Materials and methods for enhanced biological production processes

Abstract

Provided herein are methods for reducing lactate spikes and increasing titer in a fed-batch process for producing a protein of interest, the method comprising reducing pyruvate (e.g., sodium pyruvate) concentration in one or more feeds to cells in a bioreactor in the fed-batch process.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Patent Application No. 63 / 352,969, filed June 16, 2022, and U.S. Patent Application No. 63 / 305,226, filed January 31, 2022, the disclosures of each of which are incorporated by reference in their entireties herein. Summary of the Invention

[0002] In one aspect, provided herein is a method for reducing lactate spikes in a fed-batch process for producing a protein of interest, the method comprising reducing a pyruvate concentration in one or more feeds to cells comprising a nucleic acid encoding the protein in a bioreactor in the fed-batch process. In another aspect, provided herein is a method for increasing the titer of a protein of interest produced by cells in a fed-batch process, the method comprising reducing a pyruvate concentration in one or more feeds to cells in a bioreactor in the fed-batch process. In some embodiments, the method further comprises increasing a concentration of one or more amino acids in the one or more feeds. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. In some embodiments, the one or more amino acids are glutamic acid, valine, or a combination thereof. In some embodiments, the amino acid concentration is increased by about 0% to about 100%. In some embodiments, the pyruvate concentration is reduced by about 65% to about 100%. In some embodiments, the cells are CHO cells. In certain embodiments, the protein of interest is a recombinant protein. In some embodiments, the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. In some embodiments, the protein of interest is an antibody, a cytokine, or an antigen.In some embodiments, the antibody is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1, In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, a bacteria, a fungus, or a parasite. In some embodiments, the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17.

[0003] In another aspect, provided herein is a method of producing a protein of interest, comprising: (a) feeding culture cells comprising a nucleic acid encoding the protein in a bioreactor under conditions sufficient for the cells to produce the protein, the feeding culture comprising adding a volume of one or more feeds (e.g., one or more composite feeds) having a pyruvate concentration 65% to 100% lower than the pyruvate concentration used in feeding culture of the cells under the same conditions where a lactate spike is observed; and (b) purifying the protein from the cells or liquid culture medium. In some embodiments, the concentration of one or more amino acids in the one or more feeds is increased by about 0% to about 100% compared to feeding culture of the cells under the same conditions where a lactate spike is observed. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. In some embodiments, the one or more amino acids are glutamic acid, valine, or a combination thereof. In some embodiments, the cell is a CHO cell. In certain embodiments, the protein of interest is a recombinant protein. In some embodiments, the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. In some embodiments, the protein of interest is an antibody, a cytokine, or an antigen.In some embodiments, the antibody is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1, In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, a bacteria, a fungus, or a parasite. In some embodiments, the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17.

[0004] In another aspect, provided herein is a method for providing a digital computer simulation of a fed-batch process for producing a protein of interest, the method being performed by one or more computing devices and including receiving data characterizing the fed-batch process, initializing a model of a mammalian cell line, simulating the fed-batch process using the initialized model and the received data to characterize lactate spikes, identifying at least one factor that contributes to the lactate spikes based on the simulation, and providing data characterizing the identified at least one factor. In some embodiments, the provided data characterizes one or more of the product composition, initial conditions, and nutrient additions of the fed-batch process. In some embodiments, the model is a metabolic model. In some embodiments, the model includes one or more machine learning models. In some embodiments, the one or more machine learning models include a neural network. In some embodiments, the method further includes training the neural network using data extracted from representative processes that do not exhibit lactate spikes and representative processes that exhibit lactate spikes. In some embodiments, the extracted data characterizes one or more of the product composition, initial conditions, and nutrient additions of the fed-batch process. In some embodiments, providing the data comprises one or more of displaying the identified at least one factor in a graphical user interface, physically persistently storing the identified at least one factor, loading the identified at least one factor into a memory, or transmitting the identified at least one factor over a network to a remote computing system. In some embodiments, the identified at least one factor indicates a need to reduce pyruvate concentration in one or more feeds to cells in a bioreactor in a fed-batch process to reduce lactate spikes.In some embodiments, the at least one identified factor indicates that the concentration of one or more amino acids in one or more feeds to cells in a bioreactor in a fed-batch process needs to be increased to reduce lactate spikes.

[0005] In another aspect, provided herein is a method for producing a protein of interest as part of a fed-batch process, the method comprising: receiving data characterizing the fed-batch process; initializing a model of a mammalian cell line; simulating the fed-batch process using the initialized model and the received data to characterize a lactate spike; identifying at least one factor that contributes to the lactate spike based on the simulation; and modifying one or more operating parameters of the fed-batch process based on the identified at least one factor. In some embodiments, the modification comprises decreasing a pyruvate concentration in one or more feeds to cells in a bioreactor in the fed-batch process. In some embodiments, the modification comprises increasing one or more amino acids in one or more feeds to cells in a bioreactor in the fed-batch process.

[0006] In another aspect, provided herein is a method for reducing lactate spikes during a fed-batch culture of cells, the method comprising feeding-batch culturing cells comprising a nucleic acid encoding a protein of interest in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culturing comprising adding a volume of a first feed (e.g., a first composite feed) within 0-6 days after initiation of fed-batch culturing of the cells, the first feed having a first pyruvate concentration, the first pyruvate concentration being about 65% to about 100% lower than a pyruvate concentration used in the same volume of the first feed in a second fed-batch culture of the cells under the same conditions in which lactate spikes are observed. In some embodiments, the volume of the first feed is within 0-3 days. In some embodiments, the volume of the first feed is within 3-5 days. In some embodiments, the volume of the first feed is within 3-6 days. In some embodiments, the volume of the first feed is within 4-6 days. In some embodiments, the volume of the first feed is within 5-6 days. In some embodiments, the concentration of one or more amino acids is increased. In some embodiments, the increase in the concentration of the one or more amino acids is from about 0% to about 100%. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. In some embodiments, the one or more amino acids are glutamic acid, valine, or a combination thereof. In some embodiments, the cell is a CHO cell. In certain embodiments, the protein of interest is a recombinant protein. In some embodiments, the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. In some embodiments, the protein of interest is an antibody, a cytokine, or an antigen.In some embodiments, the antibody is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1, In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, a bacteria, a fungus, or a parasite. In some embodiments, the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17.

[0007] In another aspect, a method for identifying a pyruvate concentration for use in one or more feeds in a fed-batch process includes: (a) feeding-batch culturing cells comprising a nucleic acid encoding a protein of interest in a first bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culturing comprising adding a volume of one or more feeds (e.g., one or more composite feeds) having a first pyruvate concentration; and (b) feeding-batch culturing the same cells in a second bioreactor under the same conditions used in the fed-batch culturing of step (a), except that the one or more feeds have a second pyruvate concentration, the second pyruvate concentration being about 65% to about 100% (e.g., about 65% to about 75%, about 75% to about 85%, about 85% to about 95%, about 80% to about 95%, or about 85% to about 100%) lower than the first pyruvate concentration. (c) measuring the lactate concentration in the fed-batch culture of step (a) within about 12 to about 72 hours (e.g., about 12 hours, about 18 hours, about 24 hours, about 48 hours, or about 72 hours) after each feeding and measuring the lactate concentration in the fed-batch culture of step (b) within about 12 to about 72 hours (e.g., about 12 hours, about 18 hours, about 24 hours, about 48 hours, or about 72 hours) after each feeding; and (d) comparing the lactate concentration measured for the fed-batch culture of step (a) with the lactate concentration measured for the fed-batch culture of step (b), wherein a decrease in the lactate concentration for the fed-batch culture of step (b) compared to the lactate concentration for the fed-batch culture of step (a) indicates that the pyruvate concentration used in the one or more feeds in the fed-batch culture of step (b) is more favorable for fed-batch cultivation of cells in a bioreactor. In some embodiments, the method further comprises (e) performing production of the protein by fed-batch culturing the cells comprising the nucleic acid under conditions sufficient for the cells to produce the recombinant protein, the fed-batch culturing comprising adding a volume of one or more feeds having a second pyruvate concentration. In some embodiments, the cells are CHO cells. In certain embodiments, the protein of interest is a recombinant protein.In some embodiments, the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. In some embodiments, the protein of interest is an antibody, a cytokine, or an antigen. In some embodiments, the antibody is an antigen selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCA), prostate-associated antigen (PSA ... In some embodiments, the antibody binds to an antigen of a pathogen. In some embodiments, the pathogen is a virus, a bacterium, a fungus, or a parasite. In some embodiments, the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17. For further information and embodiments regarding proteins of interest, see Section 4.1.1.

[0008] In another aspect, provided herein is a method for reducing lactate spikes during fed-batch culture of cells, the method comprising feeding cells comprising a nucleic acid encoding a protein of interest in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culture comprising adding one or more feeds (e.g., one or more complex feeds) during growth of the cells and decreasing a pyruvate concentration in the one or more additional feeds (e.g., one or more complex feeds) prior to a cell density peak, the pyruvate concentration in the one or more additional feeds being decreased by 65% ​​to 100% compared to the pyruvate concentration in the one or more feeds during growth of the cells. In some embodiments, the cell density peak is about day 6 to about day 7 of the fed-batch culture of the cells, and the decrease in the pyruvate concentration in the one or more additional feeds is about day 3 to 5 (e.g., about day 3, about day 4, or about day 5) of the fed-batch culture of the cells. In some embodiments, the concentration of one or more amino acids in the one or more additional feeds is increased prior to a cell density peak. In some embodiments, the concentration of one or more amino acids in the one or more additional feeds is increased by 0% to 100% compared to the concentration of the one or more amino acids in the one or more feeds during cell growth and prior to the cell density peak. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. In some embodiments, the one or more amino acids are glutamic acid, valine, or a combination thereof. In some embodiments, the cells are CHO cells. [Brief description of the drawings]

[0009] [Figure 1] Bioreactors for a fed-batch process to produce antibody A show lactate spikes, increased glucose consumption during the lactate spikes, decreased glucose consumption after the lactate spikes, and increased osmolality with base addition. No differences in viable cell density, viability, and glutamine concentration were observed. [Diagram 2] Bioreactors for fed-batch processes to produce antibody B and antibody C show lactate spikes. No differences in viable cell density were observed. [Diagram 3] Lactate production and glucose consumption per cell in bioreactors for the fed-batch process to produce antibody A, antibody B, and antibody C. [Figure 4A] Viable cell density (Figure 4A), titer (Figure 4B), and lactate (Figure 4C) of cell culture processes operated under comparable conditions showing very rapid lactate accumulation (solid line), moderately rapid lactate accumulation (dashed line), or no rapid lactate accumulation (dashed line) with no obvious growth or titer differences. [Figure 4B] Viable cell density (Figure 4A), titer (Figure 4B), and lactate (Figure 4C) of cell culture processes operated under comparable conditions showing very rapid lactate accumulation (solid line), moderately rapid lactate accumulation (dashed line), or no rapid lactate accumulation (dashed line) with no obvious growth or titer differences. [Figure 4C] Viable cell density (Figure 4A), titer (Figure 4B), and lactate (Figure 4C) of cell culture processes operated under comparable conditions showing very rapid lactate accumulation (solid line), moderately rapid lactate accumulation (dashed line), or no rapid lactate accumulation (dashed line) with no obvious growth or titer differences. [Figure 5A] Schematic of the digital twin (i.e., model). Figure 5A) The digital twin contains three separate models that together describe the dynamics of the cell culture process. Figure 5B) The individual models of the DT are mapped to physical systems where cells (circles), extracellular metabolites (squares), and volumetric manipulations (arrows) correspond to the respective models in Figure 5A (i.e., the dynamic cell model, the extracellular reaction model, and the reactor model, respectively). [Figure 5B]Schematic of the digital twin (i.e., model). Figure 5A) The digital twin contains three separate models that together describe the dynamics of the cell culture process. Figure 5B) The individual models of the DT are mapped to physical systems where cells (circles), extracellular metabolites (squares), and volumetric manipulations (arrows) correspond to the respective models in Figure 5A (i.e., the dynamic cell model, the extracellular reaction model, and the reactor model, respectively). [Figure 6] Two representative processes exhibiting "no lactate spikes" (M19L059) or "lactate spikes" (M19L062) were used to train the metabolic model. The updated model parameters resulted in an accurate fit of the model (solid line) to the process data (dots) for both phenotypes. [Figure 7] Sensitivity analysis of feed medium components on lactate formation. Positive correlation (outlined with dashed line) = increasing concentration increases lactate formation. Negative correlation (outlined with solid line) = decreasing concentration decreases lactate formation. [Figure 8] Diagram of the TCA cycle entry points for medium components most likely to affect lactate formation. Components from the sensitivity analysis are underlined. Dashed lines indicate multiple reactions before the entry point. [Figure 9A] Experimental validation results of model-identified levers. Figure 9A) Lactate concentrations compared to the previous lactate spiking process (dashed-dotted line). Conditions containing lower pyruvate (thin solid line) consistently resulted in lower lactate concentrations. Figure 9B) Normalized titer results. Each condition (n > 2) was normalized to the average titer of the lactate spiking process shown in Figure 4C. Error bars represent standard deviation. [Figure 9B] Experimental validation results of model-identified levers. Figure 9A) Lactate concentrations compared to the previous lactate spiking process (dashed-dotted line). Conditions containing lower pyruvate (thin solid line) consistently resulted in lower lactate concentrations. Figure 9B) Normalized titer results. Each condition (n > 2) was normalized to the average titer of the lactate spiking process shown in Figure 4C. Error bars represent standard deviation. [Figure 10A]The digital twin calculated the flux distribution in µmoles / cell / day at 120 h (time of lactate spike). Figure 10A) Glycolytic flux for a high lactate process where 55% of glucose is converted to lactate. Figure 10B) Low pyruvate glycolytic flux where 22% of glucose is converted to lactate and most of the carbon enters the TCA cycle. [Figure 10B] The digital twin calculated the flux distribution in µmoles / cell / day at 120 h (time of lactate spike). Figure 10A) Glycolytic flux for a high lactate process where 55% of glucose is converted to lactate. Figure 10B) Low pyruvate glycolytic flux where 22% of glucose is converted to lactate and most of the carbon enters the TCA cycle. [Figure 11] Proposed mechanisms of lactate spikes and their alleviation: 1) High concentrations of extracellular pyruvate activate glucose uptake. 2) High lactate concentrations allosterically inhibit PFK, thus reducing glycolytic flux. [Figure 12] Experimental conditions for validation of sensitivity analysis. As mentioned above, the experimental conditions refer to those described in FIG. 9B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 4.1 Fed-batch processing The present disclosure relates, in part, to methods, systems, and compositions for reducing lactate accumulation during a fed-batch process for producing a protein using a genome-based metabolic model. For example, the present disclosure addresses the phenomenon of rapid lactate accumulation in the middle of a fed-batch process for the production of a protein of interest (referred to herein as "lactate spike"). In particular, it has been observed that during fed-batch production of a protein of interest, cells sporadically produce 2-3 g / L of lactate over a 24-hour period near the cell density peak, followed by excess lactate consumption over the remainder of the fed-batch process. When this phenomenon occurs, reduced glucose metabolism, reduced product expression, and reduced product quality have been observed. In particular, the following have been observed with respect to the lactate spike phenomenon: (1) increased glucose consumption during the lactate spike, followed by reduced glucose consumption; (2) reduced oxygen consumption; and (3) increased culture osmolality with base addition. The inventors have surprisingly found that low sodium pyruvate in the composite feed eliminates lactate spikes while improving, for example, the titer of the protein of interest.

[0011] In certain embodiments, the methods described herein for fed-batch processes for the production of a protein of interest are environmentally friendly since they can improve the titer of the protein produced and thus reduce the number of processes that need to be performed to achieve the amount of desired protein.

[0012] As used herein, the terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0013] Fed-batch processes are commonly known in the art and are used to optimize protein production (see, e.g., YM Huang et al., Biotechnol Prog. 2010 Sep-Oct;26(5):1400-10). As used herein, the terms "fed-batch process", "fed-batch culture" and similar terms are used to refer to techniques for culturing cells (e.g., mammalian cell lines) in a bioreactor. In general, a fed-batch process is a biotechnology process in which one or more nutrients are discretely or continuously fed to a bioreactor during the culture of cells (e.g., mammalian cell lines) and the protein of interest remains in the bioreactor until the end of the run. A fed-batch culture is usually terminated at some point and the cells and / or components in the medium are harvested and optionally purified.

[0014] A fed-batch process typically includes a growth phase and a production phase. Typically, the growth phase (also known as seed train or seed culture) is the stage where all components for cell culture are fed into the bioreactor at the beginning of the culture process and the cell mass is then grown to production scale. Thus, the bioreactor is inoculated with cells at an inoculation density appropriate for the initial cell growth phase, depending on the starting cells (e.g., cell line). Typically, the production phase is the stage where the protein is produced and recovered. For information and embodiments regarding purification, see section 4.1.5.

[0015] In fed-batch processes, the medium is typically replenished at intervals during the cell culture. These feeds (e.g., complex feeds) are typically used during the production phase. The feeds may be spaced apart, with a frequency of multiple times per day, daily, or every 2-3 days, during the duration of the production culture. In some embodiments, the feeds may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 8 times throughout the duration of the production culture of at least 1 week and up to 2 weeks or more of culture. For example, the feeds may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 8 times throughout the duration of the production culture of 1-2 weeks (e.g., 1 week or 2 weeks). In another example, the feeds may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 8 times throughout the duration of the production culture of 2-4 weeks (e.g., 2 weeks, 3 weeks, or 4 weeks). In some embodiments, supplemental feeding may occur daily during the culture period. Alternative culture feeding schedules are also contemplated. The feeds generally contain nutrients that are depleted during cell culture. In some embodiments, the first feed is on days 0-3.

[0016] As used herein, the term "nutrient" can refer to any compound, molecule, or substance that an organism uses to survive, grow, produce a protein of interest, or otherwise add to biomass. Examples of nutrients can include carbohydrate sources (e.g., simple sugars such as glucose, galactose, maltose, or fructose, or more complex sugars), amino acids, and vitamins (e.g., B vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, and biotin).

[0017] The term "amino acid" may refer to any of the 20 standard amino acids (i.e., glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid), their single stereoisomers, and racemic mixtures thereof. In some embodiments, the term "amino acid" refers to any of the 20 standard amino acids or their stereoisomers. In some embodiments, the amino acid is a single stereoisomer (e.g., the L-stereoisomer) of any of the 20 standard amino acids. The term "amino acid" may also refer to known non-standard amino acids (e.g., 4-hydroxyproline, hydroxyproline, s-phosphocysteine, phosphotyrosine, eN,N,N-trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, g-carboxyglutamate, eN-acetyllysine, co-N-methylarginine, N-acetylserine, N,N,N-trimethylalanine, N-formylmethionine, g-aminobutyric acid, histamine, dopamine, thyroxine, citrulline, ornithine, b-cyanoalanine, homocysteine, azaserine, and S-adenosylmethionine).

[0018] As used herein, the term "pyruvate" includes the free form of pyruvic acid as well as acid salts, including sodium pyruvate and other acid salts. In certain embodiments, the pyruvate is sodium pyruvate.

[0019] In one aspect, provided herein is a method of producing a protein of interest, the method comprising: a) feeding culture cells comprising a nucleic acid encoding the protein in a bioreactor under conditions sufficient for the cells to produce the protein, the feeding culture comprising adding one or more feeds (e.g., one, two, three, four or more feeds (e.g., a combined feed)) having a pyruvate concentration reduced by about 65% to about 100%; and (b) purifying the protein from the cells or liquid culture medium. In some embodiments, the pyruvate concentration is reduced by at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%. In some embodiments, the pyruvate concentration is reduced by at least 90%, at least 95%, or at least 98%. In some embodiments, the pyruvate concentration is reduced by about 65% to about 80%, about 70% to about 80%, about 75% to about 85%, or about 80% to about 95%. In certain embodiments, the reduction in pyruvate concentration is the same except for the pyruvate concentration, compared to the pyruvate concentration used in the fed-batch process in which lactate spikes are observed. In certain embodiments, the reduction in pyruvate concentration is the same except for the pyruvate concentration and / or the concentration of one or more amino acids, compared to the pyruvate concentration used in the fed-batch process in which lactate spikes are observed. In some embodiments, the concentration of one or more amino acids in one or more feeds (e.g., one or more composite feeds) is increased by 0% to about 100%. In some embodiments, the concentration of one or more amino acids in one or more feeds is increased by 0% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, or 80% to 100%. In some embodiments, the concentration of one or more amino acids in one or more of the feeds is increased by about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to about 95%. In some embodiments, the concentration of more amino acids in one or more feeds is increased by 0% to 40%. In some embodiments, the concentration of more amino acids in one or more feeds is increased by at least 5%, at least 10%, at least 20%, at least 30%, or at least 40%.In some embodiments, the concentration of one or more amino acids in one or more feeds is increased by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. In some embodiments, the one or more amino acids are glutamic acid, valine, or a combination thereof.

[0020] In some embodiments, the concentration of one or more amino acids is reduced by at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%. In some embodiments, the concentration of one or more amino acids is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%. In some embodiments, the concentration of one or more amino acids is reduced by at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%. In some embodiments, the pyruvate concentration is reduced by at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the concentration of one or more amino acids is reduced by about 5% to about 10%, about 10% to about 25%, about 20% to about 40%, about 25% to about 50%, about 40% to 60%, or about 50% to about 65%. In some embodiments, the concentration of one or more amino acids is reduced by about 65% to about 80%, about 70% to about 80%, about 75% to about 85%, about 80% to about 95%, or about 90% to about 100%. In some embodiments, the one or more amino acids reduced is asparagine. The volume of the feed (e.g., composite feed) can be 0.1 mL to about 1 mL, about 1 mL to about 20 mL, about 20 mL to about 50 mL, about 50 mL to about 100 mL, about 100 mL to about 500 mL, or about 500 mL to about 1000 mL. The volume of the feed (e.g., composite feed) can be about 1 L to about 10 L, about 10 L to about 50 L, about 50 L to about 100 L, about 100 L to about 500 L, or about 500 L to about 2000 L. One of skill in the art will appreciate that the volume of the feed (e.g., composite feed) may range from 0.05% to 10% of the bioreactor working volume.

[0021] In one aspect, provided herein is a method for reducing / mitigating lactate spikes in a fed-batch process for producing a protein of interest, comprising reducing pyruvate (e.g., sodium pyruvate) concentration in one or more feeds (e.g., one or more composite feeds) to cells in a bioreactor in the fed-batch process. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more feeds is reduced by about 10% to about 30% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in the one or more feeds in the same fed-batch process in which lactate spikes are observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more feeds is reduced by about 25% to about 50% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in the one or more feeds in the same fed-batch process in which lactate spikes are observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 25% to about 75% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 50% to about 75% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 65% to about 100% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 75% to about 95% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed.In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 85% to about 98% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 10%, about 15%, about 25%, about 30%, about 35%, or about 40% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed.

[0022] In some embodiments, in addition to decreasing the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds (e.g., one or more complex feeds) of a fed-batch process for producing a protein of interest, the one or more feeds include an increased concentration of one or more amino acids. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, aspartic acid, isoleucine, and threonine, or combinations thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, aspartic acid, isoleucine, and threonine. In some embodiments, the one or more amino acids are glutamic acid, valine, and leucine. In some embodiments, the one or more amino acids are glutamic acid and valine. In some embodiments, the increase in the concentration of the amino acid is about 0% to about 100% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0.5% to about 10% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 6%, about 7%, about 8%, about 9%, or about 10% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0.5% to about 100% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in concentration of the amino acid is between about 5% and about 100%, between about 5% and about 75%, between about 5% and about 50%, or between about 5% and about 25% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which a lactate spike is observed.In some embodiments, the increase in the concentration of the amino acid is about 25% to about 100%, about 25% to about 75%, or about 25% to about 50% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0% to about 20%, about 20% to about 40%, about 40% to about 60%, or about 20% to about 60% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0% to about 40%, about 1% to about 40%, about 60% to about 80%, or about 80% to about 100% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is at least 0.05%, at least 1%, at least 5%, at least 10%, at least 20%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed.

[0023] In certain embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition). In some embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition) between days 5 and 6 of culture. In some embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition) between days 4 and 7 of culture. In some embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition) between days 3 and 8 of culture. In some embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition) between days 2 and 10 of culture. In some embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition) between days 4 and 5 of culture. In some embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition) between days 6 and 7 of culture. In some embodiments, lactate spikes are mitigated by feed addition (e.g., composite feed addition) between days 7 and 8 of culture. In some embodiments, the lactate spike is mitigated by a composite feed addition between days 8 and 9 of the culture. In some embodiments, the lactate spike is mitigated by a feed addition (e.g., a composite feed addition) between days 9 and 10 of the culture. In some embodiments, the lactate spike is mitigated by a feed addition (e.g., a composite feed addition) on day 3 of the culture. In some embodiments, the lactate spike is mitigated by a composite feed addition on day 4 of the culture. In some embodiments, the lactate spike is mitigated by a feed addition (e.g., a composite feed addition) on day 5 of the culture. In some embodiments, the lactate spike is mitigated by a feed addition (e.g., a composite feed addition) on day 6 of the culture. In some embodiments, the lactate spike is mitigated by a composite feed addition on day 7 of the culture. In some embodiments, the lactate spike is mitigated by a feed addition (e.g., a composite feed addition) on day 8 of the culture.

[0024] In some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian host cell lines suitable for use in the present disclosure include Chinese hamster ovary (CHO) cells, mouse myeloma-derived NS0 and Sp2 / 0 cells, human embryonic kidney cells (HEK293), and human embryonic retina-derived PER.C6 cells. In some embodiments, the mammalian cell line is a CHO cell line. For further information and embodiments regarding cells, see Section 4.1.2.

[0025] In certain embodiments, the media and feeds (e.g., complex feeds) used in a fed-batch process are suitable for the cells used. In certain embodiments, the media and feeds (e.g., complex feeds) used in a fed-batch process allow the cells used to grow and produce the protein of interest. For further information and embodiments regarding media and feeds, see Section 4.1.4.

[0026] The bioreactor used for fed-batch culture has an appropriate volume that allows the cultivation and growth of biological cells capable of producing a protein of interest. For example, the volume of the bioreactor can be about 10 milliliters (mL) to about 25,000 L. In another example, the volume of the bioreactor can be about 0.5 liters (L) to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 10 mL. In some embodiments, the volume of the bioreactor can be about 10 mL to about 100 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 200 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 300 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 500 mL. In some embodiments, the volume of the bioreactor can be about 500 mL to about 750 mL. In some embodiments, the volume of the bioreactor can be about 500 mL to about 1000 mL. In some embodiments, the volume of the bioreactor may be about 500 mL to about 2 L. In some embodiments, the volume of the bioreactor may be about 1 L to about 5 L. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be about 0.5 liters (L) to about 250 L. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 50 L. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 25 L. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 1 L or less. In some embodiments, the volume of the bioreactor may be about 1 L. In some embodiments, the volume of the bioreactor may be about 2 L. In some embodiments, the volume of the bioreactor may be about 3L.In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 100 L or less. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be 1,000 L or more. In some embodiments, the volume of the bioreactor may be about 1,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 10,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 1,000 L. In some embodiments, the volume of the bioreactor may be about 2,000 L. In some embodiments, the volume of the bioreactor may be about 5,000 L or less. In some embodiments, the volume of the bioreactor can be about 10,000 L or less. In some embodiments, the volume of the bioreactor can be about 15,000 L or less. In some embodiments, the volume of the bioreactor can be about 25,000 L or less. For more information and embodiments regarding bioreactors, see Section 4.1.3.

[0027] In some embodiments, the lactate spike occurs between the 5th and 6th days of culture. In some embodiments, the lactate spike occurs between the 4th and 7th days of culture. In some embodiments, the lactate spike occurs between the 3rd and 8th days of culture. In some embodiments, the lactate spike occurs between the 2nd and 10th days of culture. In some embodiments, the lactate spike occurs between the 4th and 5th days of culture. In some embodiments, the lactate spike occurs between the 6th and 7th days of culture. In some embodiments, the lactate spike occurs between the 7th and 8th days of culture. In some embodiments, the lactate spike occurs between the 8th and 9th days of culture. In some embodiments, the lactate spike occurs between the 9th and 10th days of culture. In some embodiments, the lactate spike occurs on the 3rd day of culture. In some embodiments, the lactate spike occurs on the 4th day of culture. In some embodiments, the lactate spike occurs on the 5th day of culture. In some embodiments, the lactate spike occurs on the 6th day of culture. In some embodiments, the lactate spike occurs on the 7th day of culture. In some embodiments, the lactate spike occurs on day 8 of culture.

[0028] In some embodiments, the lactate spike is attenuated by one or more of: (a) a decrease in asparagine, pyruvate, or a combination thereof; and / or (b) an increase in glutamate, isoleucine, leucine, aspartate, valine, threonine, or a combination thereof. In some embodiments, the lactate spike is attenuated by an increase in one or more amino acids selected from the group consisting of glutamate, valine, leucine, threonine, aspartate, and isoleucine, or a combination thereof. In certain embodiments, the lactate spike is attenuated by a decrease in pyruvate. In some embodiments, the lactate spike is attenuated by an increase in glutamate, valine, leucine, or a combination thereof. In some embodiments, the lactate spike is attenuated by an increase in glutamate, valine, or a combination thereof. In certain embodiments, the lactate spike is attenuated by an increase in glutamate, valine, or a combination thereof. In some embodiments, the lactate spike is attenuated by an increase in glutamate, valine, or a combination thereof. In some embodiments, lactate spikes are mitigated by increasing glutamate, valine, or a combination thereof.

[0029] As provided herein, mitigation of lactate spikes can improve productivity and / or product quality (e.g., capillary isoelectric focusing (cIEF), purity, glycans). In some embodiments, the productivity of the bioreactor process is increased by about 5%. In some embodiments, the productivity of the bioreactor process is increased by about 10%. In some embodiments, the productivity of the bioreactor process is increased by about 20%. In some embodiments, the productivity of the bioreactor process is increased by about 30%. In some embodiments, the productivity of the bioreactor process is increased by about 40%. In some embodiments, the productivity of the bioreactor process is increased by about 50%. In some embodiments, the productivity of the bioreactor process is increased by about 60%. In some embodiments, the productivity of the bioreactor process is increased by about 70%. In some embodiments, the productivity of the bioreactor process is increased by about 80%. In some embodiments, the productivity of the bioreactor process is increased by about 90%. In some embodiments, the productivity of the bioreactor process is increased by more than 100%.

[0030] In some embodiments, product quality is increased by about 5%. In some embodiments, product quality is increased by about 10%. In some embodiments, product quality is increased by about 20%. In some embodiments, product quality is increased by about 30%. In some embodiments, product quality is increased by about 40%. In some embodiments, product quality is increased by about 50%. In some embodiments, product quality is increased by about 60%. In some embodiments, product quality is increased by about 70%. In some embodiments, product quality is increased by about 80%. In some embodiments, product quality is increased by about 90%. In some embodiments, product quality is increased by more than 100%.

[0031] In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest. In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest of about 5% to about 30%. In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest of about 5% to about 20%. In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest of about 5% to about 15%. In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest of about 5% to about 10%. In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest of about 10% to about 30%. In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest of about 10% to about 20%. In some embodiments, the method for reducing lactate spikes results in an increase in the titer of the protein of interest of about 20% to about 30%. In some embodiments, the methods for reducing lactate spikes result in about a 5%, about a 10%, about a 15%, about a 20%, about a 25%, or about a 30% increase in the titer of the protein of interest. In some embodiments, the methods for reducing lactate spikes result in about a 25% to about a 75% increase in the titer of the protein of interest. In some embodiments, the methods for reducing lactate spikes result in about a 50% to about a 75% increase in the titer of the protein of interest. In some embodiments, the methods for reducing lactate spikes result in about a 75% to about a 95% increase in the titer of the protein of interest.

[0032] In some embodiments, the increase in titer is about a 10% increase. In some embodiments, the increase in titer is about a 20% increase. In some embodiments, the increase in titer is about a 30% increase. In some embodiments, the increase in titer is about a 40% increase. In some embodiments, the increase in titer is about a 50% increase. In some embodiments, the increase in titer is about a 60% increase. In some embodiments, the increase in titer is about a 70% increase. In some embodiments, the increase in titer is about an 80% increase. In some embodiments, the increase in titer is about a 90% increase. In some embodiments, the increase in titer is about a 100% increase. In some embodiments, the increase in titer is greater than 100%.

[0033] In some embodiments, the protein production yield or titer, expressed in grams of protein product per liter of culture medium, from cells cultured according to the 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.

[0034] In certain embodiments, reducing pyruvate (e.g., sodium pyruvate) increases the titer by about 0.5 g / L. In certain embodiments, reducing pyruvate (e.g., sodium pyruvate) increases the titer by about 1 g / L. In certain embodiments, reducing pyruvate (e.g., sodium pyruvate) increases the titer by about 1.5 g / L. In a specific embodiment, reducing pyruvate (e.g., sodium pyruvate) increases the titer by about 2 g / L. In a specific embodiment, reducing pyruvate (e.g., sodium pyruvate) increases the titer by about 2.5 g / L. In a specific embodiment, reducing pyruvate (e.g., sodium pyruvate) increases the titer by about 3 g / L. In a specific embodiment, reducing pyruvate (e.g., sodium pyruvate) increases the titer by 3.5 g / L. In a specific embodiment, reducing pyruvate (e.g., sodium pyruvate) increases the titer by about 4 g / L. In a specific embodiment, a reduction in pyruvate (e.g., sodium pyruvate) increases the titer by 4.5 g / L. In a specific embodiment, a reduction in pyruvate (e.g., sodium pyruvate) increases the titer by about 5 g / L. In a specific embodiment, a reduction in pyruvate (e.g., sodium pyruvate) increases the titer by 5.5 g / L. In a specific embodiment, a reduction in pyruvate (e.g., sodium pyruvate) increases the titer by about 6 g / L. In some embodiments, a reduction in pyruvate (e.g., sodium pyruvate) increases the titer by about 0.5 g / L to about 6 g / L. In some embodiments, a reduction in pyruvate (e.g., sodium pyruvate) increases the titer by about 1 g / L to about 6 g / L, about 2 g / L to about 6 g / L, or about 2 g / L to about 4 g / L. In some embodiments, a reduction in pyruvate (e.g., sodium pyruvate) increases titer by about 3 g / L to about 6 g / L, about 4 g / L to about 6 g / L, about 3 g / L to about 5 g / L, about 3 g / L to about 4 g / L, or about 4 g / L to about 5 g / L.

[0035] In some embodiments, the method for reducing lactate spikes results in an increase in viable cell density (VCD). In some embodiments, the method for reducing lactate spikes results in an increase in VCD of about 5% to about 30%. In some embodiments, the method for reducing lactate spikes results in an increase in VCD of about 5% to about 20%. In some embodiments, the method for reducing lactate spikes results in an increase in VCD of about 5% to about 15%. In some embodiments, the method for reducing lactate spikes results in an increase in VCD of about 5% to about 10%. In some embodiments, the method for reducing lactate spikes results in an increase in VCD of about 10% to about 30%. In some embodiments, the method for reducing lactate spikes results in an increase in VCD of about 10% to about 20%. In some embodiments, the method for reducing lactate spikes results in an increase in VCD of about 20% to about 30%. In some embodiments, the methods for reducing lactate spikes result in an increase in VCD of about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%.

[0036] In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 5% to about 30%. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 5% to about 20%. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 5% to about 15%. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 5% to about 10%. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 10% to about 30%. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 10% to about 20%. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 20% to about 30%. In some embodiments, the method for reducing lactate spikes results in an increase in cell productivity of about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%.

[0037] In some embodiments, the method for reducing lactate spikes results in an increase in purity (e.g., cSDS purity). In some embodiments, the method for reducing lactate spikes results in an increase in purity (e.g., cSDS purity) of about 10% to 40%. In some embodiments, the method for reducing lactate spikes results in an increase in purity (e.g., cSDS purity) of about 20% to 40%. In some embodiments, the method for reducing lactate spikes results in an increase in purity (e.g., cSDS purity) of about 30% to 40%. In some embodiments, the reduction in sodium pyruvate results in an increase in capillary sodium dodecyl sulfate gel electrophoresis (cSDS) purity.

[0038] In some embodiments, the method for reducing lactate spikes results in increased flux into the tricarboxylic acid (TCA) cycle, for example as described in the Examples below. In some embodiments, the method for reducing lactate spikes balances the rates of glycolytic flux and TCA flux. In some embodiments, the method for reducing lactate spikes results in one, two, or more, or all of the effects described in the Examples below.

[0039] In some embodiments, the method for reducing a lactic acid spike results in a reduction in glycation. In some embodiments, the method for reducing a lactic acid spike results in about a 5% to about 30% reduction in glycation. In some embodiments, the method for reducing a lactic acid spike results in about a 5% to about 20% reduction in glycation. In some embodiments, the method for reducing a lactic acid spike results in about a 5% to about 10% reduction in glycation. In some embodiments, the method for reducing a lactic acid spike results in about a 10% to about 20% reduction in glycation. In some embodiments, the method for reducing a lactic acid spike results in about a 10% to about 30% reduction in glycation. In some embodiments, the method for reducing a lactic acid spike results in about a 20% to about 30% reduction in glycation.

[0040] In some embodiments, reducing pyruvate (e.g., sodium pyruvate) results in improved glycosylation and capillary isoelectric focusing (cIEF) profiles. In some embodiments, reducing pyruvate (e.g., sodium pyruvate) results in increased glycan complexity. For example, as provided herein, reducing pyruvate (e.g., sodium pyruvate) can reduce G0F / G0F-GlcNac and increase G1F / G2F compared to high pyruvate (e.g., sodium pyruvate) conditions.

[0041] In one aspect, as provided herein, a reduction in lactate spiking increases the titer of a bioreactor culture. In some embodiments, the increase in titer is about a 5% increase. In some embodiments, the increase in titer is about a 10% increase. In some embodiments, the increase in titer is about a 20% increase. In some embodiments, the increase in titer is about a 30% increase. In some embodiments, the increase in titer is about a 40% increase. In some embodiments, the increase in titer is about a 50% increase. In some embodiments, the increase in titer is about a 60% increase. In some embodiments, the increase in titer is about a 70% increase. In some embodiments, the increase in titer is about an 80% increase. In some embodiments, the increase in titer is about a 90% increase. In some embodiments, the increase in titer is about a 100% increase. In some embodiments, the increase in titer is greater than 100%.

[0042] In some embodiments, the protein production yield or titer, expressed in grams of protein product per liter of culture medium, from cells cultured according to the 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.

[0043] In one aspect, as provided herein, a reduction in lactate spikes increases the yield of a bioreactor culture. In some embodiments, the increase in yield is about a 5% increase. In some embodiments, the increase in yield is about a 10% increase. In some embodiments, the increase in yield is about a 20% increase. In some embodiments, the increase in yield is about a 30% increase. In some embodiments, the increase in yield is about a 40% increase. In some embodiments, the increase in yield is about a 50% increase. In some embodiments, the increase in yield is about a 60% increase. In some embodiments, the increase in yield is about a 70% increase. In some embodiments, the increase in yield is about an 80% increase. In some embodiments, the increase in yield is about a 90% increase. In some embodiments, the increase in yield is about a 100% increase. In some embodiments, the increase in yield is greater than 100%.

[0044] In one aspect, as provided herein, a reduction in lactate spikes increases the rate of a bioreactor culture. In some embodiments, the increase in rate is about a 5% increase. In some embodiments, the increase in rate is about a 10% increase. In some embodiments, the increase in rate is about a 20% increase. In some embodiments, the increase in rate is about a 30% increase. In some embodiments, the increase in rate is about a 40% increase. In some embodiments, the increase in rate is about a 50% increase. In some embodiments, the increase in rate is about a 60% increase. In some embodiments, the increase in rate is about a 70% increase. In some embodiments, the increase in rate is about an 80% increase. In some embodiments, the increase in rate is about a 90% increase. In some embodiments, the increase in rate is about a 100% increase. In some embodiments, the increase in rate is greater than 100%.

[0045] The protein of interest can be any protein that one of skill in the art desires to produce using a fed-batch process. In certain embodiments, the protein of interest can be any protein that can be recombinantly expressed (e.g., any recombinant protein). In some embodiments, the protein of interest is an antibody or an antigen-binding fragment thereof. The antibody can be monospecific, bispecific, or multispecific (e.g., trispecific). In some embodiments, the protein of interest is an antibody. The antibody can be monoparatopic, biparatopic, or multiparatopic. The antibody can be an antibody fusion protein. The antibody or antigen-binding fragment thereof can bind to any antigen (e.g., an infectious disease antigen, a cancer antigen, or an antigen associated with another disease or disorder). In some embodiments, the protein of interest is a cytokine. In some embodiments, the protein of interest is an antigen. In certain embodiments, the protein of interest is a secreted protein. In some embodiments, the protein of interest is an enzyme (e.g., human N-acetylgalactosamine-6-sulfatase (rhGALNS)) or glucocerebrosidase). The enzyme may be an enzyme used in the food industry, such as, for example, an enzyme used as an emulsifier in dairy production, bakery production, brewing, or wine production, or an enzyme used in sweetener production. The enzyme may be used in the pharmaceutical industry, such as, for example, a recombinant enzyme used as a biocatalyst for the preparation of a chiral drug intermediate, or a recombinant enzyme drug (e.g., a recombinant enzyme used in enzyme replacement). In some embodiments, the protein of interest is an enzyme used in enzyme replacement, such as a lysosomal storage disease enzyme. In some embodiments, the protein of interest is agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, or alglucosidase alpha.The protein of interest can be tissue plasminogen activator (tpa), an interferon (e.g., IFN-α, IFN-β, or IFN-γ), or an anti-nerve agent (e.g., recombinant human butyrylcholinesterase for protection against organophosphate poisoning). In some embodiments, the protein of interest is a membrane-bound protein. In some embodiments, the protein of interest is a protein expressed intracellularly. See Section 4.1.1 for further information and embodiments regarding proteins of interest.

[0046] In certain embodiments, the media and feeds used in a fed-batch process are suitable for the cells used. In certain embodiments, the media and feeds used in a fed-batch process allow the cells used to grow and produce the protein of interest. For further information and embodiments regarding media and feeds, see section 4.1.4.

[0047] In another aspect, provided herein is a method for increasing the titer of a protein of interest in a fed-batch process for producing the protein, comprising decreasing the pyruvate (e.g., sodium pyruvate) concentration in one or more feeds (e.g., one or more composite feeds) to cells in a bioreactor in the fed-batch process. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more feeds is decreased by about 10% to about 30% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in the one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more feeds is decreased by about 25% to about 50% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in the one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 25% to about 75% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 50% to about 75% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 75% to about 95% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 85% to about 98% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed.In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 10%, about 15%, about 25%, about 30%, about 35%, or about 40% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by at least 90%, at least 95%, or at least 98% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 65% to about 80%, about 70% to about 80%, about 75% to about 85%, about 80% to about 95%, or about 65% to about 100% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed.In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds is reduced by about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 90% to about 95%, or about 95% to about 100% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in one or more feeds in the same fed-batch process in which a lactate spike is observed.

[0048] In some embodiments, in addition to decreasing the concentration of pyruvate (e.g., sodium pyruvate) in one or more feeds (e.g., one or more complex feeds) of a fed-batch process for producing a protein of interest, the one or more feeds include an increased concentration of one or more amino acids. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, isoleucine, and threonine, or a combination thereof. In some embodiments, the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. In some embodiments, the one or more amino acids are glutamic acid, valine, or a combination thereof. In some embodiments, the one or more amino acids do not include threonine. In some embodiments, the increase in the concentration of the amino acid is about 0% to about 100% or about 0.5% to about 10% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 6%, about 7%, about 8%, about 9%, or about 10% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0.5% to about 100% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in concentration of the amino acid is between about 5% and about 100%, between about 5% and about 75%, between about 5% and about 50%, or between about 5% and about 25% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which a lactate spike is observed.In some embodiments, the increase in the concentration of the amino acid is about 25% to about 100%, about 25% to about 75%, or about 25% to about 50% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0% to about 20%, about 20% to about 40%, about 40% to about 60%, or about 20% to about 60% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0% to about 40%, about 1% to about 40%, about 60% to about 80%, or about 80% to about 100% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is about 0% to about 40% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is at least 0.05%, at least 1%, at least 5%, at least 10%, at least 20%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in the concentration of the amino acid is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which the lactate spike is observed. In some embodiments, the increase in concentration of the amino acid is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to the concentration of the amino acid found in one or more feeds in the same fed-batch process in which a lactate spike is observed. For further information and embodiments regarding proteins of interest, see Section 4.1.1.

[0049] In some embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a mammalian cell line. Non-limiting examples of mammalian host cell lines suitable for use in the present disclosure include Chinese Hamster Ovary (CHO) cells, mouse myeloma-derived NS0 and Sp2 / 0 cells, human embryonic kidney cells (HEK293), and human embryonic retinoblast-derived PER.C6 cells. In some embodiments, the mammalian cell line is a CHO cell line. For further information and embodiments regarding cells, see Section 4.1.2.

[0050] In certain embodiments, the media and feeds (e.g., complex feeds) used in a fed-batch process are suitable for the cells used. In certain embodiments, the media and feeds (e.g., complex feeds) used in a fed-batch process allow the cells used to grow and produce the protein of interest. For further information and embodiments regarding media and feeds, see Section 4.1.4.

[0051] The bioreactor used for fed-batch culture has an appropriate volume that allows the cultivation and growth of biological cells capable of producing a protein of interest. For example, the volume of the bioreactor can be about 10 milliliters (mL) to about 25,000 L. In another example, the volume of the bioreactor can be about 0.5 liters (L) to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 10 mL. In some embodiments, the volume of the bioreactor can be about 10 mL to about 100 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 200 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 300 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 500 mL. In some embodiments, the volume of the bioreactor can be about 500 mL to about 750 mL. In some embodiments, the volume of the bioreactor can be about 500 mL to about 1000 mL. In some embodiments, the volume of the bioreactor may be about 500 mL to about 2 L. In some embodiments, the volume of the bioreactor may be about 1 L to about 5 L. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be about 0.5 liters (L) to about 250 L. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 50 L. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 25 L. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 1 L or less. In some embodiments, the volume of the bioreactor may be about 1 L. In some embodiments, the volume of the bioreactor may be about 2 L. In some embodiments, the volume of the bioreactor may be about 3L.In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 100 L or less. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be 1,000 L or more. In some embodiments, the volume of the bioreactor may be about 1,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 10,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 1,000 L. In some embodiments, the volume of the bioreactor may be about 2,000 L. In some embodiments, the volume of the bioreactor may be about 5,000 L or less. In some embodiments, the volume of the bioreactor can be about 10,000 L or less. In some embodiments, the volume of the bioreactor can be about 15,000 L or less. In some embodiments, the volume of the bioreactor can be about 25,000 L or less. For more information and embodiments regarding bioreactors, see Section 4.1.3.

[0052] In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 20%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 15%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 10%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 10% to about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 10% to about 20%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 20% to about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 25% to about 75%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 50% to about 75%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 75% to about 95%.

[0053] In some embodiments, the increase in titer is about a 10% increase. In some embodiments, the increase in titer is about a 20% increase. In some embodiments, the increase in titer is about a 30% increase. In some embodiments, the increase in titer is about a 40% increase. In some embodiments, the increase in titer is about a 50% increase. In some embodiments, the increase in titer is about a 60% increase. In some embodiments, the increase in titer is about a 70% increase. In some embodiments, the increase in titer is about an 80% increase. In some embodiments, the increase in titer is about a 90% increase. In some embodiments, the increase in titer is about a 100% increase. In some embodiments, the increase in titer is greater than 100%.

[0054] In some embodiments, the protein production yield or titer, expressed in grams of protein product per liter of culture medium, from cells cultured according to the 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.

[0055] In some embodiments, the method results in an increase in VCD. In some embodiments, the method results in an increase in VCD of about 5% to about 30%. In some embodiments, the method results in an increase in VCD of about 5% to about 20%. In some embodiments, the method results in an increase in VCD of about 5% to about 15%. In some embodiments, the method results in an increase in VCD of about 5% to about 10%. In some embodiments, the method results in an increase in VCD of about 10% to about 30%. In some embodiments, the method results in an increase in VCD of about 10% to about 20%. In some embodiments, the method results in an increase in VCD of about 20% to about 30%. In some embodiments, the method results in an increase in VCD of about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%.

[0056] In some embodiments, the method results in an increase in cell productivity. In some embodiments, the method results in about a 5% to about a 30% increase in cell productivity. In some embodiments, the method results in about a 5% to about a 20% increase in cell productivity. In some embodiments, the method results in about a 5% to about a 15% increase in cell productivity. In some embodiments, the method results in about a 5% to about a 10% increase in cell productivity. In some embodiments, the method results in about a 10% to about a 30% increase in cell productivity. In some embodiments, the method results in about a 10% to about a 20% to about a 30% increase in cell productivity. In some embodiments, the method results in about a 5%, about 10%, about 15%, about 20%, about 25%, or about 30% increase in cell productivity.

[0057] In some embodiments, the method results in an increase in purity (e.g., cSDS purity). In some embodiments, the method results in an increase in purity (e.g., cSDS purity) of about 10%-40%. In some embodiments, the method results in an increase in purity (e.g., cSDS purity) of about 20%-40%. In some embodiments, the method results in an increase in purity (e.g., cSDS purity) of about 30%-40%.

[0058] In some embodiments, the methods result in a decrease in glycation. In some embodiments, the methods result in about a 5% to about 30% decrease in glycation. In some embodiments, the methods result in about a 5% to about 20% decrease in glycation. In some embodiments, the methods result in about a 5% to about 10% decrease in glycation. In some embodiments, the methods result in about a 10% to about 20% decrease in glycation. In some embodiments, the methods result in about a 10% to about 30% decrease in glycation. In some embodiments, the methods result in about a 20% to about 30% decrease in glycation.

[0059] In some embodiments, the method results in improved glycosylation and capillary isoelectric focusing (cIEF) profiles. In some embodiments, the method increases glycan complexity. For example, as provided herein, the method reduces G0F / G0F-GlcNac and increases G1F / G2F compared to high pyruvate (e.g., sodium pyruvate) conditions.

[0060] In another aspect, provided herein is a method for reducing lactate spikes during fed-batch culture of cells, the method comprising feeding cells comprising a nucleic acid encoding a protein of interest in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culture comprising adding one or more feeds (e.g., one or more complex feeds) during a growth phase of the cells, and reducing a pyruvate concentration in the one or more additional feeds (e.g., one or more complex feeds) prior to a cell density peak, the pyruvate concentration in the one or more additional feeds being reduced by 65% ​​to 100% compared to the pyruvate concentration in the one or more feeds during the growth phase of the cells. This is based at least in part on observations of when lactate spikes occur in fed-batch cultures. As shown in Section 5, lactate spikes occur during the growth phase and prior to the cells reaching a viable cell density peak (see, e.g., Figures 1, 2, and 4A-4C). In certain embodiments, provided herein is a method for reducing lactate spikes during a fed-batch culture of cells, the method comprising feeding-batch culturing cells comprising a nucleic acid encoding a protein of interest in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culturing comprising adding one or more feeds (e.g., one or more complex feeds) during growth of the cells and decreasing a pyruvate concentration in the one or more additional feeds (e.g., one or more additional complex feeds) prior to a cell density peak, the pyruvate concentration in the one or more additional feeds being decreased by 65% ​​to 100% compared to the pyruvate concentration in the one or more feeds during the growth phase of the cells. In some embodiments, the cell density peak is at about day 6 to about day 7 of the fed-batch culture of the cells, and the decrease in the pyruvate concentration in the one or more additional feeds is at about day 3 to day 5 (e.g., about day 3, about day 4, or about day 5) of the fed-batch culture of the cells. In some embodiments, the concentration of one or more amino acids is increased prior to the cell density peak.

[0061] In some embodiments, the concentration of pyruvate in the one or more additional feeds is increased by about 65% to about 95% compared to the pyruvate concentration in the one or more feeds during cell growth. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more additional feeds is reduced by about 75% to about 95% compared to the concentration of pyruvate (e.g., sodium pyruvate) found in the one or more feeds relative to the pyruvate concentration during cell growth. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more additional feeds is reduced by about 85% to about 98% compared to the pyruvate concentration in the one or more feeds during cell growth. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more additional feeds is reduced by about 65%, or about 70%, or about 65%, or about 85% compared to the pyruvate concentration in the one or more feeds during cell growth. In some embodiments, the concentration of pyruvate (e.g., sodium pyruvate) in the one or more additional feeds is reduced by about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% compared to the pyruvate concentration in the one or more feeds during growth of the cells.

[0062] In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is 0% to 100% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is about 0.5% to about 10% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is about 0.5%, about 1%, about 2%, about 3%, about 4% or about 5% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is about 6%, about 7%, about 8%, about 9%, or about 10% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is about 0.5% to about 100% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is about 5% to about 100%, about 5% to about 75%, about 5% to about 50%, or about 5% to about 25% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is about 25% to about 100%, about 25% to about 75%, or about 25% to about 50% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is about 0% to about 20%, about 20% to about 40%, about 40% to about 60%, or about 20% to about 60% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is from about 0% to about 40%, from about 1% to about 40%, from about 60% to about 80%, or from about 80% to about 100%, compared to the concentration of the amino acid in the one or more feeds during growth of the cell.In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is at least 0.05%, at least 1%, at least 5%, at least 10%, at least 20%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% compared to the amino acid concentration in the one or more feeds during cell growth. In some embodiments, the increase in the concentration of the amino acid in the one or more additional feeds is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to the amino acid concentration in the one or more feeds during cell growth.

[0063] In some embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a mammalian cell line. Non-limiting examples of mammalian host cell lines suitable for use in the present disclosure include Chinese Hamster Ovary (CHO) cells, mouse myeloma-derived NS0 and Sp2 / 0 cells, human embryonic kidney cells (HEK293), and human embryonic retinoblast-derived PER.C6 cells. In some embodiments, the mammalian cell line is a CHO cell line. For further information and embodiments regarding cells, see Section 4.1.2.

[0064] In certain embodiments, the media and feeds (e.g., complex feeds) used in a fed-batch process are suitable for the cells used. In certain embodiments, the media and feeds (e.g., complex feeds) used in a fed-batch process allow the cells used to grow and produce the protein of interest. For further information and embodiments regarding media and feeds, see Section 4.1.4.

[0065] The bioreactor used for fed-batch culture has an appropriate volume that allows the cultivation and growth of biological cells capable of producing a protein of interest. For example, the volume of the bioreactor can be about 10 milliliters (mL) to about 25,000 L. In another example, the volume of the bioreactor can be about 0.5 liters (L) to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 10 mL. In some embodiments, the volume of the bioreactor can be about 10 mL to about 100 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 200 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 300 mL. In some embodiments, the volume of the bioreactor can be about 100 mL to about 500 mL. In some embodiments, the volume of the bioreactor can be about 500 mL to about 750 mL. In some embodiments, the volume of the bioreactor can be about 500 mL to about 1000 mL. In some embodiments, the volume of the bioreactor may be about 500 mL to about 2 L. In some embodiments, the volume of the bioreactor may be about 1 L to about 5 L. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be about 0.5 liters (L) to about 250 L. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 50 L. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 25 L. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 1 L or less. In some embodiments, the volume of the bioreactor may be about 1 L. In some embodiments, the volume of the bioreactor may be about 2 L. In some embodiments, the volume of the bioreactor may be about 3L.In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 100 L or less. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be 1,000 L or more. In some embodiments, the volume of the bioreactor may be about 1,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 10,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 1,000 L. In some embodiments, the volume of the bioreactor may be about 2,000 L. In some embodiments, the volume of the bioreactor may be about 5,000 L or less. In some embodiments, the volume of the bioreactor can be about 10,000 L or less. In some embodiments, the volume of the bioreactor can be about 15,000 L or less. In some embodiments, the volume of the bioreactor can be about 25,000 L or less. For more information and embodiments regarding bioreactors, see Section 4.1.3.

[0066] In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 20%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 15%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5% to about 10%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 10% to about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 10% to about 20%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 20% to about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 25% to about 75%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 50% to about 75%. In some embodiments, the method results in an increase in the titer of the protein of interest of about 75% to about 95%.

[0067] In some embodiments, the increase in titer is about a 10% increase. In some embodiments, the increase in titer is about a 20% increase. In some embodiments, the increase in titer is about a 30% increase. In some embodiments, the increase in titer is about a 40% increase. In some embodiments, the increase in titer is about a 50% increase. In some embodiments, the increase in titer is about a 60% increase. In some embodiments, the increase in titer is about a 70% increase. In some embodiments, the increase in titer is about an 80% increase. In some embodiments, the increase in titer is about a 90% increase. In some embodiments, the increase in titer is about a 100% increase. In some embodiments, the increase in titer is greater than 100%.

[0068] In some embodiments, the protein production yield or titer, expressed in grams of protein product per liter of culture medium, from cells cultured according to the 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.

[0069] In some embodiments, the method results in an increase in VCD. In some embodiments, the method results in an increase in VCD of about 5% to about 30%. In some embodiments, the method results in an increase in VCD of about 5% to about 20%. In some embodiments, the method results in an increase in VCD of about 5% to about 15%. In some embodiments, the method results in an increase in VCD of about 5% to about 10%. In some embodiments, the method results in an increase in VCD of about 10% to about 30%. In some embodiments, the method results in an increase in VCD of about 10% to about 20%. In some embodiments, the method results in an increase in VCD of about 20% to about 30%. In some embodiments, the method results in an increase in VCD of about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%.

[0070] In some embodiments, the method results in an increase in cell productivity. In some embodiments, the method results in about a 5% to about a 30% increase in cell productivity. In some embodiments, the method results in about a 5% to about a 20% increase in cell productivity. In some embodiments, the method results in about a 5% to about a 15% increase in cell productivity. In some embodiments, the method results in about a 5% to about a 10% increase in cell productivity. In some embodiments, the method results in about a 10% to about a 30% increase in cell productivity. In some embodiments, the method results in about a 10% to about a 20% to about a 30% increase in cell productivity. In some embodiments, the method results in about a 5%, about 10%, about 15%, about 20%, about 25%, or about 30% increase in cell productivity.

[0071] In some embodiments, the method results in an increase in purity (e.g., cSDS purity). In some embodiments, the method results in an increase in purity (e.g., cSDS purity) of about 10%-40%. In some embodiments, the method results in an increase in purity (e.g., cSDS purity) of about 20%-40%. In some embodiments, the method results in an increase in purity (e.g., cSDS purity) of about 30%-40%.

[0072] In some embodiments, the methods result in a decrease in glycation. In some embodiments, the methods result in about a 5% to about 30% decrease in glycation. In some embodiments, the methods result in about a 5% to about 20% decrease in glycation. In some embodiments, the methods result in about a 5% to about 10% decrease in glycation. In some embodiments, the methods result in about a 10% to about 20% decrease in glycation. In some embodiments, the methods result in about a 10% to about 30% decrease in glycation. In some embodiments, the methods result in about a 20% to about 30% decrease in glycation.

[0073] In some embodiments, the method results in improved glycosylation and capillary isoelectric focusing (cIEF) profiles. In some embodiments, the method increases glycan complexity. For example, as provided herein, the method reduces G0F / G0F-GlcNac and increases G1F / G2F compared to high pyruvate (e.g., sodium pyruvate) conditions.

[0074] In another aspect, provided herein is a protein of interest produced by a method described herein (e.g., in Section 5). In certain aspects, provided herein is a fed-batch process as described in Section 5, in which lactate spiking is reduced. The fed-batch processes described herein can be used to recombinantly produce a protein of interest.

[0075] 4.1.1 Protein of interest The protein of interest can be any protein that one of skill in the art desires to produce using a fed-batch process. In certain embodiments, the protein of interest can be any protein that can be recombinantly expressed (e.g., any recombinant protein). In some embodiments, the protein of interest is an antigen-binding protein. In some embodiments, the protein of interest is an antibody or an antigen-binding fragment thereof. In some embodiments, the protein of interest is an antibody. The antibody can be monospecific, bispecific, or multispecific (e.g., trispecific). The antibody can be monoparatopic, biparatopic, or multiparatopic. The antibody can be an antibody fusion protein. The antibody or antigen-binding fragment thereof can bind to any antigen (e.g., an infectious disease antigen, a cancer antigen, or an antigen associated with another disease or disorder). In some embodiments, the protein of interest is a cytokine. In some embodiments, the cytokine is IL-1β, IL-2, IL-5, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, IL23, TNF-α, or an interferon (e.g., IFN-α, IFN-β, or IFN-γ). In some embodiments, the protein of interest is a fusion protein. In some embodiments, the protein of interest is an antigen. In some embodiments, the protein of interest is an enzyme (e.g., human N-acetylgalactosamine-6-sulfatase (rhGALNS) or glucocerebrosidase). The enzyme may be an enzyme used in the food industry, such as, for example, an enzyme used as an emulsifier in dairy, bakery, brewing, or winemaking, or an enzyme used in sweetener production. The enzyme may be used in the pharmaceutical industry, such as, for example, a recombinant enzyme used as a biocatalyst for the preparation of a chiral drug intermediate, or a recombinant enzyme drug (e.g., a recombinant enzyme used in enzyme replacement). In some embodiments, the protein of interest is an enzyme used in enzyme replacement, such as a lysosomal storage disease enzyme.In some embodiments, the protein of interest is agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, or alglucosidase alpha. The protein of interest can also be tissue plasminogen activator (tpa), interferon (e.g., IFN-α, IFN-β, or IFN-γ), clotting factors, erythropoietin, or anti-nerve agents (e.g., recombinant human butyrylcholinesterase for protection against organophosphate poisoning). In certain embodiments, the protein of interest is a secreted protein. In some embodiments, the protein of interest is a membrane-bound protein. In some embodiments, the protein of interest is a protein expressed intracellularly.

[0076] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included in the definition are polypeptides that contain one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can occur as a single chain or as two or more related chains.

[0077] As used herein, the term "antibody" is used in a broad sense and specifically includes, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific, trispecific antibodies, so long as they exhibit the desired biological activity), including, but not limited to, fusion molecules (e.g., IgG-scFv, scFv-Fc-scFv, VHH-Fc, Fc-VHH, Fc-scFv, HC-VHH, scFv-Fc-VHH, HC-scFv), single chain antibodies, and fragments thereof (e.g., domain antibodies). Antibodies may be human, humanized, chimeric, and / or affinity matured, and may also be antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides capable of binding to a specific molecular antigen and composed of two identical paired polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the respective amino-terminal portions of each chain containing a variable region of about 100 to about 130 or more amino acids, and the respective carboxy-terminal portions of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997).

[0078] Antibodies also include, but are not limited to, synthetic antibodies, nanobodies, recombinantly produced antibodies, antibodies derived from Camelidae species (e.g., llamas and alpacas) or humanized variants thereof, endogenous antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, and refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that contain an antigen-binding site that binds an antigen (e.g., one or more complementarity-determining regions (CDRs) of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibody can be an agonist antibody or an antagonist antibody.The antibody may be neither an agonist nor an antagonist.

[0079] In some embodiments, antibodies may have modifications including cross-linking agents, glycosylation, conjugated drugs, or thio-modified thiol linkages.

[0080] As used herein, the term "antigen" has its ordinary meaning in the art. An "antigen" includes a structure to which an antigen-binding protein (e.g., an antibody) can bind. An antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, an antigen is a polypeptide. In certain embodiments, an antigen is associated with a cell, e.g., present on or within a cell. In some embodiments, an antigen is associated with a cancer cell, e.g., present on or within a cancer cell. In certain embodiments, an antigen is associated with a pathogen, such as a virus, bacteria, fungus, or parasite.

[0081] As used herein, the term "antigen-binding protein" refers to a protein that binds to an antigen. An antibody is an example of an antigen-binding protein. Antigen-binding proteins include, but are not limited to, single-chain antibodies, nanobodies, multi-domain antibodies, scFv, Fab, and diabodies.

[0082] The term "binding" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site, such as an antigen-binding site on an antibody, and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody for that epitope. The dissociation rate (k off ) and association rate (k on) and the ratio (k off / k on ) is the dissociation constant K D and is inversely related to affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different complexes of antibody and antigen. on and k off The dissociation constant K of the antibodies provided herein depends on both D can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of multiple interactions between such a multivalent antibody and an antigen is called avidity.

[0083] Antigen binding proteins that bind or specifically bind to an antigen can be identified, for example, by immunoassays (e.g., ELISA, radioimmunoassays and electrochemiluminescence immunoassays), Octet®, surface plasmon resonance (e.g., BiacoreBIACore®), or other techniques known to those of skill in the art. In some embodiments, a binding protein binds or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISA). Typically, a specific or selective response is at least twice the signal or noise over background, and may be more than 10 times over background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of an antigen binding protein to a "non-target" protein is less than about 10% of the binding of the antigen binding protein to its particular target antigen, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or RIA. Antigen binding proteins that bind to an antigen include those that are capable of binding to an antigen with sufficient affinity such that the antigen binding protein is useful, for example, as an antigen-targeted therapeutic and / or diagnostic agent. In certain embodiments, an antigen binding protein that binds to an antigen has a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. D In certain embodiments, the antigen binding protein binds to an epitope of an antigen that is conserved among antigens of different species.

[0084] In certain embodiments, antigen binding proteins may include "chimeric" sequences in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567, and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may include humanized sequences.

[0085] In certain embodiments, an antigen binding protein may comprise a portion of a "humanized" form of a non-human (e.g., camelid, murine, non-human primate) antibody that comprises sequences from a human immunoglobulin (e.g., recipient antibody) in which native complementarity determining region (CDR) residues are replaced by residues from a corresponding CDR of a non-human species (e.g., donor antibody) such as camel, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. The heavy or light chain of a humanized antibody can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol., 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0086] In certain embodiments, an antigen binding protein can comprise a "fully human antibody" or a portion of a "human antibody," which terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. An antigen binding protein can comprise an antibody sequence. In specific embodiments, these terms refer to an antibody that comprises a variable region and a constant region of human origin. A "fully human" antibody can also encompass, in certain embodiments, an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies that have variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao, et al., Nature Protocols, 1:755-68 (2006)).In addition, human monoclonal antibodies can be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUS™ technology. See, e.g., Li, et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006) (regarding human antibodies made with human B cell hybridoma technology).

[0087] In certain embodiments, an antigen binding protein may comprise a portion of a "recombinant human antibody," which phrase includes human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector introduced into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD, et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0088] In certain embodiments, an antigen binding protein may comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts, and well-known post-translational modifications such as isomerization and deamidation of amino acids, oxidation of methionine, and deamidation of asparagine and glutamine, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for making the antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0089] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked by a H chain with one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at the other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. The structure and properties of different classes of antibodies are described, for example, in Basic and Clinical Immunology 71 (Stites, et al. eds., 8th ed. 1994), and Immunobiology (Janeway, et al. eds., 5th ed. 1995). th ed. 2001).

[0090] As used herein, the terms "Fab" and "Fab region" have their usual meanings in the art. Typically, the term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. A conventional IgG usually contains two Fab regions, each in one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen-binding capability in accordance with the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be present on the same polypeptide and oriented in a different order, as described in more detail in the following section.

[0091] As used herein, the terms "variable region" and "variable domain" in the context of an antibody have their usual meaning in the art. Typically, the terms "variable region", "variable domain", "V region", or "V domain" refer to a portion of an antibody's light or heavy chain that is located generally at the amino terminus of the light or heavy chain, has a length of about 120-130 amino acids for the heavy chain and about 100-110 amino acids for the light chain, and is used in the binding and specificity of each particular antibody to its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to certain segments of the variable region that vary widely in sequence between antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, the variability is not uniform across the 110 amino acid span of the variable region. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions", each about 9-12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs, which are mostly in a β-sheet structure and are connected by three hypervariable regions, which form loops that connect the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions within each chain are held in close proximity together by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0092] The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refers to the numbering system used in the heavy or light chain variable regions of the compilation of antibodies of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the sequence of that antibody with the "standard" Kabat numbering sequence at the regions of homology. The Kabat numbering system is generally used when referring to residues of the variable domain (residues 1-107 for the light chain, and residues 1-113 for the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0093] As used herein, the term "heavy chain" in the context of an antibody has its usual meaning in the art. Typically, the term "heavy chain" when used with respect to an antibody refers to a polypeptide chain of about 50-70 kDa, the amino-terminal portion of which contains a variable region of about 120-130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five distinct types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains vary in size, with α, δ, and γ containing about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to five well-known classes (i.e., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM (which also include the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4). The sequences of heavy chains from various species are known in the art (see, e.g., IMGT®, the international ImMunoGeneTics information system®, imgt.org).

[0094] As used herein, the term "light chain" in the context of an antibody has its usual meaning in the art. Typically, the term "light chain" when used with respect to an antibody refers to a polypeptide chain of about 25 kDa, the amino terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. The sequences of light chains from various species are known in the art (see, e.g., IMGT®, the international ImMunoGeneTics information system®, imgt.org). As used herein, the term "constant region" or "constant domain" in the context of an antibody has its usual meaning in the art. Typically, the term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as interaction with Fc receptors. Typically, the term refers to the portion of the immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portions of the immunoglobulin, the variable regions that contain the antigen-binding site. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0095] As used herein, the term "framework" or "FR" in the context of an antibody has its normal meaning in the art. Typically, the term "framework" or "FR" refers to the variable region residues adjacent to the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain, diabodies, linear, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0096] As used herein, the term "Fc region" of an antibody has its ordinary meaning in the art. Typically, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is often defined as extending from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies may include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, a variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example at least about 95% homology thereto.

[0097] In certain embodiments, homology is determined by sequence similarity. Modern protein sequence databases are very comprehensive. Widely used similarity search programs, such as BLAST (Altschul et al. (1997); units 3.3 and 3.4), PSI-BLAST (Altschul et al., 1997), SSEARCH (Smith and Waterman (1981); Pearson (1991), unit 3.10), FASTA (Pearson and Lipman (1988) unit 3.9) and HMMER3 (Johnson et al., 2010) programs, generate accurate statistical estimates and ensure that protein sequences that share significant similarity also have similar structures.

[0098] The term "specificity" in the context of an antibody or other antibody-binding protein refers to the selective recognition of the antigen-binding protein for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecific" as used herein refers to an antigen-binding protein having two or more antigen-binding sites, at least two of which bind to different antigens. "Bispecific" as used herein refers to an antigen-binding protein having two different antigen-binding specificities. The term "monospecific" antibody as used herein refers to an antigen-binding protein having one or more binding sites, each of which binds to the same antigen.

[0099] A variety of commercially available antibodies are provided below in Table 1 and are suitable for production in a fed-batch process as described herein, either as intact antibodies, antibody fragments, or as part of a multispecific antibody (e.g., fusion protein). In addition, antibodies that bind to the same targets as those shown in Table 1 are suitable for production in a fed-batch process as described herein.

[0100] [Table 1-1]

[0101] [Table 1-2]

[0102] [Table 1-3]

[0103] [Table 1-4]

[0104] Antibodies that bind to antigens of the organisms shown in Table 2 are suitable for production in a fed-batch process as described herein. In addition, antigens of the organisms shown in Table 2 are for production in a fed-batch process as described herein. In some embodiments, antigens of interest produced in a fed-batch process as described herein are those identified in Table 3.

[0105] [Table 2-1]

[0106] [Table 2-2]

[0107] [Table 2-3]

[0108] [Table 3]

[0109] The protein of interest may be used in vitro, ex vivo, or in vivo. For example, the protein of interest may have therapeutic or diagnostic uses. In another example, the protein of interest (e.g., an antigen) may be used as an immunogen. In another example, the commercially available antibodies listed in Table 1 may be used for the indications listed in Table 1 or another approved indication. In another example, the protein of interest may be used in the food industry, for example, enzymes used as emulsifiers in dairy, bakery, brewing, or wine making, or enzymes used in sweetener production. In another example, the protein of interest may be used in the pharmaceutical industry, for example, recombinant enzymes used as biocatalysts for the preparation of chiral drug intermediates, or recombinant enzyme drugs (e.g., recombinant enzymes used in enzyme replacement). In another example, the protein of interest may be used as a coagulant.

[0110] 4.1.2 Cells The cells used in the fed-batch process can be any cell type, for example, any animal cell type (e.g., non-human mammalian cells or human cells). In some embodiments, the cells are mammalian cells. In certain embodiments, the cells are mammalian cell lines. Non-limiting examples of mammalian cell lines suitable for use in the present disclosure include Chinese hamster ovary (CHO) cells, mouse myeloma-derived NS0 and Sp2 / 0 cells, human embryonic kidney cells (HEK293), and human embryonic retina-derived PER.C6 cells. In some embodiments, the mammalian cell line is a CHO cell line as used in Section 5. In some embodiments, the Chinese hamster ovary (CHO) cells are -DHFR (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)). Other examples of mammalian cell lines include SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells; and a human hepatoma line (Hep G2).

[0111] The cells may be engineered to express a protein of interest using techniques known to those of skill in the art. In some embodiments, the cells are recombinantly engineered to express one protein of interest. In some embodiments, the cells are recombinantly engineered to express two or more proteins of interest (e.g., two, three, four, or five proteins of interest). Conventional techniques of molecular biology, microbiology, and immunology that are within the skill of the artisan may be used. Such techniques are fully described in the literature. Examples of particularly suitable texts for reference include: Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, ed., DNA Cloning, Volumes I and II (1985); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, NY, 2d ed. 1987); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed.2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds.,2d ed.2010).In some embodiments, cells are transfected or transformed (e.g., stably transformed) with a nucleic acid molecule that encodes a protein of interest. A cell that has been transfected or transformed with a nucleic acid molecule includes the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell that was transfected or transformed with the nucleic acid molecule due to possible mutations or environmental influences that may occur in subsequent generations, or due to integration of the nucleic acid sequence into the genome of the cell.

[0112] 4.1.3 Bioreactor The bioreactor can be any culture vessel manufactured or designed to manipulate or control environmental conditions. Such culture vessels are well known in the art. For example, the bioreactor can be a stainless steel stirred tank bioreactor (STR), an airlift reactor, a disposable bioreactor, or a combination thereof (e.g., a disposable bioreactor in combination with a STR).

[0113] Bioreactor processes and systems have been developed, for example, to optimize gas exchange, provide sufficient oxygen to maintain cell growth and productivity, and remove CO. Maintaining efficient gas exchange is a key criterion to ensure successful scale-up of cell culture and protein production. Such systems are well known to those skilled in the art.

[0114] The bioreactor used in the fed-batch process described herein has an appropriate volume that allows for the cultivation and growth of biological cells capable of producing a protein of interest. For example, the volume of the bioreactor can be from about 10 milliliters (mL) to about 25,000 L. In another example, the volume of the bioreactor can be from about 0.5 liters (L) to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 10 mL. In some embodiments, the volume of the bioreactor can be from about 10 mL to about 100 mL. In some embodiments, the volume of the bioreactor can be from about 100 mL to about 200 mL. In some embodiments, the volume of the bioreactor can be from about 100 mL to about 300 mL. In some embodiments, the volume of the bioreactor can be from about 100 mL to about 500 mL. In some embodiments, the volume of the bioreactor can be from about 500 mL to about 750 mL. In some embodiments, the volume of the bioreactor can be from about 500 mL to about 1000 mL. In some embodiments, the volume of the bioreactor may be about 500 mL to about 2 L. In some embodiments, the volume of the bioreactor may be about 1 L to about 5 L. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be about 0.5 liters (L) to about 250 L. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 50 L. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 25 L. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 1 L or less. In some embodiments, the volume of the bioreactor may be about 1 L. In some embodiments, the volume of the bioreactor may be about 2 L. In some embodiments, the volume of the bioreactor may be about 3L.In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 100 L or less. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be 1,000 L or more. In some embodiments, the volume of the bioreactor may be about 1,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 10,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor may be about 1,000 L. In some embodiments, the volume of the bioreactor may be about 2,000 L. In some embodiments, the volume of the bioreactor may be about 5,000 L or less. In some embodiments, the volume of the bioreactor can be about 10,000 L or less. In some embodiments, the volume of the bioreactor can be about 15,000 L or less. In some embodiments, the volume of the bioreactor can be about 25,000 L or less.

[0115] 4.1.4 Culture medium In certain embodiments, the medium used in the fed-batch process described herein is suitable for the cells used. In certain embodiments, the medium used in the fed-batch process allows the cells used to grow and produce the protein of interest. In certain embodiments, the feed (e.g., a complex feed) of the fed-batch process comprises a medium that allows the cells used to grow and / or produce the protein of interest. In certain embodiments, one or more feeds are complex feeds. For example, commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) may be used in the fed-batch process. The medium may be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES or NaHCO3), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., Gentamicin™ agents), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source (e.g., another carbohydrate). The medium may be serum-free. Buffers are generally known in the art. The present disclosure is not limited to any particular buffer, and one of skill in the art can select an appropriate buffer or buffer system for use with a particular cell line producing a particular protein. Culture conditions such as temperature, pH, etc. are those previously used with the cells selected for expression and will be apparent to one of skill in the art. Typically, mammalian cells are grown in culture at a near-neutral pH, for example, about pH 6.5 to about pH 7.5. Typically, the temperature of the culture is controlled (e.g., the temperature is typically a physiological 37°C, but can be as low as 30°C). Typically, the oxygen concentration of the culture is between 20-100% air saturation. In certain embodiments, the culture is free of contaminants.

[0116] In certain embodiments, the liquid culture medium may be a chemically defined liquid culture medium (i.e., a liquid culture medium in which all chemical components are known), an animal-derived component-free liquid culture medium (i.e., a liquid culture medium that does not contain mammalian derived components (e.g., proteins or serum), a serum-free liquid culture medium (i.e., a liquid culture medium that does not contain mammalian serum), or a serum-containing liquid culture medium used in a fed-batch process. In some examples, one or more (e.g., one, two, or three) of the liquid culture medium, the first feed medium (e.g., complex feed), and the second feed medium (e.g., complex feed) are chemically defined liquid culture media that are animal-component-free. In some embodiments, each of the feeds comprises a chemically defined liquid culture medium that is animal-component-free. In some examples, each of the liquid culture medium, the first feed medium (e.g., complex feed), and the second feed medium (e.g., complex feed) are different. In some embodiments, each of the feed media (e.g., complex feed) are the same. In some embodiments, the feed medium (e.g., complex feed) differs between the feeds. In some embodiments, some of the feed media (eg, a complex feed) are different from other feed media.

[0117] Examples of liquid culture media that can be used in the fed-batch processes described herein include, for example, CD CHO (ThermoFisher Scientific), CD-C4 (Ecplaza), CD OptiCHO™ Medium (ThermoFisher Scientific), CD OptiCHO™ Medium, BalanCD™ CHO Feed 2, BalanCD™ CHO Feed 4, and HyClone™ ActiPro™.

[0118] The cells may be cultured in a bioreactor containing about 100 to about 200 mL of medium, about 300 to about 1000 mL of medium, about 500 mL to about 3000 mL of medium, about 2000 mL to about 8000 mL of medium, or about 4000 mL to about 15000 mL of medium. In some embodiments, the cells may be cultured in a bioreactor containing about 10,000 to about 20,000 mL of medium, about 15,000 to about 20,000 mL of medium, or about 20,000 to 30,000 mL of medium.

[0119] 4.1.5 Purification of the protein of interest The protein of interest can be isolated or purified from the fed-batch culture using techniques known to those skilled in the art. For example, the protein of interest can be purified using hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofractionation, SDS-PAGE, and ammonium sulfate precipitation, can also be used to purify the protein of interest depending on the protein (e.g., antibody) to be recovered. Following any preliminary purification steps, the mixture containing the protein of interest (e.g., antibody) and contaminants can be subjected to low pH hydrophobic interaction chromatography. In some embodiments, at least or about 5% by weight, e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50% by weight of the protein of interest may be isolated from one or more other components present in the cell culture medium (e.g., mammalian cell or culture medium proteins) or one or more other components present in a mammalian cell lysate (e.g., DNA, RNA, or other proteins). In some embodiments, at least or about 55%, 60%, 65%, 70%, or 75% by weight of the protein of interest may be isolated from one or more other components present in the cell culture medium (e.g., mammalian cell or culture medium proteins) or one or more other components present in a mammalian cell lysate (e.g., DNA, RNA, or other proteins). In some embodiments, at least or about 80%, 85%, 90%, or 95% by weight of the protein of interest may be isolated from one or more other components present in a cell culture medium (e.g., mammalian cell or culture medium proteins) or one or more other components present in a mammalian cell lysate (e.g., DNA, RNA, or other proteins).

[0120] 4.2 Metabolic model In another aspect, a metabolic model of a mammalian cell line is used to model lactate spikes and identify factors capable of reducing lactate spikes. For example, in certain embodiments, the metabolic model is an in silico model. In certain embodiments, the in silico model is a digital twin genome-scale metabolic model (see, for example, U.S. Patent Application Publication No. 2007 / 0298484, the entire contents of which are incorporated herein by reference). In some embodiments, an existing metabolic model of a mammalian cell line is used to model lactate spikes and identify factors capable of reducing lactate spikes. In some embodiments, a metabolic model of a mammalian cell line is created de novo and used to model lactate spikes and identify factors capable of reducing lactate spikes. In some embodiments, the model is a model described in the Examples.

[0121] As provided herein, the metabolic model can be used to retrain with additional experimental data sets, e.g., those that exhibit lactate spikes and those that do not. Once trained, the model is able to predict spikes and is tested to identify one or more factors that can reduce lactate spikes. For example, as provided herein, the model can be used to predict metabolic factors that can reduce lactate spikes. In some embodiments, the metabolic factor is selected from the group consisting of pyruvate and an amino acid. In certain embodiments, the amino acid is selected from the group consisting of asparagine (Asn), glutamic acid (Glu), isoleucine (Ile), leucine (Leu), aspartic acid (Asp), valine (Val), and threonine (Thr). In some embodiments, the model is trained as described in the Examples.

[0122] In one aspect, the model provided herein can identify one or more metabolic factors that should be modified to reduce lactate spikes. For example, the model can predict that one or more metabolic factors should be increased and / or that one or more metabolic factors should be decreased. In some embodiments, the model identifies, for example, that pyruvate concentration should be decreased, and that glutamate, isoleucine, aspartate, threonine, leucine, and valine concentrations should be increased. In some embodiments, the model identifies, for example, that pyruvate concentration should be decreased, but that glutamate, leucine, and valine concentrations should be increased.

[0123] In further aspects, the modeling predictions can be applied to a bioreactor and the output (e.g., titer, rate, and / or yield) can be measured. The bioreactor can have any suitable volume that allows for the culture and growth of biological cells capable of producing a desired protein. For example, the volume of the bioreactor can be about 0.5 liters (L) to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 250 L or less. In some embodiments, the volume of the bioreactor can be about 0.5 liters (L) to about 250 L. In some embodiments, the volume of the bioreactor can be about 50 L or less. In some embodiments, the volume of the bioreactor can be about 1 L to about 50 L. In some embodiments, the volume of the bioreactor can be about 1 L to about 10 L. In some embodiments, the volume of the bioreactor can be about 1 L to about 5 L. In some embodiments, the volume of the bioreactor can be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 1 L to about 25 L, in some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 1 L or less. In some embodiments, the volume of the bioreactor may be about 1 L. In some embodiments, the volume of the bioreactor may be about 2 L. In some embodiments, the volume of the bioreactor may be about 3 L. In some embodiments, the volume of the bioreactor may be about 5 L or less. In some embodiments, the volume of the bioreactor may be about 10 L or less. In some embodiments, the volume of the bioreactor may be about 25 L or less. In some embodiments, the volume of the bioreactor may be about 50 L or less. In some embodiments, the volume of the bioreactor may be about 100 L or less. In some embodiments, the volume of the bioreactor may be about 250 L or less. In some embodiments, the volume of the bioreactor may be about 1,000 L or more.In some embodiments, the volume of the bioreactor can be about 1,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 10,000 L to about 25,000 L. In some embodiments, the volume of the bioreactor can be about 1,000 L, in some embodiments, the volume of the bioreactor can be about 2,000 L, in some embodiments, the volume of the bioreactor can be about 5,000 L or less. In some embodiments, the volume of the bioreactor can be about 10,000 L or less. In some embodiments, the volume of the bioreactor can be about 15,000 L or less. In some embodiments, the volume of the bioreactor can be about 25,000 L or less.

[0124] In some embodiments, a digital computer simulation of a fed-batch process for producing a protein of interest is performed by one or more computing devices. Data characterizing the fed-batch process is received. Further, a model of the mammalian cell line is initialized to enable the simulation of the fed-batch process to characterize lactate spikes based on the received data. The simulation identifies at least one factor that contributes to the lactate spikes. Data characterizing the identified at least one factor is provided. The model can take a variety of forms, including a metabolic model that optionally includes one or more machine learning models, such as a neural network. In some embodiments, the machine learning model (e.g., a neural network, etc.) is trained using data extracted from representative processes that do not exhibit lactate spikes and representative processes that exhibit lactate spikes. The received and / or extracted data, in some embodiments, characterize one or more of the product composition, initial conditions, and nutrient additions of the fed-batch process. Providing the data can take a variety of forms, including displaying the identified at least one factor in a graphical user interface, physically persistently storing the identified at least one factor, loading the identified at least one factor into a memory, or transmitting the identified at least one factor over a network to a remote computing system. Further, in some embodiments, the identified at least one factor indicates a need to reduce pyruvate concentration in one or more feeds (e.g., one or more composite feeds) to cells in a bioreactor in a fed-batch process to reduce lactate spikes.

[0125] In some embodiments, the protein of interest is produced as part of a fed-batch process. Data characterizing the fed-batch process is received. Further, a model of the mammalian cell line is initialized to enable a simulation of the fed-batch process to characterize lactate spikes based on the received data. The simulation identifies at least one factor that contributes to the lactate spikes. One or more operating parameters of the fed-batch process are then modified based on the identified at least one factor. The modification may include decreasing a pyruvate concentration in one or more feeds to the cells in the bioreactor in the fed-batch process. Additionally or alternatively, the modification may include increasing one or more amino acids in one or more feeds to the cells in the bioreactor in the fed-batch process. EXAMPLES

[0126] 5.1 Example 1: Lactic acid spikes observed in multiple fed-batch cultures As shown in Figures 1 and 2, fed-batch cultures of CHO cells recombinantly expressing three different antibodies (Antibody A, Antibody B, and Antibody C) exhibited lactate spike proteins. Antibody A cultures exhibited lactate spikes on days 5-6 of culture, Antibody B cultures exhibited lactate spikes on days 8-9 of culture, and Antibody C exhibited lactate spikes on days 7-8 of culture. As shown in Figure 1, an increase in glucose consumption during the lactate spike followed by a decrease in glucose consumption, a decrease in oxygen consumption, and an increase in culture osmolality with base addition were observed. However, there were no differences in the observed VCD, viability, or glutamine concentrations. Bioreactor conditions for Antibody A were 2 × 10 6 The bioreactor conditions for antibody B included an inoculation density of 0.5×10 vc / mL and a bioreactor (Brx) period of 12 days. 6 Bioreactor conditions for antibody C included an inoculation density of 1.5×10 6The incubation times included an inoculation density of 100 vc / mL and a Brx period of 12 days. The basal medium for each of the three antibodies was different, and the composite feed medium was different for two antibodies. As shown in Figure 3, all cell lines showed a similar magnitude increase in lactate production per cell, and a decrease in glucose consumption per cell was observed for two to three cell lines after lactate spiking.

[0127] 5.2 Example 2: Using Genomic-Based Metabolic Modeling to Identify Process Solutions to Address Rapid Lactic Acid Accumulation While working with a fed-batch process for a late-stage clinical asset, a phenomenon occurred during the high productivity process where cells producing antibody A sporadically produced 2-3 g / L of lactate over a 24-hour period near the cell density peak, followed by excess lactate consumption over the remainder of the fed-batch process. Cultures sporadically exhibited rapid lactate production during days 5-6 of culture. This phenomenon of rapid lactate accumulation midway through the fed-batch process had not been seen before and was termed a "lactate spike." When this phenotype occurred, glucose metabolism was reduced, product expression was reduced, and product quality was compromised. Notably, an increase in glucose consumption during the lactate spike, followed by a decrease in glucose consumption, a decrease in oxygen consumption, and an increase in culture osmolality with base addition were observed. All cell lines tested showed a similar magnitude increase in lactate production per cell, and a decrease in glucose consumption per cell was observed for two to three cell lines following the lactate spike. No differences in viable cell density (VCD), viability, and glutamine concentration in the culture medium were observed.

[0128] We have attempted various approaches to reduce or eliminate this lactate spike by adjusting the glucose feeding strategy and the rate of the composite feed informed by spent medium analysis, but these were unsuccessful.To understand and eliminate the sudden lactate accumulation and identify target hypotheses that can be pursued to reduce the experimental burden, we modeled the lactate spike using an existing metabolic model of the CHO cell line.In particular, we retrained the existing genome-based metabolic model of the cell line with two additional experimental data sets, one showing lactate spikes and one not.Once trained, the model was able to predict spikes and was used to perform a sensitivity analysis on which medium components were most likely to contribute to lactate accumulation.

[0129] Model-based sensitivity analysis identified pyruvate along with glutamate, leucine, threonine, asparagine, aspartate, isoleucine, and valine as key feed components associated with rapid lactate accumulation. The model predicted that aspartate, threonine, glutamate, leucine, isoleucine, and valine concentrations should be increased while asparagine and pyruvate concentrations should be decreased. When these results were tested in a 3L bioreactor, decreasing pyruvate concentration in the combined feed was the largest contributor to eliminating the lactate spike and simultaneously increasing titer by 20%. These two covarying results of decreased lactate and increased titer are mutually exclusive, since a decrease in carbon flux through the pyruvate metabolic node would be expected to decrease flux to both the lactate and TCA cycles. Although a decrease in flux to the TCA cycle (tricarboxylic acid cycle) would result in a decrease in titer due to oxidative phosphorylation, these results indicate that a decrease in pyruvate improves the redox balance of the cells, resulting in decreased lactate and increased productivity of the culture. To explain this counterintuitive result, we investigated these results using a metabolic model using flux analysis.

[0130] 5.3 Example 3: Using the Digital Twin to Identify Process Solutions to Resolve Rapid Lactic Acid Buildup This example demonstrates that reducing pyruvate concentration in the feeding medium eliminates the sudden formation of lactate. This example proposes that the lactate spike occurs due to the activation of glucose uptake by high concentrations of extracellular pyruvate. The sudden decrease in lactate occurs due to the inhibitory effect of lactate on pfk, which limits glycolytic flux. This example demonstrates that lower pyruvate concentration in the medium balances the rates of glycolytic flux and TCA flux.

[0131] Background and Objectives: During process optimization, a cell culture process sporadically produced 2-3 g / L of lactate over a 24-hour period (i.e., a "lactate spike"). The lactate spike reduced glucose metabolism, decreased product expression, and reduced product quality. To reduce or eliminate the lactate spike, various approaches (e.g., adjusting the rate of glucose feed and composite feed addition informed by spent medium analysis) were attempted. These approaches were unsuccessful in mitigating lactate accumulation, and a metabolic model of the cell line was used to model the lactate spike and identify target hypotheses that could be pursued to eliminate the sudden lactate accumulation.

[0132] Antibody A cultures with a lactate spike phenotype (Figures 4A-C, (solid lines) and Figure 1) showed rapid lactate accumulation followed by reduced glucose consumption, reduced oxygen uptake and increased osmolality (due to base addition). Antibody B and Antibody C cultures also showed lactate spikes and changes in glucose consumption after lactate spikes (Figures 2 and 3). Combined feed and glucose feeding strategies as well as adjustments to copper concentrations did not prevent the possible occurrence of a lactate spike.

[0133] Experimental approach and results: Use of the in silico digital twin to identify target solutions. The digital twin developed specifically for the clone was retrained with two additional experimental data sets, one showing lactate spikes and one not. See Figures 5A and 5B. In particular, data (e.g., features, etc.) extracted from two representative processes showing "no lactate spikes" (M19L059) or "lactate spikes" (M19L062) were used to train one or more machine learning models that form part of the metabolic model (Figure 6). The digital twin is composed of three elements: a reactor model that considers all component concentrations and volumes added to or removed from the bioreactor, an extracellular reaction model that considers chemical reactions occurring outside the cell, and a dynamic cell model that describes concentration changes due to cellular metabolism. The dynamic cell model is composed of a metabolic network model (see, e.g., Hefzi H., et. al., "A Consensus Genome-Scale Reconstruction of Chinese Hamster Ovary Cell Metabolism", Cell Systems, 3:434-443) and a recurrent neural network (RNN) model. Model inputs (i.e., extracted features, etc.) included product composition, initial conditions (VCD, volume, pH, etc.), and nutrient additions. The digital twin is trained by first defining the number of hyperparameters in the RNN and then using an 80 / 20 split of the process data in training (80%) and testing (20%) proportions. The best hyperparameter values ​​are then used to cross-validate the model by redistributing the process data to the training-testing split. The average of the five cross-validation metrics is used to determine the predictive quality of the model. After training the models with similar inputs, a sensitivity analysis was performed on the medium components most likely to contribute to lactate accumulation. In particular, the digital twin was used for sensitivity analysis on the feed medium components most likely to contribute to rapid lactate formation. This analysis can be used to modify one or more operating parameters of the system / process (eg, a fed-batch process, etc.).For example, the sensitivity analysis can identify at least one factor that indicates a need to decrease the concentration of pyruvate in one or more feeds to cells in a bioreactor in a fed-batch process to reduce lactate spikes. As another example, the sensitivity analysis can identify at least one factor that indicates a need to increase the concentration of one or more amino acids in one or more feeds to cells in a bioreactor in a fed-batch process to reduce lactate spikes.

[0134] Sensitivity Analysis. A local sensitivity analysis was performed on single feed components. The heat map in Figure 7 identifies components whose concentrations correlate with rapid lactate formation. Table 4 provides an overview of manipulations to reduce lactate formation. The model-based sensitivity analysis identified pyruvate, glutamate, leucine, isoleucine, asparagine, aspartate, threonine, and valine as the key medium components most closely associated with rapid lactate accumulation.

[0135] [Table 4]

[0136] Experimental validation of the sensitivity analysis. Media components with the strongest correlations were mapped to entry points into the TCA cycle (Figure 8). The experiment was designed to target multiple entry points into the TCA cycle to increase the chances of successfully reducing lactate spikes. See Figure 12.

[0137] Experimental Validation Results. Results were tested in a 3L bioreactor and reducing pyruvate concentration in the composite feed was the largest contributor to eliminating lactate spikes. Reducing pyruvate concentration in the feed medium consistently reduced lactate and increased titer (approximately 20%). Reducing pyruvate concentration increased flux to the TCA cycle and increased titer by 20%. See Figures 9A-B and 10A-B and Table 5.

[0138] [Table 5]

[0139] Consideration: The key difference between the control and low pyruvate medium processes is that the glucose uptake rate is significantly higher (1.5-fold) in the control process. Surprisingly, the TCA cycle flux in both processes is similar. Thus, reducing the pyruvate concentration in the medium reduces lactate production, primarily due to a decrease in the glucose uptake rate.

[0140] Despite the higher pyruvate uptake by the control process, the flux from glucose to lactate is higher in the control (60%) than in the low pyruvate medium process (25%). Thus, the lactate spike in the control process is caused by the higher glucose uptake.

[0141] High glucose uptake and glycolytic flux are driven by cytoplasmic NAD + The cell consumes NAD faster than the asp-mal cycle can replenish it. + , resulting in a lactate spike.

[0142] Finally, the mechanism by which high concentrations of pyruvate in the medium cause cells to consume more glucose is unknown. However, without wishing to be bound by theory, it is hypothesized that this effect is regulatory. The rapid decrease in lactate may be due to the inhibitory effect of lactate on the enzyme pfk, which reduces flux through glycolysis. This increases the NAD+ / NADH ratio, allowing cells to consume lactate as a substrate again. See Figure 11.

[0143] Decreasing the pyruvate concentration in the feeding medium eliminated the sudden formation of lactate. It is proposed that the lactate spike occurs due to activation of glucose uptake by high concentrations of extracellular pyruvate. The sudden lactate decrease occurs due to the inhibitory effect of lactate on pfk, which limits glycolytic flux. The lower pyruvate concentration in the medium balanced the rates of glycolytic and TCA fluxes. Studies are planned to monitor the intracellular and extracellular redox environment to confirm their role in the lactate spike.

[0144] References for Example 2 1.Wilkens CA, Gerdtzen ZP.PLoS ONE.2015, 10(3):1-15 2. Hartley, F., et al., Biotech and Bioeng.2018, 115:1890-1903. 3. Mulukutla, BC, et al., Trends in Biotechnol, 2016, 34(8):638-651. 4. Moller, J., et al., Eng Life Sci., 2021, 100-114.

[0145] 6. Embodiment The present invention provides the following non-limiting embodiments. 1. A method for reducing lactate spikes in a fed-batch process for producing a protein of interest, comprising reducing pyruvate concentration in one or more feeds to cells containing a nucleic acid encoding the protein in a bioreactor in the fed-batch process. 2. A method for increasing the titer of a protein of interest produced by cells in a fed-batch process, comprising decreasing the pyruvate concentration in one or more feeds to cells in a bioreactor in the fed-batch process. 3. The method of embodiment 1, further comprising increasing the concentration of one or more amino acids in the one or more feeds. 4. The method of embodiment 2, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. 5. The method of embodiment 2, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. 6. The method of embodiment 2, wherein the one or more amino acids are glutamic acid, valine, or a combination thereof. 7. The method of any one of embodiments 3 to 6, wherein the amino acid concentration is increased by about 0% to about 100%. 8. The method of any one of embodiments 1 to 7, wherein the pyruvate concentration is reduced by about 65% to about 100%. 9. The method of any one of embodiments 1 to 8, wherein the cell is a CHO cell. 10. The method of any one of embodiments 1 to 9, wherein the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. 11. Antibodies include glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCA-1), prostate-specific antigen (PSA ... 11. The method of embodiment 10, wherein the antibody binds to the IL-10 antibody, IL-10 antigen-1, PCTA-1, CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin. 12. The method of embodiment 10, wherein the antibody binds to an antigen of the pathogen. 13. The method of embodiment 12, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite. 14. The method of embodiment 10, wherein the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17. 15. A method for producing a protein of interest, comprising: (a) feeding-batch culturing cells comprising a nucleic acid encoding a protein in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culturing comprising adding a volume of one or more feeds having a pyruvate concentration that is 65% to 100% lower than the pyruvate concentration used in feeding-batch culturing of the cells under the same conditions under which a lactate spike is observed; (b) purifying the protein from the cells or liquid culture medium; and A method comprising: 16. The method of embodiment 15, wherein the concentration of one or more amino acids in the one or more feeds is increased by about 0% to about 100% compared to a fed-batch culture of the cells under the same conditions in which a lactate spike is observed. 17. The method of embodiment 16, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. 18. The method of embodiment 16, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. 19. The method of embodiment 16, wherein the one or more amino acids are glutamic acid, valine, or a combination thereof. 20. The method of any one of embodiments 15 to 19, wherein the cell is a CHO cell. 21. The method according to any one of embodiments 15 to 20, wherein the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. 22. Antibodies include glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCA-1), and HER2 / neu. 22. The method of embodiment 21, wherein the antibody binds to the IL-10 antibody, IL-10 antigen-1, PCTA-1, CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin. 23. The method of embodiment 21, wherein the antibody binds to an antigen of the pathogen. 24. The method of embodiment 23, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite. 25. The method of embodiment 21, wherein the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17. 26. A method for providing a digital computer simulation of a fed-batch process for producing a protein of interest, the method being implemented by one or more computing devices, comprising: Receiving data characterizing the fed-batch process; Initializing a model of a mammalian cell line; simulating the fed-batch process using the initialized model and the received data to characterize the lactate spike; Identifying at least one factor that contributes to the lactate spike based on the simulation; and providing data characterizing at least one identified factor; A method comprising: 27. The method of embodiment 26, wherein the data provided characterizes one or more of the product composition, initial conditions, and nutrient additions of the fed-batch process. 28. The method of embodiment 26 or 27, wherein the model is a metabolic model. 29. The method of any one of embodiments 26 to 28, wherein the model includes one or more machine learning models. 30. The method of embodiment 29, wherein the one or more machine learning models include a neural network. 31. Training a neural network using data extracted from representative processes that do not exhibit lactate spikes and representative processes that do exhibit lactate spikes. 31. The method of embodiment 30, further comprising: 32. The method of embodiment 31, wherein the extracted data characterizes one or more of product composition, initial conditions, and nutrient additions of a fed-batch process. 33. Providing data is Displaying the identified at least one factor in a graphical user interface, physically persistently storing the identified at least one factor, loading the identified at least one factor into a memory, or transmitting the identified at least one factor over a network to a remote computing system. The method of any one of embodiments 26 to 32, comprising one or more of: 34. The method of any one of embodiments 26 to 33, wherein the at least one identified factor indicates that the pyruvate concentration in one or more feeds to the cells in a bioreactor in a fed-batch process needs to be reduced in order to reduce lactate spikes. 35. The method of any one of embodiments 26 to 34, wherein the at least one identified factor indicates that the concentration of one or more amino acids in one or more feeds to the cells in a bioreactor in a fed-batch process needs to be increased to reduce lactate spikes. 36. A method for producing a protein of interest as part of a fed-batch process, comprising: Receiving data characterizing the fed-batch process; Initializing a model of a mammalian cell line; simulating the fed-batch process using the initialized model and the received data to characterize the lactate spike; Identifying at least one factor that contributes to the lactate spike based on the simulation; and modifying one or more operating parameters of the fed-batch process based on the at least one identified factor; A method comprising: 37. The method of embodiment 36, wherein the modification comprises reducing the pyruvate concentration in one or more feeds to the cells in the bioreactor in the fed-batch process. 38. The method of embodiment 36 or 37, wherein the modification comprises increasing one or more amino acids in one or more feeds to the cells in the bioreactor in a fed-batch process. 39. A method for reducing lactate spikes during a fed-batch culture of cells, the method comprising: feeding-batch culturing cells, the cells comprising a nucleic acid encoding a protein of interest, in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culturing comprising adding a volume of a first complex feed within 0-3 days after initiation of the fed-batch culture of the cells, the first complex feed having a first pyruvate concentration, the first pyruvate concentration being about 65% to about 100% lower than a pyruvate concentration used for the fed-batch culture of the cells under the same conditions at which lactate spikes are observed. 40. The method of embodiment 39, wherein the concentration of one or more amino acids is increased. 41. The method of embodiment 40, wherein the increase in concentration of one or more amino acids is from about 0% to about 100%. 42. The method of embodiment 40 or 41, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. 43. The method of embodiment 40 or 41, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. 44. The method of embodiment 40 or 41, wherein the one or more amino acids are glutamic acid, valine, or a combination thereof. 45. The method according to any one of embodiments 39 to 44, wherein the cell is a CHO cell. 46. ​​The method of any one of embodiments 39 to 45, wherein the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. 47. Antibodies include glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCA-1), and thyroglobulin. 47. The method of embodiment 46, wherein the antibody binds to the IL-10 antibody, IL-10 antigen-1, PCTA-1, CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin. 48. The method of embodiment 46, wherein the antibody binds to an antigen of the pathogen. 49. The method of embodiment 48, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite. 50. The method of embodiment 46, wherein the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17. 51. A method for identifying a pyruvate concentration to be used in one or more complex feeds in a fed-batch process, comprising: (a) fed-batch culturing cells comprising a nucleic acid encoding a protein of interest in a first bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culturing comprising adding a volume of one or more complex feeds having a first pyruvate concentration; (b) feeding-batch culturing the same cells in a second bioreactor under the same conditions used in the fed-batch culturing of step (a), except that the one or more composite feeds have a second pyruvate concentration, the second pyruvate concentration being about 65%-100% lower than the first pyruvate concentration; (c) measuring the lactic acid concentration in the fed-batch culture of step (a) within about 12 to about 72 hours after each composite feed, and measuring the lactic acid concentration in the fed-batch culture of step (b) within about 12 to about 72 hours after each composite feed; (d) comparing the lactate concentration measured for the fed-batch culture of step (a) with the lactate concentration measured for the fed-batch culture of step (b), wherein a decrease in the lactate concentration for the fed-batch culture of step (b) compared to the lactate concentration for the fed-batch culture of step (a) indicates that the pyruvate concentration used in the one or more composite feeds in the fed-batch culture of step (b) is more favorable for the fed-batch culture of cells in a bioreactor; (e) performing production of the protein by fed-batch culturing the cells containing the nucleic acid under conditions sufficient for the cells to produce the recombinant protein, the fed-batch culturing comprising adding a volume of one or more complex feeds having a second pyruvate concentration; A method comprising: 52. The method of embodiment 51, wherein the cell is a CHO cell. 53. The method of embodiment 51 or 52, wherein the protein of interest is an antibody, a cytokine, an antigen, an enzyme, or a coagulant. 54. Antibodies include glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCA-1), and HER2 / neu. 54. The method of embodiment 53, wherein the antibody binds to one or more of the following: antigen-1, PCTA-1, CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin. 55. The method of embodiment 53, wherein the antibody binds to an antigen of the pathogen. 56. The method of embodiment 55, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite. 57. The method of embodiment 53, wherein the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17. 58. A method for reducing lactate spikes during fed-batch culture of cells, the method comprising: feeding-batch culturing cells comprising a nucleic acid encoding a protein of interest in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culturing comprising adding one or more feeds during growth of the cells and reducing a pyruvate concentration in the one or more additional composite feeds prior to a cell density peak, wherein the pyruvate concentration in the one or more additional composite feeds is reduced by 65% ​​to 100% compared to the pyruvate concentration in the one or more composite feeds during growth of the cells. 59. The method of embodiment 58, wherein the cell density peak is from about day 6 to about day 7 of the fed-batch culture of the cells, and the decrease in pyruvate concentration in the one or more additional complex feeds is from about day 3 to about day 5 of the fed-batch culture of the cells. 60. The method of embodiment 58 or 59, wherein the concentration of one or more amino acids in the one or more additional feeds is increased prior to the cell density peak. 61. The method according to embodiment 60, wherein the concentration of one or more amino acids in the one or more additional complex feeds is increased by 0% to 100% compared to the concentration of one or more amino acids in the one or more complexes during growth of the cells. 62. The method of embodiment 60 or 61, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof. 63. The method of embodiment 60 or 61, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, and leucine, or a combination thereof. 64. The method of embodiment 60 or 61, wherein the one or more amino acids are glutamic acid, valine, or a combination thereof. 65. The method of any one of embodiments 58 to 64, wherein the cell is a CHO cell.

[0146] In the above description and in the claims, phrases such as "at least one of" or "one or more of" may be used with a conjunctive list of the preceding elements or features. The term "and / or" may also be used with a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such phrases are intended to mean any of the listed elements or features individually, or any of the listed elements or features in combination with any of the other listed elements or features. For example, the phrases "at least one of A and B," "one or more of A and B," and "A and / or B" are each intended to mean "A alone, B alone, or A and B together." A similar interpretation is also intended for lists containing more than two items. For example, the phrases "at least one of A, B, and C," "one or more of A, B, and C," and "A, B, and / or C" are each intended to mean "A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together." Use of the term "based on" above and in the claims is intended to mean "based at least in part on," such that unrecited features or elements are also permissible.

[0147] The subject matter described herein, including the initialized model and any resulting execution of such model, may be implemented using a variety of computing devices including computing systems that include back-end components (e.g., as a data server), or that include middleware components (e.g., an application server), or that include front-end components (e.g., a client computer having a graphical user interface or web browser through which a user may interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, or front-end components. The components of the system may be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include a local area network ("local area network, LAN"), a wide area network ("wide area network, WAN"), and the Internet. These various embodiments may include the implementation of one or more computer programs executable and / or interpretable on a programmable system including at least one programmable processor, which may be a special purpose or general purpose processor connected to receive data and instructions from and transmit data and instructions to a storage system, at least one input device, and at least one output device.

[0148] The subject matter described herein may be implemented as a system, an apparatus, a method, and / or an article, depending on the desired configuration. The implementations described in the above description do not represent all implementations consistent with the subject matter described herein. Rather, they are merely some examples consistent with aspects related to the described subject matter. Although some variations have been described in detail above, other modifications or additions are contemplated. In particular, further features and / or variations may be provided in addition to those described herein. For example, the implementations described above may relate to various combinations and subcombinations of the disclosed features and / or combinations and subcombinations of some further features disclosed above. In addition, the logic flow described herein does not necessarily require the particular order shown, or sequential order, to achieve desirable results. Other embodiments may be within the scope of the following claims.

Claims

1. 1. A method for reducing lactate spikes in a fed-batch process for producing a protein of interest, comprising reducing the pyruvate concentration in one or more feeds to cells comprising a nucleic acid encoding the protein in a bioreactor in the fed-batch process.

2. The method described in claim 1, which increases the titer of a target protein.

3. 10. The method of claim 1, further comprising increasing the concentration of one or more amino acids in the one or more feeds.

4. 3. The method of claim 2, wherein the one or more amino acids are selected from the group consisting of glutamic acid, valine, leucine, threonine, aspartic acid, and isoleucine, or a combination thereof.

5. 4. The method of claim 3, wherein the amino acid concentration is increased by about 0% to about 100%.

6. 10. The method of claim 1, wherein the pyruvate concentration is reduced by about 65% to about 100%.

7. 10. The method of claim 1, wherein the cells are CHO cells.

8. The method of claim 1 , wherein the protein of interest is an antibody, cytokine, antigen, enzyme, or coagulant.

9. (a) The antibody is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, and prostate-carcinoma tumor antigen-1. antigen-1, PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin; (b) the antibody binds to an antigen of the pathogen; (c) the antibody binds to an antigen of a pathogen, and the pathogen is a virus, bacterium, fungus, or parasite; or (d) The method of claim 8, wherein the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17.

10. (a) feeding the cells in a bioreactor under conditions sufficient for the cells to produce the protein, the fed-batch culture comprising adding a volume of one or more composite feeds having a pyruvate concentration that is 65% to 100% lower than the pyruvate concentration used in feeding the cells under the same conditions at which a lactate spike is observed; (b) purifying the protein from the cells or liquid culture medium; The method of claim 1 , comprising:

11. 1. A method for providing a digital computer simulation of a fed-batch process for producing a protein of interest, the method being implemented by one or more computing devices and comprising: receiving data characterizing the fed-batch process; initializing a model of a mammalian cell line; simulating the fed-batch process using the initialized model and the received data to characterize lactate spikes; identifying at least one factor that contributes to the lactate spike based on the simulation; and providing data characterizing the identified at least one factor; A method comprising:

12. 12. The method of claim 11, wherein the provided data characterizes one or more of product composition, initial conditions, and nutrient additions of the fed-batch process.

13. The method of claim 11 , wherein the model is a metabolic model.

14. The method of claim 11 , wherein the model comprises one or more machine learning models.

15. The method of claim 14 , wherein the one or more machine learning models include a neural network.

16. training said neural network using data extracted from representative processes that do not exhibit lactate spikes and representative processes that do exhibit lactate spikes; 16. The method of claim 15, further comprising:

17. Providing the data comprises: displaying the identified at least one factor in a graphical user interface, physically persistently storing the identified at least one factor, loading the identified at least one factor into a memory, or transmitting the identified at least one factor over a network to a remote computing system. The method of claim 11 , comprising one or more of:

18. 12. The method of claim 11, wherein the identified at least one factor indicates (a) a need to decrease the concentration of pyruvate in one or more feeds to cells in a bioreactor in the fed-batch process to reduce the lactate spike, or (b) a need to increase the concentration of one or more amino acids in one or more feeds to cells in a bioreactor in the fed-batch process to reduce the lactate spike.

19. 1. A method for producing a protein of interest as part of a fed-batch process, comprising: receiving data characterizing the fed-batch process; initializing a model of a mammalian cell line; simulating the fed-batch process using the initialized model and the received data to characterize lactate spikes; identifying at least one factor that contributes to the lactate spike based on the simulation; and modifying one or more operating parameters of the fed-batch process based on the identified at least one factor; A method comprising:

20. 20. The method of claim 19, wherein the modification comprises (a) decreasing the pyruvate concentration in one or more feeds to cells in a bioreactor in the fed-batch process, or (b) increasing one or more amino acids in one or more feeds to cells in a bioreactor in the fed-batch process.

21. The method of claim 1, wherein the method comprises feeding culture the cells in a bioreactor under conditions sufficient for the cells to produce the protein, the feeding culture comprising adding a volume of a first composite feed within 0 to 3 days after initiation of the fed-batch culture of the cells, the first composite feed having a first pyruvate concentration, the first pyruvate concentration being about 65% to about 100% lower than a pyruvate concentration used in feeding culture of the cells under the same conditions at which a lactate spike is observed.

22. 1. A method for identifying a pyruvate concentration to use in one or more complex feeds in a fed-batch process, comprising: (a) fed-batch culturing cells comprising a nucleic acid encoding a protein of interest in a first bioreactor under conditions sufficient for the cells to produce the protein, wherein the fed-batch culturing comprises adding a volume of one or more complex feeds having a first pyruvate concentration; (b) feeding the same cells in a second bioreactor under the same conditions as used in the fed-batch cultivation of step (a), except that the one or more complex feeds have a second pyruvate concentration, wherein the second pyruvate concentration is about 65% to 100% lower than the first pyruvate concentration; (c) measuring the lactic acid concentration in the fed-batch culture of step (a) within about 12 to about 72 hours after each composite feed, and measuring the lactic acid concentration in the fed-batch culture of step (b) within about 12 to about 72 hours after each composite feed; (d) comparing the lactate concentration measured for the fed-batch culture of step (a) with the lactate concentration measured for the fed-batch culture of step (b), wherein a decrease in the lactate concentration for the fed-batch culture of step (b) compared to the lactate concentration for the fed-batch culture of step (a) indicates that the pyruvate concentration used in the one or more complex feeds in the fed-batch culture of step (b) is more favorable for the fed-batch culture of the cells in the bioreactor; (e) performing production of the protein by fed-batch culturing the cells containing the nucleic acid under conditions sufficient for the cells to produce the recombinant protein, the fed-batch culturing comprising adding a volume of one or more complex feeds having the second pyruvate concentration; A method comprising:

23. 23. The method of claim 22, wherein the cell is a CHO cell.

24. 23. The method of claim 22, wherein the protein of interest is an antibody, cytokine, antigen, enzyme, or coagulant.

25. (a) The antibody is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), B-cell maturation antigen (BCMA), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, GPRC5D, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, and prostate-carcinoma tumor antigen-1. antigen-1, PCTA-1), CD70, CD20, MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, or mesothelin; (b) the antibody binds to an antigen of the pathogen; (c) the antibody binds to an antigen of a pathogen, and the pathogen is a virus, bacterium, fungus, or parasite; or (d) the cytokine is IL-12, IL-23, IL-1β, IL-6, IL-15, IL-2, IL-5, TNF-α, IL-9, or IL-17.

26. The method of claim 1, wherein the method comprises feeding the cells in a bioreactor under conditions sufficient for the cells to produce the protein, the feeding comprising adding one or more complex feeds during growth of the cells and reducing the pyruvate concentration in the one or more additional complex feeds prior to a cell density peak, wherein the pyruvate concentration in the one or more additional complex feeds is reduced by 65% ​​to 100% compared to the pyruvate concentration in the one or more complex feeds during growth of the cells.

27. 27. The method of claim 26, wherein the cell density peak is from about day 6 to about day 7 of the fed-batch culture of the cells and the decrease in pyruvate concentration in the one or more additional feeds is from about day 3 to about day 5 of the fed-batch culture of the cells.

28. 27. The method of claim 26, wherein the concentration of one or more amino acids in the one or more additional feeds is increased prior to a cell density peak.

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