Asparagine supply strategies to improve cell culture outcomes and reduce asparagine sequence variants

Abstract

Methods for culturing eukaryotic cells for improved cell culture performance are provided. The methods generally include growing or maintaining eukaryotic cells in a synthetic cell culture medium, wherein the synthetic cell culture medium is supplemented with asparagine in an amount of from about 2.6 mM to about 43.2 mM during an initial fed-batch cell culture and from about 2.6 mM to about 21.6 mM during a later fed-batch cell culture, and maintaining the cells in the asparagine-supplemented cell culture medium for at least a portion of the initial fed-batch cell culture and at least a portion of the later fed-batch cell culture, wherein cell culture performance is improved by the asparagine supplementation compared to a similar method in which the initial and / or later fed-batch cell cultures are supplemented with less asparagine.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 072,740, filed on August 31, 2020, and U.S. Provisional Application No. 63 / 072,745, filed on August 31, 2020, the contents of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Field of the Invention The present invention relates to a method for culturing cells to improve cell culture performance. Specifically, the present invention relates to a method for culturing cells that uses asparagine supplementation to improve cell culture performance and reduce asparagine sequence variants, and a method for the production of protein biopharmaceuticals.

Background Art

[0003] Background of the Invention Biological agents, particularly proteins and polypeptides, are often developed as novel biopharmaceuticals. Engineered cells that produce high levels of a specific target protein have become extremely important for the successful commercial production of these biopharmaceuticals. The control and optimization of cell culture conditions are diverse and have a significant impact on the levels and quality of therapeutic proteins produced in cell culture.

[0004] It is customary to produce proteins via cell culture in batch or fed-batch processes. The initial stages of growth of the inoculum after vial thawing include culturing cells in seed culture. Typically, to progressively increase the size and / or volume of the cell population, cells are grown at an exponential growth rate in a seed-train bioreactor or similar. After the cell mass has scaled up through several bioreactor stages, the cells are then transferred to a fed-batch production bioreactor while the cells are still in exponential growth (logarithmic phase) (Gambhir, A. et al., 2003, J Bioscience Bioeng 95(4):317-327 (Non-Patent Literature 1)).

[0005] After transitioning to fed-batch culture, cells are cultured for a certain period, while the culture medium composition is monitored and controlled to enable the production of the target protein or polypeptide. After reaching a specific yield, or when it is determined that the culture should be terminated due to cell viability, waste accumulation, or nutrient depletion, the produced protein or polypeptide is isolated. Over the past decade, many significant advances have been made with the aim of improving recombinant protein yields, which now reach several grams of titer per liter. Advances in protein production processes, as well as cell line engineering, and cell culture medium and feed development, have contributed to the improvement in protein yield. For example, schemes for optimizing cell culture mediums and feeds include nutrient supplementation and the design of serum-free media with known compositions to support continuous cell growth and optimal product secretion.

[0006] However, there is still a need in the art for cell culture media and methods that enable healthy and robust cell growth and maintenance, as well as high titer production of recombinant proteins. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Gambhir, A. et al., 2003, J Bioscience Bioeng 95(4):317-327 [Overview of the project]

[0008] In one embodiment, a method for culturing eukaryotic cells for improved cell culture outcomes is provided. This method generally comprises the steps of: growing or maintaining eukaryotic cells in a synthetic cell culture medium, wherein the synthetic cell culture medium is supplemented with asparagine in amounts of approximately 3.6 mM to approximately 43.2 mM during the initial Fed-batch cell culture and approximately 3.6 mM to approximately 21.6 mM during the later Fed-batch cell culture; and maintaining the cells in the asparagine-supplemented cell culture medium for at least a portion of the initial Fed-batch cell culture and at least a portion of the later Fed-batch cell culture, wherein at least one cell culture outcome parameter is improved by asparagine supplementation compared to a similar method with less asparagine supplementation in the initial and / or later Fed-batch cell culture or without asparagine supplementation.

[0009] In certain embodiments, the asparagine supplement may be supplied to the cell culture (both early and late phases) as a bolus feed supplement (e.g., one bolus feed supplement during the early phase and one bolus feed supplement during the late phase), as multiple bolus feed supplements over at least a portion of the cell culture (e.g., two, three, four, or five bolus feed supplements during the early phase and two, three, four, or five bolus feed supplements during the late phase), or continuously over at least a portion of the cell culture.

[0010] In certain embodiments, at least one cell culture performance parameter is selected from the group consisting of increased cell viability, increased cell growth rate, increased cell density, increased titer of the recombinant protein of interest, increased yield of the recombinant protein of interest, reduced depletion of essential amino acids in at least a portion of the cell culture, reduced formation of at least one cell culture byproduct in at least a portion of the cell culture, and improvement of at least one protein quality index. In certain embodiments, at least one cell culture byproduct is selected from the group consisting of ammonium ions and alanine. In certain embodiments, at least one protein quality index is a reduction in protein sequence variants.

[0011] In certain embodiments, the eukaryotic cell may be a mammalian cell, an avian cell, an insect cell, or a yeast cell. In certain embodiments, the eukaryotic cell may be a CHO cell. In other embodiments, the recombinant protein may be selected from an Fc-fusion protein, a receptor-Fc-fusion protein (TRAP), an antibody, an antibody fragment, or an ScFv-Fc-fusion protein.

[0012] In other embodiments, a method is provided for preventing asparagine sequence variants in a target polypeptide expressed from mammalian cells in cell culture. In a particular embodiment, the method includes growing or maintaining mammalian cells in a synthetic cell culture medium, wherein the synthetic cell culture medium is supplemented with asparagine in amounts of about 3.6 mM to about 43.2 mM during the initial fed-batch cell culture and about 3.6 mM to about 21.6 mM during the later fed-batch cell culture; and maintaining the cells in the asparagine-supplemented cell culture medium under conditions sufficient for the expression of the target polypeptide for at least a portion of the initial and later fed-batch cell cultures. In a particular embodiment, extracellular asparagine levels are maintained above a depletion limit of about 0.1 mM in the cell culture medium for at least the initial fed-batch cell culture, such that the target polypeptide expressed by the mammalian cells contains less than 0.30% asparagine sequence variants at all individual sequence variant loci.

[0013] In another embodiment, a method is provided for detecting asparagine sequence variants in a recombinant polypeptide of interest expressed from eukaryotic cells in cell culture. In a particular embodiment, the method may include the steps of: growing or maintaining eukaryotic cells in a synthetic cell culture medium; expressing a recombinant protein of interest from the eukaryotic cells; measuring the intracellular and / or extracellular concentrations of one or more asparagine-related amino acids in the synthetic cell culture medium; and correlating the measured concentrations of one or more asparagine-related amino acids with the amount of asparagine sequence variants present in the expressed recombinant protein of interest. The measured concentrations of one or more asparagine-related amino acids are inversely correlated with the amount of asparagine sequence variants.

[0014] In yet other aspects, a method for monitoring and controlling cell culture media conditions is provided. This method generally uses one or more of freezing point depression, electrochemistry, digital imaging, photometry, a bioprocess analyzer, or Raman spectroscopy to measure one or more cell culture parameters in a cell culture in situ, and includes the steps of measuring one or more cell culture parameters in the cell culture, comparing the measured one or more cell culture parameters with a predetermined set value regarding the cell culture parameters to determine whether the one or more cell culture parameters are within a predetermined threshold range, and adjusting one or more of the cell culture parameters if it is determined that the cell culture parameters are outside the predetermined threshold range. In certain embodiments, the ammonium ion concentration can be monitored, and if it is determined that the ammonium ion concentration is outside a predetermined threshold range for a particular cell culture, the asparagine supplement feed can be adjusted so that the cell culture produces less ammonium while still receiving adequate asparagine.

[0015] [Invention 1001] A method for culturing eukaryotic cells for improved cell culture results, wherein the method is A step of growing or maintaining eukaryotic cells in a synthetic cell culture medium, wherein approximately 3.6 mM to approximately 43.2 mM of asparagine is added to the synthetic cell culture medium during the initial Fed-batch cell culture, and approximately 3.6 mM to approximately 21.6 mM of asparagine is added during the later Fed-batch cell culture, and the cells are grown or maintained. The steps include maintaining the cells in the asparagine-supplemented cell culture medium for at least a portion of the initial and late fed batch cell cultures. Includes, The method wherein, compared to a similar method with a lower amount of asparagine supplementation in early and / or late-stage fed-batch cell culture, or without asparagine supplementation, at least one cell culture outcome parameter is improved by the asparagine supplementation. [Invention 1002] The method of the present invention 1001, wherein the asparagine supplement is provided as part of the bulk feed or as a separate asparagine supplement feed in early and / or late-stage fed-batch cell cultures. [Invention 1003] The method of the present invention 1001 or 1002, wherein an asparagine supplement is continuously or as a bolus in early and / or late-stage fed-batch cell cultures. [Invention 1004] Any of the prior art methods of the present invention, wherein approximately 7.2 mM to approximately 21.6 mM of asparagine is added to the synthetic cell culture medium during the initial fed-batch cell culture, and approximately 3.6 mM to approximately 10.8 mM of asparagine is added during the later fed-batch cell culture. [Invention 1005] Any prior art method of the present invention further comprising the step of expressing a target recombinant protein from the eukaryotic cells during the aforementioned fed batch cell culture. [Invention 1006] The method of the present invention 1005, wherein the at least one cell culture performance parameter is selected from the group consisting of increased cell viability, increased cell growth rate, increased cell density, increased titer of the recombinant protein of the objective, increased yield of the recombinant protein of the objective, reduced depletion of essential amino acids in at least a portion of the cell culture, reduced formation of at least one cell culture byproduct in at least a portion of the cell culture, and improvement of at least one protein quality index. [Invention 1007] The method of the present invention 1006, wherein at least one cell culture byproduct is selected from the group consisting of ammonium ions and alanine. [Invention 1008] The method of the present invention 1006, wherein the at least one protein quality indicator is the reduction of protein sequence variants. [Invention 1009] The method of the present invention 1006, wherein the titer of the recombinant protein of the objective is at least 3%, 5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or at least 20% higher than the titer of cells cultured with less asparagine supplementation in early and / or late-stage fed-batch cell culture. [Invention 1010] The method of the present invention 1006, wherein the yield of the recombinant protein of the subject is increased by at least 0.1 g / L, at least 0.5 g / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, at least 2.2 g / L, at least 2.4 g / L, or at least 2.5 g / L compared to a similar method in which cells are cultured with a smaller amount of asparagine supplementation in early and / or late-stage fed-batch cell culture. [Invention 1011] The method of the present invention 1006, wherein the cell culture maintains a viable cell count of at least about 10 to 50 M cells / ml during at least a portion of the late-stage fed-batch cell culture. [Invention 1012] Any of the prior art methods, wherein the cells are maintained under asparagine-supplemented cell culture conditions for at least two days, at least three days, at least four days, at least five days, or for the duration of the initial and / or late fed-batch cell culture. [Invention 1013] The method according to any one of the 1001 to 1011 of the present invention, wherein the asparagine supplement is provided at least once, at least twice, at least three times, at least four times, at least five times daily, or continuously for at least a portion of the initial fed batch cell culture. [Invention 1014] The method according to any one of the 1001 to 1011 of the present invention, wherein the asparagine supplement is provided at least once, at least twice, at least three times, at least four times, at least five times daily, or continuously for at least a portion of the late-stage fed batch cell culture. [Invention 1015] The method according to any one of the 1001 to 1011 of the present invention, wherein the asparagine supplement is continuously supplied for at least two days, at least three days, at least four days, at least five days, or for the duration of the initial fed batch cell culture. [Invention 1016] The method according to any one of the 1001 to 1011 of the present invention, wherein the asparagine supplement is continuously supplied for at least two days, at least three days, at least four days, at least five days, or for the duration of the late-stage fed batch cell culture. [Invention 1017] The method of the present invention 1016, wherein the asparagine supplement is started on or after the fifth day of the Fed batch cell culture and subsequently supplied continuously for at least a portion of the later Fed batch cell culture. [Invention 1018] The method of the present invention 1016, wherein the continuous asparagine supplementation is discontinued after the 10th day of the fed batch of cell culture. [Invention 1019] Any method 1001 to 1011 of the present invention, wherein the asparagine supplement is continuously supplied in early and / or late-stage fed batch cell cultures. [Invention 1020] The method of the present invention 1019, wherein the asparagine supplement is provided as part of a continuous bulk feed. [Invention 1021] The method of the present invention 1019, wherein the asparagine supplement is provided as part of a separate, continuous asparagine supplement feed. [Invention 1022] Any of the prior art methods, wherein the extracellular amino acid depletion of asparagine, aspartic acid, and glutamic acid in the cell culture is delayed by at least one day, at least two days, at least three days, or at least four days or more compared to a similar method in which asparagine supplementation is provided as a bolus feed in early and / or late fed-batch cell cultures. [Invention 1023] The method of any prior art, wherein the eukaryotic cell is a mammalian cell. [Invention 1024] The method of the present invention 1023, wherein the eukaryotic cell is a CHO cell. [Invention 1025] Any method of the prior art, wherein the cells are a high-asparagine-consuming cell line, such that the eukaryotic cells consume at least about 1.8 mM / day to about 9.3 mM / day of asparagine. [Invention 1026] Any method of the prior art, wherein the cells are a low-asparagine-consuming cell line, such that the eukaryotic cells consume at least about 0.32 mM / day to about 1.8 mM / day of asparagine. [Invention 1027] Any method of the prior art, wherein the eukaryotic cells are a high-producing cell line such that the recombinant protein of the target is produced in a yield of at least 4 g / L, at least 5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L, or at least 10 g / L, at least 11 g / L, at least 12 g / L, at least 13 g / L, or at least 14 g / L. [Invention 1028] The method of the present invention 1005, wherein the target recombinant protein is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific antibody, bispecific antibody, antigen-binding antibody fragment, single-chain antibody, diabody, triabody or tetrabody, Fab fragment or F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. [Invention 1029] The method of the present invention 1005, wherein the recombinant protein for the purpose contains an Fc domain. [Invention 1030] The method of the present invention 1005, wherein the recombinant protein of the objective is selected from the group consisting of Fc-fusion proteins, receptor-Fc-fusion proteins (TRAPs), antibodies, antibody fragments, and ScFv-Fc fusion proteins. [Invention 1031] The recombinant protein of interest is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody. , anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD 1005 of the invention, wherein the method is selected from the group consisting of: 48 antibody, anti-CD3 / CD20 bispecific antibody, anti-CD3 / anti-MUC16 bispecific antibody, and anti-CD3 / anti-PSMA bispecific antibody. [Invention 1032] The method of the present invention 1005, wherein the recombinant protein of the objective is selected from the group consisting of anti-influenza virus antibody, anti-respiratory syncytial virus (RSV) antibody, anti-Middle East respiratory syndrome (MERS) virus, anti-Ebola virus antibody, anti-Zika virus antibody, anti-Severe Acute Respiratory Syndrome (SARS) antibody, and anti-COVID-19 antibody. [Invention 1033] The method of the present invention 1005, wherein the recombinant protein of the objective is selected from at least one alirocumab, sarilumab, facinumab, nesbakumab, dupilumab, trevoglumab, evinakumab, linukumab, cacirivimab, or imudevimab. [Invention 1034] A method for preventing asparagine sequence variants in target polypeptides expressed from mammalian cells in cell culture, wherein the method is A step of growing or maintaining mammalian cells in a synthetic cell culture medium, wherein approximately 3.6 mM to approximately 43.2 mM of asparagine is added to the synthetic cell culture medium during the initial Fed-batch cell culture, and approximately 3.6 mM to approximately 21.6 mM of asparagine is added during the later Fed-batch cell culture, and the cells are grown or maintained. The steps include maintaining the cells in the asparagine-supplemented cell culture medium under conditions sufficient for the expression of the target polypeptide for at least a portion of the initial and late fed-batch cell cultures, and Includes, The method, wherein the extracellular asparagine level is maintained above a depletion threshold of about 0.1 mM in the cell culture medium for at least the initial fed batch of cell culture, such that the target polypeptide expressed by the mammalian cells contains less than 0.30% asparagine sequence variants at all individual sequence variant loci. [Invention 1035] The method of the present invention 1034, wherein the target polypeptide expressed by the mammalian cells contains less than 0.30% asparagine sequence variants at all individual sequence variant loci after 12 days of cell culture. [Invention 1036] The method of the present invention 1034 or 1035, wherein the asparagine supplement is provided as part of a bulk feed or as a separate asparagine supplement feed in early and / or late-stage fed-batch cell cultures. [Invention 1037] Any method of the present invention 1034 to 1036, wherein an asparagine supplement is provided continuously or as a bolus in early and / or late-stage fed-batch cell cultures. [Invention 1038] A method according to any one of the present invention 1034 to 1037, wherein asparagine is added to the synthetic cell culture medium in an amount of approximately 7.2 mM to approximately 21.6 mM during the initial Fed-batch cell culture, and in an amount of approximately 3.6 mM to approximately 10.8 mM during the later Fed-batch cell culture. [Invention 1039] Any method of the present invention 1034 to 1038, wherein the cells are maintained under asparagine-supplemented cell culture conditions for at least two days, at least three days, at least four days, at least five days, or for the duration of the initial and / or late fed-batch cell culture. [Invention 1040] Any method 1034 to 1038 of the present invention, wherein the asparagine supplement is provided at least once, at least twice, at least three times, at least four times, at least five times, daily, or continuously for at least a portion of the initial fed batch cell culture. [Invention 1041] The method of any of the present invention 1034 to 1038, wherein the asparagine supplement is provided at least once, at least twice, at least three times, at least four times, at least five times, daily, or continuously for at least a portion of the late-stage fed-batch cell culture. [Invention 1042] The method according to any one of the present invention 1034 to 1038, wherein the asparagine supplement is continuously supplied for at least two days, at least three days, at least four days, at least five days, or for the duration of the initial fed batch cell culture. [Invention 1043] The method according to any one of the present invention 1034 to 1038, wherein the asparagine supplement is continuously supplied for at least two days, at least three days, at least four days, at least five days, or for the duration of the late-stage fed-batch cell culture. [Invention 1044] The method of the present invention 1043, wherein the asparagine supplement is started on or after the fifth day of the Fed batch cell culture and subsequently supplied continuously for at least a portion of the later Fed batch cell culture. [Invention 1045] The method of the present invention 1043, wherein the continuous asparagine supplementation is discontinued after the 10th day of the fed batch of cell culture. [Invention 1046] Any method 1034 to 1038 of the present invention, wherein the asparagine supplement is continuously supplied in early and / or late-stage fed-batch cell cultures. [Invention 1047] The method of the present invention 1046, wherein the asparagine supplement is provided as part of a continuous bulk feed. [Invention 1048] The method of the present invention 1046, wherein the asparagine supplement is provided as part of a separate, continuous asparagine supplement feed. [Invention 1049] Any method of the present invention 1034 to 1048, wherein the asparagine sequence variant is the misincorporation of serine instead of asparagine during translation of the target polypeptide. [Invention 1050] A method for detecting asparagine sequence variants in target polypeptides expressed from mammalian cells in cell culture, The steps include: growing or maintaining mammalian cells in a synthetic cell culture medium; The steps include expressing the target recombinant protein from the eukaryotic cells, The steps include measuring the intracellular and / or extracellular concentrations of asparagine or one or more asparagine-related amino acids in the aforementioned cell culture or synthetic cell culture medium, The steps include correlating the measured concentration of asparagine or one or more asparagine-related amino acids with the presence of an asparagine sequence variant in the expressed target polypeptide. Methods that include... [Invention 1051] The method of the present invention 1050, wherein the measured concentration of the asparagine or the one or more asparagine-related amino acids is inversely correlated with the presence of the asparagine sequence variant. [Invention 1052] The method of the present invention 1050 or 1051, wherein the synthetic cell culture medium is supplemented with asparagine in an amount of approximately 3.6 mM to approximately 43.2 mM during the initial Fed-batch cell culture and approximately 3.6 mM to approximately 21.6 mM during the late Fed-batch cell culture, and the cells are maintained in the asparagine-supplemented cell culture medium for at least a portion of the initial and late Fed-batch cell cultures. [Invention 1053] The method according to any one of the present invention 1050 to 1052, wherein the one or more asparagine-related amino acids are selected from aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), and combinations thereof. [Invention 1054] The method of the present invention 1053, wherein the one or more asparagine-related amino acids are glutamic acid (Glu). [Invention 1055] Any method 1050 to 1054 of the present invention, wherein one or more asparagine-related amino acids can be measured during late-stage fed batch cell culture. [Invention 1056] The method of the present invention 1055, wherein one or more asparagine-related amino acids can be measured 5, 6, 7, 8, 9, or 10 days after the cell culture. [Invention 1057] A method according to any one of the present invention 1050 to 1056, wherein one or more asparagine-related amino acids are measured intracellularly. [Invention 1058] A method according to any of the present invention 1050 to 1057, wherein one or more asparagine-related amino acids are measured extracellularly. [Invention 1059] A method for monitoring and controlling one or more cell culture parameters, The process involves measuring one or more cell culture parameters in a cell culture in situ using one or more of the following methods: freezing point depression, electrochemistry, digital imaging, photometric analysis, bioprocess analyzer, or Raman spectroscopy. The steps include: comparing the measured one or more cell culture parameters with predetermined set values ​​for the cell culture parameters to determine whether the one or more cell culture parameters are within a predetermined threshold range; If it is determined that the cell culture parameters are outside the predetermined threshold range, the step of adjusting one or more of the cell culture parameters is performed. Methods that include... [Invention 1060] The method of the present invention 1059, wherein one or more of the cell culture parameters are selected from the group consisting of cell growth rate, cell density, cell viability, asparagine concentration, asparagine-related amino acid concentration, ammonium ion concentration, and alanine concentration. [Invention 1061] The method of the present invention 1059, wherein one or more of the cell culture parameters are selected from asparagine concentration and ammonium ion concentration. [Invention 1062] The method of the present invention 1061, wherein the asparagine feed input is adjusted if it is determined that the ammonium ion concentration is outside a predetermined threshold range. While several embodiments are disclosed, further embodiments of this disclosure will become apparent to those skilled in the art from the following detailed description illustrating and describing exemplary embodiments of this disclosure. As realized, the present invention can be modified in all various aspects without departing from the spirit and scope of this disclosure. Therefore, the detailed description should be considered as illustrative and not limiting in nature. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1A shows the extracellular essential amino acid consumption during fed-batch cell culture using a standard supply strategy, while Figure 1B shows the consumption of asparagine during fed-batch cell culture using a standard supply strategy. [Figure 2] The embodiments of this disclosure demonstrate the consumption of extracellular amino acids (Figure 2A) and asparagine (Figure 2B) in fed-batch cell cultures using various supply strategies. [Figure 3A] Embodiments of this disclosure show a series of initial fed-batch asparagine supplements having low, medium, and high doses of supplements. [Figure 3B] This embodiment of the disclosure demonstrates increased cell culture growth associated with increased amounts of asparagine supplement. [Figure 3C] This embodiment of the disclosure demonstrates the increase in cell culture titer associated with an increase in asparagine supplementation. [Figure 4] Figure 4A shows low and high asparagine supplementation in late feed according to embodiments of the present disclosure. According to embodiments of the present disclosure, Figure 4C shows that overall cell culture productivity is not negatively affected, while Figure 4B shows increased byproduct formation. [Figure 5A] An improved asparagine supplementation strategy according to embodiments of this disclosure is shown. In accordance with embodiments of this disclosure, Figure 5A shows the extracellular essential amino acid concentrations in fed-batch cell cultures. [Figure 5B] An improved asparagine supplementation strategy according to embodiments of this disclosure is shown. In accordance with embodiments of this disclosure, Figure 5B shows the extracellular asparagine concentration in fed-batch cell culture. [Figure 5C] An improved asparagine supplementation strategy according to an embodiment of this disclosure is shown. Figure 5C shows increased cell culture growth according to an embodiment of this disclosure. [Figure 5D] An improved asparagine supplementation strategy according to embodiments of this disclosure is shown. In accordance with embodiments of this disclosure, Figure 5D shows an increase in cell culture titer. [Figure 5E] The cell titer after asparagine supplementation in another exemplary cell line according to embodiments of this disclosure is shown. [Figure 5F] The number of viable cells after asparagine supplementation in another exemplary cell line according to embodiments of this disclosure is shown. [Figure 5G] The cell viability after asparagine supplementation in another exemplary cell line according to embodiments of this disclosure is shown. [Figure 6] According to embodiments of the present disclosure, Figure 6A shows asparagine consumption after asparagine supplementation in an early fed-batch cell culture, while Figure 6B shows asparagine consumption after asparagine supplementation in a late fed-batch cell culture. [Figure 7] The embodiments of this disclosure demonstrate asparagine consumption rates across a range of asparagine supply strategies for exemplary high-consumption cell lines. [Figure 8] This shows the synthesis pathway for asparagine using aspartic acid and glutamic acid. [Figure 9]The effects of asparagine levels in late-stage fed-batch cell culture of exemplary high-consumption cell lines according to embodiments of this disclosure are shown in Figures 9A-9B, which show extracellular asparagine (Figure 9A) and extracellular glutamate (Figure 9B) concentrations, and Figures 9C-9D, which show intracellular asparagine (Figure 9C) and intracellular glutamate (Figure 9D) concentrations. [Figure 10] The effects of asparagine levels in late-stage fed-batch cell culture of another exemplary high-consumption cell line according to embodiments of the present disclosure are shown in Figures 10A-10B, which show extracellular asparagine (Figure 10A) and extracellular glutamate (Figure 10B) concentrations, and Figures 10C-10D, which show intracellular asparagine (Figure 10C) and intracellular glutamate (Figure 10D) concentrations. [Figure 11] The effects of asparagine levels in late-stage fed-batch cell culture of exemplary high-consumption cell lines according to embodiments of this disclosure are shown in Figures 11A-11B, which show extracellular asparagine (Figure 11A) and extracellular glutamate (Figure 11B) concentrations, and Figures 11C-11D, which show intracellular asparagine (Figure 11C) and intracellular glutamate (Figure 11D) concentrations. [Figure 12] The effects of high (3x in the early stage, 1.5x in the later stage) and very high (6x in the early stage, 3x in the later stage) asparagine levels in late-stage Fed batch cell cultures according to embodiments of this disclosure are shown in Figure 12A, Figure 12B, Figure 12C, Figure 12D, Figure 12E, Figure 12F, Figure 12F. Figure 12A shows the asparagine concentration, Figure 12B shows the aspartic acid concentration, Figure 12C shows the titer, Figure 12D shows the glutamic acid concentration, Figure 12E shows the glutamine concentration, and Figure 12F shows the ammonium concentration. [Figure 13-1] The effects of bolus and continuous asparagine feeding according to embodiments of the present disclosure are shown in Figure 13A, Figure 13B, Figure 13C, Figure 13D, Figure 13D. Figure 13A shows the number of viable cells, Figure 13B shows the titer, Figure 13C shows ammonium formation, and Figure 13D shows alanine formation. [Figure 13-2] According to embodiments of this disclosure, continuous asparagine supplementation feeds slow extracellular asparagine depletion (Figure 13E), aspartic acid depletion (Figure 13F), and glutamic acid depletion (Figure 13G) compared to bolus asparagine supplementation feeds having the same total amount of asparagine supplementation. [Figure 14] The effects of bolus and continuous asparagine feeding according to embodiments of this disclosure are shown. Figure 14A shows the consumption of asparagine, while Figures 14B-14C show the consumption of asparagine-related metabolites (aspartic acid, Figure 14B, and glutamic acid, Figure 14C). Figure 14D shows the formation of ammonium, a cell culture byproduct. [Figure 15] An embodiment of the present disclosure illustrates a hybrid supply approach in an exemplary cell line (continuous asparagine supplement feed combined with a bolus asparagine supplement feed), where Figure 15A shows extracellular asparagine, Figure 15B shows extracellular aspartic acid, Figure 15C shows extracellular glutamic acid, Figure 15D shows the number of viable cells, Figure 15E shows the titer, Figure 15F shows ammonium formation, and Figure 15G shows alanine formation. [Figure 16] An embodiment of the present disclosure illustrates a hybrid feeding approach in another exemplary cell line (continuous asparagine supplement feed combined with a bolus asparagine supplement feed), where Figure 16A shows extracellular asparagine, Figure 16B shows the number of viable cells, Figure 16C shows the titer, Figure 16D shows ammonium formation, and Figure 16E shows alanine formation. [Figure 17] According to embodiments of this disclosure, Figure 17A shows asparagine consumption in fed-batch cell culture according to an asparagine supply strategy, and Figure 17B shows the reduction of asparagine sequence variant formation. [Figure 18] Figure 18B shows asparagine consumption in fed-batch cell culture according to an asparagine supply strategy according to an embodiment of the disclosure. According to an embodiment of the disclosure, Figure 18B shows intracellular glutamate levels, and Figure 18C shows the reduction of asparagine sequence variant formation. [Figure 19]The correlations between asparagine, asparagine-related amino acids, and asparagine sequence variants in exemplary cell lines according to embodiments of this disclosure are shown. Figures 19B–19D show extracellular asparagine (Figure 19B), aspartic acid (Figure 19C), and glutamic acid (Figure 19D), while Figures 19E–19G show exemplary intracellular asparagine (Figure 19E), aspartic acid (Figure 19F), and glutamic acid (Figure 19G). [Figure 20] The correlations between asparagine, asparagine-related amino acids, and asparagine sequence variants in another exemplary cell line (high-consumption cell line) according to embodiments of this disclosure are shown. Figures 20B-20D show extracellular asparagine (Figure 20B), intracellular aspartic acid (Figure 20C), and intracellular glutamic acid (Figure 20D). [Figure 21A] Figure 21A shows the asparagine sequence variant trends for exemplary cell lines according to embodiments of this disclosure. Figure 21A shows a high-asparagine initial supply strategy (Figure 21A, amount of sequence variant determined by mass spectrometry). [Figure 21B] Figure 21B shows the asparagine sequence variant trends for exemplary cell lines according to embodiments of this disclosure. Figure 21B illustrates a low-asparagine initial supply strategy (Figure 21B, amount of sequence variant determined by mass spectrometry). [Figure 21C] The asparagine sequence variant trends for exemplary cell lines according to embodiments of this disclosure are shown. Figure 21C shows a high-asparagine initial supply strategy (Figure 21C, intracellular glutamate concentration). [Figure 21D] The asparagine sequence variant trends for exemplary cell lines according to embodiments of this disclosure are shown. Figure 21D shows a low asparagine initial supply strategy (Figure 21D, intracellular glutamate concentration). [Figure 21E] The asparagine sequence variant trends for exemplary cell lines according to embodiments of this disclosure are shown. Figure 21E shows intracellular glutamate levels for one exemplary cell line. [Figure 21F]Figure 21F shows the asparagine sequence variant trends for exemplary cell lines according to embodiments of this disclosure. Figure 21F shows the intracellular glutamate profile for another exemplary cell line. [Figure 22] The correlations of asparagine, asparagine-related amino acids, and asparagine sequence variants for exemplary cell lines are shown. Figures 22A–22D show extracellular asparagine (Figure 22A), extracellular aspartic acid (Figure 22B), extracellular glutamic acid (Figure 22C), and extracellular glutamine (Figure 22D) for exemplary cell lines for high and low asparagine supply strategies. Figures 22E–22H show extracellular asparagine (Figure 22E), extracellular aspartic acid (Figure 22F), extracellular glutamic acid (Figure 22G), and extracellular glutamine (Figure 22H) for other exemplary cell lines for high and low asparagine supply strategies. [Figure 23] This further illustrates that extracellular glutamine can act as a surrogate for asparagine sequence variants in late-stage fed-batch cell cultures. Figure 23A shows glutamine production via a high-asparagine supply strategy in an exemplary cell line. Similarly, Figure 23B shows glutamine production via a high-asparagine supply strategy in an exemplary cell line. Finally, Figure 23C shows that when asparagine is not supplemented on days 6 and 8, asparagine sequence variants are detected and extracellular glutamine falls below the depletion threshold (i.e., insufficient glutamine is produced via the asparagine supply strategy). [Modes for carrying out the invention]

[0017] Detailed description of the invention According to aspects of this disclosure, it was unexpectedly found that performing cell culture operations using an optimized asparagine supply strategy can improve cell culture outcomes, including improved cell growth and improved protein production by cells, compared to cell culture operations without such an optimized asparagine supply strategy.

[0018] As a non-limiting example, improvements in cell culture performance can be determined by evaluating cell growth rate, cell density, cell viability, protein production, production of cell growth byproducts (e.g., ammonium ion concentration, alanine concentration, etc.), and various combinations thereof. Improvements can be evaluated compared to cell culture operations without an optimized asparagine supply strategy, as disclosed herein.

[0019] Asparagine is a non-essential amino acid that is often consumed at high levels by cells during culture, playing a central role in cell culture metabolism. In certain embodiments of this disclosure, the role of asparagine in cell culture outcomes of fed-batch cell cultures was investigated along with metabolic studies of intracellular and extracellular asparagine and related metabolites.

[0020] As shown in Figures 1A and 1B, extracellular essential amino acids (Figure 1A) were found to be highly consumed and largely depleted at the end of fed-batch cell culture, while extracellular asparagine (Figure 1B), a non-essential amino acid, was depleted throughout the entire timeline of fed-batch cell culture following a standard bolus asparagine supply strategy.

[0021] Embodiments of this disclosure relate to cell culture methods that utilize asparagine supplementation to minimize, delay, and prevent amino acid depletion while improving cell culture performance. For example, referring to Figures 2A-2B, it was found that extracellular essential amino acid depletion can be prevented by essential amino acid supplementation (Figure 2A), but it was suggested that if the amount of asparagine supplementation is high during the growth phase (early stage) and fixation / disintegration phase (late stage) of the production phase of fed-batch cell culture, extracellular asparagine depletion in exemplary high-productivity cell lines is not prevented, and the asparagine consumption rate exceeds the stoichiometric requirement (Figure 2B). Therefore, according to embodiments of this disclosure, the effects of asparagine supplementation during and after the early, growth, and late stages of fed-batch cell culture were evaluated to select a balanced asparagine supply strategy as described herein.

[0022] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. Methods and techniques described herein are generally performed in accordance with prior art methods known in the art unless otherwise indicated, and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Julio E. Celis, Cell Biology: A Laboratory Handbook, 2nd ed., Academic Press, New York, NY (1998); and Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1995). All publications mentioned throughout this disclosure are incorporated herein by reference in their entirety.

[0023] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out the present invention, but specific methods and materials are described below.

[0024] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably throughout and refer to molecules containing two or more amino acid residues linked to each other by peptide bonds. Peptides, polypeptides, and proteins may also include modifications such as glycosylation, lipid attachment, sulfated substances, gamma-carboxylation, alkylation, hydroxylation, and ADP-ribosylation of glutamate residues. Peptides, polypeptides, and proteins may be of scientific or commercial interest, including protein-based drugs. Peptides, polypeptides, and proteins include, among other things, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are produced by recombinant animal cell lines using cell culture methods.

[0025] As used herein, the term “heterogeneic polynucleotide sequence” refers to nucleic acid polymers that encode a protein of interest, such as a chimeric protein (like a trap molecule), an antibody, or an antibody moiety (e.g., VH, VL, CDR3), produced as a biopharmaceutical active pharmaceutical ingredient. Heterogeneic polynucleotide sequences can be manufactured by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intron-free sequences, etc.) and may exist as episomes or be introduced into cells, which may be incorporated into the cellular genome. Heterogeneic polynucleotide sequences may be naturally occurring sequences introduced into ectopic sites within the producing cell genome. Heterogeneic polypeptide sequences may be naturally occurring sequences of another organism, such as sequences encoding human orthologues.

[0026] An "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are dotted with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes references to both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass. The term “antibody” includes antibody molecules prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express antibodies. The term “antibody” also includes bispecific antibodies, which include heterotetrameric immunoglobulins capable of binding to two or more different epitopes. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010 / 0331527, which is incorporated by reference in this application.

[0027] The term "antigen-binding moiety" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding moiety" of an antibody include: (i) the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) the F(ab′)2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) the Fd fragment, consisting of VH and CH1 domains; (iv) the Fv fragment, consisting of the VL and VH domains of a single arm of the antibody; (v) the dAb fragment, consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) the isolated CDR; and (vii) the scFv fragment, consisting of the two domains of the Fv fragment, VL and VH, which are joined by a synthetic linker to form a single protein chain in which VL and VH pair to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also included under the term "antibody" (see, for example, Holliger et al. (1993) PNAS USA 90:6444-6448 and Poljak et al. (1994) Structure 2:1121-1123).

[0028] Furthermore, an antibody or its antigen-binding moiety may be part of a larger immunoadhesion molecule formed by the covalent or non-covalent association of the antibody or antibody moiety with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to construct tetrameric scFv molecules (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to construct divalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol.Immunol.31:1047-1058). Antibody moieties such as Fab and F(ab')2 fragments can be prepared from the whole antibody using conventional techniques, such as through papain or pepsin digestion of the whole antibody. Furthermore, antibodies, antibody moieties, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques commonly known in the art (see Sambrook et al., 1989).

[0029] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include, for example, amino acid residues in the CDR, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been transplanted into a human framework sequence.

[0030] As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells; antibodies isolated from recombinant combinatorial human antibody libraries; antibodies isolated from animals transgenic to human immunoglobulin genes (e.g., mice) (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies prepared, expressed, created, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are used for in vitro mutagenesis (or, if transgenic animals are used for human Ig sequences, for in vivo somatic cell mutagenesis), so the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, while they may not naturally exist within the in vivo human antibody germline repertoire.

[0031] An "Fc-fusion protein" comprises part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, which is otherwise not found together in nature. Preparations of fusion proteins containing certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) are described, for example, in Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991, Byrn et al., Nature 344:677, 1990, and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. In some embodiments, a "receptor Fc-fusion protein" comprises one or more extracellular domains of a receptor ligated to the Fc portion containing the hinge region of an immunoglobulin, followed by CH2 and CH3 domains. In some embodiments, the Fc-fusion protein contains two or more different receptor chains that bind to one or more ligands. For example, the Fc-fusion protein is a trap, such as an IL-1 trap (e.g., lilonacept containing an IL-1RAcP ligand-binding domain fused to an IL-1R1 extracellular domain fused to the Fc of hIgG1, see U.S. Patent No. 6,927,004), or a VEGF trap (e.g., aflibercept containing an Ig domain 2 of VEGF receptor Flt1 fused to an Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1, see U.S. Patents No. 7,087,411 and 7,279,159).

[0032] Asparagine supplementation In certain embodiments of this disclosure, it has been found that supplementing Fed-batch cell cultures with asparagine affects cell culture outcomes. However, it has also been found that asparagine depletion in Fed-batch cell cultures cannot be avoided regardless of the amount of asparagine supplemented. In this regard, but not limited to certain embodiments of this disclosure, it has been unexpectedly found that while asparagine depletion in Fed-batch cell cultures can adversely affect cell culture outcomes, excessively high levels of asparagine can also adversely affect cell culture outcomes, for example, by forming byproducts such as ammonium.

[0033] In a particular embodiment, a method for culturing eukaryotic cells for improved cell culture outcomes is provided. This method generally comprises the steps of: growing or maintaining eukaryotic cells in a synthetic cell culture medium, wherein the synthetic cell culture medium is supplemented with asparagine in amounts of about 2.6 mM to about 28.6 mM during the initial Fed-batch cell culture and about 2.6 mM to about 21.6 mM during the later Fed-batch cell culture; and maintaining the cells in the asparagine-supplemented cell culture medium for at least a portion of the initial Fed-batch cell culture and at least a portion of the later Fed-batch cell culture, wherein the cell culture outcomes are improved by asparagine supplementation compared to similar methods with smaller amounts of asparagine supplementation in the initial and / or later Fed-batch cell culture.

[0034] As will be described in more detail herein, asparagine supplements may be supplied to cell culture (both early and late phases) as a bolus feed supplement (e.g., one bolus feed supplement during the early phase and one bolus feed supplement during the late phase), as multiple bolus feed supplements over at least a portion of the cell culture (e.g., two, three, four, or five bolus feed supplements during the early phase and two, three, four, or five bolus feed supplements during the late phase), or continuously over at least a portion of the cell culture.

[0035] In certain embodiments, the asparagine supplement may be provided as part of the bulk feed or as a separate supplement feed to the bulk feed. More specifically, the supplement may be provided to cell culture (both early and late phases) in a bolus feed or continuous feed, either as part of the bulk feed or as a separate supplement feed to the bulk feed.

[0036] In certain embodiments, the cell culture density and / or titer are increased compared to similar methods with less asparagine supplementation in the early and / or late fed-batch cell culture. In certain embodiments, the cell culture can maintain a viable cell count of at least about 10–50 M cells / ml, e.g., about 10–35 M cells / ml, during at least a portion of the late fed-batch cell culture after asparagine supplementation.

[0037] In certain embodiments, the eukaryotic cells are a high-asparagine-consuming cell line, such that the eukaryotic cells consume at least about 1.8 mM / day to about 9.3 mM / day of asparagine. In other embodiments, the eukaryotic cells are a low-asparagine-consuming cell line, such that the eukaryotic cells consume at least about 0.32 mM / day to about 1.8 mM / day of asparagine.

[0038] In certain embodiments, the method further comprises expressing the recombinant protein of interest from eukaryotic cells during fed-batch cell culture. The titer of the recombinant protein of interest may be at least 3%, 5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or at least 20% higher than that of cells cultured in non-asparagine supplemented culture. The yield of the recombinant protein of interest may be increased by at least 0.1 g / L, at least 0.5 g / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, at least 2.2 g / L, at least 2.4 g / L, or at least 2.5 g / L compared to a similar method in which cells are cultured in non-asparagine supplemented culture. In certain embodiments, the eukaryotic cell line is a high-productivity cell line such that the recombinant protein of interest is produced in yields of at least 4 g / L, at least 5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L, or at least 10 g / L, at least 11 g / L, at least 12 g / L, at least 13 g / L, or at least 14 g / L.

[0039] In certain embodiments, synthetic cell culture media is supplemented with asparagine in amounts such as approximately 2.6 mM to 28.6 mM, 3.0 mM to 25.0 mM, 5.0 mM to 23.0 mM, 6.0 mM to 22.0 mM, 7.2 mM to 21.6 mM, and 10.6 mM during the initial fed-batch cell culture, and approximately 2.6 mM to 21.6 mM, 2.6 mM to 20.0 mM, 2.6 mM to 18.0 mM, 2.6 mM to 16.0 mM, 2.6 mM to 14.4 mM, and 5.3 mM during the later fed-batch cell culture. In other embodiments, the synthetic cell culture medium is supplemented with approximately 7.2 mM to 21.6 mM of asparagine during the initial fed batch and approximately 2.6 mM to 14.4 mM during the later fed batch. In other embodiments, the synthetic cell culture medium is supplemented with approximately 10.6 mM of asparagine during the initial fed batch and approximately 5.3 mM during the later fed batch.

[0040] In certain embodiments, cells may be maintained under asparagine-supplemented cell culture conditions for at least two, three, four, or five days of a fed batch cell culture, or for the duration of the fed batch cell culture. The asparagine supplement may be supplied daily or continuously at least once, twice, three times, four times, or five times during at least a portion of the initial fed batch cell culture. Similarly, the asparagine supplement may be supplied daily or continuously at least once, twice, three times, four times, or five times during at least a portion of the later fed batch cell culture.

[0041] In certain embodiments, cells may be maintained under asparagine-supplemented cell culture conditions in cell cultures having volumes such as at least 500 liters, at least 750 liters, at least 1,000 liters, at least 1,500 liters, at least 2,000 liters, at least 2,500 liters, at least 5,000 liters, at least 7,500 liters, at least 10,000 liters, at least 12,500 liters, at least 15,000 liters, at least 20,000 liters, at least 25,000 liters, or 500 liters to 25,000 liters.

[0042] In certain embodiments, asparagine supplements may be continuously supplied in early and / or late-stage fed-batch cell cultures. In certain embodiments, continuous asparagine supplementation in cell culture results in a delay of at least one, at least two, at least three, or at least four days or more in extracellular amino acid depletion of asparagine, aspartic acid, and glutamic acid compared to similar methods in which asparagine supplementation is provided as a bolus feed in early and / or late-stage fed-batch cell cultures.

[0043] More specifically, in certain embodiments, as described herein, the asparagine supplement may be continuously supplied for at least two days, at least three days, at least four days, at least five days, or throughout the period of the initial fed-batch cell culture. Similarly, in certain embodiments, the asparagine supplement may be continuously supplied for at least two days, at least three days, at least four days, at least five days, or throughout the period of the late fed-batch cell culture.

[0044] In certain embodiments, but not limited to, the asparagine supplement may be continuously supplied from day 2 of the Fed batch cell culture and thereafter for at least a portion of the initial Fed batch cell culture, or from day 5 of the Fed batch cell culture and thereafter for at least a portion of the late Fed batch cell culture. Such late Fed batch continuous asparagine supplementation may be discontinued from day 10 of the late Fed batch cell culture.

[0045] Asparagine supplementation to mitigate asparagine sequence variants In other embodiments, it has been found that asparagine (Asn) depletion during fed-batch cell culture can lead to the development of asparagine sequence variants (SVs) in the polypeptide of interest expressed from mammalian cells in cell culture. For background, as disclosed in U.S. Patent No. 9,096,879, incorporated herein by reference, depletion of a specific amino acid during recombinant expression of the polypeptide of interest in mammalian cells is known to cause misuptake of the amino acid. For example, depletion of a specific amino acid during recombinant expression of the polypeptide of interest in mammalian cells can lead to the substitution of that amino acid with a second amino acid during translation of the polypeptide of interest in mammalian cells. An Asn→Ser asparagine sequence variant (i.e., misuptake of serine instead of asparagine during translation of the polypeptide of interest) is observed, but can be minimized by supplementing the cell culture medium with asparagine.

[0046] In this regard, it has been found that supplementing Fed-batch cell cultures with asparagine can minimize the formation of asparagine SVs. According to embodiments of the present disclosure, it has been unexpectedly found that asparagine SVs can be minimized by maintaining extracellular asparagine levels in the cell culture medium above depletion limits such as approximately 1.0 mM, approximately 0.5 mM, approximately 0.25 mM, approximately 0.2 mM, and approximately 0.1 mM, at least during the initial Fed-batch cell culture. In certain embodiments, extracellular asparagine levels in the cell culture medium can be maintained above such depletion limits for at least the first 6 days, at least the first 5 days, at least the first 4 days, at least the first 3 days, and at least the first 2 days of the initial Fed-batch cell culture. For example, such maintenance periods exceeding the depletion limit may include providing asparagine supplements at least once, at least twice, at least three times, daily, continuously, etc., during the initial stage of fed-batch cell culture.

[0047] According to certain embodiments of the present disclosure, supplementation of fed batch cell cultures with asparagine can reduce, or prevent, the generation of asparagine SV, such that the amount of asparagine SV in the target polypeptide is less than approximately 0.50%, less than approximately 0.45%, less than approximately 0.40%, less than approximately 0.35%, less than approximately 0.30%, less than approximately 0.25%, less than approximately 0.20%, less than approximately 0.15%, less than approximately 0.10%, or no asparagine SV is detected.

[0048] In a particular embodiment, supplementation of fed batch cell cultures with asparagine can reduce asparagine SV formation to such a level during late fed batch cell cultures, for example, after day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, and day 14 of cell culture.

[0049] In certain embodiments, asparagine as commonly described herein may be supplemented into cell cultures in amounts such as approximately 2.6 mM to approximately 28.6 mM, approximately 7.2 mM to approximately 21.6 mM, and approximately 10.6 mM during early fed-batch cell cultures, and approximately 2.6 mM to approximately 21.6 mM, for example, approximately 2.6 mM to approximately 14.4 mM and approximately 5.3 mM during late fed-batch cell cultures. In certain embodiments, the asparagine supplement may be provided as part of the bulk feed or as a separate asparagine supplement feed.

[0050] In certain embodiments, it was unexpectedly found that asparagine SV formation could be mitigated if sufficient asparagine supplementation was provided during the initial stages of fed-batch cell culture (e.g., a second bolus feed in certain embodiments) to prevent asparagine depletion until at least day 4 of fed-batch cell culture. In certain embodiments, supplementation in the initial stages of fed-batch cell culture to prevent depletion of aspartic acid (Asp) and glutamic acid (Glu) (or to produce sufficient glutamine (Gln) (i.e., greater than 100 mg / L)) while balancing the formation of excess ammonium, followed by sufficient asparagine supplementation in subsequent and later stages of fed-batch cell culture, provided an optimized reduction of asparagine SV.

[0051] In certain embodiments, it was unexpectedly found that providing sufficient asparagine supplementation during the initial stages of Fed-Batch cell culture (e.g., until at least day 4 of Fed-Batch cell culture) to maintain levels above the depletion limit, and then providing sufficient asparagine supplementation during the later stages of Fed-Batch cell culture to minimize the formation of undesirable by-products (e.g., ammonium), was beneficial. As a non-limiting example, asparagine supplementation during the initial and later stages of Fed-Batch cell culture may meet any of the following criteria for providing a low risk of asparagine SV.

[0052] Non-restrictive, exemplary initial FED batch conditions TIFF0007899162000001.tif26150

[0053] Non-restrictive, exemplary late-stage FED batch conditions TIFF0007899162000002.tif53150

[0054] Asparagine-related amino acids In other aspects of this disclosure, it has been found that targeted asparagine depletion may affect asparagine-related non-essential amino acids via cellular metabolic pathways. For context, asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), and glutamine (Gln) can be interconverted to each other via cellular metabolic pathways. When asparagine is needed, cells can synthesize supplemental asparagine by increasing the utilization of aspartic acid and glutamic acid. Higher intracellular / extracellular concentrations of aspartic acid, glutamic acid, and glutamine may indicate that the cell has sufficient intracellular asparagine to support its needs. However, decreased concentrations of aspartic acid, glutamic acid, and glutamine may indicate asparagine limitation.

[0055] Asparagine-related amino acids as surrogate markers for asparagine sequence variants (SVs) In other embodiments, asparagine-related amino acids, including aspartic acid (Asp), glutamic acid (Glu), and glutamine (Gln), have been found to be usable as surrogate markers for asparagine sequence variants formed during the production of the polypeptide of interest. According to certain embodiments of this disclosure, intracellular and extracellular concentrations of asparagine-related amino acids have been found to function as qualitative indicators of asparagine sequence variant formation.

[0056] Without intending to limit ourselves to theory, higher intracellular and / or extracellular concentrations of asparagine-related amino acids generally indicate that cells have sufficient intracellular asparagine to support their cellular needs. Such sufficient intracellular asparagine will result in minimal asparagine sequence variants in the target polypeptide produced. Conversely, lower intracellular and / or extracellular concentrations of asparagine-related amino acids generally indicate that cells have insufficient intracellular asparagine to support their cellular needs. Such insufficient intracellular asparagine will result in increased asparagine sequence variants in the target polypeptide produced. Therefore, asparagine-related amino acids can be used as surrogate markers for asparagine sequence variants based on a general inverse correlation. The strength of these surrogate correlations depends on the effect of targeted asparagine supplementation on the extracellular and intracellular amino acid profiles of the related amino acids, and can therefore be partially determined by the cell line and asparagine concentration.

[0057] In certain embodiments, intracellular and extracellular asparagine and asparagine-related amino acid concentrations may be used as surrogate markers for asparagine sequence variants. Without being limited, it has been unexpectedly found that asparagine SVs can be particularly minimized by supplementing with sufficient asparagine in early-stage fed-batch cell cultures, and that asparagine SV formation can be monitored using intracellular and extracellular asparagine and asparagine-related amino acid concentrations. As an example, asparagine SV formation can be monitored in situ by monitoring intracellular or extracellular concentrations of asparagine or asparagine-related amino acids. In certain embodiments, extracellular asparagine (Asn) may be monitored in early-stage fed-batch cell cultures as a surrogate for asparagine SVs, while extracellular asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), and glutamine (Gln) may be monitored in later-stage fed-batch cell cultures. In alternative embodiments, intracellular Asn, Asp, Glu, and Gln may be monitored in late-stage fed-batch cell cultures.

[0058] In certain embodiments, a method is provided for detecting, measuring, or screening asparagine sequence variants in a polypeptide of interest. This method generally includes the steps of: growing or maintaining eukaryotic cells in a synthetic cell culture medium; expressing a recombinant protein of interest from the eukaryotic cells; measuring intracellular and / or extracellular concentrations of asparagine or one or more asparagine-related amino acids in the synthetic cell culture medium; and correlating the measured concentrations of asparagine or one or more asparagine-related amino acids with the presence of asparagine sequence variants in the expressed recombinant protein of interest. In some embodiments, the measured concentrations of asparagine or one or more asparagine-related amino acids are inversely correlated with the presence of asparagine sequence variants.

[0059] In certain embodiments, asparagine may be added to synthetic cell culture media as described herein. In certain embodiments, the asparagine-related amino acid may be selected from aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), and combinations thereof. In some embodiments, the asparagine-related amino acid is glutamic acid. In some embodiments, the asparagine-related amino acid may be measured intracellularly.

[0060] In some embodiments, one or more asparagine-related amino acids may be measured during late-stage Fed batch cell culture, for example, 5, 6, 7, 8, 9, 10 days after Fed batch cell culture.

[0061] In certain embodiments, a method for detecting, measuring, or screening asparagine sequence variants using asparagine-related amino acids as surrogate markers may provide a high-throughput screening and optimization technique for identifying optimal cell culture parameters for a particular polypeptide of interest. For example, this method may be used to target the optimal asparagine feed level that minimizes asparagine sequence variants for a particular polypeptide of interest.

[0062] Optimization, in-situ monitoring, and control of asparagine supplementation. In certain embodiments, methods are provided for monitoring and controlling cell culture parameters, including asparagine supplementation. Such methods can monitor and control a variety of cell culture process parameters to provide optimal asparagine supplementation for a target cell line and / or polypeptide of interest. Exemplary cell culture process parameters that can be monitored and controlled include, but are not limited to, asparagine concentration, asparagine-related amino acid concentration, live cell number, dead cell number, protein titer, ammonium ions, alanine, osmotic pressure, and combinations thereof.

[0063] In certain embodiments, the monitoring and control methods of this disclosure may be used to optimize and prevent asparagine depletion in cell culture media while simultaneously controlling the formation of cell culture byproducts such as ammonium ions and alanine. Such monitoring and control can thereby provide improved control of cell culture performance parameters, such as viable cell count and protein titer.

[0064] In certain embodiments, a method is provided for monitoring and controlling cell culture medium conditions. This method may generally include the steps of: measuring one or more cell culture parameters in a cell culture using in situ freezing point depression, electrochemistry, digital imaging, photometrics, bioprocess analyzers, or Raman spectroscopy; comparing the measured one or more cell culture parameters to predetermined setpoints for the cell culture parameters to determine whether one or more cell culture parameters are within a predetermined threshold range; and, if it is determined that the cell culture parameters are outside the predetermined threshold range, adjusting one or more of the cell culture parameters. In certain embodiments, ammonium ion concentration may be monitored, and if it is determined that the ammonium ion concentration is outside a predetermined threshold range for a particular cell culture, the asparagine supplement feed may be adjusted so that the cell culture produces less ammonium while still receiving adequate asparagine. Such a method may be used to provide optimal and maximum asparagine supplementation while minimizing the production of cellular byproducts such as ammonium ions and alanine.

[0065] In certain embodiments, the monitoring and control methods of the present disclosure may utilize in situ freezing point depression, electrochemistry, digital imaging, photometrics, bioprocess analyzers, or Raman spectroscopy, or other suitable in situ cell culture parameter measurement methodologies and metric chemical modeling techniques for real-time evaluation of cell culture parameters, combined with signal processing techniques, for accurate and continuous feedback and model predictive control of cell culture process parameters. In situ freezing point depression, electrochemistry, digital imaging, photometrics, bioprocess analyzers, or Raman spectroscopy of bioreactor contents allows for the analysis of one or more process variables without physically removing the sample for testing. Such techniques can provide real-time feedback control of cell culture process parameters, including asparagine supplementation.

[0066] In one embodiment, predetermined maximum setpoints for ammonium ions and / or alanine may be set. Similarly, predetermined setpoints for the continuous asparagine replenishment flow rate may be set. Continuous asparagine replenishment may be initiated at a desired point in time during fed-batch cell culture (e.g., initial fed-batch timeframes: day 1, day 2, day 3, day 4; later fed-batch timeframes: day 5, day 6, day 7, day 8, day 9, day 10, etc.). One or more concentrations of asparagine, asparagine-related amino acids, ammonium ions, and / or alanine may be monitored during the cell culture operation by freezing point depression, electrochemistry, digital imaging, photometrics, bioprocess analyzer, or Raman spectroscopy. In certain embodiments, data from freezing point depression, electrochemistry, digital imaging, photometrics, bioprocess analyzer, or Raman spectroscopy may be acquired approximately every 10 minutes to 2 hours to ensure that the raw spectral data is continuously up-to-date. In another embodiment, data may be acquired approximately every 15 minutes to 1 hour. In yet another embodiment, data may be acquired approximately every 20 to 30 minutes.

[0067] Monitoring of one or more cell culture process parameters can be performed using any commercially available analyzer that enables in-situ analysis. The in-situ analyzer must be able to acquire raw data within the cell culture (for example, the analyzer must be equipped with a probe that can be inserted into a bioreactor). Suitable analyzers include, but are not limited to, the RamanRXN2 and RamanRXN4 analyzers (Kaiser Optical Systems, Inc. Ann Arbor, Mich.) or the BioProfile Flex automated cell culture analyzer.

[0068] Raw spectral data acquired by an in-situ analyzer can be compared to offline measurements of specific process parameters that are monitored or controlled (e.g., offline asparagine concentration measurements) to correlate the data within the process parameters. Offline measurement data can be collected by any suitable analytical method. Furthermore, any type of multivariate software package, e.g., SIMCA13 (MKS Data Analytic Solutions, Umea, Sweden), can be used to correlate the data with offline measurements of specific process variables that are monitored or controlled. However, in some embodiments, it may be necessary to preprocess the raw data with filters to remove any fluctuating baselines. For example, the raw data can be preprocessed with any type of point smoothing or normalization technique. Normalization may be necessary, for example, to correct for any power fluctuations and exposure times. In one embodiment, the raw data is 21 cm -1 It can be processed by point smoothing, such as the first derivative having point smoothing, and normalization, such as the standard normalized variable (SNV).

[0069] Quantitative chemical modeling can also be performed on the obtained spectral data. In certain embodiments, but not limited to, one or more multivariate methods, including partial least squares (PLS), principal component analysis (PCA), orthogonal partial least squares (OPLS), multivariate regression, canonical correlation, factor analysis, cluster analysis, and graphical procedures, can be used on the spectral data. In one embodiment, the acquired spectral data is used to create a PLS regression model. The PLS regression model can be created by projecting the predicted and observed variables into a new space. In this embodiment, the PLS regression model can be created using measurements obtained from Raman analysis and offline measurements. The PLS regression model provides predicted process values, for example, predicted nutrient concentration values.

[0070] After quantitative chemical modeling, signal processing techniques may be applied to predicted process values ​​(e.g., predicted asparagine concentration values). In one embodiment, the signal processing techniques include noise reduction techniques. In a particular embodiment, one or more noise reduction techniques may be applied to the predicted process parameters. Any noise reduction technique known to those skilled in the art may be utilized. For example, noise reduction techniques may include data smoothing and / or signal rejection. Smoothing is achieved by a series of smoothing algorithms and filters, while signal rejection uses signal characteristics to identify data that should not be included in the analyzed spectral data. In one embodiment, the predicted process values ​​are noise reduced by the noise reduction filter. The noise reduction filter provides the final filtered values ​​(e.g., final filtered nutrient concentration values). In this embodiment, the noise reduction technique combines the raw measurement with a model-based estimate of what the measurement should obtain according to the model. In one embodiment, the noise reduction technique combines the current predicted process value with its uncertainty. The uncertainty may be determined by the predictiveness of the predicted process value and the reproducibility of the current process conditions. When the next predicted process value is observed, the estimate of the predicted process value (e.g., predicted nutrient concentration value) is updated using a weighted average, where estimates with higher certainty are given more weight. Using an iterative approach, the final process value may be updated based on previous measurements and current process conditions. In this embodiment, the algorithm must be recursive and real-time executable to utilize the current predicted process value, previous values, and experimentally determined constants. Noise reduction techniques improve the robustness of measurements received from Raman analysis and PLS predictions by reducing the noise on which the automatic feedback controller acts.

[0071] Once the final filtered process values ​​(e.g., final filtered nutrient concentration values) are obtained, these final values ​​may be transmitted to an automatic feedback controller. The automatic feedback controller may be used to control and maintain process parameters (e.g., monitoring ammonium concentration and controlling asparagine concentration) below predetermined setpoints. In one embodiment, if an ammonium concentration exceeding a maximum setpoint is detected, the automatic feedback controller may prompt to reduce the amount of asparagine feed within a threshold range. The automatic feedback controller may include any type of controller capable of calculating an error value as the difference between a desired setpoint (e.g., a predetermined setpoint) and the measured process parameter, and automatically applying accurate and responsive corrections. The automatic feedback controller should also have a control device that can be modified in real time from a platform interface. For example, the automatic feedback controller should have a user interface that allows adjustment of a predetermined setpoint. The automatic feedback controller should be capable of responding to changes in a predetermined setpoint.

[0072] In one embodiment, the automatic feedback controller may be a proportional-integral-derivative (PID) controller. In this embodiment, the PID controller is operable to calculate the difference between a predetermined setpoint and a measured process variable (e.g., a measured asparagine concentration) and to automatically apply an accurate correction. For example, when controlling nutrient concentrations in a cell culture, the PID controller may be operable to calculate the difference between the filtered nutrient value and a predetermined setpoint and to provide a correction for the nutrient amount. In this embodiment, the PID controller may be operably connected to a nutrient pump on a bioreactor so that the corrected nutrient amount can be pumped to the bioreactor.

[0073] By using real-time analysis and feedback control, the method of this disclosure can provide optimal asparagine supplementation for a target cell line and / or polypeptide. That is, the method of the present invention can provide optimal asparagine supplementation for cell culture while minimizing the production of cell culture byproducts, including harmful cell culture byproducts.

[0074] Cell culture medium The terms "cell culture medium" and "culture medium" typically refer to a nutrient solution used to grow cells, such as eukaryotic cells, that provides essential nutrients for improving cell growth, including carbohydrate energy sources, essential (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) and non-essential (e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine) amino acids, trace elements, energy sources, lipids, and vitamins. Cell culture mediums may contain extracts that supply raw materials to support cell growth, such as serum or peptone (hydrolyzate). The medium may contain yeast-derived extracts or soybean extracts instead of animal-derived extracts. A known-composition medium refers to a cell culture medium in which all chemical components are known (i.e., have known chemical structures). Generally, a known-composition medium does not contain serum or animal-derived peptone, as well as animal-derived components such as yeast and soybean extracts. In one embodiment, the culture medium is a culture medium of known composition.

[0075] The culture medium may also contain components such as hormones and growth factors that improve the growth rate and / or viability to a level above the minimum growth rate and / or viability. The culture medium is preferably formulated to have an optimal pH and salt concentration for cell survival and proliferation.

[0076] In certain embodiments, the cell culture medium may be serum-free. “Serum-free” applies to cell culture media that do not contain animal serum, such as fetal bovine serum. Serum-free media may contain hydrolysates, such as soybean hydrolysate, at a concentration of 16 g / L or less. This disclosure also provides known-composition media that are not only serum-free but also hydrolysate-free. “Hydrolysate-free” applies to cell culture media that do not contain exogenous protein hydrolysates, such as animal or plant protein hydrolysates like peptone or tryptone.

[0077] "Basic medium" is the initial medium in which cells grow (e.g., present in the seed train and / or on day 0 of cell culture production) and contains all necessary nutrients, including a basic mixture of amino acids. Various recipes (i.e., formulations) of basic medium can be manufactured or purchased in commercial lots. Similarly, "basic feed medium" contains a mixture of supplemental nutrients that is commonly consumed during production culture and utilized in feeding strategies (for so-called "fed-batch" culture). Various basic feed media are commercially available. "Feeding" includes scheduled additions or additions to the medium at regular intervals, such as according to protocols involving continuous feed culture systems, as found in chemostats (see C. Altamirano et al., Biotechnol Prog. 2001 November-December; 17(6): 1032-41), or according to a fed-batch process (see YMHuang et al., Biotechnol Prog. 2010 September-October; 26(5): 1400-10). For example, the culture may be supplied once a day, once every two days, once every three days, or when the concentration of a particular culture medium component being monitored is outside the desired range.

[0078] Without being intended to limit the scope, this disclosure may be carried out using any one or more of a variety of basic media or combinations thereof. Basic media are generally known in this art, and include, in particular, Eagle's MEME (Minimum Essential Medium) (Eagle, Science, 1955, 112(3168):501-504), Ham's F12 (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, 53:288-293), F-12 K medium, Dulbecco's medium, Dulbecco's modified Eagle medium (Proc. Natl. Acad. Sci. USA, 1952 August; 38(8):747-752), DMEM / Ham's F12 1:1, Trowell's T8, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, 10:389-401), Waymouth medium (Davidson and Waymouth, Biochem. J., 1945, 39(2): 188-199), Williams E medium (William's et al., Exp. Cell Res., 1971, 69: 105 and below), RPMI 1640 (Moore et al., J. Amer. Med. Assoc., 1967, 199: 519-524), MCDB 104 / 110 medium (Bettger et al., Proc. Nat'l. Acad. Sci. USA, 1981, 78(9):5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al. al., Appl. Microbiol., 1973, 25(1):49-54), RPMI-1640 medium, RPMI-1641 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz's L-15 medium, and, if serum is not included, for example, EX-CELL® 300 series (JRH Biosciences, Lenexa, Kans.), Protamine Zinc Insulin Medium (Weiss et al.(1974, U.S. Patent No. 4,072,565), Biotin Folate Medium (Cartaya, 1978, U.S. Re30,985), Transferrin Fatty Acid Medium (Baker, 1982, U.S. Patent No. 4,560,655), Transferrin-EGF Medium (Hasegawa, 1982, U.S. Patent No. 4,615,977; Chessebeu, 1984, U.S. Patent No. 4,786,599), and permutations of other media (Inlow, U.S. Patent No. 6,048,728; Drapeau, U.S. Patent No. 7,294) See also U.S. Patent Nos. 484; Mather, No. 5,122,469; Furukawa, No. 5,976,833; Chen, No. 6,180,401; Chen, No. 5,856,179; Etcheverry, No. 5,705,364; Etcheverry, No. 7,666,416; Ryll, No. 6,528,286; Singh, No. 6,924,124; Luan, No. 7,429,491, etc.

[0079] Cell culture media may also be supplied periodically (as in so-called "fed-batch" cultures), with or without additional components such as polyamines, or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements, depending on the requirements of the cells being cultured or the desired cell culture parameters.

[0080] In certain embodiments, the cell culture medium may be depleted of amino acids over the course of recombinant protein production if no additional amino acid supplementation is provided, or it may be “non-depleted” (as described below) if amino acid supplementation is provided for the depleted amino acids.

[0081] In one embodiment, the culture medium further contains 100 μM ± 15 μM of ornithine, or 300 μM ± 45 μM of ornithine, or 600 μM ± 90 μM of ornithine, or 900 μM ± 135 μM of ornithine. In another embodiment, the culture medium contains at least about 29 μM ± 1 μM of ornithine, or at least about 59 μM ± 12 μM of ornithine, 80 μM ± 13 μM of ornithine, or at least about 296 μM ± 44 μM of ornithine, or at least about 593 μM ± 89 μM of ornithine, or at least about 889 μM ± 133 μM of ornithine.

[0082] Optionally, putrescine may be added to the supplemented medium. Putrescine is included as an ingredient in several cell culture medium formulations at low concentrations, e.g., 0.01 to 120 mg / L, for example, WO2005 / 028626, U.S. Patent No. 5,426,699 (0.08 mg / L), U.S. Patent No. RE30,985 (0.16 mg / L), U.S. Patent No. 5,811,299 (0.27 mg / L), U.S. Patent No. 5,122,469 (0.5635 mg / L), U.S. Patent No. 5,063,157 (1 mg / L), WO2008 / 154014 (approximately 100 mg / L). See U.S. Patent Application No. 2007 / 0212770 (0.5-30 mg / L polyamines, 2 mg / L putrescine, 2 mg / L putrescine + 2 mg / L ornithine, 2 mg / L putrescine + 10 mg / L ornithine) for concentrations of 82 M to approximately 1000 μM.

[0083] In some embodiments, the cell culture medium is further supplemented with a combination of ornithine and putrescine, where the putrescine may be at a concentration of at least about 150–720 μM. In some embodiments, the medium is further supplemented with putrescine at a concentration of about 170–230 μM. In one embodiment, the medium contains 90 μM ± 15 μM or more of ornithine in addition to 200 μM ± 30 μM of putrescine. In one embodiment, the medium contains 89 μM ± 13 μM or less of ornithine in addition to 186 μM ± 28 μM or less of putrescine. In another embodiment, the medium contains 89 μM ± 13 μM or more of ornithine in addition to 186 μM ± 28 μM or more of putrescine (see International Publication No. 2014 / 144198A1, published on 18 September 2014, which is incorporated herein by reference in its entirety).

[0084] In further embodiments, ornithine is present in the culture medium at concentrations ranging from 0.09±0.014 mM to 0.9±0.14 mM, for example, 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM. In some embodiments, the culture medium also contains at least 0.20±0.03 mM of putrescine. In some embodiments, the additional putrescine is present at concentrations ranging from 0.20±0.03 mM to 0.714±0.11 mM, for example, 0.20±0.03 mM, 0.35±0.06 mM, or 0.714±0.11 mM.

[0085] In yet another embodiment, the culture medium may be supplemented with taurine at a concentration of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM (expressed as millimoles per liter).

[0086] Various other supplements may be added to the culture medium, and determining more appropriate conditions is within the scope of the art of the art. In certain embodiments, the supplement may be an asparagine supplement as described herein. In some embodiments, the culture medium is supplemented with a mixture of amino acids selected from the group consisting of aspartic acid, cysteine, glutamic acid, glycine, lysine, phenylalanine, proline, serine, threonine, valine, arginine, histidine, asparagine, glutamine, alanine, isoleucine, leucine, methionine, tyrosine, and tryptophan, either to prevent depletion or as supplemental nutrients are needed (i.e., bulk feed).

[0087] In one embodiment, the culture medium is further supplemented with approximately 170 μM to 175 μM of nucleoside. In one embodiment, the culture medium contains a purine derivative at a cumulative concentration of at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, or at least 105 μM. In one embodiment, the culture medium contains approximately 100 μM to 110 μM of purine derivative. Examples of purine derivatives include hypoxanthine, nucleoside adenosine, and guanosine. In one embodiment, the culture medium contains a pyrimidine derivative at a cumulative concentration of at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, or at least 65 μM. In one embodiment, the culture medium contains approximately 65 μM to 75 μM of a pyrimidine derivative. Examples of pyrimidine derivatives include nucleoside thymidine, uridine, and cytidine. In a particular embodiment, the culture medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0088] In addition to containing any of the above additives, in one embodiment, the culture medium is further supplemented with micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment, the fatty acids include one or more of the following: linoleic acid, linolenic acid, thiotic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. In one embodiment, the culture medium contains tocopherol, linoleic acid, and thioctic acid.

[0089] In one embodiment, the culture medium may be further supplemented with a mixture of vitamins containing other nutrients and essential nutrients at a cumulative concentration of at least about 700 μM or at least about 2 mM. In one embodiment, the vitamin mixture contains one or more of the following: D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12. In one embodiment, the vitamin mixture contains all of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12.

[0090] In certain embodiments, the cell culture medium may be of a known composition and may include an amino acid mixture as discussed herein; CaCl22H2O, KCl, MgSO4, NaCl; Na2HPO4 or other phosphates; pyruvate; D-biotin; choline chloride; folic acid; myo-inositol; niacinamide; pyridoxine HCl; D-pantothenic acid; riboflavin; thiamine HCl; vitamin B12; ρ-aminobenzoic acid; ethanolamine HCl; poloxamer 188; DL-α-tocopherol phosphate; linoleic acid; Na2SeO3; thiotic acid; one or more buffers; and glucose; and optionally adenine; guanosine; cytidine; uric acid; thymidine; and hypoxanthine disodium.

[0091] In one embodiment, the initial osmotic pressure of the medium of the Disclosure is 200–500, 250–400, 275–350, or about 300 mOsm. During cell growth in the medium of the Disclosure, particularly after any supply by a fed-batch protocol, the osmotic pressure of the culture may increase to a maximum of about 350, 400, 450, 500, or up to about 550 mOsm.

[0092] In some embodiments where the osmotic pressure of the culture medium is less than about 300, the osmotic pressure can be adjusted to about 300 by adding one or more salts in amounts exceeding a specified amount. In one embodiment, the osmotic pressure is increased to a desired level by adding one or more osmoregulators selected from sodium chloride, potassium chloride, magnesium salts, calcium salts, amino acid salts, fatty acid salts, sodium bicarbonate, sodium carbonate, potassium carbonate, chelating agents which are salts, sugars (e.g., galactose, glucose, sucrose, fructose, fucose, etc.), and combinations thereof. In one embodiment, the osmoregulator is added in amounts exceeding its concentration in components already present in the synthetic culture medium (e.g., sugars are added in amounts exceeding a specified concentration relative to the sugar component).

[0093] cell culture One aspect of the present disclosure provides a cell culture comprising a cell line expressing a recombinant protein of interest cultured with an asparagine supplement, as described herein. Examples of cell lines routinely used to produce recombinant proteins include, among others, primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells, and chicken embryo cells. In one embodiment, the cell line is a CHO cell line, or one or more of several specific CHO cell variants optimized for large-scale protein production, such as CHO-K1.

[0094] Another aspect of the present disclosure relates to a method of culturing cells using an asparagine supplement as described herein, wherein the use of such asparagine supplement enhances the proliferation of eukaryotic cells and, at the same time, improves the titer of one or more recombinant proteins of interest by such cells compared to similar methods with lower amounts of asparagine supplementation in early and / or late-stage fed-batch cell cultures, and maintains cell viability, particularly when used in early and / or late-stage fed-batch cell cultures.

[0095] In some embodiments, recombinant protein titers are improved compared to cells grown with lower amounts of asparagine supplementation in early and / or late-stage fed-batch cell cultures. In some embodiments, the protein titer obtained from cell cultures grown with less asparagine supplementation in early and / or late-fed batch cell cultures is at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, or at least about 29% greater than the protein titer (yield) from cells cultured with less asparagine supplementation in early / late-fed batch cell cultures. In some embodiments, the protein titer obtained from cell cultures with the asparagine supplement of this disclosure is greater than that of similar or identical cells cultured with less asparagine supplementation in early and / or late-batch cell cultures.

[0096] In some embodiments, cell growth (e.g., double-accelerated), viable cell density, cell viability, and combinations thereof are improved compared to cells grown with less asparagine supplementation in early and / or late-stage fed-batch cell cultures.

[0097] In some embodiments, the doubling acceleration of viable cells cultured with the asparagine supplement of the present disclosure is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 3 times greater than the doubling acceleration of cells cultured with less asparagine supplementation in early and / or late fed-batch cell cultures. In some embodiments, the doubling acceleration of live cells cultured with the asparagine supplement of this disclosure is about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% greater than the doubling acceleration of live cells cultured with less asparagine supplementation in early and / or late fed-batch cell cultures.

[0098] In some embodiments, the doubling time of actively cycling mammalian cells, when cultured with the asparagine supplement of this disclosure, is less than 30 hours, less than 29 hours, less than 28 hours, less than 27 hours, less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, or less than 18 hours, compared to cells cultured with less asparagine supplement in early and / or late-fed batch cell cultures. In some embodiments, the doubling time of actively growing mammalian cells, when cultured with the asparagine supplement of this disclosure, is less than 28 hours, compared to cells cultured with less asparagine supplement in early and / or late-fed batch cell cultures. In some embodiments, the doubling time of mammalian cells, when cultured with the asparagine supplement of this disclosure, is approximately 27±1 hours, approximately 26±1 hours, approximately 25±1 hours, approximately 24±1 hours, approximately 23±1 hours, approximately 22±1 hours, or approximately 21±1 hours, compared to cells cultured with less asparagine supplementation in early and / or late-fed batch cell cultures. In some embodiments, the doubling time of actively cycling mammalian cells, when cultured with the asparagine supplement of this disclosure, is approximately 24±1 hours, compared to cells cultured with less asparagine supplementation in early and / or late-fed batch cell cultures. In some embodiments, the doubling time of actively dividing cells, when cultured with the asparagine supplement of this disclosure, is at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or at least 25% shorter than the doubling time of actively cycling cells cultured with less asparagine supplementation in early and / or late-fed batch cell cultures.

[0099] With respect to cell viability, cells cultured with the asparagine supplement of this disclosure exhibit viability at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, or at least three times greater than the viability of cells cultured with less asparagine supplementation in early and / or late-batch cell cultures.

[0100] In a production culture vessel or bioreactor, following the seed culture or growth phase, basal culture medium and cells are supplied to the culture vessel. In certain embodiments, the cell supernatant or cell lysate is collected after production culture. In other embodiments, the polypeptide or protein of interest is recovered from the culture medium or cell lysate, or whatever it may be, depending on the location of the protein of interest, using techniques well known in the art.

[0101] A "cell line" refers to cells derived from a specific lineage through continuous subculturing or subculture of cells. The term "cells" is used interchangeably with "cell population."

[0102] The term "cell" includes any cell suitable for expressing recombinant nucleic acid sequences. Cells include non-human animal cells, mammalian cells, human cells, avian cells, insect cells, yeast cells, or eukaryotic cells such as cell fusions, such as hybridomas or quadromas. In certain embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In other embodiments, the cells are selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC5, Colo25, HB 8065, HL-60, lymphocytes (e.g., Jurkat (T lymphocyte) or Daudi (B lymphocyte)), A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells include one or more viral genes, e.g., retinal cells expressing viral genes (e.g., PER.C6® cells). In some embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0103] In the recombinant protein production phase, “fed batch cell culture” or “fed batch culture” refers to a batch culture in which animal cells and culture medium are initially supplied to the culture vessel, with or without periodic cell and / or product harvesting before the end of the culture, and additional culture nutrients are slowly supplied to the culture in continuous or separate increments during the culture. Fed batch culture includes “semi-continuous fed batch culture,” in which the entire culture (which may include cells and culture medium) is periodically removed and replaced with fresh culture medium. Fed batch culture is distinguished from simple “batch culture,” in which all components for cell culture (including animal cells and all culture nutrients) are supplied to the culture vessel at the start of the culture process in batch culture. Fed batch culture can be further distinguished from perfusion culture unless the supernatant is removed from the culture vessel during the process, in which cells are inhibited in the culture, for example by filtration, and culture medium is continuously or intermittently introduced and removed from the culture vessel. However, removal of samples for experimental purposes is intended during fed batch cell culture. The fed batch process continues until it is determined that the maximum working volume and / or protein production has been reached.

[0104] As used herein, the term “continuous cell culture” typically refers to techniques used to continuously grow cells in a particular growth phase. For example, cell culture may require maintenance in a particular growth phase if a constant supply of cells is required, or if the production of a polypeptide or protein of a particular purpose is required. Therefore, conditions must be continuously monitored and adjusted in order to maintain the cells in that particular phase.

[0105] One aspect of the present disclosure relates to fed-batch producing cell cultures in which proteins are produced and harvested. Prior to the production phase, there is typically a growth phase (also known as seed train or seed culture) in which all components for cell culture are supplied to the culture vessel at the start of the culture process, and the cell population is then expanded until it is ready for production scale. Thus, the culture vessel is inoculated with cells at a suitable seeding density for the initial cell growth phase, depending on the starting cell line. In some aspects, the asparagine supplement of the present disclosure may be used in conjunction with fed-batch producing cell cultures as further described herein.

[0106] Culture vessels include, but are not limited to, well plates, T-flasks, vibrating flasks, stirring vessels, spinner flasks, hollow fiber vessels, and air-lift bioreactors. A preferred cell culture vessel is a bioreactor. A bioreactor refers to a culture vessel manufactured or designed to manipulate or control environmental conditions. Such culture vessels are well known in the art.

[0107] Bioreactor processes and systems have been developed to optimize gas exchange to supply sufficient oxygen and remove CO2 to maintain cell growth and productivity. Maintaining gas exchange efficiency is a critical criterion for ensuring the successful scale-up of cell culture and protein production. Such systems are well known to those skilled in the art.

[0108] In one embodiment, the culture medium is replenished at intervals during cell culture according to a fed-batch process. Fed-batch culture is commonly known in the art and is used to optimize protein production (YMHuang et al., Biotechnol Prog. 2010 September-October;26(5):1400-10). The fed-batch process is typically used during the production phase.

[0109] Supplementary supply may be provided daily or at intervals of every 2-3 days during the production culture period, containing additional nutrients, such as vitamins, amino acids, and other nutrients as described herein. Supplementary supply (addition of supplemented medium containing nutrients) may be provided at least twice, or at least eight times, throughout the production culture period for cultures of two weeks or longer. In another embodiment, supplementary supply may be provided daily during the culture period. Alternative culture supply schedules are also envisioned.

[0110] Non-depleted media may be provided by supplementing with additional amino acids, the depleted amino acids being determined according to methods known in the art and described herein. When this regime is used, the additional amino acids are supplemented or added at intervals, preferably daily or every 2-3 days, during the period of production culture, depending on the determination of amino acid depletion. In one embodiment, a mixture of additional amino acids to maintain non-depleted cell culture medium is added to the culture on day 1 or about day 1, day 2 or about day 2, day 3 or about day 3, day 4 or about day 4, day 5 or about day 5, day 6 or about day 6, day 7 or about day 7, day 8 or about day 8, day 9 or about day 9, day 10 or about day 10, day 11 or about day 11, day 12 or about day 12, day 13 or about day 13, and day 14 or about day 14 for culture of two weeks or more. Alternative culture supply schedules are also envisioned.

[0111] Eukaryotic cells such as CHO cells can be cultured on a small scale, for example, in a 125 mL container with about 25 mL of medium, a 250 mL container with about 50–100 mL of medium, a 250 mL container with about 150–240 mL of medium, or a 500 mL container with about 100–200 mL of medium. Alternatively, cultures can be on a large scale, for example, in a 1000 mL container with about 300–1000 mL of medium, a 3000 mL container with about 500–3000 mL of medium, an 8000 mL container with about 2000–8000 mL of medium, and a 15000 mL container with about 4000–15000 mL of medium. Cultures for production can contain 10,000 L or more of medium. Large-scale cell cultures, such as those for the clinical production of protein therapeutics, are typically maintained for several days or even several weeks while the cells produce the desired protein. During this period, the culture may be supplemented with a concentrated feed medium containing nutrients and components such as amino acids that are consumed during the culture process. The concentrated feed medium can be based on any cell culture medium formulation. Such a concentrated feed medium may contain most of the components of the cell culture medium at, for example, about 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800, or even about 1000 times the normal useful amount. Concentrated feed medium is often used in fed-batch culture processes.

[0112] In some embodiments, during the process of cell growth or protein production, the cell culture may be further supplemented with “use-point additives,” use-point components, or use-point chemicals, also known as additives. Examples of use-point additives include one or more of the following: growth factors or other proteins, buffers, energy sources, salts, amino acids, metals, and chelating agents. Other proteins include transferrin and albumin. Growth factors, including cytokines and chemokines, are commonly known in the art and are known to stimulate cell growth, or optionally, cell differentiation. Growth factors are typically proteins (e.g., insulin), small peptides, or steroid hormones such as estrogen, DHEA, and testosterone. In some cases, growth factors may be non-natural chemicals that promote cell proliferation or protein production, such as tetrahydrofolate (THF) and methotrexate. Non-exclusive examples of protein and peptide growth factors include angiopoietin, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), transition-stimulating factors, and mi Examples include ostatin (GDF), nerve growth factor (NGF), other neurotrophic factors, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF), WNT signaling pathway agonists, placental growth factor (PIGF), fetal bovine somatotropin (FBS), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7. In one embodiment, the cell culture medium is supplemented with use-point-added growth factor insulin.In one embodiment, the concentration of insulin in the culture medium, i.e., the amount of insulin in the cell culture medium after addition, is approximately 0.1 μM to 10 μM.

[0113] Buffers are generally known in the art. The present invention is not limited to any particular buffer, and those skilled in the art can select a suitable buffer or buffering system for use with a particular cell line that produces a particular protein. In one embodiment, the buffer added at the point of use is NaHCO3. In another embodiment, the buffer is HEPES. In yet another embodiment, the buffer added at the point of use includes both NaHCO3 and HEPES.

[0114] Energy sources for use as use-point additives in cell culture are also well known in the art. In one embodiment, but not limited to, the use-point additive energy source is glucose. Depending on the specific cell line and the protein to be produced and the specific requirements, in one embodiment glucose may be added to the culture medium at a concentration of about 1 to 20 mM. In some cases, glucose may be added at high levels of 20 g / L or more.

[0115] Chelating agents are also well known in cell culture and protein production techniques. EDTA tetrasodium dihydrate and EDTA tetrasodium citrate are two common chelating agents used in the art, but other chelating agents may be employed in the implementation of the present invention. In one embodiment, the use-point chelating agent is EDTA tetrasodium dihydrate. In one embodiment, the use-point chelating agent is a citrate such as Na3C6H5O7.

[0116] In one embodiment, the cell culture medium may be supplemented with one or more use-point added amino acids as an energy source, such as glutamine. In one embodiment, the cell culture medium is supplemented with use-point added glutamine at a final concentration of about 1 mM to 13 mM.

[0117] Other use-point additives include one or more of various metal salts, such as iron, nickel, zinc, and copper salts. In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, ferrous chloride, and nickel sulfate.

[0118] Protein production In certain embodiments, the Disclosure provides a method for improving cell culture performance, comprising improving recombinant protein titer in the production of recombinant proteins by culturing eukaryotic cells. In some embodiments, the eukaryotic cells contain stably integrated nucleic acids encoding recombinant proteins. In other embodiments, the method of the Disclosure provides improved cell growth (e.g., doubled acceleration), viable cell density, cell viability, and combinations thereof.

[0119] In some embodiments, the method of the present disclosure includes the steps of: providing the asparagine supplement of the present disclosure; culturing eukaryotic cells in the asparagine supplement; expressing a recombinant protein of interest from the eukaryotic cells; and producing a recombinant protein of higher titer from eukaryotic cells cultured in the asparagine supplement compared to similar or identical eukaryotic cells cultured in less asparagine supplementation or without asparagine supplementation in early and / or late fed-batch cell cultures.

[0120] In some embodiments, the protein production rate or titer, which can be expressed in grams of protein producible per liter of culture medium, from cells cultured with the asparagine supplement of the present disclosure is at least 100 mg / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, at least 2.5 g / L, at least 3 g / L, at least 3.5 g / L, at least 4 g / L, at least 4.5 g / L, at least 5 g / L, at least 5.5 g / L, at least 6 g / L, at least 6.5 g / L, at least 7 g / L, at least 7.5 g / L, at least 8 g / L, at least 8.5 g / L, at least 9 g / L, at least 9.5 g / L, at least 10 g / L, at least 15 g / L, or at least 20 g / L.

[0121] In some embodiments, the protein titer obtained from cells cultured with the asparagine supplement of the present disclosure is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, or at least about 29% greater than the protein titer (yield) from similar or identical cells cultured with less asparagine supplementation or without asparagine supplementation in early and / or late fed-batch cell cultures.

[0122] In some embodiments, the protein titer (yield) of mammalian cells cultured with the asparagine supplement of the present disclosure is at least 100 mg / L, at least 0.5 g / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, and at least 2.5 g / L greater than the protein titer of similar or identical cells cultured with less asparagine supplementation or no asparagine supplementation in early and / or late fed-batch cell cultures.

[0123] The methods disclosed herein are useful for improving protein production through cell culture processes. The cell lines used in the present invention may be genetically engineered to express recombinant proteins of commercial or scientific interest. Genetically engineering a cell line involves transfecting, transforming, or transducing a host cell with a recombinant polynucleotide molecule, or otherwise modifying it (e.g., by homologous recombination and gene activation, or by fusion of recombinant cells with non-recombinant cells), to express a desired recombinant polypeptide in the host cell. Methods and vectors for genetically engineering cells or cell lines to express a polypeptide of interest are well known to those skilled in the art, and various techniques are shown, for example, in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates), Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Laboratory Press, 1989), and Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial vendors.

[0124] In some embodiments, the protein product (the protein of interest) is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific antibody, bispecific antibody, antigen-binding antibody fragment, single-chain antibody, diabody, triabody, or tetrabody, Fab fragment or F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, or a combination thereof. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0125] In some embodiments, the antibody may be an anti-programmed cell death 1 antibody (e.g., the anti-PDI antibody described in U.S. Patent Publication No. 2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., the anti-PD-L1 antibody described in U.S. Patent Publication No. 2015 / 0203580A1), an anti-DII4 antibody, an anti-angiopoietin-2 antibody (e.g., the anti-ANG2 antibody described in U.S. Patent No. 9,402,898), or an anti-angiopoietin-like 3 antibody (e.g., described in U.S. Patent No. 9,018,356). Anti-AngPtl3 antibodies, anti-platelet-derived growth factor receptor antibodies (e.g., anti-PDGFR antibody described in U.S. Patent No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibody described in U.S. Patent No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibody described in U.S. Patent Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibody described in U.S. Patent No. 9,132,192), or Anti-EGFRvIII antibody as described in U.S. Patent Application Publication 2015 / 0259423A1), anti-proprotein convertase subtilisin kexin-9 antibody (e.g., anti-PCSK9 antibody as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication 2014 / 0044730A1), anti-growth and differentiation factor-8 antibody (e.g., anti-GDF8 antibody as described in U.S. Patent No. 8,871,209 or 9,260,515, also known as anti-myostatin antibody), anti-glucagon receptor (e.g. For example, anti-GCGR antibodies described in U.S. Patent Application Publication No. 2015 / 0337045A1 or No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (for example, anti-IL4R antibodies described in U.S. Patent Application Publication No. 2014 / 0271681A1 or U.S. Patent No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (for example, U.S. Patent No. 7,582,298, No. 8,043,617 or No. 9,173,Anti-IL6R antibody (as described in Patent No. 880), anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., anti-IL33 antibody as described in U.S. Patent Application Publication No. 2014 / 0271658A1 or 2014 / 0271642A1), anti-differentiation antigen group 3 (e.g., U.S. Patent Application Publication No. 2014 / 0088295A1 and 2015 / 026696) Anti-CD3 antibody as described in U.S. Patent No. 6A1 and U.S. Patent Application No. 62 / 222,605), anti-differentiation antigen group 20 (e.g., anti-CD20 antibody as described in U.S. Patent Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1 and U.S. Patent No. 7,879,984), anti-CD19 antibody, anti-CD28 antibody, anti-differentiation antigen group 48 (e.g., anti-CD48 antibody as described in U.S. Patent No. 9,228,014), anti-Fel d1 antibody (e.g., as described in U.S. Patent No. 9,079,948), anti-influenza virus antibody, anti-respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Patent Publication No. 2014 / 0271653A1), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS-CoV antibody as described in U.S. Patent Publication No. 2015 / 0337029A1), anti-Ebola virus antibody (e.g., U.S. Patent Publication No. 2016 / (as described in Patent No. 0215040), anti-Zika virus antibodies, anti-Severe Acute Respiratory Syndrome (SARS) antibodies (e.g., anti-SARS-CoV antibodies, anti-COVID-19 antibodies (e.g., anti-SARS-CoV-2 antibodies), anti-lymphocyte activator gene 3 antibodies (e.g., anti-LAG3 antibodies or anti-CD223 antibodies), anti-nerve growth factor antibodies (e.g., U.S. Patent Application Publication No. 2016 / 0017029 and U.S. Patents No. 8,309,088 and No. 9,353),The anti-NGF antibody (as described in Patent No. 176) and the anti-activin A antibody are selected from the group comprising these. In some embodiments, the bispecific antibody is selected from the group comprising the anti-CD3 × anti-CD20 bispecific antibody (as described in U.S. Patent Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1), the anti-CD3 × anti-mucin 16 bispecific antibody (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and the anti-CD3 × anti-prostate-specific membrane antigen bispecific antibody (e.g., anti-CD3 × anti-PSMA bispecific antibody). In one embodiment, the target protein comprises any of the above combinations.

[0126] In some embodiments, the target protein is selected from the group consisting of anti-influenza virus antibodies, anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibody as described in U.S. Patent Application Publication 2014 / 0271653A1), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS-CoV antibody as described in U.S. Patent Application Publication 2015 / 0337029A1), anti-Ebola virus antibodies (e.g., as described in U.S. Patent Application Publication 2016 / 0215040), anti-Zika virus antibodies, anti-severe acute respiratory syndrome (SARS) antibodies (e.g., anti-SARS-CoV antibody), and anti-COVID-19 antibodies (e.g., anti-SARS-CoV-2 antibody). In one embodiment, the target protein includes any combination of the above.

[0127] In some embodiments, the target protein is selected from the group consisting of alirocumab, sarilumab, facinumab, nesbakumab, dupilumab, trevoglumab, evinacumab, and linucumab. In one embodiment, the target protein includes any combination of the above.

[0128] In some embodiments, the protein of interest is a recombinant protein containing an Fc moiety and another domain (e.g., an Fc-fusion protein). In some embodiments, the Fc-fusion protein is a receptor Fc-fusion protein containing one or more extracellular domains of a receptor linked to the Fc moiety. In some embodiments, the Fc moiety contains a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc-fusion protein contains two or more distinct receptor chains that bind to a single ligand or to a plurality of ligands. For example, the Fc-fusion protein is a TRAP protein, such as an IL-1 trap (e.g., lilonacept containing an IL-1RAcP ligand-binding domain fused to the II-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept containing the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patents No. 7,087,411 and 7,279,159). In other embodiments, the Fc-fusion protein is an ScFv-Fc fusion protein containing one or more antigen-binding domains, such as variable heavy chain fragments and variable light chain fragments of an antibody, linked to the Fc portion.

[0129] Again, this disclosure is not limited to any particular type of cell for recombinant protein production. Examples of cell types suitable for recombinant protein production include mammalian cells, insect cells, avian cells, bacterial cells, and yeast cells. The cells may be stem cells or recombinant cells transformed with a vector for recombinant gene expression, or cells transfected with a virus for the production of a viral product. The cells may contain a recombinant heterologous polynucleotide construct encoding the protein of interest. The construct may be an episome, or it may be an element that is physically integrated into the cell's genome. The cells may also produce the protein of interest without having the protein encoded on a heterologous polypeptide construct. In other words, the cells may naturally encode the protein of interest, such as antibody-producing B cells. The cells may also be primary cells, or primary cell lines, such as chicken embryo cells.

[0130] Examples of useful cells include CHO, COS, retinal cells, Vero, CV1, kidney cells, HeLa, HepG2, WI38, MRC5, Colo25, HB 8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. In various embodiments, the cell line is a CHO cell derivative, such as CHO-K1, CHO DUX B-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, or CHO Iec mutant.

[0131] The production phase can be carried out at any scale of culture, from shaker flasks or wave bags to 250 mL bioreactors, 1 liter bioreactors, and large industrial bioreactors. Similarly, the seed train expansion phase can be carried out at any scale of culture, from shaker flasks or wave bags to 250 mL bioreactors, 1 liter or larger bioreactors. Large-scale processes can be carried out in volumes of approximately 100 liters to 20,000 liters or more. One or more of several means may be used to control protein production, such as temperature shifts or chemical induction. The growth phase may occur at a higher temperature than the production phase. For example, the growth phase may occur at a first temperature of approximately 35°C to 38°C, and the production phase may occur at a second temperature of approximately 29°C to 37°C, optionally approximately 30°C to 36°C, or approximately 30°C to 34°C. Furthermore, chemotherapeutic agents for protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetoamide (HMBA), may be added simultaneously with, before, or after, the temperature shift. If the inducers are added after the temperature shift, they may be added 1 to 5 days after the temperature shift, such as 1 to 2 days after the temperature shift. Cell culture of producing cells may be carried out as a continuous feed culture system, such as in a chemostat (see C. Altamirano et al., 2001 above), or according to a fed-batch process (see Huang, 2010 above).

[0132] This disclosure is not limited to the specific embodiments described herein, which are intended as examples of individual aspects or embodiments of the invention. Functionally equivalent methods and components are within the scope of the invention. Various modifications of the invention beyond those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are within the scope of the invention.

[0133] All publications referenced throughout this disclosure are incorporated herein by reference in their entirety. [Examples]

[0134] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0135] Fed batch methodology As described herein, fed-batch supply development is carried out via an automated AMBR250, 24-way, parallel bioreactor system. The AMBR250 high-throughput workstation provides integrated parallel control of 24 disposable bioreactors. More specifically, the AMBR250 (24-way) is a fully integrated high-throughput system consisting of 24 bioreactors, each with a working capacity of up to 250 ml under fully automated control, allowing cell culture processes to proceed in parallel. Individual continuous control and monitoring of each bioreactor vessel is provided, including temperature, impeller speed, pH and dissolved oxygen (DO), and off-gas analysis. The system provides fully automated medium filling, inoculation, sampling, and supply. The system uses bioreactors equipped with impellers, spargers, or headspace gas, pH and DO probes, and up to four supply lines.

[0136] In implementing the supply strategy development described herein, the AMBR250 bioreactor (Sartorious Stem) is operated at a maximum working volume of 250 mL. Proprietary, known-composition base and feed media are used. Feed media and supply strategies vary by cell line. Glucose is supplied daily, and levels are controlled based on the target. The bioreactor is inoculated with cell cultures. Oxygen is maintained by control, and pure oxygen is added through a sparger at varying flow rates during the study. Similarly, pH is maintained by control via CO2 spraying for the upper pH range, while the lower pH range is uncontrolled. Inoculation cell density, dissolved oxygen, temperature, pH, and dead zone are kept constant. Air and oxygen agitation and flow rates vary by scale.

[0137] Unless otherwise specified, high-asparagine-consuming CHO cell line 1 expressing exemplary polypeptide A is used in all examples.

[0138] Extracellular amino acid measurement Extracellular levels of amino acids may be measured as described herein. In certain embodiments, such levels are measured according to the following procedure. Used cell culture medium samples were derivatized and prepared using the Waters AccQ·Tag protocol and Waters AccQ·Tag reagent kit (Waters, Milford, MA). Prior to derivatization, the samples were diluted 10-fold and spiked with a 100 mg / L sarcosine internal standard (Millipore Sigma, Burlington, MA). Separation and subsequent measurement of derivatized amino acids were performed using a UPLC equipped with an AccQ·Tag Ultra C18 column (1.7 μM, 2.1 × 10 mm), and data were collected at a wavelength of 260 nm. The mobile phase and gradient followed the Waters AccQ·Tag protocol.

[0139] Intracellular amino acid measurement As described herein, intracellular levels of amino acids may be measured. In certain embodiments, such levels are measured by the following procedure: Cells are harvested from the bioreactor, centrifuged at room temperature, and the supernatant is separated from the cell pellet. The cell pellet is washed with 1×PBS and centrifuged. The PBS is removed, and the cell pellet is cold-quenched with liquid nitrogen. The study sample is spiked with a stable, labeled internal standard, extracted, and subjected to protein precipitation with an organic solvent. After centrifugation, aliquots of the supernatant are diluted and injected into an Agilent 1290 / AB Sciex QTrap 5500LC-MS / MS system equipped with a C18 reversed-phase UHPLC column. The mass spectrometer is operated in positive mode using electrospray ionization (ESI). The peak area of ​​the parent ion of each analyte is measured against the peak area of ​​the parent ion of the corresponding internal standard in pseudo-MRM mode. Quantification is performed using weighted least-squares regression analysis produced from enhanced calibration criteria prepared immediately before each run. LC-MS / MS raw data is collected and processed using AB SCIEX software Analyst 1.6.2. Data organization is performed using Microsoft Excel for Office 365 v.16.

[0140] Asparagine sequence variant analysis As described herein, asparagine sequence variants of the polypeptide of interest may be measured. Any suitable method for measuring such sequence variants may be used, but the quantitative amount of asparagine sequence variants may be determined as follows: A rapid and sensitive LC-MS-based assay focusing on three peptides out of approximately 50 possible peptides may be used as a screening tool to identify examples of substitutions and obtain a quantitative estimate of the degree of substitution. The polypeptide of interest may be purified on a small scale using appropriate purification methodologies. The polypeptide of interest may then be reduced, denatured, and subsequently digested enzymatically. In this way, a triplicate map of the polypeptide of interest may be produced, and the protein sequence may be analyzed to determine whether any variation from the expected sequence is present. HPLC gradient-based separation may then be performed, and individual peptides may be analyzed by a Q-TOF MS system. Certain peptides may be analyzed in detail by mass spectrometry. Changes in the peptide sequence may be analyzed. As an example, a 27Da shift is observed for asparagine to serine substitution.

[0141] Example 1 - Effects of asparagine on cell culture and supply strategy development Fed batch cell culture performance and supply strategies were evaluated using exemplary CHO cell lines and various asparagine supplements. Fed batch cell culture performance and supply strategy development can be carried out using the AMBR250 bioreactor system and amino acid measurements, as described herein.

[0142] Referring to Figures 3A-3C, it was found that in certain embodiments, increasing asparagine in early fed-batch cell cultures leads to increased cell growth and does not adversely affect culture productivity when balanced with the needs of other cellular nutrients. Figure 3A shows a series of early fed-batch asparagine supplements with low, medium, and high amounts of supplementation (e.g., ranging from approximately 1.8 mM to approximately 5.4 mM arginine per supply), showing that such asparagine supplements do not prevent late fed-batch asparagine depletion. Figure 3B shows increased cell culture growth with increasing amounts of asparagine supplementation, while Figure 3C shows increased cell culture potency with increasing asparagine supplementation until a plateau is reached due to depletion of several essential amino acids.

[0143] Referring to Figures 4A-4C, it was also found that increased asparagine levels in late-fed batch cell cultures did not significantly affect cell culture productivity, despite increased byproduct formation such as ammonium. More specifically, Figure 4A shows low and high asparagine supplementation in late feeds (ranging from approximately 1.8 mM to 7.2 mM per feed), but such asparagine supplementation does not prevent late-fed batch asparagine depletion. Overall cell culture productivity is not adversely affected (Figure 4C), but higher amounts of asparagine supplementation in late-fed batch cell cultures may lead to the formation of excess byproducts (Figure 4B), suggesting that while more asparagine can be supplied to the cell culture as needed, preventing depletion remains difficult.

[0144] According to embodiments of this disclosure, an improved asparagine supplementation supply strategy (i.e., a “novel supply platform” having 3x asparagine in the initial fed batch feed and 1.5x asparagine in the later fed batch feed) was developed, which prevented depletion of essential amino acids and asparagine in the initial fed batch cell culture and resulted in an overall improvement in cell culture performance. As shown in Figures 5A–5D, initial fed batch asparagine supplementation prevented initial fed batch asparagine depletion and increased cell culture growth. Furthermore, as shown, the increase in asparagine-related cell culture growth, along with the elimination of essential amino acid depletion, improved cell culture productivity, extended the productive fed batch time, and resulted in higher titer. However, higher asparagine supplementation in the later fed batch cell culture still could not avoid asparagine depletion in the later fed batch. Referring to Figures 5E–5G, cell titer, viable cell count, and cell viability were all improved with higher asparagine supplementation in another exemplary cell line.

[0145] Referring to Figure 6A, supplementing with varying amounts of asparagine in early Fed-batch cell cultures leads to depletion at similar points in time. However, increasing the amount of asparagine supplementation increases the rate of asparagine consumption (see Figure 7), suggesting that cells are not efficiently utilizing asparagine. Referring to Figure 6B, supplementing with increased amounts of asparagine in late Fed-batch cell cultures does not prevent late Fed-batch asparagine depletion and may even lead to excessive byproduct formation, again suggesting that cells are not efficiently utilizing asparagine.

[0146] In this regard, Figure 7 shows the asparagine consumption rates across a range of asparagine supply strategies for exemplary high-consumption cell lines. As shown, the novel platform supply strategies of the present disclosure result in fed-batch cell cultures that consume less asparagine over the duration of cell culture compared to previously known platform supply strategies. Furthermore, as shown, the high-asparagine supply strategies result in higher asparagine consumption rates compared to the low-asparagine supply strategies. This data suggests that the novel platform supply strategies of the present disclosure provide the most efficient use of asparagine.

[0147] In summary, asparagine was identified as being depleted in high-consumption cell lines. Supplementation in early fed-batch cell cultures affects cell culture outcomes. Supplementation in late fed-batch cell cultures increases the production of by-products, such as ammonium. Unexpectedly, a balanced asparagine supply strategy that prevents essential amino acid depletion, overcomes asparagine depletion in early fed-batch cells, and minimizes the effects of ammonium was found to improve cell culture productivity.

[0148] Example 2 - Effects of asparagine on asparagine-related amino acids in fed-batch cell culture The interrelationships between asparagine and asparagine-related non-essential amino acids via metabolic pathways can be investigated using the AMBR250 bioreactor system and amino acid assays, as described herein.

[0149] Referring to Figure 8, when asparagine is needed, cells may synthesize supplemental asparagine by increasing the utilization of aspartic acid and glutamic acid. Higher intracellular / extracellular concentrations of aspartic acid, glutamic acid, and glutamine may indicate that cells have enough intracellular asparagine to support their cellular needs. However, decreased concentrations of aspartic acid, glutamic acid, and glutamine may indicate asparagine limitation.

[0150] Referring to Figures 9A–9D, the effects of asparagine levels in late-stage fed-batch cell culture of an exemplary high-consumption cell line 1 are shown. As shown, low asparagine affects the intracellular profile of relevant non-essential amino acids (intracellular glutamate decreases with low asparagine).

[0151] Referring to Figures 10A–10D, the effects of asparagine levels in late fed-batch cell cultures of another exemplary high-consumption cell line 2 are shown. However, as shown, low asparagine affects the intracellular profile of relevant non-essential amino acids to a lower degree than that observed in exemplary high-consumption cell line 1 (intracellular glutamate decreases with low asparagine).

[0152] Referring to Figures 11A–11D, the effects of asparagine levels in late-stage Fed batch cell cultures of exemplary low-consumption cell lines are shown. As shown, low asparagine levels do not affect the extracellular or intracellular profiles of relevant non-essential amino acids in this exemplary cell line (the intracellular profile of glutamate is not reduced with low asparagine). As shown, high levels of intracellular asparagine and glutamate are present at the end of the Fed batch cycle.

[0153] Finally, as shown in Figures 12A–12F, increased extracellular asparagine can increase asparagine-related amino acids, but also have effects on high (3x in the early stages, 1.5x later) and very high (6x in the early stages, 3x later) levels, indicating that higher levels of supplementation asparagine (Figure 12A) can lead to increased asparagine-related amino acids (Figures 12B, 12D, and 12E), but the resulting increase in undesirable cell culture byproducts (i.e., Figure 12F, ammonium) can lead to decreased cell culture performance (i.e., Figure 12C, titer).

[0154] Figures 9A–9D, 10A–10D, 11A–11D, and 12A–12F collectively show that the asparagine-related amino acid profiles and cell culture outcomes differ among cell lines for different asparagine supply levels, suggesting that asparagine supply requirements can be targeted to the specific needs of cell lines.

[0155] In summary, intracellular concentrations of asparagine-related amino acids suggest specific asparagine requirements for particular cell lines. More specifically, the effects of asparagine supplementation on the extracellular and intracellular amino acid profiles of the relevant amino acids can target the specific needs of cell lines.

[0156] Example 3 - Overcoming Asparagine Depletion Improved asparagine supply strategies and platforms optimized to overcome asparagine depletion while maintaining cell culture performance can be developed using the AMBR250 bioreactor system and amino acid measurements, as described herein.

[0157] Table 1 below shows exemplary asparagine supply strategies that were investigated.

[0158] [Table 1]

[0159] As shown in Table 1, a standard fed-batch bolus asparagine feed was compared with several asparagine supply strategies, namely continuous asparagine supplement feed (separate from bolus cell culture bulk feed), a combination of continuous bulk + asparagine feed, and a hybrid supply strategy utilizing both bolus asparagine feed and continuous asparagine supplement feed (separate from bolus cell culture bulk feed), so that twice the total amount of asparagine supplement was supplied to the cell culture.

[0160] Referring to Figures 13A–13D, it was found that bolus feeding and continuous asparagine feeding yielded comparable cell culture results (number of viable cells shown in Figure 13A, and culture productivity shown in Figure 13B) for the same total amount of asparagine supplement. Furthermore, cell culture byproduct formation (Figure 13C shows ammonium formation, and Figure 13D shows alanine formation) was relatively constant with bolus and continuous asparagine feeding. However, referring to Figures 13E–13G, continuous asparagine supplement feeding slowed the depletion of extracellular asparagine (Figure 13E), aspartic acid (Figure 13F), and glutamic acid (Figure 13G) compared to bolus asparagine supplement feeding with the same total amount of asparagine supplement. According to aspects of this disclosure, it was unexpectedly found that the same total amount of asparagine supplement, when supplied continuously, reduced the consumption rates of asparagine, aspartic acid, and glutamic acid. In this regard, the improved amino acid depletion profile with comparable cell culture performance suggests more efficient asparagine consumption by cell culture.

[0161] Referring to Figures 14A-14D, it was found that asparagine supplementation via continuous, independent asparagine feed (separate from bolus bulk feed) did not prevent extracellular asparagine depletion for the same total amount of asparagine supplement (Figure 14A), but reduced cell culture byproduct formation (Figure 14D) and regulated the consumption rate of asparagine-related metabolites (Figures 14B and 14C). All illustrated supply strategies yielded comparable cell culture outcomes.

[0162] A hybrid supply approach (continuous asparagine supplement feed combined with bolus asparagine supplement feed) was found to slow amino acid depletion compared to bolus asparagine supplementation, but to increase byproduct formation across multiple exemplary cell lines.

[0163] Referring to FIGS. 15A-15G, in the first exemplary cell line, the hybrid supply approach delays the depletion of asparagine and related metabolites (FIG. 15A shows extracellular asparagine, FIG. 15B shows extracellular aspartic acid, and FIG. 15C shows extracellular glutamic acid), and it was found that comparable cell culture performance (the number of viable cells shown in FIG. 15D, the culture productivity shown in FIG. 15E) can be obtained, but it was also found that it increases the formation of by-products (FIG. 15F shows ammonium formation, and FIG. 15G shows alanine formation).

[0164] Referring to FIGS. 16A-16E, in the second exemplary cell line, the hybrid supply approach was found to delay asparagine (FIG. 16A shows extracellular asparagine), but it led to a decrease in cell culture productivity (the number of viable cells shown in FIG. 16B, the culture productivity shown in FIG. 16C). The hybrid supply approach was also found to increase the formation of by-products (FIG. 16D shows ammonium formation, and FIG. 16E shows alanine formation).

[0165] In summary, the impact of extracellular asparagine depletion in late fed-batch was addressed by identifying a new asparagine supplement supply strategy. It was unexpectedly found that continuous asparagine supplementation is a more efficient platform for supplying asparagine. In particular, continuous asparagine supplementation as an independent separate asparagine feed reduces the consumption rate of asparagine and related metabolites.

[0166] Example 4 - Reduction and control of asparagine sequence variants in fed-batch cell culture, and use of asparagine-related amino acids as surrogate markers for asparagine sequence variants The use of asparagine supplements to reduce asparagine sequence variants and the use of asparagine-related amino acids as surrogate markers for asparagine sequence variants in the polypeptide of interest can be investigated using the AMBR250 bioreactor system and amino acid measurements as described herein.

[0167] Referring back to FIGS. 10A - 10D showing the effect of asparagine levels in the late fed - batch cell culture of exemplary high - consuming cell line 2, asparagine sequence variants were analyzed by mass spectrometry on the final day of the fed - batch cell culture. In the low - asparagine - supplemented cell culture, 0.30% of asparagine sequence variants were found to be present, and in the high - asparagine - supplemented cell culture, 0.24% of asparagine sequence variants were found to be present. These SV values are relatively low and are consistent with the higher levels of intracellular glutamate observed in cell culture, as shown in FIG. 10D.

[0168] Similarly, referring to FIGS. 11A - 11D showing the effect of asparagine levels in the late fed - batch cell culture of an exemplary low - consuming cell line, asparagine sequence variants were analyzed by mass spectrometry on the final day of the fed - batch cell culture. In the low - asparagine - supplemented cell culture, 0.11% of asparagine sequence variants were found to be present, and in the high - asparagine - supplemented cell culture, 0.04% of asparagine sequence variants were found to be present. Again, these SV values are relatively low and are consistent with the higher levels of intracellular glutamate observed in cell culture, as shown in FIG. 11D.

[0169] Referring to FIGS. 17A - 17B, it has been found that extracellular asparagine depletion in the initial stage of the fed - batch can be an indicator of the formation of Asn→Ser asparagine sequence variants. As shown in FIG. 17A, a high - asparagine supply strategy (triple in the initial stage at 10.8 mM and 1.5 - fold in the late stage) that maintains extracellular asparagine levels above the depletion limit of 0.1 mM (15 mg / L) until at least the 4th day of the initial stage of cell culture (e.g., up to Feed 2) can reduce the formation of asparagine sequence variants to a level of less than about 0.20% SV (FIG. 17B).

[0170] Referring to Figure 18A, it has been found that lower asparagine levels in the later feed increase the Asn→Ser, asparagine sequence variant, particularly after the peak viable cell concentration. In the experiment shown in Figure 18A, the asparagine levels in the initial feed were the same. Figure 18B shows intracellular glutamate (Glu) levels for the high asparagine later feeding strategy compared to the low asparagine later feeding strategy. Furthermore, it was unexpectedly found that the addition of Gln to the cell culture medium further reduced the formation of asparagine sequence variants (Figure 18C).

[0171] As shown, intracellular glutamate concentration is inversely correlated with the presence of asparagine sequence variants. Higher intracellular glutamate levels are observed in cell cultures with a low incidence of asparagine sequence variants, while lower intracellular glutamate levels are observed in cell cultures with a high incidence of asparagine sequence variants. Thus, intracellular glutamate can be used as a surrogate marker for the amount of asparagine sequence variants in the polypeptide of interest.

[0172] Figures 19A–19G show the correlations between asparagine, asparagine-related amino acids, and asparagine sequence variants for exemplary cell line 1. Figures 19B–19D show extracellular asparagine (Figure 19B), aspartic acid (Figure 19C), and glutamic acid (Figure 19D), while Figures 19E–19G show exemplary intracellular asparagine (Figure 19E), aspartic acid (Figure 19F), and glutamic acid (Figure 19G). Figure 19G shows that late-stage fed-batch intracellular glutamic acid (Glu) can serve as a surrogate marker for asparagine sequence variants for polypeptides produced using the high and low asparagine supply strategies in Figure 19A. As shown, a high asparagine supplement feed results in high late-stage fed-batch intracellular glutamic acid and low asparagine sequence variants. A low asparagine supplement feed results in low late-stage intracellular glutamic acid and higher asparagine sequence variants. Surrogate intracellular glutamate measurement is consistent with asparagine sequence variant analysis by mass spectrometry. However, there is no clear trend regarding extracellular or intracellular asparagine or aspartate, or extracellular glutamate, for this particular cell line.

[0173] Figures 20A–20D show the correlations between asparagine, asparagine-related amino acids, and asparagine sequence variants for another exemplary cell line (high-consumption cell line). Figures 20B–20D show extracellular asparagine (Figure 20B), intracellular aspartic acid (Figure 20C), and intracellular glutamic acid (Figure 20D). Figures 20C and 20D show that both late-stage fed-batch intracellular aspartic acid and intracellular glutamic acid can serve as surrogate markers for asparagine sequence variants for polypeptides produced using the high and low asparagine supply strategies in Figure 20A. As shown, a high asparagine supplement feed results in high late-stage fed-batch intracellular aspartic acid, high late-stage fed-batch intracellular glutamic acid, and low asparagine sequence variants. A low asparagine supplement feed results in low late-stage fed-batch intracellular aspartic acid, low late-stage fed-batch intracellular glutamic acid, and higher asparagine sequence variants. Measurements of surrogate intracellular aspartate and intracellular glutamate are consistent with asparagine sequence variant analysis by mass spectrometry.

[0174] Figures 21A–21F show that the trend of asparagine sequence variants in exemplary cell line 1 coincides with the trend of intracellular glutamate, indicating that intracellular asparagine-related amino acids can be used as surrogates for asparagine sequence variants. Figures 21A and 21C illustrate a high asparagine initial supply strategy (Figure 21A, amount of sequence variant determined by mass spectrometry; Figure 21C, intracellular glutamate concentration). Figures 21B and 21D illustrate a low asparagine initial supply strategy (Figure 21B, amount of sequence variant determined by mass spectrometry; Figure 21D, intracellular glutamate concentration). Figure 21E shows intracellular glutamate levels in exemplary cell line 4 that correlate with higher and lower incidences of asparagine sequence variants, and Figure 21F shows a similar intracellular glutamate profile for another exemplary cell line 5.

[0175] Figures 22A–22H show the correlations between asparagine, asparagine-related amino acids, and asparagine sequence variants for exemplary cell lines. Figures 22A–22D show extracellular asparagine (Figure 22A), extracellular aspartic acid (Figure 22B), extracellular glutamic acid (Figure 22C), and extracellular glutamine (Figure 22D) for exemplary cell line 1, relating to high and low asparagine supply strategies. As shown, no clear trends were observed for the formation of extracellular asparagine, aspartic acid, or glutamic acid, and asparagine sequence variants. However, a correlation exists between late-stage fed-batch extracellular glutamine and the formation of asparagine sequence variants (particularly considering the replenishment of asparagine beyond the depletion limit in early-stage fed-batch cell cultures). Similar trends for exemplary cell line 4 are shown in Figures 22E–22H, illustrating extracellular asparagine (Figure 22E), extracellular aspartic acid (Figure 22F), extracellular glutamic acid (Figure 22G), and extracellular glutamine (Figure 22H) in relation to high and low asparagine supply strategies.

[0176] Figures 23A–23C further demonstrate that extracellular glutamine can act as a surrogate for asparagine sequence variants in late-stage fed-batch cell cultures. Figure 23A shows that sufficient glutamine can be produced via a high-asparagine supply strategy in exemplary cell line 1 (e.g., exceeding the glutamine depletion limit). Similarly, Figure 23B shows that sufficient glutamine can be produced via a high-asparagine supply strategy in exemplary cell line 1, correlating with embodiments where asparagine sequence variants were not detected by mass spectrometry. Finally, Figure 23C shows that when asparagine is not supplemented on days 6 and 8, asparagine sequence variants are detected and extracellular glutamine falls below the depletion limit (i.e., insufficient glutamine is produced via the asparagine supply strategy). However, when sufficient glutamine is produced via a high-asparagine supply strategy, extracellular glutamine correlates with asparagine sequence variant formation.

[0177] All publications and patent applications cited herein are incorporated herein by reference in the same manner as each publication or patent application is specifically and individually incorporated by reference.

[0178] While the present invention has been described with reference to exemplary embodiments, those skilled in the art will understand that various modifications may be made without departing from the scope of the invention, and that equivalents may be substituted for its elements. In addition, many modifications may be made to adapt the teaching to specific situations or materials without departing from its essential scope. Thus, the present invention is not limited to the specific embodiments disclosed as the best mode intended for carrying out the invention, and the invention is intended to include all embodiments that fall within the scope of the appended claims.

Claims

1. A method for culturing mammalian cells for improved cell culture results, wherein the method is A step of growing or maintaining mammalian cells in a cell culture medium, wherein 3.6 mM to 43.2 mM of asparagine is added to the cell culture medium during the initial Fed-batch cell culture, and 2.6 mM to 21.6 mM of asparagine is added during the later Fed-batch cell culture, the amount of asparagine added during the initial Fed-batch cell culture is greater than the amount of asparagine added during the later Fed-batch cell culture, the addition begins after the first day of the initial Fed-batch cell culture, and the addition is carried out at two-day intervals; The steps include maintaining the cells in the asparagine-supplemented cell culture medium for at least a portion of the initial and late fed batch cell cultures. Includes, The method wherein, compared to supplementing with asparagine at a dose of less than 3.6 mM in early and / or late-stage fed-batch cell culture, or a similar method without asparagine supplementation, at least one cell culture performance parameter is improved by the asparagine supplementation.

2. The method according to claim 1, wherein the asparagine supplement is provided as part of a bulk feed or as a separate asparagine supplement feed in early and / or late-stage fed-batch cell cultures.

3. The method according to claim 1 or 2, wherein 7.2 mM to 21.6 mM of asparagine is added to the cell culture medium during the initial fed-batch cell culture, and 3.6 mM to 10.8 mM of asparagine is added during the later fed-batch cell culture.

4. The method according to any one of claims 1 to 3, further comprising the step of expressing a target recombinant protein from the mammalian cells during the fed batch cell culture.

5. The method according to claim 4, wherein the at least one cell culture performance parameter is selected from the group consisting of increased cell viability, increased cell growth rate, increased cell density, increased titer of the recombinant protein of the objective, increased yield of the recombinant protein of the objective, reduced depletion of essential amino acids in at least a portion of the cell culture, reduced formation of at least one cell culture byproduct in at least a portion of the cell culture, and improvement of at least one protein quality index.

6. The method according to claim 5, wherein the at least one cell culture byproduct is selected from the group consisting of ammonium ions and alanine.

7. The method according to claim 5, wherein the at least one protein quality indicator is a reduction in protein sequence variants.

8. The method according to claim 5, wherein the titer of the recombinant protein of the objective is at least 3%, 5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or at least 20% higher than the titer of cells cultured with less asparagine supplementation in early and / or late-stage fed-batch cell culture.

9. The method according to claim 5, wherein the yield of the recombinant protein of the objective is increased by at least 0.1 g / L, at least 0.5 g / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, at least 2.2 g / L, at least 2.4 g / L, or at least 2.5 g / L compared to a similar method in which cells are cultured with less asparagine supplementation in early and / or late-stage fed-batch cell culture.

10. The method according to claim 5, wherein the cell culture maintains a viable cell count of at least 10 to 50 M cells / mL during at least a portion of the late-stage fed-batch cell culture.

11. The method according to any one of claims 1 to 10, wherein the cells are maintained under asparagine-supplemented cell culture conditions for at least two days, at least three days, at least four days, at least five days, or for the duration of the initial and / or late fed-batch cell culture.

12. The method according to any one of claims 1 to 11, wherein the asparagine supplementation is provided at least once, at least twice, at least three times, at least four times, or at least five times for at least a portion of the initial fed batch cell culture.

13. The method according to any one of claims 1 to 11, wherein the asparagine supplementation is provided at least once, at least twice, at least three times, at least four times, or at least five times for at least a portion of the late-stage fed batch cell culture.

14. The method according to any one of claims 1 to 13, wherein the extracellular amino acid depletion of asparagine, aspartic acid, and glutamic acid in the cell culture is delayed by at least one day, at least two days, at least three days, or at least four days or more compared to a similar method in which asparagine is supplemented in an amount of less than 3.6 mM in the early and / or late fed-batch cell culture, or in which asparagine is not supplemented.

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

16. The method according to any one of claims 1 to 15, wherein the mammalian cells are a high-asparagine-consuming cell line such that the mammalian cells consume 1.8 mM / day to 9.3 mM / day of asparagine.

17. The method according to any one of claims 1 to 15, wherein the mammalian cells are a low-asparagine-consuming cell line such that the mammalian cells consume 0.32 mM / day to 1.8 mM / day of asparagine.

18. The method according to any one of claims 1 to 17, wherein the mammalian cells are a high-producing cell line so that the recombinant protein of the target is produced in a yield of at least 4 g / L, at least 5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L, at least 10 g / L, at least 11 g / L, at least 12 g / L, at least 13 g / L, or at least 14 g / L.

19. The method according to claim 4, wherein the recombinant protein of the objective is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody or tetrabody, a Fab fragment or F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

20. The method according to claim 4, wherein the recombinant protein for the purpose comprises an Fc domain.

21. The method according to claim 4, wherein the recombinant protein of the objective is selected from the group consisting of Fc-fusion proteins, receptor-Fc-fusion proteins (TRAP), antibodies, antibody fragments, and ScFv-Fc fusion proteins.

22. The recombinant protein of interest is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti- TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody , anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD 5. The method of claim 4, wherein the method is selected from the group consisting of: 48 antibody, anti-CD3 / CD20 bispecific antibody, anti-CD3 / anti-MUC16 bispecific antibody, and anti-CD3 / anti-PSMA bispecific antibody.

23. The method according to claim 4, wherein the recombinant protein for the purpose is selected from the group consisting of anti-influenza virus antibody, anti-respiratory syncytial virus (RSV) antibody, anti-Middle East Respiratory Syndrome (MERS) virus, anti-Ebola virus antibody, anti-Zika virus antibody, anti-Severe Acute Respiratory Syndrome (SARS) antibody, and anti-COVID-19 antibody.

24. The method according to claim 4, wherein the recombinant protein of the objective is selected from the group consisting of alirocumab, sarilumab, facinumab, nesbakumab, dupilumab, trevoglumab, evinakumab, linukumab, casirivimab, and imudevimab.

25. A method for preventing asparagine sequence variants in target polypeptides expressed from mammalian cells in cell culture, wherein the method is A stage of growing or maintaining mammalian cells in a cell culture medium, wherein 3.6 mM to 43.2 mM of asparagine is added to the cell culture medium during the initial Fed-batch cell culture, and 2.6 mM to 21.6 mM of asparagine is added during the later Fed-batch cell culture, the amount of asparagine added during the initial Fed-batch cell culture is greater than the amount of asparagine added during the later Fed-batch cell culture, the addition begins after the first day of the initial Fed-batch cell culture, and the addition is carried out at two-day intervals; The steps include maintaining the cells in the asparagine-supplemented cell culture medium under conditions sufficient for the expression of the target polypeptide for at least a portion of the initial and late fed-batch cell cultures, and Includes, The method, wherein the extracellular asparagine level is maintained above a 0.1 mM depletion limit in the cell culture medium for at least the initial fed batch of cell culture, such that the target polypeptide expressed by the mammalian cells contains less than 0.30% asparagine sequence variants at all individual sequence variant loci.

26. The method according to claim 25, wherein the target polypeptide expressed by the mammalian cells contains less than 0.30% asparagine sequence variants at all individual sequence variant loci after 12 days of cell culture.

27. ​​The method according to claim 25 or 26, wherein the asparagine supplement is provided as part of a bulk feed or as a separate asparagine supplement feed in early and / or late-stage fed-batch cell cultures.

28. The method according to any one of claims 25 to 27, wherein 7.2 mM to 21.6 mM of asparagine is added to the cell culture medium during the initial fed-batch cell culture, and 3.6 mM to 10.8 mM of asparagine is added during the later fed-batch cell culture.

29. The method according to any one of claims 25 to 28, wherein the cells are maintained under asparagine-supplemented cell culture conditions for at least two days, at least three days, at least four days, at least five days, or for the duration of the initial and / or late fed-batch cell culture.

30. The method according to any one of claims 25 to 29, wherein the asparagine supplementation is provided at least once, at least twice, at least three times, at least four times, or at least five times for at least a portion of the initial fed batch cell culture.

31. The method according to any one of claims 25 to 29, wherein the asparagine supplementation is provided at least once, at least twice, at least three times, at least four times, or at least five times for at least a portion of the late-stage fed-batch cell culture.

32. The method according to any one of claims 25 to 31, wherein the asparagine sequence variant is the misincorporation of serine instead of asparagine during translation of the target polypeptide.

33. A method for producing a target recombinant protein, wherein the method is A step of growing or maintaining CHO cells in a synthetic cell culture medium, wherein the CHO cells express the target recombinant protein, and asparagine is added to the cell culture medium in amounts of 3.6 mM to 43.2 mM during the initial Fed-batch cell culture and 2.6 mM to 21.6 mM during the later Fed-batch cell culture, wherein the amount of asparagine added during the initial Fed-batch cell culture is greater than the amount of asparagine added during the later Fed-batch cell culture, and the addition begins after the first day of the initial Fed-batch cell culture and is carried out at 2-day intervals; To produce the aforementioned recombinant protein, the cells are maintained in the asparagine-supplemented cell culture medium for at least a portion of the initial and late fed-batch cell cultures. Includes, The method wherein the recombinant protein of interest has reduced sequence variants compared to the recombinant protein of interest produced by a method using less or no asparagine supplementation in early and / or late-stage fed-batch cell culture.

34. The method according to claim 33, wherein the asparagine supplement is provided as part of a bulk feed or as a separate asparagine supplement feed in early and / or late-stage fed-batch cell cultures.

35. The method according to claim 33 or 34, wherein 7.2 mM to 21.6 mM of asparagine is added to the synthetic cell culture medium during the initial fed-batch cell culture, and 3.6 mM to 10.8 mM of asparagine is added during the later fed-batch cell culture.

36. The method according to any one of claims 33 to 35, wherein the recombinant protein of the objective is an Fc-fusion protein, a receptor-Fc-fusion protein, an antibody, an antibody fragment, or an ScFv-Fc fusion protein.

37. The method according to any one of claims 33 to 36, wherein, compared to a method in which less or no asparagine supplementation is used in early and / or late-stage fed-batch cell cultures, at least one additional cell culture performance parameter is improved by the asparagine supplementation, the at least one additional cell culture performance parameter being selected from the group consisting of increased cell viability, increased cell growth rate, increased cell density, increased titer of the recombinant protein of interest, increased yield of the recombinant protein of interest, reduced depletion of essential amino acids in at least a portion of the cell culture, reduced formation of at least one cell culture byproduct in at least a portion of the cell culture, and improvement of at least one protein quality index.

38. The method according to claim 37, wherein the at least one cell culture byproduct is selected from the group consisting of ammonium ions and alanine.

39. The method according to any one of claims 33 to 38, wherein the extracellular amino acid depletion of asparagine, aspartic acid, and glutamic acid in the cell culture is delayed by at least one day, at least two days, at least three days, or at least four days or more compared to a similar method in which asparagine supplementation is provided as a bolus feed in early and / or late fed-batch cell cultures.

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