minutes, and the supernatant was analyzed using a protein A UPLC method. Normalized titer, expressed as normalized wt / L, is equal to the true titer (g / L) at each time point divided by the average day 10 titer (g / L) for mAb-1, as described in our previous report (Xu et al. 2021b). Normalized volumetric productivity, expressed as normalized wt / L / day, was calculated as the difference in normalized titer divided by the duration between the two time points.

[0174] For quality attribute assays, all supernatants, both CpB-treated and non-CpB-treated, were thawed at -80°C and initially purified by protein A chromatography. Quality attribute assays were performed as previously described (Yongky et al. 2019). Charge variant species (acidic, major, and basic) were measured by imaging capillary isoelectric focusing. Both the major peak and basic species for mAb-8 and mAb-3 were normalized to the mAb-1 standard for the major peak as 100%, as previously described (Xu et al. 2021b). N-glycan profiles (e.g., G0, G0F, G1F, G2F, and Man5) were measured using Waters' commercially available RapiFluor-MS N-Glycan kit. SEC was used to measure SEC impurities (e.g., high molecular weight (HMW), low molecular weight (LMW), and monomeric).

[0175] statistical analysis Unless otherwise stated, data in figures are sample means with standard deviations. Student's t-test analysis was performed using Microsoft Excel, and the significance level was set at p-value <0.05.

[0176] result Cell culture performance and critical quality attributes of mAb-8 in an industrial CHO GS cell line before and after in vitro CpB treatment For mAb-8 production using the industrial CHO GS cell line, a 12-day platform fed-batch cell culture process was used in 250 mL shake flasks (n=3). The initial inoculum VCD was 1.65±0.13×10 6 On day 6, the mean cell count was 25.83 ± 0.71 × 10 cells / mL (Figure 11A). 6 By day 9, the VCD peak of approximately 25 x 10 cells / mL was reached. 6 The VCD then decreased from day 9 to day 12, with the final VCD being 19.83 ± 0.65 × 10 cells / mL. 6The initial cell viability was 98.1±0.65% and was well maintained throughout the entire period, with a final cell viability of 97.0±0.15% (FIG. 11A).

[0177] The normalized titer increased linearly from 13.5 ± 0.0% on day 6 to 43.1 ± 0.0% on day 10, with an average productivity of 7.41% normalized wt / L / day. Subsequently, the titer gradually plateaued, with a final normalized titer of 48.5 ± 0.0% on day 12 and an average productivity of 2.69% between days 10 and 12 (Figure 11A).

[0178] Metabolite profiles for fed-batch production of mAb-8 are shown in Figure 11B. Glucose concentrations were maintained at 1–6 g / L throughout the entire period. As expected, lactate increased during the early cell growth phase, peaking at 3.16 ± 0.01 g / L on day 5, and then switched to the lactate consumption phase, decreasing to 1.2–1.4 g / L on days 10–12 (Figure 11B). Ammonium increased to a peak of 3.60 ± 0.02 mM on day 4 during the early cell growth phase, decreased to a minimum of 1.12 ± 0.04 mM on day 7, and gradually increased again from days 7 to 12, reaching a final concentration of 2.70 ± 0.02 mM (Figure 11B). This pattern of ammonium, increasing early in cell culture, decreasing in the middle, and then increasing again late, is typical for fed-batch cell culture and is consistent with our previous reports on different mAb production by other CHO cell lines (Xu et al. 2020b; Xu et al. 2020c).

[0179] Charge variants and other important quality characteristics of mAb-8 production with and without CpB treatment are shown in Figures 12A, 12B, and 12C (n = 3). The control condition, which was not subjected to CpB, showed a mean normalized basic species content of 47.6 ± 1.60%. Notably, in the CpB-treated condition, the normalized basic species content significantly decreased to 29.0 ± 0.37% (p < 0.0001) (Figure 12A). A corresponding increase in the mean normalized main peak content to 108.8 ± 0.31% was observed for the CpB-treated condition, a significant increase from the normalized main peak content of 93.9 ± 1.12% shown in the control (p < 0.0001) (Figure 12A). Thus, the majority of the basic species reduced by CpB treatment (14.8% of 18.6%) was converted to the main peak, and the remaining 3.8% of the basic species reduced by CpB treatment was converted to acidic species. CpB treatment significantly increased the main peak of mAb-8, whereas SEC impurities (Figure 12B) and N-glycans (Figure 12C) were unaffected by the treatment, suggesting that this CpB treatment reduces basic charge variants of the mAb without affecting other important quality attributes.

[0180] Cell culture performance and critical quality attributes of mAb-3 in industrial CHO DG44 cell line before and after in-vitro CpB treatment Similar to mAb-8, mAb-3 was produced in an industrial CHO DG44 cell line using a different seed and basal medium than those used for mAb-8, using a similar platform fed-batch cell culture process for 12 days in 250 mL shake flasks (n=3), as described in the Methods section. The initial inoculum VCD was 1.51 ± 0.08 × 10 6 cells / mL, and on day 10, 27.43 ± 1.00 × 10 6 A peak VCD of 10 cells / mL was reached (Figure 13A). The VCD decreased from day 10 to day 12, with a final VCD of 24.03 ± 1.01 × 10 6The cell viability was 97.5 ± 0.53% on day 0, which was well maintained throughout most of the culture period, with cell viability at day 10 of 94.6 ± 0.62%. However, viability decreased from day 10 to harvest on day 12, with a final cell viability of 77.3 ± 1.51% (Figure 13A).

[0181] The normalized titer increased from 13.5 ± 0.0% on day 6 to 26.9 ± 0.0% on day 8, with an average productivity of 6.73% normalized wt / L / day, with the highest average productivity of 9.43% observed between days 8 and 10. The average productivity leveled off between days 10 and 12 at 5.84% normalized wt / L / day, paralleling a final normalized titer of 57.5 ± 0.8% (Figure 13A).

[0182] Metabolite profiles for fed-batch production of mAb-3 are shown in Figure 13B. Glucose concentrations were maintained between 1 and 9 g / L throughout the entire period. Lactate increased throughout the period, peaking at 2.67 ± 0.07 g / L on day 12 for mAb-3 (Figure 13B), which was lower than the peak lactate concentration of 3.16 g / L for mAb-8 (Figure 11B). Despite the absence of a lactate-consuming phase for mAb-3, the low lactate peak of 2.67 g / L suggests that lactate itself should not affect the performance of fed-batch cell cultures. Ammonium increased during the early cell growth phase to a peak concentration of 5.31 ± 0.03 mM on day 7. Ammonium then decreased during the late cell growth phase to a lowest level of 1.76 ± 0.02 mM on day 10, but increased slightly again to approximately 2.5 mM on days 11 and 12 (Figure 13B).

[0183] Similar to mAb-8, in vitro CpB treatment of whole cell cultures increased the main peak of mAb-3 without affecting other important quality attributes (Figures 14A, 14B, and 14C, n = 3). The mean normalized basic species in the CpB-treated condition was 14.3 ± 0.44%, which was significantly lower than the untreated control's 51.8 ± 0.25% (p < 0.0001) (Figure 14A). Meanwhile, the mean normalized main peak in the CpB-treated condition was 114.6 ± 0.37%, which was significantly higher than the control's 92.1 ± 0.51% (p < 0.001) (Figure 14A). This means that after CpB treatment, 22.5% of the 37.5% basic species were converted to the main peak, and the remaining 15% of the basic species were converted to acidic species. Importantly, although CpB treatment significantly increased the major peak of mAb-3 compared to the untreated control, other important quality attributes, including SEC impurities (Figure 14B) and N-glycosylation (Figure 14C), were not affected by CpB treatment.

Claims

1. 1. A method for producing an antibody or an Fc fusion protein, wherein said antibody or said Fc fusion protein has a lysine residue at the C-terminus, said method comprising: a) culturing host cells expressing said antibody or said Fc-fusion protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase B (CPB) to the cultured cells; c) recovering the clarified bulk; and d) subjecting the clarified bulk to a purification process.

2. 1. A method for improving the productivity of an antibody or an Fc fusion protein, wherein said antibody or said Fc fusion protein has a lysine residue at its C-terminus, said method comprising: a) culturing host cells expressing said antibody or said Fc-fusion protein in a bioreactor under optimized culture conditions for at least 6 days; b) adding carboxypeptidase B (CPB) to the cultured cells; c) recovering the clarified bulk; and d) subjecting the clarified bulk to a purification process.

3. 3. The method of claim 1 or 2, wherein the bioreactor is a production bioreactor.

4. 4. The method of claim 3, wherein the cells are cultured for 14 days.

5. 3. The method of claim 1 or 2, wherein carboxypeptidase B (CPB) is added to the cultured cells 2 to 24 hours before the step of collecting the clarified bulk.

6. 6. The method of claim 5, wherein carboxypeptidase B (CPB) is added to the cultured cells 2 hours prior to the step of collecting the clarified bulk.

7. 3. The method of claim 1 or 2, wherein the cells are in a perfusion, batch or fed-batch cell culture.

8. 3. The method of claim 1 or 2, wherein carboxypeptidase B is added at a CPB:said antibody or said Fc-fusion protein ratio of between 0.001% and 1% w / w.

9. The method of claim 1 or 2, wherein the antibody is a monoclonal antibody (mAb).

10. 10. The method of claim 9, wherein carboxypeptidase B is added in an amount sufficient to reduce basic species of the mAb of interest.

11. 3. The method of claim 1 or 2, wherein the host cell is a CHO cell.

12. 10. The method of claim 9, wherein the antibody binds to an antigen selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, GITR, CXCR4, CD73 HER2, VEGF, CD20, CD40, CD11a, tissue factor (TF), PSCA, IL-8, IL-13, EGFR, SARS-CoV-2 spike protein, HER3, and HER4.

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