Cell culture strategy for modulating protein glycosylation

Abstract

The presently disclosed subject matter relates to cell culture media and cell culture strategies for modulating the glycosylation pattern, e.g., fucosylation and / or galactosylation pattern, of a glycoprotein of interest, e.g., an antibody, as well as cell cultures and glycoprotein compositions prepared using such matrices and / or strategies.

Description

[0001] This application is a divisional application of international application number PCT / US2019 / 045900, with an international application date of August 9, 2019, and a date of entry into the Chinese national phase of March 25, 2021. The Chinese national application number is 201980063223.3, and the name of the invention is “Cell culture strategy for regulating protein glycosylation”.

[0002] Cross-references to related patent applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 717,751, filed on August 10, 2018, the contents of which are incorporated by reference in their entirety. Technical Field

[0004] The presently disclosed subject matter relates to cell culture media and cell culture strategies for modulating the glycosylation pattern (e.g., fucosylation and / or galactosylation pattern) of a glycoprotein (e.g., an antibody) of interest, as well as cell culture and glycoprotein compositions prepared using such media and / or strategies. Background Art

[0005] N-linked glycosylation can affect the physicochemical properties of recombinant glycoproteins, including monoclonal antibodies (mAbs). These properties include protein folding, solubility, binding, stability, immunogenicity, and pharmacokinetics (Varki A. (1993), Glycobiology, 3 (2), 97-130). Depending on the mechanism of action of the therapeutic mAb, the efficacy of the mAb may depend on complement dependent cytotoxicity (CDC) activity and / or antibody dependent cell-mediated cytotoxicity (ADCC) activity. In some studies, mAbs with higher terminal galactosylation (which refers to the addition of terminal galactose residues to N-acetyl-glucosamine (GlcNAc)) have higher CDC activity (Boyd et al., (1995) Mol. Immunology 32, 1311-1318; Hodoniczky et al., (2005), Biotechnol. Prog. 21, 1644-1652; Tsuchiya et al., (1989) J. Rheumatol., 16, 285-290). Therefore, for mAb products with CDC as a mechanism of action, an optimal and consistent level of galactosylation may be highly desirable. In other studies, mAbs with lower core fucosylation (which refers to the addition of fucose residues to the oligosaccharide core) have higher ADCC activity (Ferrara et al., (2011), Proc. Natl. Acad. Sci., 108, 12669-12674; Shields et al., (2002), J. Biol. Chem., 277, 26733-26740; Shinkawa et al., (2003) J. Biol. Chem., 278, 3466-3473; Thomann et al., (2016) Molecular Immunology, 73, 60-75). Therefore, for mAb products with ADCC as a mechanism of action, an optimal and consistent level of non-fucosylation (i.e., lack of core fucose on N-linked glycans) may be highly desirable.Strategies for modulating mAb glycosylation (e.g., galactosylation and / or fucosylation) during cell culture generally fall into one of four categories: (1) genetic engineering of recombinant cell lines (Louie et al., (2016), Biotechnol Bioeng, 114(3), 632-644; Yamane-Ohnuki et al., (2004), Biotechnol Bioeng, 87(5), 614-622); (2) addition of enzyme inhibitors (Allen et al., (2016), ACS Chem Biol, 11(10), 2734-2743; Okeley et al., (2013), PNAS, 110(14), 5404-5409); (3) modification of the levels of cofactors and substrates for glycosylation, including supplementation with alternative sugars (Hossler et al., (2014), Biotechnol Prog, 30(6), 1419-1431; Hossler et al., (2017), mAbs, 9(4), 715-734); and (4) fine-tuning of culture process parameters (Konno et al., (2012), Cytotechnology, 64, 249-265). Although many cell culture process parameters are known to affect galactosylation (Hossler et al., (2009), Glycobiology, 19(9), 936-949), fewer parameters have been identified to control fucosylation. Summary of the invention

[0006] The subject matter disclosed herein relates to regulating the glycosylation pattern (e.g., galactosylation and / or fucosylation pattern) of a recombinant glycoprotein of interest. For example, but not by way of limitation, embodiments described herein relate to regulating glycosylation to achieve or maintain a desired glycoprotein glycosylation pattern (e.g., galactosylation and / or fucosylation pattern). Methods by which glycosylation can be regulated according to the present disclosure include, but are not limited to: (1) controlling the manganese (Mn) concentration of the cell culture medium and / or cell culture, e.g., Mn concentration analysis of the feedstock, supplementation of Mn to the cell culture medium and / or during cell culture, and / or minimizing Mn loss from the cell culture by establishing a reduced pH target or set point for substrate pH adjustment prior to high temperature, short time (HTST) heat treatment of the substrate; (2) controlling process parameters during cell culture, e.g., pCO2, substrate storage duration, culture duration, culture temperature, and osmotic pressure / Na + ;And (3) control the cell culture medium and / or cell culture galactose and / or fucose concentration.The subject matter of the present disclosure also relates to cell culture and glycoprotein compositions prepared when such process parameters are controlled as described herein.

[0007] In certain embodiments, the present disclosure relates to a method for regulating the glycosylation pattern of a glycoprotein of interest in cell culture, comprising: regulating the following parameters in a cell culture medium and / or in a cell culture environment, alone or in any combination: Mn concentration from about 1 nM to about 20,000 nM under high partial pressure CO2 (pCO2) conditions; Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; pCO2 from about 10 mmHg to about 250 mmHg; cell culture medium storage duration from about 0 hours to about 120 hours before inoculation at a temperature of about 25°C to 39°C; cell culture duration from about 0 days to about 150 days; Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C.

[0008] In certain embodiments, the cell culture environment is in a bioreactor with or without cells. In certain embodiments, the low pCO2 condition is from about 10 mmHg to about 100 mmHg, and the high pCO2 condition is from about 20 mmHg to about 250 mmHg.

[0009] In certain embodiments, the duration of pCO2 regulation covers at least the first half of the cell culture duration.

[0010] In certain embodiments, the glycoprotein of interest is a recombinant protein. In certain embodiments, the recombinant protein is an antibody or antibody fragment, scFv (single chain variable fragment), BsDb (bispecific diabody), scBsDb (single chain bispecific diabody), scBsTaFv (single chain bispecific tandem variable domain), DNL-(Fab)3 (docking locked trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody). In certain embodiments, the antibody is a chimeric antibody, a humanized antibody or a human antibody. In certain embodiments, the antibody is an anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody is ocrelizumab. In certain embodiments, the antibodies or antibody fragments exhibit: a G0-F% (percentage of non-fucosylated glycoprotein) of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; or a normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; and / or a G0% (percentage of non-galactosylated glycoprotein) of about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%.

[0011] In certain embodiments, the glycosylation is modulated to achieve: increased afucosylation (e.g., G0-F (afucosylated G0)) while decreasing agalactosylation (e.g., G0 (fucosylated, nongalactosylated G0)); or decreased afucosylation (e.g., G0-F) while increasing agalactosylation (e.g., G0); or increased or decreased afucosylation (e.g., G0-F) without affecting agalactosylation (e.g., G0); or increased or decreased afucosylation (e.g., G0) without affecting afucosylation (e.g., G0-F).

[0012] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; and the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: a cell culture medium storage duration from about 0 hours to about 120 hours before inoculation at a temperature of about 25°C to 39°C; a cell culture duration from about 0 days to about 150 days; a Na+ concentration from about 0 mM to about 300 mM; an osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration from about 0 mM to about 60 mM; a fucose concentration from about 0 mM to about 60 mM; and a culture temperature from about 29°C to about 39°C.

[0013] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; and the following parameters in the cell culture medium and / or in the cell culture environment: a cell culture medium storage duration from about 0 hours to about 120 hours before inoculation at a temperature of about 25°C to 39°C; and a cell culture duration from about 0 days to about 150 days.

[0014] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; and the following parameters in the cell culture medium and / or in the cell culture environment: galactose concentration from about 0 mM to about 60 mM; and / or, fucose concentration from about 0 mM to about 60 mM.

[0015] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the cell culture medium storage duration before inoculation and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: Mn concentration from about 1 nM to about 20,000 nM under high partial pressure CO2 (pCO2) conditions; Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; pCO2 from about 10 mmHg to about 250 mmHg; cell culture duration from about 0 days to about 150 days; Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C; wherein at a temperature of about 25°C to 39°C, the cell culture medium storage duration is from about 0 hours to about 120 hours.

[0016] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the cell culture medium storage duration before inoculation and the following parameters in the cell culture medium and / or in the cell culture environment: under high partial pressure CO2 (pCO2) conditions, the Mn concentration is from about 1 nM to about 20,000 nM; or, under low pCO2 conditions, the Mn concentration is from about 1 nM to about 30,000 nM; the pCO2 is from about 10 mmHg to about 250 mmHg; and, the cell culture duration is from about 0 days to about 150 days; wherein at a temperature of about 25°C to 39°C, the cell culture medium storage duration is from about 0 hours to about 120 hours.

[0017] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the cell culture duration and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C, wherein the cell culture duration is from about 0 days to about 150 days.

[0018] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the Na+ concentration to about 0 nM to about 300 nM and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29° C. to about 39° C., wherein the Na+ concentration is from about 0 mM to about 300 mM.

[0019] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises adjusting the Na+ concentration and the pCO2 from about 10 mmHg to about 250 mmHg.

[0020] In certain embodiments, the step of regulating the glycosylation pattern of the glycoprotein of interest comprises: regulating the osmotic pressure and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29° C. to about 39° C., wherein the osmotic pressure is from about 250 mOsm / kg to about 550 mOsm / kg.

[0021] In certain embodiments, the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; adjusting the Na+ concentration from about 0 mM to about 300 mM; and adjusting the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours.

[0022] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises: adjusting the osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg, and the pCO2 from about 10 mmHg to about 250 mmHg.

[0023] In certain embodiments, the regulation of the glycosylation pattern of the glycoprotein of interest comprises regulating the following: the culture temperature from about 29°C to about 39°C, and, the galactose concentration from about 0 mM to about 60 mM; and / or the fucose concentration from about 0 mM to about 60 mM.

[0024] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises: adjusting the pCO2 from about 10 mmHg to about 250 mmHg; and the fucose concentration from about 0 mM to about 60 mM.

[0025] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises adjusting the fucose concentration from about 0 mM to about 60 mM and the culture temperature from about 29°C to about 39°C.

[0026] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises: regulating the pCO2 concentration and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: Mn concentration from about 1 nM to about 20,000 nM under high partial pressure CO2 (pCO2) conditions; Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; cell culture medium storage duration from about 0 hours to about 120 hours before inoculation at a temperature of about 25°C to 39°C; cell culture duration from about 0 days to about 150 days; Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C, wherein the pCO2 concentration is from about 10 mmHg to about 250 mmHg.

[0027] In certain embodiments, the Mn concentration is: from about 1 nM to about 20,000 nM in high pCO2 culture; from about 1 nM to about 10,000 nM, from about 1 nM to about 5,000 nM, from about 1 nM to about 4,000 nM, from about 1 nM to about 3,000 nM, from about 1 nM to about 2,000 nM, from about 1 nM to about 1000 nM in high pCO2 culture; In the culture medium, from about 1 nM to about 500 nM, from about 1 nM to about 100 nM, from about 1 nM to about 50 nM, from about 1 nM to about 20 nM, from about 20 nM to about 2000 nM, from about 20 nM to about 3000 nM, from about 20 nM to about 10000 nM, from about 20 nM to about 20,000 nM, from about 20 nM to about 300 nM, about 30 nM to about 110 nM.

[0028] In certain embodiments, the Mn concentration is: from about 1 nM to about 30,000 nM in low pCO2 culture; from about 1 nM to about 20,000 nM, from about 1 nM to about 10,000 nM, from about 1 nM to about 5,000 nM, from about 1 nM to about 4,000 nM, from about 1 nM to about 3,000 nM, from about 1 nM to about 2000 nM, from about 1 nM to about 1000 nM, from about 1 nM to about 500 nM, from about 1 nM to about 1000 nM, nM, from about 1 nM to about 50 nM, from about 1 nM to about 20 nM, from about 20 nM to about 100 nM, from about 20 nM to about 300 nM, from about 20 nM to about 500 nM, from about 20 nM to about 1000 nM, from about 20 nM to about 2000 nM, from about 20 nM to about 3000 nM, from about 20 nM to about 5000 nM, from about 20 nM to about 10000 nM, from about 20 nM to about 20000 nM, or from about 30 nM to about 110 nM.

[0029] In certain embodiments, adjusting the Mn concentration comprises determining the Mn content in the cell culture feedstock and selecting a feedstock batch to adjust the Mn concentration.

[0030] In certain embodiments, the adjustment of the Mn concentration comprises (i) controlling the material in contact with the cell culture medium or cell culture; or (ii) calculating the concentration of Mn leached in the cell culture medium or during cell culture; or a combination of (i) and (ii) to adjust the Mn concentration. In certain embodiments, the leached Mn is produced by contact of the cell culture and / or cell culture medium with: (i) a filter; (ii) a matrix preparation, storage or culture container; or (iii) a combination of (i) and (ii). In certain embodiments, filters include, but are not limited to, depth filters, columns, membranes, and disks. In certain embodiments, filter materials include, but are not limited to, diatomaceous earth, hollow fibers, or resins.

[0031] In certain embodiments, the cell culture medium is a basal matrix, a reconstituted matrix, a feed matrix, a hydrolysate, a supplement, a serum, or an additive.

[0032] In certain embodiments, the cell culture medium is supplemented during the production phase of the cell culture.

[0033] In certain embodiments, the cell culture medium is supplemented prior to the production phase of the cell culture.

[0034] In certain embodiments, the cell culture medium includes one or more of: Mn, fucose, galactose, and / or Na+, and wherein the supplementation is based on a predetermined schedule or standard.

[0035] In certain embodiments, one or more of Mn, fucose, galactose, and Na+ are supplemented as a single dose, intermittent supplementation, continuous supplementation, semi-continuous supplementation, feedback loop-based supplementation, or a combination of one or more thereof.

[0036] In certain embodiments, the cell culture medium consists essentially of one or more of: i) Mn, ii) fucose, iii) galactose, and / or iv) Na+.

[0037] In certain embodiments, the adjustment of the Mn concentration comprises employing a cell culture medium pH of about 6.1 to about 7.3, or about 6.3 to about 7.3, prior to high temperature, short time (HTST) heat treatment.

[0038] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of pCO2.

[0039] In certain embodiments, the cell culture or cell culture medium is in a bioreactor, and wherein regulation of pCO2 is achieved by regulating: the bioreactor working volume; the bioreactor gas sparging strategy; the bioreactor agitation strategy; the bioreactor matrix exchange strategy, the bioreactor perfusion strategy, the bioreactor feeding strategy, or any combination thereof.

[0040] In certain embodiments, the pCO2 regulation comprises establishing a high pCO2 culture. In certain embodiments, the pCO2 is about 20 mmHg to about 250 mmHg, about 20 mmHg to about 250 mmHg, about 20 mmHg to about 150 mmHg, or about 30 mmHg to about 250 mmHg.

[0041] In certain embodiments, the pCO2 regulation comprises establishing a low pCO2 culture. In certain embodiments, the pCO2 is about 10 mmHg to about 100 mmHg, 10 mmHg to about 80 mmHg, about 20 mmHg to about 70 mmHg, or about 30 mmHg to about 60 mmHg.

[0042] In certain embodiments, said pCO2 adjustment occurs on day 0 of said culturing.

[0043] In certain embodiments, the pCO2 regulation occurs for about a majority of the cell culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0044] In certain embodiments, the pCO2 regulation occurs for about a majority of the production culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0045] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the pre-plating cell culture medium storage duration, wherein the pre-plating cell culture medium storage duration is from about 0 hours to about 120 hours, from about 0 hours to about 72 hours, from about 0 hours to about 48 hours, or from about 0 hours to about 24 hours.

[0046] In certain embodiments, the temperature of the matrix during storage of the pre-seeding cell culture medium is about 25°C to about 39°C, about 30°C to about 39°C, about 35°C to about 39°C, or about 36°C to about 39°C.

[0047] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the duration of the cell culture, wherein the duration of the cell culture is from about 0 days to about 150 days, from about 0 days to about 15 days, from about 0 days to about 12 days, from 0 days to about 7 days, or from about 0 days to about 5 days.

[0048] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the Na+ concentration, wherein the Na+ concentration is about 0 mM to about 300 mM, about 20 mM to about 20 mM, about 30 mM to about 150 mM, or about 40 mM to about 130 mM.

[0049] In certain embodiments, the regulation of the Na+ concentration comprises supplementing the cell culture with a Na compound, wherein the Na compound includes but is not limited to Na2CO3, NaHCO3, NaOH, NaCl, or a combination thereof.

[0050] In certain embodiments, the regulation of the glycosylation pattern of the target glycoprotein comprises regulating the osmotic pressure, wherein the osmotic pressure is about 250mOsm / kg to about 550mOsm / kg, about 300mOsm / kg to about 450mOsm / kg, or about 325mOsm / kg to about 425mOsm / kg. In certain embodiments, the regulation of the osmotic pressure comprises supplementing the cell culture with an osmotic pressure-adjusting matrix component. In certain embodiments, the osmotic pressure-adjusting matrix component is NaCl, KCl, sorbitol, an osmotic protectant, or a combination thereof. In certain embodiments, the osmotic pressure-adjusting matrix component is supplemented as a single dose, an intermittent supplement, a continuous supplement, a semi-continuous supplement, a supplement based on a feedback loop, or a combination of one or more thereof.

[0051] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the galactose concentration, wherein the galactose concentration is from about 0 mM to about 60 mM or from about 0 mM to about 50 mM.

[0052] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the fucose concentration, wherein the fucose concentration is about 0 mM to about 60 mM, 0 mM to about 40 mM, about 0 mM to about 20 mM, or about 0 mM to about 10 mM.

[0053] In certain embodiments, the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture temperature, wherein the cell culture temperature is about 29°C to about 39°C, about 30°C to about 39°C, about 31°C to about 38°C, or about 34°C to about 38°C.

[0054] In certain embodiments, the cell culture temperature is regulated during a production phase of the cell culture.

[0055] In certain embodiments, the cell culture temperature is adjusted prior to a production phase of the cell culture.

[0056] In certain embodiments, the cell culture temperature is adjusted based on a predetermined schedule or criteria.

[0057] In certain embodiments, the cell culture comprises eukaryotic cells. In certain embodiments, the eukaryotic cells are insect cells, avian cells, fungal cells, plant cells or mammalian cells. In certain embodiments, the fungal cells are yeast cells, Pichia cells or any filamentous fungal cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae cells. In certain embodiments, the mammalian cells are CHO cells.

[0058] In certain embodiments, the cell culture is in a bioreactor, including but not limited to single use technology (SUT) bags or bioreactors, WAVE bioreactors, stainless steel bioreactors, flasks, test tubes, and chambers.

[0059] In certain embodiments, the volume of the cell culture is from 1 mL to 10 ml, from 1 mL to 50 ml, from 1 mL to 100 ml, from 1 mL to 200 ml, from 1 mL to 300 ml, from 1 mL to 500 ml, from 1 mL to 1000 ml, from 1 mL to 2000 ml, from 1 mL to 3000 ml, from 1 mL to 4000 ml, from 1 mL to 5000 ml, from 1 mL to 1 L, from 1 mL to 2 L, from 1 mL to 3 L, from 1 mL to 4 L, from 1 mL to 5 L, from 1 mL to 6 L, from 1 mL to 10 L, from 1 mL to 20 L, from 1 m L to 30L, from 1mL to 40L, from 1mL to 50L, from 1mL to 60L, from 1mL to 70L, from 1mL to 100L, from 1mL to 200L, from 1mL to 300L, from 1mL to 400L, from 1mL to 500L, from 1mL to 1000L, from 1mL to 2000L, from 1mL to 3000L, from 1mL to 4000L, from 1mL to 5000L, from 1mL to 10,000L, from 1mL to 20,000L, from 1mL to 30,000L, from 1mL to 30,000L, from 1mL to 35,000L.

[0060] In certain embodiments, the present disclosure relates to a method for preparing a cell culture medium, a feed matrix, a hydrolysate, or an additive, comprising adjusting one or more of the following steps: a Mn concentration in a high partial pressure CO2 (pCO2) culture from about 1 nM to about 20,000 nM; a Mn concentration in a low pCO2 culture from about 1 nM to about 30,000 nM; the pCO2 from about 10 mmHg to about 250 mmHg; a cell culture medium storage duration from about 0 hours to about 120 hours before inoculation; a cell culture duration from about 0 days to about 150 days; a Na+ concentration from about 0 mM to about 300 mM; an osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration from about 0 mM to about 60 mM; a fucose concentration from about 0 mM to about 60 mM; and a culture temperature from about 29°C to about 39°C; wherein the cell culture medium, the feed matrix, the hydrolysate, or the additive adjusts the glycosylation pattern of a glycoprotein of interest.

[0061] In certain embodiments, the method involves adjusting the pCO2 from about 10 mmHg to about 250 mmHg, the Na+ concentration from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours.

[0062] In certain embodiments, the method involves adjusting the Mn concentration from about 1 nM to about 30000 nM, the pCO2 from about 10 mmHg to about 250 mmHg, and the Na+ concentration from about 0 mM to about 300 mM.

[0063] In certain embodiments, the method involves adjusting the Mn concentration from about 1 nM to about 30,000 nM, the pCO2 from about 10 mmHg to about 250 mmHg, the Na+ concentration from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration from about 0 hours to about 72 hours.

[0064] In certain embodiments, methods involve adjusting said pCO2 from about 10 mmHg to about 250 mmHg; and said Na+ concentration from about 0 mM to about 300 mM.

[0065] In certain embodiments, methods involve adjusting said osmolarity from about 250 mOsm / kg to about 550 mOsm / kg, and said pCO2 from about 10 mmHg to about 250 mmHg.

[0066] In certain embodiments, the method involves adjusting the pCO2 from about 10 mmHg to about 250 mmHg, the Mn concentration from about 1 mM to about 30,000 mM, the cell culture duration from about 0 days to about 150 days, and the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours.

[0067] In certain embodiments, the method involves adjusting the Mn concentration from about 1 nM to about 30000 nM, and the galactose concentration from about 0 mM to about 60 mM.

[0068] In certain embodiments, the method involves adjusting the fucose concentration from about 0 mM to about 60 mM, and the Mn concentration from about 1 nM to about 30000 nM.

[0069] In certain embodiments, methods involve adjusting said fucose concentration from about 0 mM to about 60 mM, and said pCO2 from about 10 mmHg to about 250 mmHg.

[0070] In certain embodiments, methods involve adjusting said fucose concentration from about 0 mM to about 60 mM, said Mn concentration from about 1 nM to about 30000 nM, and said pCO2 from about 10 mmHg to about 250 mmHg.

[0071] In certain embodiments, methods involve adjusting said fucose concentration from about 0 mM to about 60 mM, and said cell culture temperature is about 29°C to about 39°C.

[0072] In certain embodiments, the method involves adjusting the fucose concentration from about 0 mM to about 60 mM, and the cell culture duration from about 0 days to about 150 days.

[0073] In certain embodiments, the Mn concentration is: about 1 nM to about 20,000 nM in high pCO2 culture; about 1 nM to about 1000 nM in high pCO2 culture; about 20 nM to about 300 nM in high pCO2 culture; or about 30 nM to about 110 nM in high pCO2 culture.

[0074] In certain embodiments, the Mn concentration is: about 1 nM to about 30,000 nM in low pCO2 culture; about 1 nM to about 3,000 nM in low pCO2 culture; about 20 nM to about 300 nM in low pCO2 culture; or about 30 nM to about 110 nM in low pCO2 culture.

[0075] In certain embodiments, adjusting the Mn concentration comprises determining the Mn content in the cell culture feedstock and selecting a feedstock batch to adjust the Mn concentration.

[0076] In certain embodiments, the adjustment of the Mn concentration comprises i) controlling the material in contact with the cell culture medium or cell culture; or (ii) calculating the concentration of Mn leached into the cell culture medium or during cell culture; or a combination of (i) and (ii) to adjust the Mn concentration.

[0077] In certain embodiments, the leached Mn is produced by contacting the cell culture and / or cell culture medium with: (i) a filter; (ii) a matrix preparation, storage or culture vessel; or (iii) a combination of (i) and (ii). In certain embodiments, filters include, but are not limited to, depth filters, columns, membranes, and disks. In certain embodiments, filter materials include, but are not limited to, diatomaceous earth, hollow fibers, or resins.

[0078] In certain embodiments, the adjustment of the Mn concentration comprises employing a cell culture medium pH of about 6.1 to about 7.3, or about 6.3 to about 7.3, prior to HTST treatment.

[0079] In certain embodiments, the pCO is regulated. In certain embodiments, the cell culture medium is in a bioreactor, and wherein the pCO regulation is achieved by regulating the following items: bioreactor working volume, bioreactor gas injection strategy, bioreactor stirring strategy, bioreactor feeding strategy, bioreactor perfusion strategy, bioreactor matrix exchange strategy or any combination thereof. In certain embodiments, the pCO regulation includes setting up high pCO cultivation. In certain embodiments, the pCO is about 20mmHg to about 250mmHg, about 20mmHg to about 250mmHg, about 20mmHg to about 150mmHg or about 30mmHg to about 150mmHg.

[0080] In certain embodiments, the pCO2 adjustment comprises establishing a low pCO2 culture. In certain embodiments, the pCO2 is about 10 mmHg to about 100 mmHg, 10 mmHg to about 80 mmHg, about 20 mmHg to about 70 mmHg, or about 30 mmHg to about 60 mmHg. In certain embodiments, the pCO2 adjustment occurs on day 0 of the culture. In certain embodiments, the pCO2 adjustment occurs for about a majority of the cell culture, about the first 5 days, about the first 7 days, or about the first 10 days. In certain embodiments, the pCO2 adjustment occurs for about a majority of the production culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0081] In certain embodiments, the pre-seeding cell culture medium storage duration is about 0 hours to about 120 hours, 0 hours to about 72 hours, about 0 hours to about 48 hours, or about 0 hours to about 24 hours. In certain embodiments, the temperature of the matrix during the pre-seeding cell culture medium storage period is about 25°C to about 39°C, about 30°C to about 39°C, about 35°C to about 39°C, or about 36°C to about 39°C.

[0082] In certain embodiments, the cell culture duration is about 0 days to about 150 days, about 0 days to about 15 days, about 0 days to about 12 days, about 0 days to about 7 days, or about 0 days to about 5 days.

[0083] In certain embodiments, the Na+ concentration is about 0 mM to about 300 mM, about 20 mM to about 200 mM, about 30 mM to about 150 mM, or about 40 mM to about 130 mM. In certain embodiments, the adjustment of the Na+ concentration comprises supplementing the cell culture with a Na compound, including but not limited to Na2CO3, NaHCO3, NaOH, NaCl, or a combination thereof.

[0084] In certain embodiments, the osmotic pressure is about 250mOsm / kg to about 550mOsm / kg, about 300mOsm / kg to about 450mOsm / kg, or about 325mOsm / kg to about 425mOsm / kg. In certain embodiments, the osmotic pressure is adjusted by supplementing the cell culture with an osmotic pressure-adjusting matrix component. In certain embodiments, the osmotic pressure-adjusting matrix component is NaCl, KCl, sorbitol, an osmotic protectant, or a combination thereof.

[0085] In certain embodiments, the galactose concentration is about 0 mM to about 60 mM or about 0 mM to about 50 mM.

[0086] In certain embodiments, the fucose concentration is about 0 mM to about 60 mM, 0 mM to about 40 mM, about 0 mM to about 20 mM, or about 0 mM to about 10 mM.

[0087] In certain embodiments, the cell culture temperature is about 29°C to about 39°C, about 30°C to about 39°C, about 31°C to about 38°C, or about 34°C to about 38°C.

[0088] In certain embodiments, the present disclosure relates to a eukaryotic cell fermentation process for producing a recombinant protein. In certain embodiments, the recombinant protein is an antibody or antibody fragment, scFv (single chain variable fragment), BsDb (bispecific diabody), scBsDb (single chain bispecific diabody), scBsTaFv (single chain bispecific tandem variable domain), DNL-(Fab)3 (docking locked trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody). In certain embodiments, the antibody is a chimeric antibody, a humanized antibody or a human antibody. In certain embodiments, the antibody is an anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody is ocrelizumab. In certain embodiments, the antibody or antibody fragment exhibits: G0-F% (non-fucosylated glycoprotein percentage) of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; and / or G0% (non-galactosylated glycoprotein percentage) of about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%. In certain embodiments, the eukaryotic cell is an insect cell, an avian cell, a fungal cell, a plant cell, or a mammalian cell. In certain embodiments, the fungal cell is a yeast cell, a Pichia cell, or any filamentous fungal cell. In certain embodiments, the yeast cell is a Saccharomyces cerevisiae cell. In certain embodiments, the mammalian cell is a CHO cell.

[0089] In certain embodiments, the present disclosure relates to a cell culture composition comprising: a host cell engineered to express a glycoprotein of interest; and a cell culture and / or cell culture medium adjusted to target one or more predetermined parameters selected from the following: an Mn concentration in a high partial pressure CO2 (pCO2) culture from about 1 nM to about 20,000 nM; an Mn concentration in a low pCO2 culture from about 1 nM to about 30,000 nM; the pCO2 from about 10 mmHg to about 250 mmHg; the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours; the cell culture duration from about 0 days to about 150 days; the Na+ concentration from about 0 mM to about 300 mM; the osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; the galactose concentration from about 0 mM to about 60 mM; the fucose concentration from about 0 mM to about 60 mM; and the culture temperature from about 29°C to about 39°C.

[0090] In certain embodiments, the Mn concentration is from about 1 nM to about 30000 nM, and the pre-seeding cell culture medium storage duration is from about 0 hours to about 120 hours.

[0091] In certain embodiments, the pCO2 is from about 10 mmHg to about 250 mmHg, the Na+ concentration is from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours.

[0092] In certain embodiments, the Mn concentration is from about 1 nM to about 30000 nM, the pCO2 is from about 10 mmHg to about 250 mmHg, and the Na+ concentration is from about 0 mM to about 300 mM.

[0093] In certain embodiments, the Mn concentration is from about 1 nM to about 30000 nM, the pCO2 is from about 10 mmHg to about 250 mmHg, the Na+ concentration is from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours.

[0094] In certain embodiments, the pCO2 is from about 10 mmHg to about 250 mmHg; and the Na+ concentration is from about 0 mM to about 300 mM.

[0095] In certain embodiments, the osmolality is from about 250 mOsm / kg to about 550 mOsm / kg, and the pCO2 is from about 10 mmHg to about 250 mmHg.

[0096] In certain embodiments, the pCO2 is from about 10 mmHg to about 250 mmHg, the Mn concentration is from about 1 mM to about 30000 mM, the cell culture duration is from about 0 days to about 150 days, and the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours.

[0097] In certain embodiments, the Mn concentration is from about 1 nM to about 30000 nM, and the galactose concentration is from about 0 mM to about 60 mM. In certain embodiments, the fucose concentration is from about 0 mM to about 60 mM, and the Mn concentration is from about 1 nM to about 30000 nM.

[0098] In certain embodiments, the fucose concentration is from about 0 mM to about 60 mM, and the pCO2 is from about 10 mmHg to about 250 mmHg. In certain embodiments, the fucose concentration is from about 0 mM to about 60 mM, the Mn concentration is from about 1 nM to about 30000 nM, and the pCO2 is from about 10 mmHg to about 250 mmHg. In certain embodiments, the fucose concentration is from about 0 mM to about 60 mM, and the cell culture temperature is about 29°C to about 39°C. In certain embodiments, the fucose concentration is from about 0 mM to about 60 mM, and the cell culture duration is from about 0 days to about 150 days.

[0099] In certain embodiments, the present disclosure relates to a method for producing a glycoprotein of interest in cell culture, comprising: subjecting a cell culture medium suitable for culturing eukaryotic cells to the method according to any one of the embodiments disclosed herein, inoculating the conditioned cell culture medium with the eukaryotic cells expressing the recombinant protein; and culturing the eukaryotic cells such that the recombinant protein is expressed.

[0100] In certain embodiments, the present disclosure relates to a method of modulating glycosylation of a glycoprotein of interest, the method comprising: assaying a cell culture medium to determine whether a manganese concentration of the cell culture medium is within a target range; and culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium that is within the target range; wherein the glycosylation of the glycoprotein of interest is modulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium that is outside the target range of manganese concentration.

[0101] In certain embodiments, the present disclosure relates to a composition comprising a glycoprotein of interest, wherein the preparation comprises: a cell culture medium assayed to determine whether the manganese concentration of the cell culture medium falls within a target range; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0102] In certain embodiments, the present disclosure relates to a method of modulating glycosylation of a glycoprotein of interest, the method comprising: supplementing a cell culture medium employed when culturing a host cell expressing the glycoprotein of interest with between about 10 nM and about 2000 nM manganese under high CO2 conditions; or supplementing a cell culture medium employed when culturing a host cell expressing the glycoprotein of interest with between about 10 nM and about 3000 nM manganese under low CO2 conditions; wherein the glycosylation of the glycoprotein of interest is modulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a medium that has not been so supplemented.

[0103] In certain embodiments, the present disclosure relates to a cell culture composition comprising: a cell culture medium supplemented with: between about 10 nM and about 2000 nM manganese under high CO2 conditions; or between about 10 nM and about 3000 nM manganese under low CO2 conditions; and a host cell engineered to express a glycoprotein of interest.

[0104] In certain embodiments, the present disclosure relates to a composition comprising a glycoprotein of interest, wherein the preparation comprises: a cell culture medium supplemented with manganese, wherein the culture is supplemented with: between about 10 nM and about 2000 nM manganese under high CO2 conditions; or between about 10 nM and about 3000 nM manganese under low CO2 conditions; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0105] In certain embodiments, the method of regulating the glycosylation of a glycoprotein of interest comprises: measuring a cell culture medium and / or a cell culture to determine whether the manganese concentration of the cell culture medium and / or the cell culture falls within a target range and culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium that falls within the target range. In certain embodiments, the glycosylation of the glycoprotein of interest is regulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in the culture medium and / or the cell culture that falls outside the target range of manganese concentration. In certain embodiments, the target range of manganese concentration is between about 20 nM and about 200 nM. In a non-limiting embodiment, the target range of manganese concentration is between about 30 nM and about 110 nM.

[0106] In certain embodiments, the disclosed glycoprotein of interest is an antibody. The antibody may be a chimeric antibody, a humanized antibody, or a human antibody. In a non-limiting embodiment, the antibody is ocrelizumab.

[0107] In certain embodiments, the disclosed host cells are mammalian cells. The host cells may be Chinese hamster ovary (CHO) cells.

[0108] In certain embodiments, the disclosed cell culture medium comprises determining the manganese concentration of the cell culture medium component. In certain embodiments, the disclosed cell culture medium comprises determining the manganese concentration of the cell culture medium component. The cell culture medium component is a hydrolysate or serum. The cell culture medium component may also be a complex mixture of multiple components.

[0109] In certain embodiments, the glycosylation is modulated to achieve increased afucosylation (e.g., G0-F (non-fucosylated G0)) while reducing agalactosylation (e.g., G0 (fucosylated, non-galactosylated G0)). In certain embodiments, the glycosylation is modulated to achieve reduced afucosylation (e.g., G0-F) while increasing agalactosylation (e.g., G0).

[0110] In certain embodiments, glycosylation is modulated to achieve increased or decreased afucosylation (e.g., G0-F) without affecting agalactosylation (e.g., G0). In certain embodiments, glycosylation is modulated to achieve increased or decreased agalactosylation (e.g., G0) without affecting afucosylation (e.g., G0-F).

[0111] In certain embodiments, the subject matter disclosed herein relates to a cell culture composition comprising: a cell culture medium and / or cell culture that is determined to determine whether the manganese concentration of the cell culture medium and / or cell culture falls within a target range; and a host cell engineered to express a glycoprotein of interest. In certain embodiments, the concentration of manganese is controlled by selecting or avoiding raw materials that contact the culture medium and / or cell culture and that will leach manganese (e.g., a depth filter and / or matrix filter, a matrix preparation dish and / or a storage dish, and a bioreactor). In certain embodiments, the cell culture composition further comprises the glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0112] In certain embodiments, the subject matter disclosed herein relates to a preparation comprising a glycoprotein of interest, wherein the preparation comprises a cell culture medium that is assayed to determine whether the manganese concentration of the cell culture medium falls within a target range; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest. In certain embodiments, the manganese concentration is controlled by selecting a raw material containing a desired level of manganese. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0113] In certain embodiments, the subject matter disclosed herein relates to a method of regulating glycosylation of a glycoprotein of interest, the method comprising supplementing a cell culture medium employed in culturing a host cell expressing the glycoprotein of interest with between about 10 nM and about 2000 nM manganese under high CO2 conditions; or supplementing a cell culture medium employed in culturing a host cell expressing the glycoprotein of interest with between about 10 nM and about 3000 nM manganese under low CO2 conditions; wherein the glycosylation of the glycoprotein of interest is modulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a medium that has not been so supplemented. In certain embodiments, manganese is supplemented directly to the cell culture. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell. In certain embodiments, the glycosylation is modulated to achieve increased afucosylation (e.g., G0-F (afucosylated G0)) while reducing agalactosylation (e.g., G0 (fucosylated G0)). In certain embodiments, the glycosylation is modulated to achieve reduced afucosylation (e.g., G0-F) while increasing agalactosylation (e.g., G0).

[0114] In certain embodiments, the subject matter disclosed herein relates to a cell culture composition comprising: a cell culture medium and / or a cell culture supplemented with: manganese between about 10 nM and about 2000 nM under high CO2 conditions; or manganese between about 10 nM and about 3000 nM under low CO2 conditions; and a host cell engineered to express a glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0115] In certain embodiments, the subject matter disclosed herein relates to a composition comprising a glycoprotein of interest, wherein the preparation comprises a cell culture medium supplemented with manganese, wherein the culture is supplemented with the following items: manganese between about 10nM and about 2000nM under high CO2 conditions; or manganese between about 10nM and about 3000nM under low CO2 conditions; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest. In certain embodiments, manganese is supplemented by using a raw material that leaches manganese during contact with the culture medium and / or cell culture (e.g., a depth filter and / or matrix filter, a matrix preparation dish and / or a storage dish, and a bioreactor). In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0116] In certain embodiments, the subject matter disclosed herein relates to a method for regulating glycosylation of a glycoprotein of interest, the method comprising exposing a cell culture medium having a pH target value of about 6.10 to about 7.25 to a high temperature short time (HTST) heat treatment; and culturing a host cell expressing the glycoprotein of interest in the cell culture medium; wherein the glycosylation of the glycoprotein of interest is regulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium having a pH target value of greater than 7.25 before the HTST heat treatment. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell. In certain embodiments, the glycosylation is regulated to achieve increased G0-F (non-fucosylated G0) while reducing G0 (fucosylated G0).

[0117] In certain embodiments, the subject matter disclosed herein relates to a method of regulating Mn levels in a cell culture medium and / or a cell culture, comprising employing a cell culture medium pH of about 6.1 to about 7.3 prior to high temperature, short time (HTST) heat treatment; and culturing a host cell expressing the glycoprotein of interest in the cell culture medium. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell. In certain embodiments, glycosylation is regulated to achieve increased G0-F (non-fucosylated G0) while reducing G0 (fucosylated G0).

[0118] In certain embodiments, the subject matter disclosed herein relates to a cell culture composition comprising: a cell culture medium exposed to a HTST heat treatment with a pH target of about 6.30 to about 7.25; and a host cell engineered to express a glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0119] In certain embodiments, the subject matter disclosed herein relates to a cell culture composition comprising: a cell culture medium having a target pH of about 6.3 to about 7.3 before exposure to HTST heat treatment; and a host cell engineered to express a glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0120] In certain embodiments, the subject matter disclosed herein relates to a composition comprising a glycoprotein of interest, wherein the preparation comprises a cell culture medium exposed to HTST heat treatment with a pH target of about 6.10 to about 7.25; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0121] In certain embodiments, the subject matter disclosed herein relates to a composition comprising a glycoprotein of interest, wherein the preparation comprises a cell culture medium having a target pH of about 6.1 to about 7.3 prior to exposure to HTST heat treatment; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0122] In certain embodiments, the subject matter disclosed herein relates to a method of modulating glycosylation of a glycoprotein of interest, the method comprising: culturing a host cell expressing the glycoprotein of interest in a cell culture medium, wherein the cell culture is supplemented with higher or lower levels of manganese, galactose and / or fucose (or not supplemented), exposed to high or low pCO2, the cell culture is exposed to extended or shortened substrate retention time and / or culture duration, the culture is maintained at a higher or lower culture temperature, maintained at a higher or lower osmotic pressure, and / or the cell culture includes an increased or decreased Na+ concentration, and / or any combination thereof; wherein the glycosylation of the glycoprotein of interest is modulated compared to the fucosylation and / or galactosylation of a preparation of the glycoprotein of interest expressed by the host cell in a medium exposed to low pCO2, shortened substrate retention time, and / or reduced Na+ concentration. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, for example, a CHO cell. In certain embodiments, the glycosylation is regulated to achieve increased G0-F (non-fucosylated G0) while reducing G0 (fucosylated G0) or reduced G0-F (non-fucosylated G0) while increasing G0 (fucosylated G0). In certain embodiments, glycosylation is regulated to achieve increased or decreased non-fucosylation (e.g., G0-F) without affecting non-galactosylation (e.g., G0). In certain embodiments, glycosylation is regulated to achieve increased or decreased non-galactosylation (e.g., G0) without affecting non-fucosylation (e.g., G0-F).

[0123] In certain embodiments, the subject matter disclosed herein relates to a cell culture composition comprising: a cell culture medium and / or cell culture supplemented (or not supplemented) with manganese, galactose and / or fucose, high or low pCO2, extended or shortened substrate storage time, extended or shortened culture duration, higher or lower culture temperature, higher or lower osmotic pressure, and / or increased or decreased Na+ concentration, and / or any combination thereof; and a host cell engineered to express a glycoprotein of interest. In certain embodiments, the cell culture composition further comprises the glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0124] In certain embodiments, the subject matter disclosed herein relates to a composition comprising a glycoprotein of interest, wherein the preparation comprises a cell culture medium and / or cell culture supplemented (or not supplemented) with manganese, galactose and / or fucose, high or low pCO2, extended or shortened substrate storage time, extended or shortened culture duration, higher or lower culture temperature, higher or lower osmotic pressure, and / or increased or decreased Na+ concentration, and / or any combination thereof; a host cell engineered to express a glycoprotein of interest; and the glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 Depicted are changes in Mn levels on day 0 of production culture in relation to changes in non-galactosylated, GO% (fucosylated G0, bottom) and non-fucosylated, normalized G0-F% (non-fucosylated, top) antibody species.

[0126] Figure 2A and 2B Depicted is the effect of manganese concentration on day 0 on galactosylation and fucosylation of the mAb product during ocrelizumab cell culture. Figure 2A G0 is plotted against the Mn concentration (nM) on day 0. Figure 2B A graph of normalized G0-F versus Mn concentration (nM) at day 0 is depicted in FIG.

[0127] Figure 3A and Figure 3B Depicted is the effect of Mn supplementation on day 0 on the galactosylation and fucosylation of the mAb product during ocrelizumab cell culture. Figure 3A G0 versus supplemented Mn concentration (nM) is plotted in . Figure 3B G0-F versus supplemented Mn concentration (nM) is depicted in FIG.

[0128] Figure 4A and Figure 4B Depicted is the effect of Mn concentration on day 0 on galactosylation and fucosylation of the mAb product during ocrelizumab cell culture at different levels of cell culture scale. Figure 4A A graph of normalized G0-F versus Mn concentration (nM) at day 0 is depicted in FIG. Figure 4B G0 is plotted against Mn concentration (nM) on day 0. The 2L scale-dependency factor refers to the use of a high pCO2 environment in the bioreactor.

[0129] Figure 5A and Figure 5B The effects of supplemental Mn on the galactosylation and fucosylation of the mAb product during ocrelizumab cell culture at different levels of cell culture scale are depicted. Figure 5A A plot of normalized G0-F versus supplemented Mn concentration (nM) is depicted in FIG. Figure 5B G0 versus supplemented Mn concentration (nM) is plotted in Figure 2. The 2L scale-dependency factor refers to the use of a high pCO2 environment in the bioreactor.

[0130] Fig. 6A and Figure 6B The effect of Mn supplementation on the antibody I cell culture process is depicted. Fig. 6A A graph illustrating that Mn supplementation increases total non-fucosylation (G0) is depicted in FIG. Figure 6B A graph illustrating that Mn supplementation reduces non-galactosylation (G0F%) is depicted in FIG.

[0131] Figure 7 The effect of supplemented Mn on the Antibody II cell culture process is depicted. Mn supplementation increased G0-F% (top), and decreased GO% (bottom).

[0132] Fig. 8A and Figure 8B The effect of supplemental Mn on the culture process of Antibody III cells is depicted. Fig. 8A A graph illustrating the increase in G0-F% with Mn supplementation is depicted in Figure 8B A graph illustrating the reduction in G0% with Mn supplementation is depicted in FIG.

[0133] Fig.9A and Fig. 9B The effect of Mn supplementation on the antibody IV cell culture process is depicted. Fig.9A A graph illustrating the increase in G0-F% with Mn supplementation is depicted in Fig. 9B A graph illustrating the reduction in G0% with Mn supplementation is depicted in FIG.

[0134] Fig. 10A and Fig. 10B The effect of Mn supplementation on the antibody V cell culture process is depicted. Fig. 10A A graph illustrating the increase in G0-F% with Mn supplementation is depicted in Fig. 10B A graph illustrating the reduction in G0% with Mn supplementation is depicted in FIG.

[0135] Fig.11 The effect of the timing of Mn addition on glycosylation is depicted (top: G0-F%, and bottom: GO%).

[0136] Figures 12A-12B The effect of Mn addition timing during production culture on glycosylation ( Fig. 12A) and normalized G0-F( Fig. 12B )’s impact.

[0137] Fig.13 Depicted are exemplary typical and atypical high temperature, short time (HTST) pressure (top) and flow rate characteristics (bottom) observed during ocrelizumab HTST thermal treatment.

[0138] Fig.14 Turbidity change (left) and Mn loss (right) of the matrix in a sand bath HTST screen are depicted versus pre-HTST pH adjustment of the matrix.

[0139] Fig.15 The effect of pre-HTST pH adjustment on 2L cell culture performance is depicted. Key performance indicators (final viability % (top), IVPCV (middle) and final titer (bottom)) are shown.

[0140] Fig.16 The effect of pH adjustment before HTST on 2L cell culture performance is depicted. Charge-related variants are shown (photoprotected acidic region % (top), major IE-HPLC % (middle), and basic region % (bottom)).

[0141] Fig.17 Effect of pre-HTST pH adjustment on 2L cell culture performance is depicted. Size-related variants are shown (HMWS % (top), SE-HPLC main peak (middle) and Fab % (bottom)).

[0142] Fig.18 Effect of pH adjustment before HTST on glycans from 2L bioreactor is depicted. GO%, G0-F%, normalized G0-F%, G2+NANA%, Man5%, G1 / G1'%, G2% are shown.

[0143] Figures 19A-19H Depicted is the effect of pH adjustment on the matrix prior to HTST heat treatment with Antibody III. Fig.19A A schematic diagram showing the experimental design is depicted in Fig.19B The variability of manganese concentration for substrate pH targets before HTST heat treatment is depicted in Figure . Fig.19C The variability graphs for final viability (top) and IVPCV (bottom) are depicted in Figure 2. Fig.19D Figure 1 depicts the variability of titers at day 13 (left) and day 14 (right). Fig.19E The variability of G0-F% on day 13 (left) and day 14 (right) is depicted in Figure 2. Fig.19F The variability of G0% on day 13 (left) and day 14 (right) is depicted in Figure 2. Figure 19GDepicted in Figure 5 are variability graphs of size variants at day 13 (left column) and day 14 (right column), where size-related variants include HMWS%, main peak%, and LMWS%. Fig.19H Depicted in the figure are variability graphs of charge-related variants at day 13 (left column) and day 14 (right column), where the charge-related variants include photoprotected acidic region %, main peak IE-HPLC % and basic region %.

[0144] Fig. 20A and Fig. 20B A schematic diagram of an exemplary bioreactor is depicted. Fig. 20A A high carbon dioxide partial pressure (pCO2) model is depicted in (top) and a diagram illustrating the gassing strategy used to maintain constant dissolved oxygen in the high pCO2 model (bottom). Fig. 20B A low pCO2 model is depicted in FIG and a diagram illustrating the gassing strategy used to maintain constant dissolved oxygen in the low pCO2 model (below).

[0145] Fig.21A and Fig. 21B The effects of the pCO2 model, matrix storage, and Mn supplementation, and their combination on the afucosylation of mAbs (calculated as normalized G0-F) and the pCO2 profile of the cultures at harvest (day 12) are depicted. Fig.21A A graph illustrating that Mn levels on day 0 were approximately 5-fold higher in cultures supplemented with Mn than in cultures not supplemented with Mn is depicted in FIG. Fig. 21B Graphs illustrating the pCO2 characteristics of cultures maintained in low and high pCO2 models are depicted in FIG.

[0146] Figures 22A-22D The effects of pCO2 and matrix storage on CHO cells are depicted. Fig.22A A graph illustrating the non-fucosylation of mAbs (calculated as normalized G0-F) at harvest (day 12) with increasing levels of Mn supplementation (day 0) is depicted in FIG. Fig. 22B A graph illustrating the pCO2 characteristics during cell culture is depicted in . Fig. 22C A diagram illustrating the osmotic pressure characteristics during cell culture is depicted in . Fig.22D Figure 2 depicts the Na + Characteristics diagram.

[0147] Figures 23A-23D The effects of osmotic pressure, pCO2 model, and osmotic titrant type on CHO cells are depicted. Fig.23A A graph illustrating the non-fucosylation of mAbs (calculated as normalized G0-F) at harvest (day 12) is depicted in FIG. Fig. 23B Figure 2 depicts the Na + Characteristics of the graph. Fig.23CA graph illustrating the pCO2 characteristics during cell culture is depicted in . Fig.23D A graph illustrating the osmotic pressure characteristics during cell culture is depicted in .

[0148] Fig.24A and Fig. 24B Plotted are the effects of pCO2 and osmotic pressure on intracellular pH (pH i ). Fig.24A Figure 2 illustrates different pCO2 levels while maintaining similar osmolarity (406-413 mOsm / kg) and Na + (83-87mM) level. Fig. 24B The diagram shows different osmotic pressure levels (using NaCl as osmotic pressure titrant; 46-152 mM Na + ), while maintaining similar pCO2 levels (23-28 mm Hg).

[0149] Fig.25A and Fig.25B Depicted are the effects of different culture conditions and culture duration on the non-fucosylation (calculated as normalized G0-F) of mAbs produced in a 3-L bioreactor. Fig.25A A graph showing the differences in culture conditions is shown in . Fig.25B Graphs illustrating afucosylation levels at day 7 and at harvest (day 12) are depicted in FIG.

[0150] Figures 26A-26D A whole proteome analysis is depicted. Fig.26A A schematic diagram showing the experimental design and workflow is depicted in . Fig.26B A plot illustrating principal component analysis (PCA) of samples separated by day (PC1) and cell culture treatment (PC2) is depicted in FIG. Fig.26C Ingenuity Pathway Analysis (IPA) of the canonical pathways for all cases is depicted in . Fig.26D The expression of glycolytic enzymes per treatment and day compared with cases is depicted in .

[0151] Figures 27A-27C Depicted are results evaluating the possibility that GDP-fucose may be affected in cell culture conditions that result in increased mAb afucosylation. Fig.27A The de novo and salvage pathways for GDP-fucose synthesis are depicted in . Fig.27B A heat map of the key enzymes in the GDP-fucose synthesis pathway is depicted in Fig.27C Depicted in FIG are graphs illustrating the effect of L-fucose addition (at day 0) on the level of afucosylation (calculated as normalized G0-F) at harvest (day 12).

[0152] Fig.28A and Fig.28B Describes proteomic analysis to determine the differential expression of key proteins in the glycosylation pathway under different culture conditions in a 3-L bioreactor. Fig.25A A description of the four cases (i–iv) tested in the 3-L bioreactor and the resulting afucosylation levels are provided in . Fig.28A A diagram of the glycosylation pathway in the Golgi apparatus is depicted in , illustrating only the glycosylation variants associated with non-fucosylation (Man5–G2). Fig.28B Depicted in the figure are heat maps of selected glycosylation enzymes (FUT8, MAN I, GnT II, ​​GalT3, GalT4, GalT7), intracellular and Golgi pH regulators (NHE1 and GPR89, respectively), and Mn level indicator proteins (ATP2A1, GPP13) at day 7 and day 12 of production culture.

[0153] Figures 29A-29E The performance of recombinant CHO cells cultured in a 3-L bioreactor using high and low pCO2 models is depicted. Fig.29A Growth represented by the packed cell volume (PCV) is depicted in Fig.29B A graph representing the viability of recombinant CHO cells is depicted in FIG. Fig.29C A graph representing mAb titers is depicted in FIG. Fig.29D A graph representing the charge variants (day 12) is depicted in FIG. Fig.29E A graph representing size variants (day 12) is depicted in . HWMS refers to high molecular weight species; LWMS refers to low molecular weight species.

[0154] Fig.30 Plotted are the effects of pCO2 model, matrix preservation, and Mn supplementation on the G0 of mAbs at harvest (day 12). The graph shows the effect of each factor on its own, as well as the effect on G0 in combination with other factors.

[0155] Fig.31 Depicted are the effects of L-fucose addition (Day 0) and manganese addition (Day 0) on the G0 of mAbs produced in a 3-L bioreactor at harvest (Day 12). The graph shows the effects of fucose and manganese supplementation on their own as well as their combined effects on G0.

[0156] Fig.32 The variability of manganese content in PP3 and GEM is depicted.

[0157] Figures 33A-33D The effects of matrix preservation, Mn supplementation, and their combination on G0 and G0-F are depicted. Figures 33A-33B The effect of substrate storage time at high temperature (38°C) on the non-galactosylated, G0( Fig.34A ) and non-fucosylated, normalized to G0-F ( Fig.33B )’s impact. Fig.33C The cumulative effect of matrix storage on the afucosylation (G0-F%) of Antibody III is depicted. Fig.33D The effects of matrix preservation, Mn supplementation and their combination on the afucosylation (G0-F%) of Antibody III are depicted.

[0158] Figures 34A-34B The effects of galactose and supplemented Mn, and their interaction on the non-galactosylated, G0( Fig.34A ) and non-fucosylated, normalized to G0-F ( Fig.34B )’s impact.

[0159] Figures 35A-35B The effects of galactose and Mn and their interaction on the non-galactosylated G0( Fig.35A ) and non-fucosylated, normalized to G0-F ( Fig.35B )’s impact.

[0160] Figures 36A-36B The galactose residues from non-galactosylated G0 ( Fig.36A ) and non-fucosylated, normalized to G0-F ( Fig.36B )’s impact.

[0161] Figures 37A-37B The effect of fucose supplementation on non-fucosylated, G0-F ( Fig.37A ) and non-galactosylated, G0( Fig.37B )’s impact.

[0162] Figures 38A-38B The effect of fucose addition timing on non-fucosylation, G0-F ( Fig.38A ) and non-galactosylated, G0( Fig.38B )’s impact.

[0163] Figures 39A-39B The effects of fucose concentration and temperature and their interaction on non-fucosylation, G0-F ( Fig.39A ) and non-galactosylated, G0( Fig.39B )’s impact. DETAILED DESCRIPTION

[0164] The subject matter disclosed herein relates to regulating the glycosylation (e.g., galactosylation and / or fucosylation) of a recombinant glycoprotein of interest (e.g., mAb) so that it falls within a desired quality attribute range. For example, but not by way of limitation, the subject matter disclosed herein is suitable for modifying the glycosylation characteristics of a mAb so that it falls within a narrower range of quality attributes than those obtained using conventional cell culture media, matrix preparation strategies, and / or cell culture strategies. Methods by which glycosylation can be regulated in accordance with the present disclosure include, but are not limited to: (1) controlling cell culture medium manganese (Mn) concentration, e.g., Mn concentration analysis of the feedstock, supplementation of Mn during cell culture, and / or establishment of a reduced pH set point for matrix pH regulation prior to high temperature, short time (HTST) heat treatment of the matrix; and (2) controlling process parameters during cell culture, e.g., pCO2, matrix storage duration, and osmotic pressure / Na + The presently disclosed subject matter also relates to cell cultures and glycoprotein compositions produced when such process parameters are controlled as described herein.

[0165] To make the disclosure clearer but not limiting, the detailed description is divided into the following subsections:

[0166] 1. Definition

[0167] 2. Control raw materials to regulate glycosylation

[0168] 3. Supplement manganese to regulate glycosylation

[0169] 4. Improved pH target of the substrate prior to high temperature, short time (HTST) treatment modulates glycosylation by minimizing manganese loss during HTST

[0170] 5. pCO2, Mn, substrate storage, culture duration, culture temperature and osmotic pressure / Na + To regulate glycosylation

[0171] 6. Galactose to regulate glycosylation

[0172] 7. Fucose and culture temperature and their combination to regulate glycosylation

[0173] 8. Cell Culture and Glycoprotein Composition

[0174] 1. Definition

[0175] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, the definitions included in this document shall prevail. Certain methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used to practice or test the subject matter currently disclosed. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0176] As used herein, the terms "comprises," "including," "having," "having," "may," "containing," and variations thereof are intended to be open transitional phrases, terms, or words and do not exclude the possibility of additional actions or structures. Unless expressly provided otherwise, the singular forms "a," "an," and "the" include plural references. The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of," whether or not expressly set forth, the embodiments or elements presented herein.

[0177] For enumerating the numerical ranges herein, each intermediate number with the same degree of precision therebetween is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated; and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0178] As used herein, the term "about" or "approximately" means within an acceptable error range of a value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with practice in the art, "about" can mean within 3 or more than 3 standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within a certain order of magnitude of a certain value, preferably within 5 times, more preferably within 2 times.

[0179] The term "supplementation" is used in the broadest sense and includes various types, techniques or methods for adding target molecules, materials, objects or combinations thereof. Single dose, fully continuous, semi-continuous, intermittent, time-based, feedback loop-based addition are examples of supplementation.

[0180] The term "modulate" is used herein to refer to an increase or decrease in a corresponding property.

[0181] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0182] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0183] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single V H or V L The domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen may use V domains from antibodies that bind to that antigen, respectively. H or V L domains to separate and screen for complementary V L or V H Libraries of domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0184] As used herein, the term "homologous sequence" refers to a sequence that shares significant sequence similarity, as determined by sequence alignment. For example, two sequences can be about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or about 99.9% homologous. Alignment is performed by algorithms and computer programs (including but not limited to BLAST, FASTA and HMME), which can compare sequences and calculate the statistical significance of matching based on factors such as sequence length, sequence identity and similarity and sequence mismatches and the presence and length of gaps. Homologous sequences can refer to both DNA and protein sequences.

[0185] The terms "polypeptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. These terms also encompass amino acid polymers that have been naturally modified or modified by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component). Also included within the definition are, for example, one or more analogs containing amino acids (including, for example, non-natural amino acids, etc.) and other modified polypeptides known in the art. As used herein, the terms "polypeptide" and "protein" specifically encompass antibodies.

[0186] The term "glycoprotein" refers to a polypeptide or protein coupled to at least one carbohydrate moiety, such as a polysaccharide or oligosaccharide, which is linked to the protein via an oxygen- or nitrogen-containing side chain of an amino acid residue, such as a serine or threonine residue ("O-linked") or an asparagine residue ("N-linked"). The term "glycan" refers to a polysaccharide or oligosaccharide, such as a polymer composed of monosaccharides. A glycan may be a homopolymer or heteropolymer of monosaccharide residues and may be linear or branched.

[0187] As used herein, "glycosylation pattern" and "glycosylation characteristics" of a recombinant glycoprotein of interest refer to various physical characteristics of the polysaccharides or oligosaccharides of the glycoprotein, such as the quantity and quality of the various monosaccharides present, the degree of branching, and / or attachment (e.g., N-linked or O-linked).

[0188] "Fucosylation" refers to the degree and distribution of fucose residues on polysaccharides and oligosaccharides, e.g., N-glycans, O-glycans, and glycolipids. "Non-fucosylation" refers to the lack of fucose residues on polysaccharides and oligosaccharides. G0 glycans refer to glycans lacking terminal galactose residues. Two different nomenclatures have been identified in the art for identifying fucosylated / non-fucosylated G0 glycans:

[0189] (1) wherein "G0-F" (i.e., "G0' minus 'F") is used to indicate non-fucosylated G0 glycans, and "G0" is used to indicate fucosylated G0 glycans; and

[0190] (2) where "G0" is used to indicate non-fucosylated G0 glycans, and then

[0191] "G0F" is used to indicate fucosylated G0 glycans.

[0192] Identifying which convention to use in a particular context involves analyzing the use of G0 and the use of G0-F or G0F. Therapeutic glycoproteins, such as antibodies or Fc fusion proteins, with non-fucosylated or afucosylated N-glycans exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) due to enhanced FcγRIIIa binding ability without any detectable changes in complement-dependent cytotoxicity (CDC) or antigen-binding ability. In some cases, such as cancer treatment, non-fucosylated or afucosylated antibodies are ideal because they can achieve therapeutic effects at low doses while inducing high cytotoxicity against tumor cells and triggering high effector functions of NK cells by enhancing interaction with FcγRIIIa. In other cases, such as treatment of inflammatory or autoimmune diseases, enhanced ADCC and FcγRIIIa binding are undesirable, and therefore, therapeutic glycoproteins with higher levels of fucose residues in their N-glycans may be preferred. As used herein, the term "fucose %" or "non-fucosylation %" refers to the percentage of non-fucosylated N-glycans present on the recombinant glycoprotein of interest. A higher non-fucose % or non-fucosylation % indicates a higher number of non-fucosylated N-glycans, while a lower fucose % or non-fucosylation % indicates a higher number of fucosylated N-glycans. Non-fucosylation can sometimes be expressed as normalized G0-F%, which is calculated by the following term:

[0193]

[0194] As used in the context of the present invention, the term "galactosylated" refers to the addition of galactose units to the oligosaccharide chains of glycoproteins. The term "non-galactosylated" refers to the lack of galactose units on the oligosaccharide chains of glycoproteins. As used herein, the term "galactosylated" antibody refers to an antibody wherein the N-linked glycans of the antibody include at least one galactose residue (e.g., G1 and G2 glycans). As used herein, the term "non-galactosylated" antibody refers to an antibody wherein the N-linked glycans of the antibody do not have galactose residues (e.g., G0 and G0F glycans).

[0195] As used herein, the term "expression" refers to transcription and / or translation. In certain embodiments, the level of transcription of the desired product can be determined based on the amount of the corresponding mRNA present. For example, the mRNA transcribed from the target sequence can be quantified by PCR or Northern blot hybridization. In certain embodiments, the protein encoded by the target sequence can be quantified by a variety of methods, for example, by ELISA, by measuring the biological activity of the protein, or by using antibodies that recognize and bind to the protein, using assays unrelated to this activity, such as Western blotting or radioimmunoassay.

[0196] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which it has been introduced. In certain embodiments, a vector directs the expression of a nucleic acid to which it is operably linked. Such vectors are referred to herein as "expression vectors."

[0197] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), allowing these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model (such as disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998)).

[0198] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to an antibody (of the appropriate subclass) bound to its cognate antigen. To assess complement activation, a CDC assay may be performed as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Polypeptide variants having altered Fc region amino acid sequences (polypeptides having variant Fc regions) and increased or decreased Clq binding ability are described in, for example, U.S. Patent No. 6,194,551B1 and WO 1999 / 51642. See also, for example, Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0199] "Culturing" cells means contacting the cells with a cell culture medium under conditions suitable for cell survival and / or growth and / or cell proliferation. Cell culture can be performed under a variety of conditions, including but not limited to batch, fed-batch, continuous, perfusion processes. The duration of cell culture can vary depending on the process. For example, but not by way of limitation, a fed-batch process can be run for fewer days, such as from 0 to 20 days, while a typical perfusion process can be run for up to 150 days or even more days.

[0200] "Batch culture" refers to culture in which all components for cell culture (including cells and all culture nutrients) are supplied to a culture vessel at the beginning of the culture process.

[0201] As used herein, the phrase "fed-batch cell culture" refers to a batch culture in which cells and culture medium are first supplied to a culture vessel, and additional culture nutrients are fed to the culture continuously or in discontinuous increments during the course of the culture, with or without periodic cell and / or product harvesting prior to terminating the culture.

[0202] The phrase "bioreactor agitation strategy" refers to the agitation rate and / or physical manipulation of the culture and / or medium in the bioreactor.

[0203] The phrase "bioreactor matrix exchange strategy" refers to any process in which the matrix in contact with the bioreactor and / or cells in culture is changed, including but not limited to processes in which cells are centrifuged from a cell culture sample taken from a bioreactor and resuspended in a new culture medium, which may be different from the original cell culture medium initially used to grow the cells.

[0204] "Perfusion culture" is a culture in which cells are confined in culture by, for example, filtration, encapsulation, anchoring to microcarriers, etc., and culture medium is continuously or intermittently introduced and removed from the culture vessel.

[0205] "Cultivating vessel" or "culture vessel" and "bioreactor" refer to containers used to culture cells. A culture vessel can be of any size as long as it is useful for the culture of cells. In certain embodiments, the bioreactor used in the presently disclosed methods is a stainless steel vessel. In certain embodiments, the bioreactor used in the presently disclosed methods is a shake bag. In certain embodiments, the bioreactor used in the presently disclosed methods is a disposable bioreactor.

[0206] The terms "matrix" and "cell culture medium" refer to a nutrient source for growing or maintaining cells. As will be appreciated by those skilled in the art, a nutrient source may contain components required for cell growth and / or survival, or may contain components that contribute to cell growth and / or survival. Cell culture medium also refers to any fluid supernatant for growing or maintaining cells. A matrix component refers to any component that can be added to a cell culture or cell culture medium at any stage of culture, at any time, or in any form. A culture medium component also refers to a component from a cell culture medium raw material. Vitamins, essential or non-essential amino acids, and trace elements are examples of matrix components. It should be understood that "matrix (medium and media)" are used interchangeably throughout the specification.

[0207] "Chemically defined cell medium" or "CDM" is a matrix of a specific composition that is free of animal-derived or undefined products, such as animal serum and peptone. As will be appreciated by those skilled in the art, a CDM can be used in a polypeptide production process, whereby cells contact the CDM and secrete polypeptides into the CDM. Thus, it should be understood that a composition can contain a CDM and a polypeptide product, and the presence of the polypeptide product does not render the CDM chemically undefined.

[0208] "Chemically undefined cell culture medium" refers to a medium whose chemical composition cannot be determined and may contain one or more animal-derived or undefined products, such as animal serum and peptone. As will be understood by those skilled in the art, a chemically undefined cell culture medium may contain animal-derived products as a nutrient source.

[0209] " Matrix preservation " refers to the cell culture practice in which cell culture medium is stored in a culture dish (for example, a bioreactor, a disposable bag) or a container (for example, a stainless steel tank, a disposable container) for matrix preparation or matrix storage before being used to culture cells. In cell culture operations, the culture medium can be heated, then stored at a culture temperature or close to a culture temperature, and then the cells are inoculated in a bioreactor using the matrix." Matrix preservation duration " or "matrix preservation time" refers to the time range in which the matrix is ​​preserved (for example, at a temperature higher than ambient temperature) before using the matrix to inoculate cells in a bioreactor. It should be understood that throughout the specification, "matrix preservation", "matrix preservation duration" and "matrix preservation time" are used interchangeably.

[0210] "HTST" refers to "high temperature, short time" treatment of cell culture media. This HTST treatment of cell culture media can provide an additional safety barrier against adventitious agents. (Floris et al., (2018) Appl Microbiol Biotechnol. 102(13):5495-5504; Pohlscheidt et al., (2014) Appl Microbiol Biotechnol. 98(7):2965-71. During HTST treatment of cell culture media, precipitates may form, HTST equipment may foul, and matrix components may fall out of solution. Adjustments to specific culture media parameters can be made to reduce or prevent the formation of precipitates in the matrix caused by HTST treatment.

[0211] As used herein, the phrase "low pCO2" describes an operation within a relatively narrow range of carbon dioxide, wherein the upper limit of CO2 is lower than the upper limit used in a "high pCO2" operation. Low pCO2 can be from about 10 mmHg to about 100 mmHg, about 10 mm Hg to about 80 mmHg, about 10 mmHg to about 70 mmHg, or about 10 mmHg to about 60 mmHg. In contrast, "high pCO2" is used herein to refer to a wider range of carbon dioxide, wherein the upper limit of pCO2 is higher than the upper limit used in a low pCO2 operation. High pCO2 can be from about 20 mmHg to about 250 mmHg, about 20 mmHg to about 200 mmHg, about 20 mmHg to about 150 mmHg, or about 30 mmHg to 150 mmHg. pCO2 regulation as described herein can occur for at least the first half of the duration of cell culture. For example, but not limited to, for a 20-day culture, pCO2 regulation can occur for at least the first 10 days. Depending on the varying cell culture duration, the pCO2 regulation will also vary accordingly.

[0212] 2. Control raw materials to regulate glycosylation

[0213] It is known that a variety of cell culture factors may affect the glycosylation of glycoproteins (e.g., mAbs). These factors include process parameters and matrix components, such as galactose and trace metals. The level changes of individual matrix components can be introduced into the mAb cell culture process via the use of complex raw materials. For example, but not as a limitation, cell culture media, such as base matrix or feed matrix (and its individual components, such as hydrolysates or various types of serum), can show batch-to-batch variations, which may affect mAb glycosylation. Therefore, in certain embodiments, the present disclosure relates to compositions and methods for reducing the variability of cell culture media to regulate mAb glycosylation (e.g., galactosylation and / or fucosylation). For example, but not as a limitation, Mn supplementation can be achieved by using matrix components containing Mn as impurities or raw materials that can release Mn into cell culture media or cell cultures (e.g., depth filters, stainless steel containers, or glass containers).

[0214] In certain embodiments, the present disclosure relates to strategies for screening cell culture media and / or individual components thereof in order to modulate mAb glycosylation (e.g., galactosylation and / or fucosylation). In non-limiting embodiments, cell culture media that meet specific target amounts of individual components, such as Mn concentration, galactose concentration, can be screened. For example, but not by way of limitation, cell culture media can be screened and selected based on a target range of Mn concentration of about 1 nM to about 10 μM, about 1 nM to about 1 μM, about 20 nM to about 300 nM, or about 30 nM to about 110 nM (wherein no matrix associated with the cell culture of mAbs that falls outside of this target range is employed). In certain embodiments, the cell culture media can be further supplemented with galactose up to 10 g / L (e.g., about 0 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 7 g / L, or about 10 g / L). In certain embodiments, the cell culture media can be supplemented with galactose up to about 6 g / L.

[0215] Certain embodiments described herein relate to regulating glycosylation (e.g., non-fucosylation and / or galactosylation) by screening cell culture media and / or cell cultures based on the disclosed target ranges of Mn concentrations to achieve or maintain a desired glycoprotein glycosylation pattern. In certain non-limiting embodiments, the desired glycoprotein glycosylation pattern can be regulation of non-fucosylation and / or galactosylation of glycoproteins. For example, but not by way of limitation, the target range of non-fucosylated G0 (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%. The GO-F% or normalized GO-F% can be adjusted by about 0.5%, 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%. In certain embodiments, the desired glycosylation pattern of the glycoprotein can be a decrease or increase in the fucosylation of the glycoprotein. For example, but not by way of limitation, the target range (GO%) of fucosylated, non-galactosylated GO can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. The GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0216] In certain embodiments, the desired glycosylation pattern of the glycoprotein may be a combination of modulation of the non-fucosylation and galactosylation of the glycoprotein. For example, but not by way of limitation, the target range of G0-F% or normalized G0-F% may be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range of G0% may be between about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%. GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0217] 3. Supplement manganese to regulate glycosylation

[0218] As described herein, cell culture medium Mn concentration can affect glycosylation, e.g., mAb galactosylation and / or fucosylation. Also as described herein, cell culture Mn concentration can affect glycosylation, e.g., mAb galactosylation and / or fucosylation. Thus, in certain embodiments, mAb glycosylation can be regulated not only by controlling the amount of Mn present in the cell culture medium feedstock as described above, but also by supplementing the cell culture medium with Mn. In certain embodiments, an increase in Mn concentration can increase non-fucosylation (by increasing the level of G0-F, a non-fucosylated form of G0), and / or increase galactosylation (which results in a decrease in G0, i.e., non-galactosylated and fucosylated glycan species).

[0219] Although an increase in Mn concentration in the cell culture medium and / or cell culture generally results in an increase in G0-F (non-fucosylated G0) and a decrease in G0 (fucosylated G0), as summarized in Figures 5-10, identifying this trend in six different mAbs, the extent of increase in non-fucosylation (and therefore G0-F species) and non-galactosylation (and therefore G0 species) can be improved by establishing specific Mn supplementation targets. For example, in certain embodiments, the concentration of Mn to be supplemented should be sufficient to increase G0-F and decrease G0 while not causing the resulting mAb to exceed the desired product quality specifications. In certain embodiments, Mn is supplemented to achieve a selected range in the cell culture medium and / or cell culture. In certain embodiments, the concentration of Mn supplementation is selected to be less than about 10 μM (e.g., about 10 nM, about 40 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 500 nM, about 700 nM, about 750 nM, about 1000 nM, about 1500 nM, about 2000 nM, about 3000 nM, about 5000 nM, about 8000 nM, or about 10 μM, including concentrations falling within the disclosed ranges). In certain embodiments, the concentration of Mn supplementation may be between about 20 nM and about 300 nM. In a non-limiting embodiment, the concentration of Mn supplementation may be between about 30 nM and about 110 nM. In certain embodiments, including those in which Mn supplementation occurs in a cell culture medium exposed to high CO, the concentration of Mn supplementation is selected to be less than 3000 nM (e.g., about 5 nM, 10 nM, about 30 nM, 40 nM, about 50 nM, 100 nM, about 200 nM, about 250 nM, about 500 nM, about 1000 nM, about 2000 nM, about 3000 nM, including concentrations falling within the disclosed ranges). As outlined herein, this concentration has the unexpected ability to increase non-fucosylation (and therefore G0-F glycans) and decrease non-galactosylation (and therefore G0 glycans) without causing the resulting mAb to exceed the desired product quality specifications.

[0220] In certain embodiments, the timing of supplementing the culture with Mn also affects glycosylation (e.g., galactosylation and non-fucosylation). Mn supplements can be added during the expansion culture phase prior to production and / or during the production culture phase. In certain embodiments, Mn supplementation may come from leaching of Mn from materials in contact with the cell culture medium and / or cell culture (e.g., depth filters or matrix filters, culture dishes, matrix storage dishes). In a non-limiting embodiment, Mn supplementation can be achieved by using a depth filter containing diatomaceous earth during the matrix preparation filtration process, which leaches Mn and other trace metals, thereby supplementing the culture.

[0221] In certain embodiments, mAb can be harvested after Mn supplementation. For example, but not limited to, mAb can be harvested between about the 2nd day of culture and about the 25th day of culture (e.g., about the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th or 25th day of cell culture). In a non-limiting embodiment, mAb can be harvested between about the 7th day to about the 15th day of cell culture. In certain embodiments, mAb can be harvested between about the 5th day to about the 20th day of cell culture.

[0222] In certain embodiments, the matrix compositions and cell culture processes disclosed herein can be combined with additional and / or alternative glycosylation modulating concentrations of one or more of the group consisting of fucose, ammonia, sodium, uridine, N-acetylglucosamine, N-acetylgalactosamine, cadmium, lipoic acid, divalent metal ions (such as V 2+ Cr 2+ , Fe 2+ 、Co 2+ 、Ni 2 + , Cu 2+ 、Zn 2+ , Ca 2+ Mg 2+ and kifnoline).

[0223] 4. Modification of pH of substrates prior to HTST treatment to modulate glycosylation

[0224] As disclosed herein, the concentration of Mn in a cell culture medium can modulate glycoproteins, e.g., mAbs, glycosylation. Thus, in certain embodiments, the disclosure relates to methods for controlling the concentration of Mn in a cell culture medium to modulate the glycosylation (e.g., galactosylation and / or fucosylation) of a mAb. In certain embodiments, the disclosure also relates to methods for controlling the concentration of Mn in a cell culture medium to modulate the glycosylation (e.g., galactosylation and / or fucosylation) of a mAb. For example, but not by way of limitation, the disclosure states that HTST treatment of a substrate having a pre-HTST substrate pH adjustment target value greater than about 7.0 can result in a reduction in the Mn concentration of the cell culture medium after HTST treatment. Thus, in certain embodiments, the disclosure relates to performing HTST with a substrate having a prepared pH target value less than about 7.25 (e.g., between about 6.1 and about 7.2). In certain embodiments, the disclosure relates to performing HTST with a substrate having a prepared pH target value less than about 7.3 (e.g., between about 6.1 and about 7.3). In certain embodiments, the pH target value of a substrate prepared for HTST treatment can be about 6.1, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.1, about 7.2, or about 7.3.

[0225] 5. pCO2, manganese, substrate storage, culture duration, culture temperature, and Na+ / osmotic pressure to regulate glycosylation

[0226] As disclosed herein, controlling the pCO2, matrix storage duration, culture duration, culture temperature, manganese concentration, osmotic pressure / Na+ concentration, and / or combinations thereof of a cell culture medium and / or cell culture can result in modulation of fucosylated and / or non-fucosylated G0 glycans of glycoproteins, e.g., mAbs, cultured in such a matrix. In certain embodiments, the disclosure relates to cell culture methods employing a matrix or cell culture, wherein the pCO2, manganese concentration, matrix storage duration, culture duration, culture temperature, Na+ concentration, osmotic pressure, or combinations thereof have been controlled as outlined herein.

[0227] 5.1 Matrix preservation

[0228] As described herein, the duration of matrix storage at a specific temperature or temperature range can affect glycosylation (e.g., galactosylation and / or non-fucosylation). In certain embodiments, the elevated matrix storage temperature can be between about 25°C and about 39°C, about 30°C to about 39°C, about 35°C to about 39°C, or about 36°C to about 39°C. In certain embodiments, the duration of matrix storage at a specific temperature or temperature range ranges from about 0 hours to about 12 hours, about 0 hours to about 24 hours, about 0 hours to about 36 hours, about 0 hours to about 48 hours, about 0 hours to about 60 hours, about 0 hours to about 72 hours, about 0 hours to about 96 hours or longer. In certain non-limiting embodiments, the cell culture medium is stored at a temperature between about 25°C and about 39°C for a period of about 0 hours to about 72 hours to adjust glycosylation (e.g., non-fucosylation and / or galactosylation). In certain embodiments, the cell culture preserved in this manner is based on production culture, expansion culture, or both.

[0229] Certain embodiments described herein relate to regulating or glycosylation (e.g., non-fucosylation and / or galactosylation) by applying the disclosed matrix storage time at a specific temperature or temperature range to achieve or maintain a desired glycoprotein glycosylation pattern. In certain embodiments, the desired glycoprotein glycosylation pattern can be a regulation of non-fucosylation of the glycoprotein. For example, but not limited to, the target range of non-fucosylated G0 (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%. In certain embodiments, G0-F% or normalized G0-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%. In certain embodiments, the desired glycoprotein glycosylation pattern can be the regulation of galactosylation of glycoprotein. For example, but not limited to, the target range of non-galactosylation (e.g., GO%) can be between about 40% to about 90%, about 50% to about 90%, about 55% to about 85% or about 60% to about 80%. In certain embodiments, GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40% or about 50%. In certain embodiments, the desired glycoprotein glycosylation pattern can be a combination of non-fucosylated and galactosylated regulation of glycoprotein. For example, but not by way of limitation, the target range for G0-F% or normalized G0-F% can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range for G0% can be between about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%. In certain embodiments, GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0230] 5.2 Carbon dioxide partial pressure (pCO2)

[0231] In certain embodiments, the disclosure relates to strategies for regulating the partial pressure of carbon dioxide (pCO2) in cell culture to regulate mAb glycosylation (e.g., galactosylation and / or fucosylation). In non-limiting embodiments, the level of pCO2 can be between 0mmHg and 250mmHg. The pCO2 range of the high pCO2 model is about 0mmHg to about 250mmHg, about 20mmHg to about 250mmHg, about 20mmHg to about 200mmHg, about 20mmHg to about 150mmHg, or about 30mmHg to 150mmHg, for most of the duration of culture starting from day 0. The pCO2 range of the low pCO2 model is about 10mmHg to about 100mmHg, about 10mmHg to about 80mmHg, about 10mmHg to about 70mmHg, or about 10mmHg to about 60mmHg, for most of the duration of culture starting from day 0. Certain embodiments described herein relate to regulating glycosylation by adjusting the level of pCO2 to a target range to achieve or maintain a desired glycoprotein glycosylation pattern. For example, in certain embodiments, the desired glycoprotein glycosylation pattern can be a non-fucosylated regulation of glycoprotein. In certain embodiments, the target range of non-fucosylated G0 (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%. In certain embodiments, G0-F% or normalized G0-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% or about 20%. In certain embodiments, the desired glycoprotein glycosylation pattern can be a galactosylation regulation of glycoprotein. For example, but not by way of limitation, the target range of non-galactosylation (GO%) can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, the GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%. In certain embodiments, the desired glycoprotein glycosylation pattern can be a combination of adjustments of non-fucosylation and galactosylation of the glycoprotein. For example, but not by way of limitation, the target range for G0-F% or normalized G0-F% can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range for G0% can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%.In certain embodiments, GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0232] 5.3 Sodium (Na+) concentration

[0233] In certain embodiments, the present disclosure relates to strategies for adjusting the concentration of sodium (Na+) in cell culture media and / or cell cultures to modulate mAb glycosylation (e.g., galactosylation and / or fucosylation). For example, but not by way of limitation, the cell culture media can be supplemented with Na2CO3, NaHCO3, NaCl, NaOH, and / or Na+ compounds (e.g., for pH control), or combinations thereof, to achieve a target range of Na+ concentration of about 0 mM to about 250 mM, 20 mM to about 200 mM, 30 mM to about 150 mM, or 40 mM to about 130 mM.

[0234] Certain embodiments described herein relate to regulating glycosylation by adjusting the Na+ concentration in the cell culture medium and / or cell culture to a specified target range to achieve or maintain the desired glycosylation pattern of the glycoprotein. In certain embodiments, the desired glycoprotein glycosylation pattern can be the regulation of the non-fucosylation of the glycoprotein. For example, but not limited to, the target range of non-fucosylation (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%. G0-F% or normalized G0-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% or about 20%. In certain embodiments, the desired glycoprotein glycosylation pattern can be the regulation of the galactosylation of the glycoprotein. For example, but not by way of limitation, the target range of non-galactosylation (GO%) can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. The GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%. In certain embodiments, the desired glycoprotein glycosylation pattern can be a combination of adjustments of non-fucosylation and galactosylation of the glycoprotein. For example, but not by way of limitation, the target range for G0-F% or normalized G0-F% can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range for G0% can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60%. In certain embodiments, GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0235] 5.4 Osmotic pressure

[0236] In certain embodiments, the present disclosure relates to strategies for adjusting the osmotic pressure of the culture medium to adjust mAb glycosylation (e.g., galactosylation and / or fucosylation). For example, but not by way of limitation, the osmotic pressure of the cell culture medium can be adjusted by adding sorbitol, KCl, an osmoprotectant (e.g., betaine), and / or NaCl to achieve an osmotic pressure target range of about 250 mOsm / kg to about 600 mOsm / kg, about 300 mOsm / kg to 450 mOsm / kg, about 325 mOsm / kg to 450 mOsm / kg, or about 325 mOsm / kg to 425 mOsm / kg.

[0237] Certain embodiments described herein relate to regulating glycosylation by adjusting the osmotic pressure level of the culture medium and / or cell culture to a target range to achieve or maintain the desired glycosylation pattern of the glycoprotein. In certain embodiments, the desired glycoprotein glycosylation pattern can be the regulation of the non-fucosylation of the glycoprotein. For example, but not limited to, the target range of non-fucosylation (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%. In certain embodiments, G0-F% or normalized G0-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% or about 20%. In certain embodiments, the desired glycoprotein glycosylation pattern can be the regulation of the galactosylation of the glycoprotein. For example, but not by way of limitation, the target range of non-galactosylation (GO%) can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, the GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%. In certain embodiments, the desired glycoprotein glycosylation pattern can be a combination of adjustments of non-fucosylation and galactosylation of the glycoprotein. For example, but not by way of limitation, the target range for G0-F% or normalized G0-F% can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range for G0% can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, GO-F% or normalized GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0238] 5.5 Supplemental manganese

[0239] As described above in Sections 2 and 3, the concentration of Mn in the cell culture medium and / or cell culture can affect glycosylation, e.g., mAb galactosylation and / or fucosylation. Thus, in certain embodiments, mAb glycosylation can be regulated not only by controlling the amount of Mn present in the cell culture medium feedstock as described above, but also by supplementing the cell culture medium and / or cell culture with Mn to achieve a specific Mn concentration target or target range (including in combination with one or more other parameters as outlined in this section). In certain embodiments, the concentration of Mn supplementation is selected to achieve a final target concentration or concentration range of less than 10 uM (e.g., about 10 nM, 40 nM, 100 nM, 150 nM, 200 nM, 250 nM, 500 nM, 700 nM, 750 nM, 1000 nM, 1500 nM, 2000 nM, 3000 nM, 5000 nM, 8000 nM, or 10 uM, including concentrations falling within the disclosed ranges).

[0240] Certain embodiments described herein relate to regulating glycosylation by supplementing the disclosed concentration of Mn into the cell culture medium and / or cell culture to achieve or maintain the desired glycosylation pattern of glycoproteins. In non-limiting embodiments, the desired glycosylation pattern of glycoproteins can be the regulation of the non-fucosylation of glycoproteins. For example, but not limited to, the target range of non-fucosylated G0 (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%. G0-F% or normalized G0-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% or about 20%. In certain embodiments, the desired glycosylation pattern of glycoproteins can be the regulation of galactosylation of glycoproteins. For example, but not by way of limitation, the target range for non-galactosylation (GO%) can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, the GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0241] In certain embodiments, the desired glycosylation pattern of the glycoprotein achieved by Mn supplementation can be a combination of modulation of non-fucosylation and modulation of fucosylation of the glycoprotein. For example, but not by way of limitation, the target range of G0-F% or normalized G0-F% can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5%, and the target range of G0% can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0242] 5.6 Combinations of pCO2, substrate storage, culture duration, supplemented Mn, osmotic pressure, and Na+ concentration

[0243] In certain embodiments, a combination of disclosed techniques for regulating glycosylation (e.g., non-fucosylation and / or galactosylation) of glycoproteins can be used. For example, but not by way of limitation, a combination of disclosed conditions of pCO2, substrate storage, culture duration, supplemented Mn, osmotic pressure and / or Na+ concentration can be used in cell culture media and / or cell cultures to achieve or maintain a desired glycosylation pattern of glycoproteins. In non-limiting embodiments, the disclosed substrate storage time (e.g., about 0 hours to about 72 hours) at a defined temperature or temperature range (e.g., about 25°C to about 39°C) can be applied to the culture medium together with supplemented Mn (e.g., about 1 nM to about 30000 nM), pCO2 levels (e.g., about 0 mmHg to about 250 mmHg), culture duration (e.g., about 0 days to about 25 days), Na+ concentration (e.g., about 0 mM to 250 mM) and osmotic pressure (e.g., about 250 mOsm / kg to about 600 mOsm / kg).

[0244] In certain embodiments, the disclosed combination of conditions of pCO2, matrix preservation, culture duration, supplemented Mn, osmotic pressure, and Na+ concentration can induce a combination or synergistic effect on the non-fucosylation and / or galactosylation properties of the glycoprotein. For example, but not by way of limitation, when the disclosed conditions of pCO2, matrix preservation, culture duration, supplemented Mn, osmotic pressure, and Na+ concentration are employed, a synergistic regulation (e.g., increase or decrease) of the G0-F% of the glycoprotein can occur. In addition, in non-limiting embodiments, when the disclosed conditions of pCO2, matrix preservation, culture duration, supplemented Mn, osmotic pressure, and Na+ concentration are employed, a synergistic regulation (e.g., increase or decrease) of the G0% of the glycoprotein can occur. In addition, in certain embodiments, when the disclosed conditions of pCO2, matrix preservation, culture duration, supplemented Mn, osmotic pressure, and Na+ concentration are employed, a synergistic regulation of G0-F% can occur.

[0245] Certain embodiments described herein relate to regulating glycosylation by modifying a combination of disclosed conditions (e.g., pCO2, substrate preservation, culture duration, supplemented Mn, osmotic pressure, and Na+ concentration) to achieve or maintain a desired glycoprotein glycosylation pattern. In certain non-limiting embodiments, the desired glycoprotein glycosylation pattern may be an increase or decrease in the non-fucosylation of the glycoprotein. For example, but not as a limitation, the target range of non-fucosylated G0 (G0-F% or normalized G0-F%) may be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%. In certain embodiments, G0-F% or normalized G0-F% may be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%. In certain embodiments, the desired glycoprotein glycosylation pattern can be the regulation of galactosylation of glycoprotein. For example, but not limited to, the target range (GO%) of galactosylation can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85% or about 60% to about 80%. In certain embodiments, GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40% or about 50%. In certain embodiments, the desired glycoprotein glycosylation pattern can be a combination of non-fucosylated and galactosylated regulation of glycoprotein. In certain embodiments, the target range for G0-F% or normalized G0-F% may be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range for G0% may be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0246] 6. Galactose to regulate glycosylation

[0247] In certain embodiments, galactose, Mn, or a combination thereof in the cell culture medium and / or cell culture can affect glycosylation (e.g., galactosylation and non-fucosylation). Therefore, mAb glycosylation can be regulated by supplementing galactose, Mn, or a combination thereof. For example, but not limited to, the concentration of galactose can be increased to about 10 g / L (e.g., about 0 g / L, about 1.2 g / L, about 2 g / L, about 4 g / L, about 6 g / L, about 6.8 g / L, about 8 g / L, or about 10 g / L). In non-limiting embodiments, the concentration of galactose can be increased to about 100 mM. For example, but not limited to, the concentration of galactose can be between about 0 mM to about 60 mM, about 0 mM to about 45 mM, about 0 mM to about 20 mM, or about 0 mM to about 10 mM. Cell culture can be further supplemented to achieve an Mn concentration of about 10 nM, about 40 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 500 nM, about 700 nM, about 750 nM, about 1000 nM, about 1500 nM, about 2000 nM, about 3000 nM, about 5000 nM, about 8000 nM or about 10 μ M. The target concentration range of galactose and Mn can include concentrations falling within the range. Non-limiting examples of galactose and Mn addition can include addition to production culture and / or in the amplification culture before the production culture stage.

[0248] Certain embodiments described herein relate to regulating glycosylation by supplementing the culture medium with or without the disclosed concentration of galactose of the disclosed concentration of Mn to achieve or maintain the desired glycoprotein glycosylation pattern. In non-limiting embodiments, the desired glycoprotein glycosylation pattern can be the regulation of the non-fucosylation of the glycoprotein. For example, but not limited to, the target range of non-fucosylated G0 (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%. In certain embodiments, G0-F% or normalized G0-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% or about 20%. In certain embodiments, the desired glycoprotein glycosylation pattern can be the regulation of the galactosylation of the glycoprotein. For example, but not by way of limitation, the target range of non-galactosylation (GO%) can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, the GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%. In certain embodiments, the desired glycoprotein glycosylation pattern can be a combination of adjustments of non-fucosylation and galactosylation of the glycoprotein. For example, but not by way of limitation, the target range for G0-F% or normalized G0-F% can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range for G0% can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0249] 7. Fucose and culture temperature and their combination to regulate glycosylation

[0250] In certain embodiments, the addition of L-fucose (fucose) to the cell culture medium and / or cell culture may also affect glycosylation (e.g., galactosylation and / or non-fucosylation). Therefore, in certain embodiments, mAb glycosylation can be regulated by supplementing the cell culture medium with fucose. The addition of fucose results in the regulation of non-fucosylation (e.g., GO-F), and the degree of non-fucosylation regulation can be improved by the concentration of fucose and / or the timing of fucose addition. In certain non-limiting embodiments, the concentration of the added fucose may be between about 0 g / L and about 5 g / L (e.g., about 0 g / L, about 0.05 g / L, about 0.1 g / L, about 0.25 g / L, about 0.5 g / L, about 0.75 g / L, about 1 g / L, or about 5 g / L). In certain embodiments, the concentration of fucose may be increased to about 100 mM. For example, but not by way of limitation, the concentration of fucose can be between about 0 mM to about 100 mM, about 0 mM to about 30 mM, about 0 mM to about 10 mM, or about 0 mM to about 5 mM. In certain embodiments, after inoculating the production culture with different fucose concentrations (e.g., about 0.1 g / L, about 0.5 g / L, including concentrations falling within the disclosed range), the timing of fucose addition can be between about 0 days and the end of the production culture (e.g., about 0 days, about 5 days, about 7 days, about 10 days, about 12 days, about 15 days, or about 25 days). In certain embodiments, fucose addition can be performed at a level in the range of about 0 g / L to about 1 g / L in combination with a culture temperature in the range of about 25°C to about 39°C. In certain embodiments, the culture temperature can be between about 25°C and about 39°C, about 30°C to about 39°C, about 35°C to about 39°C, or about 36°C to about 39°C. In certain embodiments, fucose addition can be performed at a level of about 0 g / L to about 1 g / L or about 0 mM to about 60 mM in combination with Mn supplementation at a level of about 0 nM to 20000 nM in a low pCO2 or high pCO2 background. Non-limiting examples of fucose addition include addition to a production culture and / or to an expansion culture prior to a production culture stage.

[0251] Certain embodiments described herein relate to regulating glycosylation by supplementing the culture medium with a disclosed concentration of fucose under disclosed conditions (e.g., pCO2, supplemented Mn, etc.) to achieve or maintain a desired glycoprotein glycosylation pattern. In a non-limiting embodiment, the desired glycoprotein glycosylation pattern that can be achieved by increasing the fucose concentration is the regulation (e.g., increase or decrease) of the non-fucosylation of the glycoprotein. For example, but not as a limitation, the target range of non-fucosylated G0 (G0-F% or normalized G0-F%) can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%. In certain embodiments, G0-F% or normalized G0-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%. In certain embodiments, the desired glycoprotein glycosylation pattern achieved by increasing the fucose concentration can be the regulation of the galactosylation of the glycoprotein. For example, but not limited to, the target range (GO%) of fucosylated GO can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85% or about 60% to about 80%. In certain embodiments, GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40% or about 50%. In certain embodiments, the desired glycoprotein glycosylation pattern achieved by increasing the fucose concentration can be a combination of the regulation of non-fucosylation and galactosylation of the glycoprotein. For example, but not by way of limitation, the target range for G0-F% or normalized G0-F% can be between about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%, and the target range for G0% can be between about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, GO-F% or normalized GO-F% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 20%, and GO% can be adjusted by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0252] 8. Cell Culture and Glycoprotein Composition

[0253] In certain embodiments, the present disclosure relates to compositions of glycoproteins (e.g., mAbs) obtained using the cell culture strategies outlined herein. Such compositions may include specific cell culture compositions defined by the properties of cell culture medium, host cells, and expressed glycoproteins. For example, but not limited to, compositions of the present disclosure relate to compositions of target glycoproteins (e.g., mAbs) exhibiting specific glycosylation characteristics (e.g., a specific amount of fucosylated and / or galactosylated G0 glycans). In certain embodiments, compositions of the present disclosure relate to compositions containing or supplemented with cell culture medium containing a favorable Mn concentration. In certain embodiments, the present disclosure may relate to combinations of such cell culture medium and such target glycoproteins exhibiting specific glycosylation characteristics.

[0254] In certain embodiments, the present disclosure relates to a composition of a glycoprotein (e.g., mAb) obtained by screening and selecting a cell culture medium to meet a specific action target or otherwise controlling the changes in the cell culture medium components. For example, but not by way of limitation, the present disclosure relates to a mAb composition, wherein the composition is produced by cell culture, wherein the cell culture medium Mn concentration is in the range of 30nM to 110nM, and wherein the matrix falling outside this range of Mn concentration is not used in conjunction with the cell culture producing mAb. The present disclosure also relates to a mAb composition, wherein the composition is produced by cell culture, wherein the cell culture Mn concentration is in the range of 30nM to 110nM, and wherein the cell culture falling outside this range of Mn concentration is not used in conjunction with the cell culture producing mAb. In certain embodiments, the compositions of the present disclosure relate to compositions of cell culture mediums in which the Mn concentration of the raw material has been screened and / or selected. In certain embodiments, the present disclosure may relate to a combination of such cell culture medium and such target glycoproteins exhibiting specific glycosylation characteristics.

[0255] In certain embodiments, the present disclosure relates to compositions of glycoproteins (e.g., mAbs) exhibiting specific glycosylation characteristics (e.g., specific amounts of fucosylated and / or galactosylated G0 glycans) obtained by controlling the Mn concentration in a cell culture medium by HTST treatment of a substrate having a pre-HTST pH adjustment target of less than about 7.3 or less than about 7.0. For example, but not by way of limitation, the pre-HTST pH adjustment target value of the substrate may be about 6.1, about 6.3, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, or about 7.3. In certain embodiments, the compositions of the present disclosure relate to compositions of cell culture media in which a HTST treatment step has been performed with a pre-HTST pH adjustment target as disclosed herein. In certain embodiments, the present disclosure may relate to combinations of such cell culture media and such glycoproteins of interest exhibiting specific glycosylation characteristics.

[0256] In certain embodiments, the present disclosure relates to compositions of glycoproteins (e.g., mAbs) exhibiting specific glycosylation characteristics (e.g., specific amounts of fucosylated and / or galactosylated G0 glycans) obtained by controlling pCO2, substrate storage duration, culture duration, culture temperature, manganese, galactose, fucose and / or osmotic pressure / Na+ concentration, or a combination thereof, during cell culture. In certain embodiments, the compositions of the present disclosure relate to compositions of cell culture media, in which pCO2, substrate storage duration, culture duration, culture temperature, manganese, galactose, fucose and / or osmotic pressure / Na+ concentration, or a combination thereof, have been controlled, as outlined herein. In certain embodiments, the present disclosure may relate to combinations of such cell culture media and such glycoproteins of interest exhibiting specific glycosylation characteristics.

[0257] In certain embodiments, various bioreactor configurations may be used for the composition of cell culture media. For example, but not by way of limitation, the volume of the bioreactor can be between about 1 L and about 20,000 L (e.g., about 1 L, about 1.5 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L, about 3000 L, about 4000 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10,000 L, about 11,000 L, about 12,000 L, about 13,000 L, about 14,000 L, about 15,000 L, about 16,000 L, about 17,000 L, about 18,000 L, about 19,000 L, or about 20,000 L). In non-limiting examples, the bioreactor configuration can be modified to adjust the level of pCO2, substrate retention duration, osmotic pressure, Na+, Mn, temperature, pH, fucose, galactose, or a combination thereof.

[0258] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of features disclosed and claimed herein. Thus, the features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. For purposes of illustration and description, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0259] It will be apparent to those skilled in the art that various modifications and variations may be made to the compositions and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Therefore, it is intended that the disclosed subject matter includes modifications and variations within the scope of the appended claims and their equivalents. Various publications, patents, and patent applications are cited herein, the contents of which are incorporated herein by reference in their entirety.

[0260] Embodiments of the present disclosure

[0261] The following are non-limiting examples of the present disclosure.

[0262] 1. A method for regulating the glycosylation pattern of a glycoprotein of interest in cell culture, comprising: regulating the following parameters in a cell culture medium and / or in a cell culture environment, alone or in any combination: Mn concentration from about 1 nM to about 20,000 nM under high partial pressure CO2 (pCO2) conditions; Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; pCO2 from about 10 mmHg to about 250 mmHg; cell culture medium storage duration from about 0 hours to about 120 hours before inoculation at a temperature of about 25°C to 39°C; cell culture duration from about 0 days to about 150 days; Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C.

[0263] 2. The method according to embodiment 1, wherein the cell culture environment is in a bioreactor with or without cells.

[0264] 3. The method of embodiment 1 or embodiment 2, wherein the low pCO2 condition is from about 10 mmHg to about 100 mmHg, and the high pCO2 condition is from about 20 mmHg to about 250 mmHg.

[0265] 4. The method according to embodiment 3, wherein the duration of pCO2 regulation covers at least the first half of the cell culture duration.

[0266] 5. The method according to any one of the preceding embodiments, wherein the target glycoprotein is a recombinant protein.

[0267] 6. A method according to any one of the preceding embodiments, wherein the recombinant protein is an antibody or antibody fragment, scFv (single-chain variable fragment), BsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domains), DNL-(Fab)3 (docked locked trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

[0268] 7. The method of any one of the preceding embodiments, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0269] 8. The method of any preceding embodiment, wherein the antibody is an anti-CD20 antibody.

[0270] 9. The method of any one of the preceding embodiments, wherein the anti-CD20 antibody is ocrelizumab.

[0271] 10. The method of embodiments 6 to 8, wherein the antibody or antibody fragment exhibits: a G0-F% (percentage of non-fucosylated glycoprotein) of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; or a normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; and / or a G0% (percentage of non-galactosylated glycoprotein) of about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%.

[0272] 11. The method of embodiments 6 to 9, wherein the glycosylation is modulated to achieve: increased non-fucosylation (e.g., G0-F (non-fucosylated G0)) while decreasing non-galactosylation (e.g., G0 (fucosylated, non-galactosylated G0)); or decreased non-fucosylation (e.g., G0-F) while increasing non-galactosylation (e.g., G0); or increased or decreased non-fucosylation (e.g., G0-F) without affecting non-galactosylation (e.g., G0); or increased or decreased non-galactosylation (e.g., G0) without affecting non-fucosylation (e.g., G0-F).

[0273] 12. The method according to any one of the preceding embodiments, wherein the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; and the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours at a temperature of about 25°C to 39°C; the cell culture duration is from about 0 days to about 150 days; the Na+ concentration is from about 0 mM to about 300 mM; the osmotic pressure is from about 250 mOsm / kg to about 550 mOsm / kg; the galactose concentration is from about 0 mM to about 60 mM; the fucose concentration is from about 0 mM to about 60 mM; and the culture temperature is from about 29°C to about 39°C.

[0274] 13. The method according to embodiment 12, wherein the step of adjusting the glycosylation pattern of the target glycoprotein comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; and the following parameters in the cell culture medium and / or in the cell culture environment: at a temperature of about 25°C to 39°C, the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours; and the cell culture duration is from about 0 days to about 150 days.

[0275] 14. A method according to embodiment 12, wherein the step of adjusting the glycosylation pattern of the target glycoprotein comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; and the following parameters in the cell culture medium and / or in the cell culture environment: the galactose concentration from about 0 mM to about 60 mM; and / or, the fucose concentration from about 0 mM to about 60 mM.

[0276] 15. The method according to any one of the preceding embodiments, wherein the step of adjusting the glycosylation pattern of the target glycoprotein comprises: adjusting the pre-inoculation cell culture medium storage duration and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: Mn concentration from about 1 nM to about 20,000 nM under high partial pressure CO2 (pCO2) conditions; Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; pCO2 from about 10 mmHg to about 250 mmHg; cell culture duration from about 0 days to about 150 days; Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C; wherein at a temperature of about 25°C to 39°C, the cell culture medium is stored for a duration of from about 0 hours to about 120 hours;

[0277] 16. The method according to embodiment 15, wherein the step of adjusting the glycosylation pattern of the target glycoprotein comprises: adjusting the storage duration of the cell culture medium before inoculation and the following parameters in the cell culture medium and / or in the cell culture environment: under high partial pressure CO2 (pCO2) conditions, the Mn concentration is from about 1 nM to about 20,000 nM; or, under low pCO2 conditions, the Mn concentration is from about 1 nM to about 30,000 nM; the pCO2 is from about 10 mmHg to about 250 mmHg; and the cell culture duration is from about 0 days to about 150 days; wherein at a temperature of about 25°C to 39°C, the cell culture medium storage duration is from about 0 hours to about 120 hours;

[0278] 17. The method according to any one of embodiments 12-15, wherein the target glycoprotein is an antibody or an antibody fragment thereof.

[0279] 18. The method of embodiment 17, wherein the antibody or antibody fragment thereof is an anti-CD20 antibody.

[0280] 19. The method of embodiment 18, wherein the anti-CD20 antibody is ocrelizumab.

[0281] 20. A method according to any one of the preceding embodiments, wherein the step of regulating the glycosylation pattern of the target glycoprotein comprises: regulating the cell culture duration and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C, wherein the cell culture duration is from about 0 days to about 150 days.

[0282] 21. A method according to any one of the preceding embodiments, wherein the step of regulating the glycosylation pattern of the target glycoprotein comprises: adjusting the Na+ concentration to about 0 nM to about 300 nM and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C, wherein the Na+ concentration is from about 0 mM to about 300 mM.

[0283] 22. The method of any one of the preceding embodiments, wherein the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises adjusting the Na+ concentration and the pCO2 from about 10 mmHg to about 250 mmHg.

[0284] 23. A method according to any one of the preceding embodiments, wherein the step of regulating the glycosylation pattern of the target glycoprotein comprises: regulating the osmotic pressure and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C, wherein the osmotic pressure is from about 250 mOsm / kg to about 550 mOsm / kg.

[0285] 24. The method of embodiment 1, wherein the step of adjusting the glycosylation pattern of the target glycoprotein comprises: adjusting the Mn concentration from about 1 nM to about 30,000 nM under low pCO2 conditions; or adjusting the Mn concentration from about 1 nM to about 20,000 nM under high pCO2 conditions; adjusting the Na+ concentration from about 0 mM to about 300 mM; and adjusting the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours.

[0286] 25. The method of embodiment 1, wherein the modulation of the glycosylation pattern of the glycoprotein of interest comprises: regulating the osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg, and the pCO2 from about 10 mmHg to about 250 mmHg.

[0287] 26. A method according to embodiment 1, wherein the regulation of the glycosylation pattern of the target glycoprotein includes adjusting the following items: the culture temperature from about 29°C to about 39°C, and, the galactose concentration from about 0 mM to about 60 mM; and / or the fucose concentration from about 0 mM to about 60 mM.

[0288] 27. The method of embodiment 1, wherein the modulation of the glycosylation pattern of the glycoprotein of interest comprises: adjusting the pCO2 from about 10 mmHg to about 250 mmHg; and the fucose concentration from about 0 mM to about 60 mM.

[0289] 28. The method of embodiment 1, wherein the modulation of the glycosylation pattern of the glycoprotein of interest comprises: adjusting the fucose concentration from about 0 mM to about 60 mM; and the culture temperature from about 29°C to about 39°C.

[0290] 29. The method according to embodiment 1, wherein the regulation of the glycosylation pattern of the target glycoprotein comprises: regulating the pCO2 concentration and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: under high partial pressure CO2 (pCO2) conditions, the Mn concentration is from about 1 nM to about 20,000 nM; under low pCO2 conditions, the Mn concentration is from about 1 nM to about 30,000 nM; at a temperature of about 25°C to 39°C, inoculation the pre-cell culture medium storage duration is from about 0 hours to about 120 hours; the cell culture duration is from about 0 days to about 150 days; the Na+ concentration is from about 0 mM to about 300 mM; the osmotic pressure is from about 250 mOsm / kg to about 550 mOsm / kg; the galactose concentration is from about 0 mM to about 60 mM; the fucose concentration is from about 0 mM to about 60 mM; and the culture temperature is from about 29° C. to about 39° C., wherein the pCO2 concentration is from about 10 mmHg to about 250 mmHg.

[0291] 30. The method according to any one of the above embodiments, wherein the Mn concentration is: from about 1 nM to about 20000 nM in high pCO2 culture; from about 1 nM to about 10000 nM, from about 1 nM to about 5000 nM, from about 1 nM to about 4000 nM, from about 1 nM to about 3000 nM, from about 1 nM to about 2000 nM, from about 1 nM to about 1000 nM in high pCO2 culture; In high pCO2 cultures, from about 1 nM to about 500 nM, from about 1 nM to about 100 nM, from about 1 nM to about 50 nM, from about 1 nM to about 20 nM, from about 20 nM to about 2000 nM, from about 20 nM to about 3000 nM, from about 20 nM to about 10000 nM, from about 20 nM to about 20,000 nM, from about 20 nM to about 300 nM, about 30 nM to about 110 nM.

[0292] 31. The method according to any of the above embodiments, wherein the Mn concentration is: from about 1 nM to about 30,000 nM in low pCO2 culture; from about 1 nM to about 20,000 nM, from about 1 nM to about 10,000 nM, from about 1 nM to about 5000 nM, from about 1 nM to about 4000 nM, from about 1 nM to about 3000 nM, from about 1 nM to about 2000 nM, from about 1 nM to about 1000 nM, from about 1 nM to about 5000 nM, from about 1 nM to about 4000 nM, from about 1 nM to about 3000 nM, from about 1 nM to about 2000 nM, from about 1 nM to about 1000 nM, from about 1 nM to about 500 nM, from about 1 nM to about 100nM, from about 1nM to about 50nM, from about 1nM to about 20nM, from about 20nM to about 100nM, about 20nM to about 300nM, from about 20nM to about 500nM, from about 20nM to about 1000nM, from about 20nM to about 2000nM, from about 20nM to about 3000nM, from about 20nM to about 5000nM, from about 20nM to about 10000nM, from about 20nM to about 20000nM, or from about 30nM to about 110nM.

[0293] 32. The method according to any one of the preceding embodiments, wherein the adjustment of the Mn concentration comprises determining the Mn content in a cell culture feedstock and selecting a feedstock batch to adjust the Mn concentration.

[0294] 33. The method according to any one of the above embodiments, wherein the adjustment of the Mn concentration comprises (i) controlling materials in contact with the cell culture medium or cell culture; or (ii) calculating the concentration of Mn leached in the cell culture medium or during cell culture; or a combination of (i) and (ii) to adjust the Mn concentration.

[0295] 34. The method of embodiment 33, wherein the leached Mn is produced by contacting the cell culture and / or cell culture medium with: (i) a filter; (ii) a matrix preparation, storage or culture vessel; or (iii) a combination of (i) and (ii).

[0296] 35. The method of embodiment 34, wherein the filter includes but is not limited to: a depth filter, a column, a membrane, and a disk.

[0297] 36. The method of embodiment 34, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resins.

[0298] 37. The method of any one of the preceding embodiments, wherein the cell culture medium is a basal matrix, a reconstituted matrix, a feed matrix, a hydrolysate, a supplement, a serum, or an additive.

[0299] 38. The method of any one of the preceding embodiments, wherein the cell culture medium is supplemented during a production phase of the cell culture.

[0300] 39. The method of any one of the preceding embodiments, wherein the cell culture medium is supplemented during a pre-production phase of the cell culture.

[0301] 40. The method of any one of the preceding embodiments, wherein the cell culture medium comprises one or more of: Mn, fucose, galactose, and / or Na+, and wherein supplementation is based on a predetermined schedule or standard.

[0302] 41. The method of any one of the preceding embodiments, wherein one or more of Mn, fucose, galactose, and Na+ is supplemented as a single dose, an intermittent supplement, a continuous supplement, a semi-continuous supplement, a feedback loop-based supplement, or a combination of one or more thereof.

[0303] 42. The method of any preceding embodiment, wherein the cell culture medium consists essentially of one or more of: i) Mn, ii) fucose, iii) galactose, and / or iv) Na+.

[0304] 43. The method of any preceding embodiment, wherein the adjustment of the Mn concentration comprises employing a cell culture medium pH of about 6.1 to about 7.3, or about 6.3 to about 7.3, prior to high temperature, short time (HTST) heat treatment.

[0305] 44. The method of any preceding embodiment, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of the pCO2.

[0306] 45. The method of any one of the preceding embodiments, wherein the cell culture or cell culture medium is in a bioreactor, and wherein regulation of pCO2 is achieved by regulating: the bioreactor working volume; the bioreactor gas sparging strategy; the bioreactor agitation strategy; the bioreactor matrix exchange strategy, the bioreactor perfusion strategy, the bioreactor feeding strategy, or any combination thereof.

[0307] 46. ​​The method of embodiment 44, wherein said pCO2 regulation comprises establishing a high pCO2 culture.

[0308] 47. The method of embodiment 46, wherein the pCO2 is about 20 mmHg to about 250 mmHg, about 20 mmHg to about 250 mmHg, about 20 mmHg to about 150 mmHg, or about 30 mmHg to about 250 mmHg.

[0309] 48. The method of any one of the preceding embodiments, wherein the pCO2 regulation comprises establishing a low pCO2 culture.

[0310] 49. The method of embodiment 48, wherein the pCO2 is about 10 mmHg to about 100 mmHg, 10 mmHg to about 80 mmHg, about 20 mmHg to about 70 mmHg, or about 30 mmHg to about 60 mmHg.

[0311] 50. The method of any one of the preceding embodiments, wherein the pCO2 adjustment occurs on day 0 of the culturing.

[0312] 51. The method of embodiment 50, wherein the pCO2 regulation occurs for: about a majority of the cell culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0313] 52. The method of embodiment 50, wherein the pCO2 regulation occurs for: about a majority of the production culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0314] 53. The method of embodiment 52, wherein the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the pre-inoculation cell culture medium storage duration, wherein the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours, from about 0 hours to about 72 hours, from about 0 hours to about 48 hours, or from about 0 hours to about 24 hours.

[0315] 54. The method of embodiment 53, wherein the temperature of the matrix during storage of the cell culture medium prior to inoculation is about 25°C to about 39°C, about 30°C to about 39°C, about 35°C to about 39°C, or about 36°C to about 39°C.

[0316] 55. A method according to any of the preceding embodiments, wherein the regulation of the glycosylation pattern of the glycoprotein of interest comprises regulating the duration of the cell culture, wherein the duration of the cell culture is from about 0 days to about 150 days, from about 0 days to about 15 days, from about 0 days to about 12 days, from 0 days to about 7 days, or from about 0 days to about 5 days.

[0317] 56. A method according to any of the preceding embodiments, wherein the regulation of the glycosylation pattern of the glycoprotein of interest comprises regulating the Na+ concentration, wherein the Na+ concentration is about 0 mM to about 300 mM, about 20 mM to about 20 mM, about 30 mM to about 150 mM, or about 40 mM to about 130 mM.

[0318] 57. A method according to any one of the preceding embodiments, wherein the regulation of the Na+ concentration comprises supplementing the cell culture with Na compounds, wherein the Na compounds include but are not limited to: Na2CO3, NaHCO3, NaOH, NaCl or a combination thereof.

[0319] 58. A method according to any one of the above embodiments, wherein the regulation of the glycosylation pattern of the glycoprotein of interest comprises regulating the osmotic pressure, wherein the osmotic pressure is about 250mOsm / kg to about 550mOsm / kg, about 300mOsm / kg to about 450mOsm / kg, or about 325mOsm / kg to about 425mOsm / kg.

[0320] 59. The method of any one of the preceding embodiments, wherein the regulation of osmotic pressure comprises supplementing the cell culture with an osmotic pressure regulating matrix component.

[0321] 60. The method of embodiment 59, wherein the osmotic pressure regulating matrix component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

[0322] 61. The method of embodiment 59, wherein the osmotic pressure regulating matrix component is supplemented as a single dose, intermittent supplementation, continuous supplementation, semi-continuous supplementation, feedback loop based supplementation, or a combination of one or more thereof.

[0323] 62. The method of any one of the preceding embodiments, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of the galactose concentration, wherein the galactose concentration is from about 0 mM to about 60 mM or from about 0 mM to about 50 mM.

[0324] 63. A method according to any of the preceding embodiments, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the fucose concentration, wherein the fucose concentration is from about 0 mM to about 60 mM, from 0 mM to about 40 mM, from about 0 mM to about 20 mM, or from about 0 mM to about 10 mM.

[0325] 64. A method according to any of the preceding embodiments, wherein regulation of the glycosylation pattern of the glycoprotein of interest comprises regulating the cell culture temperature, wherein the cell culture temperature is about 29°C to about 39°C, about 30°C to about 39°C, about 31°C to about 38°C, or about 34°C to about 38°C.

[0326] 65. The method of embodiment 64, wherein the cell culture temperature is adjusted during the production phase of the cell culture.

[0327] 66. The method of any one of the preceding embodiments, wherein the cell culture temperature is adjusted prior to a production phase of the cell culture.

[0328] 67. The method of any preceding embodiment, wherein the cell culture temperature is adjusted based on a predetermined schedule or criteria.

[0329] 68. The method of any preceding embodiment, wherein the cell culture comprises eukaryotic cells.

[0330] 69. The method of embodiment 68, wherein the eukaryotic cell is an insect cell, an avian cell, a fungal cell, a plant cell, or a mammalian cell.

[0331] 70. The method of embodiment 69, wherein the fungal cell is yeast, Pichia pastoris, or any filamentous fungal cell.

[0332] 71. The method of embodiment 70, wherein the yeast cell is a Saccharomyces cerevisiae cell.

[0333] 72. The method of embodiment 69, wherein the mammalian cell is a CHO cell.

[0334] 73. A method according to any of the preceding embodiments, wherein the cell culture is in a bioreactor, including but not limited to: single-use technology (SUT) bags or bioreactors, WAVE bioreactors, stainless steel bioreactors, flasks, test tubes and chambers.

[0335] 74. The method of any one of the preceding embodiments, wherein the volume of the cell culture is from 1 mL to 35,000 L.

[0336] 75. The method of embodiment 74, wherein the volume of the cell culture is from 1 mL to 10 ml, from 1 mL to 50 ml, from 1 mL to 100 ml, from 1 mL to 200 ml, from 1 mL to 300 ml, from 1 mL to 500 ml, from 1 mL to 1000 ml, from 1 mL to 2000 ml, from 1 mL to 3000 ml, from 1 mL to 4000 ml, from 1 mL to 5000 ml, from 1 mL to 1 L, from 1 mL to 2 L, from 1 mL to 3 L, from 1 mL to 4 L, from 1 mL to 5 L, from 1 mL to 6 L, from 1 mL to 10 L, from 1 mL to 20 L, from 1mL to 30L, from 1mL to 40L, from 1mL to 50L, from 1mL to 60L, from 1mL to 70L, from 1mL to 100L, from 1mL to 200L, from 1mL to 300L, from 1mL to 400L, from 1mL to 500L, from 1mL to 1000L, from 1mL to 2000L, from 1mL to 3000L, from 1mL to 4000L, from 1mL to 5000L, from 1mL to 10,000L, from 1mL to 20,000L, from 1mL to 30,000L, from 1mL to 30,000L, from 1mL to 35,000L.

[0337] 76. Use of the method according to any one of embodiments 1-75 for obtaining a glycoprotein of interest, wherein the glycoprotein of interest exhibits: a G0-F% (percentage of non-fucosylated glycoprotein) of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; or a normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; and / or a G0% (percentage of non-galactosylated glycoprotein) of about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%.

[0338] 77. Use of the method according to any one of embodiments 1-75, wherein the glycosylation is modulated to achieve: increased non-fucosylation (e.g., G0-F (non-fucosylated G0)) while reducing non-galactosylation (e.g., G0 (fucosylated, non-galactosylated G0)); or reduced non-fucosylation (e.g., G0-F) while increasing non-galactosylation (e.g., G0); or increased or reduced non-fucosylation (e.g., G0-F) without affecting non-galactosylation (e.g., G0); or increased or reduced non-galactosylation (e.g., G0) without affecting non-fucosylation (e.g., G0-F).

[0339] 78. A method for preparing a cell culture medium, a feed matrix, a hydrolysate or an additive, the method comprising adjusting one or more of the following steps: Mn concentration in a high partial pressure CO2 (pCO2) culture from about 1 nM to about 20,000 nM; Mn concentration in a low pCO2 culture from about 1 nM to about 30,000 nM; pCO2 from about 10 mmHg to about 250 mmHg; cell culture medium storage duration before inoculation from about 0 hours to about 120 hours; cell culture duration from about 0 days to about 150 days; Na+ concentration from about 0 mM to about 300 mM; osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration from about 0 mM to about 60 mM; fucose concentration from about 0 mM to about 60 mM; and culture temperature from about 29°C to about 39°C; wherein the cell culture medium, feed matrix, hydrolysate or additive adjusts the glycosylation pattern of a glycoprotein of interest.

[0340] 79. The method of embodiment 78, wherein the glycoprotein of interest is an antibody or an antibody fragment.

[0341] 80. The method of embodiment 79, wherein the antibody or antibody fragment exhibits: a G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; or a normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; and / or a G0% of about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%.

[0342] 81. A method according to embodiment 79, wherein the glycosylation of the antibody or antibody fragment is modulated to achieve: increased non-fucosylation (e.g., G0-F (non-fucosylated G0)) while reducing non-galactosylation (e.g., G0 (fucosylated, non-galactosylated G0)); or reduced non-fucosylation (e.g., G0-F) while increasing non-galactosylation (e.g., G0); or increased or reduced non-fucosylation (e.g., G0-F) without affecting non-galactosylation (e.g., G0); or increased or reduced non-galactosylation (e.g., G0) without affecting non-fucosylation (e.g., G0-F).

[0343] 82. The method of any one of embodiments 78-81, comprising adjusting the Mn concentration from about 1 nM to about 30000 nM, and the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours.

[0344] 83. The method of any one of embodiments 78-82, comprising adjusting the pCO2 from about 10 mmHg to about 250 mmHg, the Na+ concentration from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours.

[0345] 84. The method of any one of embodiments 78-83, comprising adjusting the Mn concentration from about 1 nM to about 30000 nM, the pCO2 from about 10 mmHg to about 250 mmHg, and the Na+ concentration from about 0 mM to about 300 mM.

[0346] 85. The method of any one of embodiments 78-84, comprising adjusting the Mn concentration from about 1 nM to about 30,000 nM, the pCO2 from about 10 mmHg to about 250 mmHg, the Na+ concentration from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration from about 0 hours to about 72 hours.

[0347] 86. The method of any one of embodiments 78-85, comprising adjusting the pCO2 from about 10 mmHg to about 250 mmHg; and the Na+ concentration from about 0 mM to about 300 mM.

[0348] 87. The method of any one of embodiments 78-86, comprising adjusting the osmotic pressure from about 250 mOsm / kg to about 550 mOsm / kg, and the pCO2 from about 10 mmHg to about 250 mmHg.

[0349] 88. The method of any one of embodiments 78-87, comprising adjusting the pCO2 from about 10 mmHg to about 250 mmHg, the Mn concentration from about 1 mM to about 30,000 mM, the cell culture duration from about 0 days to about 150 days, and the pre-inoculation cell culture medium storage duration from about 0 hours to about 120 hours.

[0350] 89. The method of any one of embodiments 78-88, comprising adjusting the Mn concentration from about 1 nM to about 30000 nM, and the galactose concentration from about 0 mM to about 60 mM.

[0351] 90. The method of any one of embodiments 78-89, comprising adjusting the fucose concentration from about 0 mM to about 60 mM, and the Mn concentration from about 1 nM to about 30000 nM.

[0352] 91. The method of any one of embodiments 78-90, comprising adjusting the fucose concentration from about 0 mM to about 60 mM, and the pCO2 from about 10 mmHg to about 250 mmHg.

[0353] 92. The method of any one of embodiments 78-91, comprising adjusting the fucose concentration from about 0 mM to about 60 mM, the Mn concentration from about 1 nM to about 30000 nM, and the pCO2 from about 10 mmHg to about 250 mmHg.

[0354] 93. The method of any one of embodiments 78-92, comprising adjusting the fucose concentration from about 0 mM to about 60 mM, and the cell culture temperature is about 29°C to about 39°C.

[0355] 94. The method of any one of embodiments 78-93, comprising adjusting the fucose concentration from about 0 mM to about 60 mM, and the cell culture duration from about 0 days to about 150 days.

[0356] 95. A method according to any one of embodiments 78-94, wherein the Mn concentration is: about 1 nM to about 20000 nM in high pCO2 culture; about 1 nM to about 1000 nM in high pCO2 culture; about 20 nM to about 300 nM in high pCO2 culture; or about 30 nM to about 110 nM in high pCO2 culture.

[0357] 96. A method according to any one of embodiments 78-95, wherein the Mn concentration is: about 1 nM to about 30,000 nM in low pCO2 culture; about 1 nM to about 3000 nM in low pCO2 culture; about 20 nM to about 300 nM in low pCO2 culture; or about 30 nM to about 110 nM in low pCO2 culture.

[0358] 97. The method of embodiment 95 or embodiment 96, wherein the adjustment of the Mn concentration comprises determining the Mn content in the cell culture feedstock and selecting a feedstock batch to adjust the Mn concentration.

[0359] 98. The method of embodiment 95 or embodiment 96, wherein the regulation of the Mn concentration comprises i) controlling materials in contact with the cell culture medium or cell culture; or (ii) calculating the concentration of Mn leached into the cell culture medium or during cell culture; or a combination of (i) and (ii) to regulate the Mn concentration.

[0360] 99. The method of embodiment 98, wherein the leached Mn is produced by contacting the cell culture and / or cell culture medium with: (i) a filter; (ii) a matrix preparation, storage or culture vessel; or (iii) a combination of (i) and (ii).

[0361] 100. The method of embodiment 99, wherein the filter includes but is not limited to: a depth filter, a column, a membrane, and a disk.

[0362] 101. The method of embodiment 99, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resins.

[0363] 102. The method of embodiment 95 or embodiment 96, wherein the adjustment of the Mn concentration comprises employing a cell culture medium pH of about 6.1 to about 7.3, or about 6.3 to about 7.3, prior to HTST treatment.

[0364] 103. The method of any one of embodiments 78-102, wherein the pCO2 is regulated.

[0365] 104. The method of embodiment 103, wherein the cell culture medium is in a bioreactor, and wherein regulation of pCO2 is achieved by regulating: the bioreactor working volume; the bioreactor gas sparging strategy; the bioreactor agitation strategy; the bioreactor feeding strategy, the bioreactor perfusion strategy, the bioreactor matrix exchange strategy, or any combination thereof.

[0366] 105. The method of embodiment 103, wherein the pCO2 regulation comprises establishing a high pCO2 culture.

[0367] 106. The method of embodiment 105, wherein the pCO2 is about 20 mmHg to about 250 mmHg, about 20 mmHg to about 250 mmHg, about 20 mmHg to about 150 mmHg, or about 30 mmHg to about 150 mmHg.

[0368] 107. The method of embodiment 103, wherein the pCO2 regulation comprises establishing a low pCO2 culture.

[0369] 108. The method of embodiment 107, wherein the pCO2 is about 10 mmHg to about 100 mmHg, 10 mmHg to about 80 mmHg, about 20 mmHg to about 70 mmHg, or about 30 mmHg to about 60 mmHg.

[0370] 109. The method of embodiment 103, wherein said pCO2 adjustment occurs on day 0 of said culturing.

[0371] 110. The method of embodiment 103, wherein the pCO2 regulation occurs for: about a majority of the cell culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0372] 111. The method of embodiment 103, wherein the pCO2 regulation occurs for: about a majority of the production culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0373] 112. The method of any one of embodiments 78-111, wherein the pre-inoculation cell culture medium storage duration is about 0 hours to about 120 hours, about 0 hours to about 72 hours, about 0 hours to about 48 hours, or about 0 hours to about 24 hours.

[0374] 113. The method of embodiment 112, wherein the temperature of the matrix during storage of the cell culture medium before inoculation is about 25°C to about 39°C, about 30°C to about 39°C, about 35°C to about 39°C, or about 36°C to about 39°C.

[0375] 114. The method of any one of embodiments 78-113, wherein the cell culture duration is about 0 days to about 150 days, about 0 days to about 15 days, about 0 days to about 12 days, 0 days to about 7 days, or about 0 days to about 5 days.

[0376] 115. The method of any one of embodiments 78-114, wherein the Na+ concentration is about 0 mM to about 300 mM, about 20 mM to about 200 mM, about 30 mM to about 150 mM, or about 40 mM to about 130 mM.

[0377] 116. A method according to embodiment 115, wherein the regulation of the Na+ concentration comprises supplementing the cell culture with Na compounds, wherein the Na compounds include but are not limited to: Na2CO3, NaHCO3, NaOH, NaCl or a combination thereof.

[0378] 117. The method of any one of embodiments 78-116, wherein the osmotic pressure is about 250 mOsm / kg to about 550 mOsm / kg, about 300 mOsm / kg to about 450 mOsm / kg, or about 325 mOsm / kg to about 425 mOsm / kg.

[0379] 118. A method according to embodiment 117, wherein the regulation of osmotic pressure comprises supplementing the cell culture with an osmotic pressure regulating matrix component.

[0380] 119. The method of embodiment 118, wherein the osmotic pressure regulating matrix component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

[0381] 120. The method of any one of embodiments 787-119, wherein the galactose concentration is about 0 mM to about 60 mM or about 0 mM to about 50 mM.

[0382] 121. The method of any one of embodiments 78-119, wherein the fucose concentration is about 0 mM to about 60 mM, 0 mM to about 40 mM, about 0 mM to about 20 mM, or about 0 mM to about 10 mM.

[0383] 122. The method of any one of embodiments 78-119, wherein the cell culture temperature is about 29°C to about 39°C, about 30°C to about 39°C, about 31°C to about 38°C, or about 34°C to about 38°C.

[0384] 123. Use of the substrate according to any one of embodiments 78-121 for producing a recombinant protein in a eukaryotic cell fermentation process.

[0385] 124. The use of the matrix according to embodiment 123, wherein the recombinant protein is an antibody or antibody fragment, scFv (single-chain variable fragment), BsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domains), DNL-(Fab)3 (docked locked trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

[0386] 125. Use of the matrix according to embodiment 124, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0387] 126. Use of the matrix according to embodiment 124, wherein the antibody is an anti-CD20 antibody.

[0388] 127. The use of the matrix according to embodiment 124, wherein the anti-CD20 antibody is ocrelizumab.

[0389] 128. Use of the matrix according to embodiment 124, wherein the antibody or antibody fragment exhibits: G0-F% (percentage of non-fucosylated glycoprotein) of about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%; normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%; and / or G0% (percentage of non-galactosylated glycoprotein) of about 40% to about 90%, about 50% to about 90%, about 55% to about 85% or about 60% to about 80%.

[0390] 129. Use of the matrix according to embodiment 124, wherein the eukaryotic cells are insect cells, avian cells, fungal cells, plant cells or mammalian cells.

[0391] 130. The use of the matrix of embodiment 129, wherein the fungal cell is yeast, Pichia pastoris, or any filamentous fungal cell.

[0392] 131. The use of the substrate of embodiment 130, wherein the yeast cells are Saccharomyces cerevisiae cells.

[0393] 132. Use of the matrix according to embodiment 129, wherein the mammalian cells are CHO cells.

[0394] 133. Use of the matrix according to any one of embodiments 123-132, wherein the cell culture is in a bioreactor, including but not limited to: single use technology (SUT) bags or bioreactors, WAVE bioreactors, stainless steel bioreactors, flasks, test tubes and chambers.

[0395] 134. Use of the matrix according to any one of embodiments 123-133, wherein the volume of the cell culture is from 1 mL to 35,000 L.

[0396] 135. Use of the matrix according to embodiment 134, wherein the volume of the cell culture is from 1 mL to 10 ml, from 1 mL to 50 ml, from 1 mL to 100 ml, from 1 mL to 200 ml, from 1 mL to 300 ml, from 1 mL to 500 ml, from 1 mL to 1000 ml, from 1 mL to 2000 ml, from 1 mL to 3000 ml, from 1 mL to 4000 ml, from 1 mL to 5000 ml, from 1 mL to 1 L, from 1 mL to 2 L, from 1 mL to 3 L, from 1 mL to 4 L, from 1 mL to 5 L, from 1 mL to 6 L, from 1 mL to 10 L, from 1 mL to 20L, from 1mL to 30L, from 1mL to 40L, from 1mL to 50L, from 1mL to 60L, from 1mL to 70L, from 1mL to 100L, from 1mL to 200L, from 1mL to 300L, from 1mL to 400L, from 1mL to 500L, from 1mL to 1000L, from 1mL to 2000L, from 1mL to 3000L, from 1mL to 4000L, from 1mL to 5000L, from 1mL to 10,000L, from 1mL to 20,000L, from 1mL to 30,000L, from 1mL to 30,000L, from 1mL to 35,000L.

[0397] 136. A cell culture composition comprising: a host cell engineered to express a glycoprotein of interest; and a cell culture and / or cell culture medium adjusted to target one or more predetermined parameters selected from the following: an Mn concentration in a high partial pressure CO2 (pCO2) culture ranging from about 1 nM to about 20,000 nM; an Mn concentration in a low pCO2 culture ranging from about 1 nM to about 30,000 nM; the pCO2 ranging from about 10 mmHg to about 250 mmHg; the pre-inoculation cell culture medium storage duration ranging from about 0 hours to about 120 hours; the cell culture duration ranging from about 0 days to about 150 days; the Na+ concentration ranging from about 0 mM to about 300 mM; the osmotic pressure ranging from about 250 mOsm / kg to about 550 mOsm / kg; the galactose concentration ranging from about 0 mM to about 60 mM; the fucose concentration ranging from about 0 mM to about 60 mM; and the culture temperature ranging from about 29°C to about 39°C.

[0398] 137. The composition of embodiment 136, wherein the cell culture environment is in a bioreactor.

[0399] 138. A composition according to any of embodiments 136-137, wherein the glycoprotein of interest is an antibody or an antibody fragment.

[0400] 139. A composition according to embodiment 138, wherein the antibody or antibody fragment exhibits: a G0-F% (percentage of non-fucosylated glycoprotein) of about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%; or a normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10% or about 1% to about 8%; and / or a G0% (percentage of non-galactosylated glycoprotein) of about 40% to about 90%, about 50% to about 90%, about 55% to about 85% or about 60% to about 80%.

[0401] 140. A composition according to embodiment 138, wherein the glycosylation of the antibody or antibody fragment is modulated to achieve: increased non-fucosylation (e.g., G0-F (non-fucosylated G0)) while reducing non-galactosylation (e.g., G0 (fucosylated, non-galactosylated G0)); or reduced non-fucosylation (e.g., G0-F) while increasing non-galactosylation (e.g., G0); or increased or reduced non-fucosylation (e.g., G0-F) without affecting non-galactosylation (e.g., G0); or increased or reduced non-galactosylation (e.g., G0) without affecting non-fucosylation (e.g., G0-F).

[0402] 141. The composition of embodiment 136, wherein the Mn concentration is from about 1 nM to about 30000 nM and the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours.

[0403] 142. The composition of embodiment 136, wherein the pCO2 is from about 10 mmHg to about 250 mmHg, the Na+ concentration is from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours.

[0404] 143. The composition of embodiment 136, wherein the Mn concentration is from about 1 nM to about 30000 nM, the pCO2 is from about 10 mmHg to about 250 mmHg, and the Na+ concentration is from about 0 mM to about 300 mM.

[0405] 144. The composition of embodiment 136, wherein the Mn concentration is from about 1 nM to about 30000 nM, the pCO2 is from about 10 mmHg to about 250 mmHg, the Na+ concentration is from about 0 mM to about 300 mM, and the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours.

[0406] 145. The composition of embodiment 136, wherein the pCO2 is from about 10 mmHg to about 250 mmHg; and the Na+ concentration is from about 0 mM to about 300 mM.

[0407] 146. The composition of embodiment 136, wherein the osmotic pressure is from about 250 mOsm / kg to about 550 mOsm / kg, and the pCO2 is from about 10 mmHg to about 250 mmHg.

[0408] 147. The composition of embodiment 136, wherein the pCO2 is from about 10 mmHg to about 250 mmHg, the Mn concentration is from about 1 mM to about 30000 mM, the cell culture duration is from about 0 days to about 150 days, and the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours

[0409] 148. The composition of embodiment 136, wherein the Mn concentration is from about 1 nM to about 30000 nM, and the galactose concentration is from about 0 mM to about 60 mM.

[0410] 149. The composition of embodiment 136, wherein the fucose concentration is from about 0 mM to about 60 mM, and the Mn concentration is from about 1 nM to about 30000 nM.

[0411] 150. The composition of embodiment 136, wherein the fucose concentration is from about 0 mM to about 60 mM, and the pCO2 is from about 10 mmHg to about 250 mmHg.

[0412] 151. The composition of embodiment 136, wherein the fucose concentration is from about 0 mM to about 60 mM, the Mn concentration is from about 1 nM to about 30000 nM, and the pCO2 is from about 10 mmHg to about 250 mmHg.

[0413] 152. The composition of embodiment 136, wherein the fucose concentration is from about 0 mM to about 60 mM and the cell culture temperature is about 29°C to about 39°C.

[0414] 153. The composition of embodiment 136, wherein the fucose concentration is from about 0 mM to about 60 mM and the cell culture duration is from about 0 days to about 150 days.

[0415] 154. The composition of any of embodiments 136-153, wherein the Mn concentration is: from about 1 nM to about 20,000 nM in high pCO2 culture; from about 1 nM to about 10,000 nM, from about 1 nM to about 5,000 nM, from about 1 nM to about 4,000 nM, from about 1 nM to about 3,000 nM, from about 1 nM to about 2,000 nM, from about 1 nM to about 10 ...10000 nM, from about 1 nM to about 5,000 nM, from about 1 nM to about 5,000 nM, from about 1 nM to about 6,000 nM, from about 1 nM to about 7,000 nM, from about 1 nM to about 8,000 nM, from about 1 nM to about 9,000 nM, from about 1 nM to about 10 in high pCO2 cultures, from about 1 nM to about 500 nM, from about 1 nM to about 100 nM, from about 1 nM to about 50 nM, from about 1 nM to about 20 nM, from about 20 nM to about 2000 nM, from about 20 nM to about 3000 nM, from about 20 nM to about 10000 nM, from about 20 nM to about 20,000 nM, from about 20 nM to about 300 nM, about 30 nM to about 110 nM.

[0416] 155. The method of any one of embodiments 136-153, wherein the Mn concentration is: from about 1 nM to about 30,000 nM in a low pCO2 culture; from about 1 nM to about 20,000 nM, from about 1 nM to about 10,000 nM, from about 1 nM to about 5,000 nM, from about 1 nM to about 4,000 nM, from about 1 nM to about 3,000 nM, from about 1 nM to about 2000 nM, from about 1 nM to about 1000 nM, from about 1 nM to about 5000 nM, from about 1 nM to about 4,000 nM, from about 1 nM to about 3,000 nM, from about 1 nM to about 2000 nM, from about 1 nM to about 1000 nM, from about 1 nM to about 500 nM, From about 1 nM to about 100 nM, from about 1 nM to about 50 nM, from about 1 nM to about 20 nM, from about 20 nM to about 100 nM, about 20 nM to about 300 nM, from about 20 nM to about 500 nM, from about 20 nM to about 1000 nM, from about 20 nM to about 2000 nM, from about 20 nM to about 3000 nM, from about 20 nM to about 5000 nM, from about 20 nM to about 10000 nM, from about 20 nM to about 20000 nM, or about 30 nM to about 110 nM.

[0417] 156. The composition of embodiment 154 or embodiment 155, wherein the adjustment of the Mn concentration comprises determining the Mn content in a cell culture feedstock and selecting a feedstock batch to adjust the Mn concentration.

[0418] 157. The composition of embodiment 154 or embodiment 155, wherein the regulation of the Mn concentration comprises (i) controlling materials in contact with the cell culture medium or cell culture; or (ii) calculating the concentration of Mn leached into the cell culture medium or during cell culture; or a combination of (i) and (ii) to regulate the Mn concentration.

[0419] 158. The composition of embodiment 157, wherein the leached Mn is produced by contacting the cell culture and / or cell culture medium with: (i) a filter; (ii) a matrix preparation, storage or culture vessel; or (iii) a combination of (i) and (ii).

[0420] 159. The composition of embodiment 158, wherein the filter includes, but is not limited to, a depth filter, a column, a membrane, and a disk.

[0421] 160. The composition of embodiment 158, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resins.

[0422] 161. The composition of embodiment 154 or embodiment 155, wherein Mn is supplemented as a component of the cell culture medium.

[0423] 162. The composition of embodiment 161, wherein the cell culture medium is a feed matrix, a hydrolysate, or a supplement.

[0424] 163. The composition of embodiment 162, wherein the feed matrix, hydrolysate, or additive comprises Mn.

[0425] 164. The composition of embodiment 162, wherein the feed matrix or additive consists essentially of Mn.

[0426] 165. The composition of embodiment 154 or embodiment 155, wherein Mn is supplemented during the production phase of the cell culture.

[0427] 166. The composition of embodiment 154 or embodiment 155, wherein the Mn is supplemented prior to the production phase of the cell culture.

[0428] 167. The composition of embodiment 154 or embodiment 155, wherein the Mn is supplemented based on a predetermined schedule or standard.

[0429] 168. The composition of embodiment 154 or embodiment 155, wherein the Mn is supplemented as a single dose, intermittent supplementation, continuous supplementation, semi-continuous supplementation, feedback loop-based supplementation, or a combination of one or more thereof.

[0430] 169. The composition of embodiment 154 or embodiment 155, wherein the adjustment of the Mn concentration comprises employing a cell culture medium pH of about 6.1 to about 7.3, or about 6.3 to about 7.3, prior to HTST heat treatment.

[0431] 170. The composition of any one of embodiments 136-153, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of the pCO2.

[0432] 171. The composition of embodiment 170, wherein the cell culture or cell culture medium is in a bioreactor, and wherein regulation of pCO2 is achieved by regulating: the bioreactor working volume; the bioreactor gas sparging strategy; the bioreactor agitation strategy; the bioreactor feeding strategy, the bioreactor perfusion strategy, the bioreactor matrix exchange strategy, or any combination thereof.

[0433] 172. The composition of embodiment 170, wherein said pCO2 regulation comprises establishing a high pCO2 culture.

[0434] 173. The composition of embodiment 172, wherein the pCO2 is about 20 mmHg to about 250 mmHg, about 20 mmHg to about 250 mmHg, about 20 mmHg to about 150 mmHg, or about 30 mmHg to about 150 mmHg.

[0435] 174. The composition of embodiment 170, wherein said pCO2 regulation comprises establishing a low pCO2 culture.

[0436] 175. The composition of embodiment 174, wherein the pCO2 is about 10 mmHg to about 100 mmHg, 10 mmHg to about 80 mmHg, about 20 mmHg to about 70 mmHg, or about 30 mmHg to about 60 mmHg.

[0437] 176. The composition of embodiment 170, wherein said pCO2 adjustment occurs on day 0 of said culturing.

[0438] 177. The composition of embodiment 170, wherein the pCO2 regulation occurs for: about a majority of the cell culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0439] 178. The composition of embodiment 170, wherein the pCO2 regulation occurs for: about a majority of the production culture, about the first 5 days, about the first 7 days, or about the first 10 days.

[0440] 179. The composition of any one of embodiments 1136-153, wherein the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the pre-inoculation cell culture medium storage duration, wherein the pre-inoculation cell culture medium storage duration is from about 0 hours to about 120 hours, from 0 hours to about 72 hours, from about 0 hours to about 48 hours, or from about 0 hours to about 24 hours.

[0441] 180. The composition of embodiment 179, wherein the temperature of the matrix during storage in the cell culture medium prior to inoculation is about 25°C to about 39°C, about 30°C to about 39°C, about 35°C to about 39°C, or about 36°C to about 39°C.

[0442] 181. A composition according to any of embodiments 136-153, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the duration of the cell culture, wherein the duration of the cell culture is from about 0 days to about 150 days, from about 0 days to about 15 days, from about 0 days to about 12 days, from 0 days to about 7 days, or from about 0 days to about 5 days.

[0443] 182. A composition according to any of embodiments 136-153, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of the Na+ concentration, wherein the Na+ concentration is from about 0 mM to about 300 mM, from about 20 mM to about 200 mM, from about 30 mM to about 150 mM, or from about 40 mM to about 130 mM.

[0444] 183. A composition according to embodiment 182, wherein the regulation of the Na+ concentration comprises supplementing the cell culture with Na compounds, wherein the Na compounds include but are not limited to: Na2CO3, NaHCO3, NaOH, NaCl or a combination thereof.

[0445] 184. The composition of embodiment 182, wherein Na+ is supplemented during the production phase of the cell culture.

[0446] 185. The composition of embodiment 182, wherein the Na+ is supplemented prior to the production phase of the cell culture.

[0447] 186. A composition according to embodiment 182, wherein the Na+ is supplemented based on a predetermined schedule or standard.

[0448] 187. The composition of embodiment 182, wherein the Na+ is supplemented as a single dose, an intermittent supplement, a continuous supplement, a semi-continuous supplement, a feedback loop based supplement, or a combination of one or more thereof.

[0449] 188. A composition according to any of embodiments 136-153, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of the osmotic pressure, wherein the osmotic pressure is about 250 mOsm / kg to about 550 mOsm / kg, about 300 mOsm / kg to about 450 mOsm / kg, or about 325 mOsm / kg to about 425 mOsm / kg.

[0450] 189. A composition according to embodiment 188, wherein the regulation of osmotic pressure comprises supplementing the cell culture with an osmotic pressure regulating matrix component.

[0451] 190. The composition of embodiment 189, wherein the osmotic pressure regulating matrix component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

[0452] 191. The composition of embodiment 189, wherein the osmotic pressure regulating matrix component is supplemented during the production phase of the cell culture.

[0453] 192. The composition of embodiment 189, wherein the osmotic pressure regulating matrix component is supplemented prior to the production phase of the cell culture.

[0454] 193. The composition of embodiment 189, wherein the osmotic pressure regulating matrix component is replenished based on a predetermined schedule or standard.

[0455] 194. The composition of embodiment 189, wherein the osmotic pressure regulating matrix component is supplemented as a single dose, an intermittent supplement, a continuous supplement, a semi-continuous supplement, a feedback loop based supplement, or a combination of one or more thereof.

[0456] 195. The composition of any one of embodiments 136-153, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of the galactose concentration, wherein the galactose concentration is from about 0 mM to about 60 mM or from about 0 mM to about 50 mM.

[0457] 196. The composition of embodiment 195, wherein galactose is supplemented as a component of the cell culture medium.

[0458] 197. The composition of embodiment 196, wherein the cell culture medium is a feed matrix, a hydrolysate, or a supplement.

[0459] 198. The composition of embodiment 197, wherein the feed matrix, hydrolysate or additive comprises galactose.

[0460] 199. The composition of embodiment 197, wherein the feed matrix or additive consists essentially of galactose.

[0461] 200. The composition of embodiment 196, wherein galactose is supplemented during the production phase of the cell culture.

[0462] 201. The composition of embodiment 196, wherein the galactose is supplemented prior to the production phase of the cell culture.

[0463] 202. The composition of embodiment 196, wherein the galactose is supplemented based on a predetermined schedule or standard.

[0464] 203. The composition of embodiment 196, wherein the galactose is supplemented as a single dose, an intermittent supplement, a continuous supplement, a semi-continuous supplement, a feedback loop based supplement, or a combination of one or more thereof.

[0465] 204. A composition according to any of embodiments 136-153, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulation of the fucose concentration, wherein the fucose concentration is from about 0 mM to about 60 mM, from 0 mM to about 40 mM, from about 0 mM to about 20 mM, or from about 0 mM to about 10 mM.

[0466] 205. The composition of embodiment 204, wherein fucose is supplemented as a component of the cell culture medium.

[0467] 206. The composition of embodiment 205, wherein the cell culture medium is a feed matrix, a hydrolysate, or a supplement.

[0468] 207. The composition of embodiment 206, wherein the feed matrix, hydrolysate, or additive comprises fucose.

[0469] 208. The composition of embodiment 206, wherein the feed matrix or additive consists essentially of fucose.

[0470] 209. The composition of embodiment 205, wherein fucose is supplemented during the production phase of the cell culture.

[0471] 210. The composition of embodiment 205, wherein the fucose is supplemented prior to the production phase of the cell culture.

[0472] 211. The composition of embodiment 205, wherein the fucose is supplemented based on a predetermined schedule or standard.

[0473] 212. The composition of embodiment 205, wherein the fucose is supplemented as: a single dose, an intermittent supplement, a continuous supplement, a semi-continuous supplement, a feedback loop based supplement, or a combination of one or more thereof.

[0474] 213. A composition according to any one of embodiments 136-153, wherein modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture temperature, wherein the cell culture temperature is about 29°C to about 39°C, about 30°C to about 39°C, about 31°C to about 38°C, or about 34°C to about 38°C.

[0475] 214. The composition of embodiment 213, wherein the cell culture temperature is adjusted during the production phase of the cell culture.

[0476] 215. The composition of embodiment 213, wherein the cell culture temperature is adjusted prior to the production phase of the cell culture.

[0477] 216. The composition of embodiment 213, wherein the cell culture temperature is adjusted based on a predetermined schedule or criteria.

[0478] 217. A composition according to any of embodiments 136-153, wherein the cell culture comprises eukaryotic cells.

[0479] 218. According to the composition of embodiment 217, the eukaryotic cell is a fungal cell or a mammalian cell.

[0480] 219. The composition of embodiment 218, wherein the fungal cells are yeast cells.

[0481] 220. The composition of embodiment 219, wherein the yeast cells are Saccharomyces cerevisiae cells.

[0482] 221. A composition according to embodiment 218, wherein the mammalian cells are CHO cells.

[0483] 222. A composition according to any of embodiments 136-221, wherein the cell culture is in a bioreactor, including but not limited to: single-use technology (SUT) bags or bioreactors, WAVE bioreactors, stainless steel bioreactors, flasks, test tubes, and chambers.

[0484] 223. A composition according to any of embodiments 136-222, wherein the volume of the cell culture is from 1 mL to 35,000L.

[0485] 224. A method for producing a glycoprotein of interest in cell culture, comprising: subjecting a cell culture medium suitable for culturing eukaryotic cells to the method according to any one of embodiments 1-75, inoculating the conditioned cell culture medium with the eukaryotic cells expressing the recombinant protein; and culturing the eukaryotic cells so that the recombinant protein is expressed.

[0486] 225. A method for producing a glycoprotein of interest in cell culture according to embodiment 224, wherein the cell culture is in a bioreactor.

[0487] 226. The method for producing a glycoprotein of interest in cell culture according to embodiment 224, wherein the low pCO2 conditions are from about 10 mmHg to about 100 mmHg, and the high pCO2 conditions are from about 20 mmHg to about 250 mmHg.

[0488] 227. The method for producing a glycoprotein of interest in cell culture according to embodiment 3, wherein the duration of pCO2 regulation covers at least the first half of the duration of the cell culture.

[0489] 228. The method of any one of embodiments 224-227, wherein the glycoprotein of interest is a recombinant protein.

[0490] 229. A method according to embodiment 228, wherein the recombinant protein is an antibody or antibody fragment, scFv (single-chain variable fragment), BsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domains), DNL-(Fab)3 (docked locked trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

[0491] 230. The method of embodiment 229, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0492] 231. The method of embodiment 229, wherein the antibody is an anti-CD20 antibody.

[0493] 232. The method of embodiment 231, wherein the anti-CD20 antibody is ocrelizumab.

[0494] 233. A method according to any one of embodiments 224-232, wherein the antibody or antibody fragment exhibits: a G0-F% (percentage of non-fucosylated glycoprotein) of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; or a normalized G0-F% of about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%; and / or a G0% (percentage of non-galactosylated glycoprotein) of about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%.

[0495] 234. The method of any one of embodiments 224-233, wherein the glycosylation is modulated to achieve: increased non-fucosylation (e.g., G0-F (non-fucosylated G0)) while decreasing non-galactosylation (e.g., G0 (fucosylated, non-galactosylated G0)); or decreased non-fucosylation (e.g., G0-F) while increasing non-galactosylation (e.g., G0); or increased or decreased non-fucosylation (e.g., G0-F) without affecting non-galactosylation (e.g., G0); or increased or decreased non-galactosylation (e.g., G0) without affecting non-fucosylation (e.g., G0-F).

[0496] 235. A method for modulating glycosylation of a glycoprotein of interest, the method comprising: assaying a cell culture medium to determine whether a manganese concentration of the cell culture medium is within a target range; and culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium within the target range; wherein the glycosylation of the glycoprotein of interest is modulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium outside the target range of manganese concentration.

[0497] 236. A method according to embodiment 235, wherein the target glycoprotein is an antibody.

[0498] 237. The method of embodiment 236, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0499] 238. A method according to any one of embodiments 236-237, wherein the antibody is an anti-CD20 antibody.

[0500] 239. The method of any one of embodiments 297-298, wherein the anti-CD20 antibody is ocrelizumab.

[0501] 240. The method of embodiment 235, wherein the host cell is a mammalian cell.

[0502] 241. The method of any one of embodiments 239-240, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

[0503] 242. The method of embodiment 235, wherein the target range of manganese concentration is between about 30 nM and about 110 nM.

[0504] 243. A method according to embodiment 235, wherein the measuring of the cell culture medium includes measuring the manganese concentration of the cell culture medium components.

[0505] 244. The method of embodiment 243, wherein the component of the cell culture medium is a hydrolysate or serum.

[0506] 245. The method of any one of embodiments 235-244, wherein the glycosylation is modulated to achieve increased G0-F (non-fucosylated G0) while decreasing G0 (fucosylated G0).

[0507] 246. A cell culture composition comprising: a cell culture medium that is assayed to determine whether the manganese concentration of the cell culture medium is within a target range; and a host cell engineered to express a glycoprotein of interest.

[0508] 247. A cell culture composition according to embodiment 246, wherein the composition further comprises the target glycoprotein.

[0509] 248. The cell culture composition of embodiment 247, wherein the glycoprotein is an antibody.

[0510] 249. A cell culture composition according to embodiment 248, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0511] 250. A cell culture composition according to any one of embodiments 248-249, wherein the antibody is an anti-CD20 antibody.

[0512] 251. The cell culture composition of any one of embodiments 248-249, wherein the anti-CD20 antibody is ocrelizumab.

[0513] 252. The cell culture composition of embodiment 246, wherein the host cell is a mammalian cell.

[0514] 253. The cell culture composition of any one of embodiments 252, wherein the host cell is a CHO cell.

[0515] 254. A cell culture composition according to embodiment 246, wherein the manganese concentration target range is between about 30 nM and about 110 nM.

[0516] 255. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a cell culture medium that is assayed to determine whether the manganese concentration of the cell culture medium is within a target range; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0517] 256. The composition of embodiment 255, wherein the glycoprotein is an antibody.

[0518] 257. The composition of embodiment 256, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0519] 258. A cell culture composition according to any one of embodiments 256-257, wherein the antibody is an anti-CD20 antibody.

[0520] 259. The cell culture composition of any one of embodiments 256-257, wherein the anti-CD20 antibody is ocrelizumab.

[0521] 260. The cell culture composition of embodiment 255, wherein the host cell is a mammalian cell.

[0522] 261. A cell culture composition according to embodiment 260, wherein the host cell is a CHO cell.

[0523] 262. A method for modulating glycosylation of a glycoprotein of interest, the method comprising: supplementing a cell culture medium employed in culturing a host cell expressing the glycoprotein of interest with between about 10 nM and about 2000 nM manganese under high CO2 conditions; or supplementing a cell culture medium employed in culturing a host cell expressing the glycoprotein of interest with between about 10 nM and about 3000 nM manganese under low CO2 conditions; wherein the glycosylation of the glycoprotein of interest is modulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium that has not been so supplemented.

[0524] 263. A method according to embodiment 262, wherein the target glycoprotein is an antibody.

[0525] 264. The method of embodiment 263, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0526] 265. The method of any one of embodiments 263-264, wherein the antibody is ocrelizumab.

[0527] 266. A method according to embodiment 262, wherein the host cell is a mammalian cell.

[0528] 267. A method according to embodiment 266, wherein the host cell is a CHO cell.

[0529] 268. The method of any one of embodiments 262-267, wherein the glycosylation is modulated to achieve increased G0-F (non-fucosylated G0) while decreasing G0 (fucosylated G0).

[0530] 269. A cell culture composition comprising: a cell culture medium supplemented with: between about 10 nM and about 2000 nM manganese under high CO2 conditions; or between about 10 nM and about 3000 nM manganese under low CO2 conditions; and a host cell engineered to express a glycoprotein of interest.

[0531] 270. The cell culture composition of embodiment 269, wherein the composition further comprises the target glycoprotein.

[0532] 271. The cell culture composition of embodiment 269, wherein the glycoprotein is an antibody.

[0533] 272. A cell culture composition according to embodiment 271, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0534] 273. The cell culture composition of any one of embodiments 271-272, wherein the antibody is ocrelizumab.

[0535] 274. The cell culture composition of embodiment 269, wherein the host cell is a mammalian cell.

[0536] 275. The cell culture composition of embodiment 274, wherein the host cell is a CHO cell.

[0537] 276. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a cell culture medium supplemented with manganese, wherein the culture is supplemented with: between about 10 nM and about 2000 nM manganese under high CO2 conditions; or between about 10 nM and about 3000 nM manganese under low CO2 conditions; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0538] 277. The composition of embodiment 276, wherein the glycoprotein is an antibody.

[0539] 278. The composition of embodiment 277, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0540] 279. The cell culture composition of any one of embodiments 276-277, wherein the antibody is ocrelizumab.

[0541] 280. The cell culture composition of embodiment 276, wherein the host cell is a mammalian cell.

[0542] 281. The cell culture composition of embodiment 280, wherein the host cell is a CHO cell.

[0543] 282. A method for regulating glycosylation of a target glycoprotein, the method comprising: exposing a cell culture medium having a target pH value of 6.30 to 7.25 to a high temperature, short time (HTST) heat treatment; and culturing a host cell expressing the target glycoprotein in the cell culture medium; wherein the glycosylation of the target glycoprotein is regulated compared to the glycosylation of the target glycoprotein expressed by the host cell in a culture medium having a target pH value greater than 7.25 before the HTST heat treatment.

[0544] 283. A method according to embodiment 282, wherein the target glycoprotein is an antibody.

[0545] 284. The method of embodiment 283, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0546] 285. The method of any one of embodiments 283-284, wherein the antibody is ocrelizumab.

[0547] 286. The method of embodiment 282, wherein the host cell is a mammalian cell.

[0548] 287. A method according to embodiment 286, wherein the host cell is a CHO cell.

[0549] 288. The method of any one of embodiments 282-287, wherein the glycosylation is modulated to achieve increased G0-F (non-fucosylated G0) while decreasing G0 (fucosylated G0).

[0550] 289. A cell culture composition comprising: a cell culture medium having a pH target of about 6.30 to about 7.25 exposed to HTST heat treatment; and a host cell engineered to express a glycoprotein of interest.

[0551] 290. A cell culture composition according to embodiment 289, wherein the composition further comprises the target glycoprotein.

[0552] 291. The cell culture composition of embodiment 290, wherein the glycoprotein is an antibody.

[0553] 292. A cell culture composition according to embodiment 291, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0554] 293. The cell culture composition of any one of embodiments 291-292, wherein the antibody is ocrelizumab.

[0555] 294. A cell culture composition according to embodiment 293, wherein the host cell is a mammalian cell.

[0556] 295. A cell culture composition according to embodiment 294, wherein the host cell is a CHO cell.

[0557] 296. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a cell culture medium exposed to HTST heat treatment with a pH target of about 6.30 to about 7.25; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0558] 297. The composition of embodiment 296, wherein the glycoprotein is an antibody.

[0559] 298. The composition of embodiment 297, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0560] 299. The cell culture composition of any one of embodiments 297-298, wherein the antibody is ocrelizumab.

[0561] 300. The cell culture composition of embodiment 296, wherein the host cell is a mammalian cell.

[0562] 301. The cell culture composition of embodiment 300, wherein the host cell is a CHO cell.

[0563] 302. A method for modulating glycosylation of a glycoprotein of interest, the method comprising: culturing a host cell expressing the glycoprotein of interest in the cell culture medium, wherein: the cell culture is exposed to high pCO2, the cell culture is exposed to extended matrix retention time, and / or the cell culture comprises increased Na+ concentration; wherein the glycosylation of the glycoprotein of interest is modulated compared to the fucosylation of a preparation of the glycoprotein of interest expressed by the host cell in a culture medium exposed to low pCO2, shortened matrix retention time, and / or reduced Na+ concentration.

[0564] 303. The method of embodiment 302, wherein the glycoprotein of interest is an antibody.

[0565] 304. The method of embodiment 303, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0566] 305. The method of any one of embodiments 303-304, wherein the antibody is ocrelizumab.

[0567] 306. The method of embodiment 302, wherein the host cell is a mammalian cell.

[0568] 307. The method of embodiment 306, wherein the host cell is a CHO cell.

[0569] 308. The method of any one of embodiments 302-307, wherein the glycosylation is modulated to achieve increased G0-F (non-fucosylated G0) while decreasing G0 (fucosylated G0).

[0570] 309. A cell culture composition comprising: a cell culture medium comprising high pCO2, extended substrate retention time and / or increased Na+ concentration; and a host cell engineered to express a glycoprotein of interest.

[0571] 310. The cell culture composition of embodiment 309, wherein the composition further comprises the target glycoprotein.

[0572] 311. The cell culture composition of embodiment 310, wherein the glycoprotein is an antibody.

[0573] 312. The cell culture composition of embodiment 311, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0574] 313. The cell culture composition of any one of embodiments 311-312, wherein the antibody is ocrelizumab.

[0575] 314. The cell culture composition of embodiment 309, wherein the host cell is a mammalian cell.

[0576] 315. The cell culture composition of any one of embodiments 314, wherein the host cell is a CHO cell.

[0577] 316. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a cell culture medium comprising high pCO2, extended substrate retention time, and / or increased Na+ concentration; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0578] 317. The composition of embodiment 316, wherein the glycoprotein is an antibody.

[0579] 318. The composition of embodiment 317, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[0580] 319. The cell culture composition of any one of embodiments 317-318, wherein the antibody is ocrelizumab.

[0581] 320. The cell culture composition of embodiment 316, wherein the host cell is a mammalian cell.

[0582] 321. The cell culture composition of embodiment 320, wherein the host cell is a CHO cell.

[0583] Examples

[0584] The following examples are merely illustrative of the presently disclosed subject matter and should not be considered limiting in any way.

[0585] Example 1: Controlling feedstock to modulate glycosylation

[0586] It is known that a variety of cell culture factors have the potential to affect the glycosylation of monoclonal antibody therapeutics. These factors include process parameters, matrix handling, and matrix components, such as galactose and trace metals. Variations in the levels of various matrix components can be introduced into the mAb cell culture process by using complex raw materials, such as Protease Peptone No. 3 (PP3) and Gene Technology Essential Matrix (GEM) powders. These sources of variability in raw materials can lead to substantial differences in Mn concentrations at the start of production culture (i.e., day 0), as summarized in Table 1.

[0587]

[0588] Table 1. Mn levels on day 0 during ocrelizumab cell culture

[0589] Changes in Mn levels on day 0 of production culture correlate with changes in G0% (fucosylated G0) and G0-F% (non-fucosylated) antibody species ( Figure 1 ).exist Figure 1 In the present invention, Groups 1 and 2 were performed using matrix depth filtration, which was performed by leaching Mn from the depth filter. Matrix depth filtration can contribute significant additional Mn to the production medium composition. Mn levels on day 0 were measured by ICP-MS. The predicted Mn levels on day 0 for Group 3 were based on the equation G0 = 109.57487-8.3488683*ln(Mn), which was derived using data from Groups 1-2 and Groups 4-6. Non-fucosylation is expressed as normalized G0-F = 100*[G0-F] / (G0+[G0-F]).

[0590] To provide improved control of Mn concentration from the feedstock, one or both of the following strategies can be implemented: (a) testing and selecting PP3 and GEM powders within a specified Mn range prior to use; and (b) controlling Mn levels on day 0 after inoculation of the production culture to within an established acceptable range (e.g., 30 nM to 110 nM).

[0591] Based on Table 2 and Fig.32 Mn ranges in select PP3 and GEM powders. These ranges were established based on the Mn at day 0 in the 2016v1.0 batch and historical data from 36 batches of PP3 and 34 batches of GEM powders, and accounted for losses during matrix preparation and handling.

[0592]

[0593] Table 2. Raw material Mn range

[0594] To further ensure process consistency and control G0, normalized G0-F, and CDC values ​​within specification, the Mn level on day 0 after inoculation can be controlled within a narrow range of 30-110 nM, with action limits of <30 nM or >110 nM.

[0595] Example 2: Manganese supplementation to regulate glycosylation

[0596] 2.1 Introduction

[0597] This example summarizes the effects of manganese supplementation on ocrelizumab and other antibodies. With increasing amounts of manganese supplementation, an increase in non-fucosylated (G0-F) and a decrease in fucosylated (G0) (non-galactosylated) species were observed.

[0598] 2.2 Evaluation of manganese supplementation with ocrelizumab

[0599] Manganese supplementation experiments were carried out for Orelizumab. Manganese (Mn) concentrations in the test cases were adjusted by adding after inoculation to the Orelizumab production culture. The manganese concentrations tested in these studies are listed in Table 3. The listed manganese concentrations represent the amount of additional manganese added to the culture based on the volume after inoculation, and due to the presence of manganese in the control process matrix, the total manganese concentration on day 0 cannot be reflected. Manganese was added using a sterile filtered solution of 0.05mM and 0.5mM manganese sulfate monohydrate, and added by a diaphragm. Parallel determinations of controls and some test cases were included in each study.

[0600]

[0601] Table 3. Manganese addition: a The manganese concentrations assessed represent additional manganese for the ocrelizumab production culture process. Low levels of manganese were present in the control matrix;b The 0.05 mM manganese sulfate solution used in these cases

[0602] Figure 2 shows the correlation between manganese concentration and non-fucosylation (normalized G0-F) and non-galactosylation (G0) on day 0 in cell culture. Figure 3 shows the effect of manganese supplementation on non-fucosylated (normalized G0-F) and fucosylated, non-galactosylated (G0) species of ocrelizumab. As manganese concentration increased, ocrelizumab G0-F increased, while G0 decreased. Figures 2 and 3 show that the trend of the effect of manganese on non-fucosylation and non-galactosylation is the same at the bioreactor scale, demonstrating the scalability of the findings at a small scale (2L). In particular, at both the 2L and 12kL bioreactor scales, increased normalized G0-F and decreased G0 were observed in Mn-supplemented cultures compared to unsupplemented cultures (no Mn addition).

[0603] 2.2 Evaluation of manganese supplementation and pCO2 for ocrelizumab

[0604] Manganese titration was performed at 0, 50, 100, 150, 250, 350, 500, 750, 1000 and 2000 nM manganese in both the scale-dependent factor model (high pCO2 model, where the 36-hour substrate was kept at 37°C) and the standard 2L model. The substrate for this study was subjected to high temperature short time (HTST) heat treatment using the following HTST conditions: 10 seconds at 102°C, 15 psig back pressure, and post-HTST cooling to 37°C. All other conditions and parameters were performed at target conditions (i.e., using the same set points).

[0605] Results for non-fucosylated (normalized G0-F) and fucosylated, non-galactosylated (G0) are shown in Figures 4 and 5. In the presence of high pCO2 levels, a greater effect on normalized G0-F and G0 was observed compared to the standard 2L model (low pCO2). The trend of this study is consistent with the manganese titration study (see 2.2 above). As shown in Figures 2-5, bioreactors with various volumes (e.g., standard 2L, scale-dependent factors of 2L, and 12,000L) can be used for the composition of the cell culture medium. The conditions of the cell culture medium can be adjusted based on the volume of the bioreactor. For example, the composition of the cell culture medium used in the 2L bioreactor can be expanded for use in the 15,000L bioreactor. In addition, the composition of the cell culture medium used in the 15,000L bioreactor can be reduced for use in the 2L bioreactor. The volume of the bioreactor can be between about 1 L and about 20,000 L (e.g., about 1 L, about 1.5 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L, about 3000 L, about 4000 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10,000 L, about 11,000 L, about 12,000 L, about 13,000 L, about 14,000 L, about 15,000 L, about 16,000 L, about 17,000 L, about 18,000 L, about 19,000 L, or about 20,000 L).

[0606] 2.3 Evaluation of manganese concentration of antibody I

[0607] A full factorial DOE (2x2x3) was designed in which three factors were considered, cell age (66 days vs. 151 days), iron concentration (20 μM vs. 75 μM), and manganese concentration (4.5 nM vs. 450 nM vs. 4500 nM). The purpose of this study was to determine the effect of higher manganese levels on glycosylation and the effect of iron concentration on charge variants. The study design is shown in Table 4. The results for G0-F (non-fucosylated) and G0 (G0F) are shown in Figure 6. Consistent with other antibodies, an increase in non-fucosylation and a decrease in G0 were observed with increasing manganese concentration. This result was consistent across all cell ages and iron concentrations, indicating that the effect of manganese was independent of the other parameters tested.

[0608] Case Cell age (d) Manganese (nM) Iron (μM) 1 66 4.5 20 2 66 4.5 75 3 66 4500 20 4 66 4500 75 5 151 4.5 20 6 151 4.5 75 7 151 4500 20 8 151 4500 75 9 66 450 20 10 151 450 20

[0609] Table 4. Antibody I cell age, manganese, and iron studies

[0610] 2.4 Evaluation of Manganese Supplementation in Antibody II

[0611] The study design consisted of a full factorial DOE incorporating three two-level variables: copper addition level, manganese addition level, and zinc addition level (Table 5). All proposed process conditions (including supplemented copper, manganese, and zinc at target levels) were tested in duplicate. The results of the study are shown in Figure 7 Manganese was estimated to have the greatest effect on both G0 and G0-F. The trends for G0 and G0-F were consistent with the other antibodies as manganese levels increased.

[0612] Case Copper(μM) Manganese (nM) Zinc (μM) 1 100 300 300 2 300 600 0 3 0 0 600 4 0 0 0 5 0 600 0 6 300 0 0 7 300 0 600 8 300 600 600 9 0 600 600 10 100 300 300

[0613] Table 5. Antibody II copper, manganese, zinc study design

[0614] 2.5 Evaluation of Manganese Supplementation in Antibody III

[0615] Manganese titration studies were performed for antibody III. Based on the final working volume calculation, 0.5mM manganese sulfate stock solution was added after production inoculation. The concentrations added except for the actual measured manganese from the test cases are shown in Table 6. Trace amounts of manganese were present in the cell culture medium, as shown in Case 1, while no additional manganese was added to the production culture. Although there was some variability in the measured manganese levels, the levels generally measured by inductively coupled plasma mass spectrometry confirmed that the correct amount of manganese stock solution was added to each bioreactor after production inoculation. All control runs were performed within the expected range. Additional manganese supplementation had no effect on growth and titer. As expected, with the increase of Mn, fucosylation (G0) (non-galactosylated) decreased, and non-fucosylated (G0-F) increased. The results are shown in Figure 8.

[0616] Case Supplemented manganese (nM) Actual Mn concentration (nM) 1 0 85 1 0 82 2 125 195 2 125 244 3 250 365 3 250 280 4 500 511 4 500 507

[0617] Table 6. Antibody III Manganese Titration Study

[0618] 2.6 Evaluation of manganese supplementation in AB II and AB V

[0619] Antibody IV and Antibody V were combined to evaluate zinc, manganese, iron and copper using a full factorial design experiment to determine their effects on the cell culture process performance and product quality of Antibody IV and Antibody V. Table 7 shows the design of the study. The results of Antibody IV are shown in Figure 9, and the results of Antibody V are shown in Figure 10. Figures 9 and 10 show the actual measured manganese. Due to the presence of manganese in the base matrix, this is different from the amount of supplemented manganese listed in Table 7. As the manganese concentration increases, the G0-F (non-fucosylated) of both Antibody IV and Antibody V increases, while the G0 (fucosylated, non-galactosylated) decreases. This effect is independent of zinc, iron and copper concentrations.

[0620] variable Low middle high Zinc 22μM - 562μM Mn 4.5nM 100nM 1000nM Fe 20μM 40μM 120μM Cu 500nM - 1000nM

[0621] Table 7. Antibody IV Metal Concentrations

[0622] 2.7 Evaluation of the timing of manganese supplementation for antibody VI

[0623] The timing of Mn addition was tested for antibody VI to evaluate the effect of different addition times on glycosylation. Fig.11 ), the cultures were supplemented with 500 nM Mn (during the previous expansion passage, or on day 0 or day 3 of production culture), or they were not supplemented. In the second study ( FIG. 12 ), the cultures were supplemented with 80 nM Mn (on day 0 or daily during production culture), or they were not supplemented. When Mn was added to the cell cultures, G0 (fucosylated, non-galactosylated) decreased, while normalized G0-F (non-fucosylated) increased, regardless of the timing of Mn supplementation.

[0624] It will be understood that the foregoing is only illustrative of the principles of the present disclosure and that various modifications may be made by those skilled in the art without departing from the scope and spirit of the disclosure. For example, but not by way of limitation, the volume of the bioreactor used in the examples can be between about 1 L and about 20,000 L (e.g., about 1 L, about 1.5 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L, about 3000 L, about 4000 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10,000 L, about 11,000 L, about 12,000 L, about 13,000 L, about 14,000 L, about 15,000 L, about 16,000 L, about 17,000 L, about 18,000 L, about 19,000 L, or about 20,000 L). Additionally, the bioreactor and its operation can be modified to adjust the level of pCO2, substrate retention duration, culture duration, osmotic pressure, Na+, Mn, culture temperature, fucose, galactose, or a combination thereof.

[0625] Example 3: pH target before high temperature short time (HTST) heat treatment

[0626] 3.1 Introduction

[0627] This example summarizes the selection of pH adjustment targets for the matrix prior to high temperature, short time (HTST) heat treatment for the preparation of ocrelizumab (rhuMAb 2H7) production medium and supporting experimental results. During HTST operation, a lower matrix pH target prior to HTST heat treatment can reduce matrix turbidity, associated precipitate formation, HTST heat transfer surface fouling, and filter plugging (see, e.g., US 9,493,744).

[0628] For several matrix products, a trend of increasing pressure and decreasing flow rate measured before the cooler was observed ( Fig.13 ). These trends were found to correlate with increased manganese (Mn) losses during the HTST and filtration operations (Table 9). In these cases, the increase in HTST pressure (before the cooler) and the decrease in HTST flow rate can be attributed to the formation of turbidity / precipitate and subsequent pressure increase on the medium filter (increased filter clogging). To mitigate this atypical HTST characteristic of the ocrelizumab manufacturing run and potentially reduce the variability of Mn losses during HTST and filtration, a lower pre-HTST pH matrix adjustment target with post-HTST pH adjustment was evaluated and implemented for the ocrelizumab production matrix.

[0629]

[0630] Table 9. Manganese measurements in HTST and Filtration Example HTST Characterization Runs

[0631] All manganese measurements were performed using inductively coupled plasma mass spectrometry (ICP-MS) determination method.

[0632] 3.2 Evaluation of Mn Loss from HTST Heat Treatment and Filtration at Different pH Adjustment Targets

[0633] A preliminary screening study using a benchtop sand bath HTST heat treatment method was used to evaluate changes in turbidity and manganese loss after heat treatment at various pH target conditions. Ocrelizumab production medium was used for this study. The sand bath HTST heat treatment method represents a worst-case scenario for HTST heat treatment, as extended heat treatment times are required to reach 102°C compared to the HTST manufacturing skid. Changes in turbidity before and after heat treatment, as well as manganese loss during heat treatment / filtration are shown in Fig.14 No significant increase in turbidity or reduction in Mn loss was observed below the target pH adjustment value of 7.00 for the production matrix prior to HTST. Larger increases in turbidity or Mn loss were observed at target pH adjustment values ​​of 7.00 or higher for the production matrix prior to HTST. This suggests that a target pH adjustment of the matrix to below 7.00 prior to HTST heat treatment is expected to reduce Mn losses during the manufacturing HTST heat treatment and filtration operations.

[0634] A pilot scale HTST study was conducted to evaluate three pre-HTST pH adjustment targets of about 6.30 (non-pH adjusted matrix), 6.70, and 7.10. Orelizumab production medium was used for this study. Mn measurements and Mn losses during HTST and filtration are shown in Table 10. Consistent with the sand bath HTST heat treatment study, the pre-HTST pH cases of about 6.30 and 6.70 demonstrated less Mn loss during HTST and filtration compared to the pH 7.10 case of HT, suggesting that lower pre-HTST pH targets can help reduce Mn losses observed in manufacturing HTST and filtration operations.

[0635]

[0636] Table 10. Manganese measurements in pilot scale HTST studies

[0637] 3.3 Effect of pH adjustment before HTST on 2L cell culture performance and product quality

[0638] The production matrix processed in the pilot scale HTST study was used for two 2L experiments. After HTST heat treatment, and before filtration, the production matrix was adjusted to a final pH target of 7.10+ / -0.10. The pH adjustment step will be carried out after HTST and filtration, and the target value is 7.15+ / -0.10. In each experiment, the control of the matrix prepared with the same protease peptone 3 (PP3) and Gene Technology Basic Matrix (GEM) powder 2 raw material batches without HTST heat treatment is included. The run is selected to be executed together with the scale-dependent 2L model, which includes 36 hours of N-1 and N matrix preservation and an improved injection strategy to generate higher pCO2 levels.

[0639] The following results from these studies were compared to 2L control runs performed during process characterization and validation (PC / PV) and historical manufacturing runs. The 2L control and manufacturing product quality data were derived from the affinity pools in the corresponding AO assays. Fig.15 The KPIs are displayed. Fig.16 – Fig.18 Product quality is shown. KPIs, charge-related variants, size-related variants, and glycans did not show significant impact from changing the pre-HTST pH target between about 6.30, 6.70, and 7.10. The study showed that cell culture performance and product quality would not be affected by changing the pre-HTST pH target between 6.30 and 7.10 with post-HTST pH adjustment to 7.10 + / - 0.10.

[0640] 3.4 Effect of pH adjustment on osmotic pressure in the matrix of ocrelizumab production

[0641] The osmotic pressure changes from sodium carbonate addition for pH adjustment for both the sand bath HTST and initial pilot scale HTST studies are shown in Tables 11 and 12. It was observed that both the osmotic pressure before HTST heat treatment at a pH of 6.90 and the final osmotic pressure after the final pH adjustment to 7.10 were within the current production matrix target osmotic pressure.

[0642]

[0643] Table 11. pH, osmotic pressure and sodium carbonate addition for ocrelizumab production matrix pH adjustment to pH 6.90

[0644]

[0645] Table 12. pH, osmotic pressure and sodium carbonate addition for ocrelizumab production matrix pH adjustment from pH 6.90 to pH 7.10

[0646] 3.5 Recommendations for pH and osmotic pressure targets and ranges before HTST in ocrelizumab production medium

[0647] Based on the results summarized in this example, the recommended pre-HTST pH adjustment target for the ocrelizumab manufacturing matrix is ​​6.90 + / - 0.10 or 6.70 + / - 0.10, with an osmolarity alarm range of 320–350 mOsm / kg. Small-scale study results showed no impact on cell culture performance or product quality and support the use of a pre-HTST pH in the range of 6.30–7.10. Additionally, historical manufacturing runs support a pre-HTST pH target of 7.15 + / - 0.10; therefore, the overall acceptable pre-HTST pH target range is 6.30–7.25. After completion of the HTST thermal treatment, filtration, and batch processing in the 12,000-L bioreactor, the ocrelizumab manufacturing matrix will require pH adjustment to a final target pH of 7.15 + / - 0.10. After the final pH adjustment in the 12,000L bioreactor, an osmolarity check should be performed to confirm that the substrate is within the target osmolarity of 340 + / - 20 mOsm / kg prior to inoculating the production culture.

[0648] 3.6 Using a Lower Pre-HTST pH Set Point for an Additional mAb

[0649] During the manufacture of the production base matrix for the additional mAb examples, Antibody III experienced the atypical HTST behavior observed with ocrelizumab ( Fig.13). To mitigate future HTST performance issues and prevent manganese loss due to atypical HTST performance, matrix pH prior to HTST heat treatment and filtration was evaluated for additional mAbs. Matrices were prepared to pH targets of 7.1, 6.6, and 6.1. Each matrix preparation was used separately with or without HTST heat treatment. Fig.19B Manganese results from matrix preparation are shown. The blue symbols show the manganese levels in the HTST heat treated matrix. There is a clear trend with increasing matrix pH target and decreasing manganese after HTST heat treatment. This indicates that at higher matrix pH, higher manganese losses are observed during HTST heat treatment and filtration. This result is consistent with studies conducted with ocrelizumab.

[0650] A 2L study was conducted to evaluate the impact of lower matrix pH prior to HTST heat treatment and filtration on cell culture performance and product quality. KPI results are shown in Fig.19C and Fig.19D The product quality results are shown in FIG. 19E to FIG. 19H This study suggests that cell culture performance and product quality of additional mAbs will not be affected by changing the pre-HTST pH target between 6.10 and 7.10.

[0651] Based on the results of this study, the pH target for additional mAbs prior to HTST heat treatment and filtration matrix was reduced to 6.6.

[0652] Example 4: pCO2, manganese, matrix preservation, osmotic pressure and Na+ to regulate fucosylation

[0653] 4.1 Introduction

[0654] Among the cell culture process parameters with unknown effects on glycosylation (e.g., galactosylation and / or afucosylation), the partial pressure of carbon dioxide in the culture medium (pCO2) is very important because pCO2 levels can vary at the bioreactor scale; therefore, maintaining comparable pCO2 characteristics is a frequently encountered challenge during process scale-up experiments. Previous studies have shown that pCO2 levels affect cell growth, productivity and recombinant protein glycosylation in mammalian cell culture (Darja et al., (2016), Journal of Biotechnology, 219, 98-109; deZengotita et al., (1998), Cytotechnology, 28, 219-227; Gray et al., (1996), Cytotechnology, 22(1-3), 65-78; Kimura et al., (1996), Biotechnol and Bioeng, 62, 152-160; Kimura et al., (1997), Biotechnol Prog, 13, 311-317; Schmelzer et al., (2002), Biotechnol Prog, 18, 346-353; Zhu et al., (2005), Biotechnol Prog, 21(1), 70-77). Although these studies did not demonstrate an effect of pCO2 on afucosylation, the pCO2 properties evaluated are not representative of those encountered in large-scale bioreactor cultures.

[0655] To better understand and establish robust control of afucosylation at the bioreactor scale, the effects of pCO2 and its potential interactions with other process levels on the afucosylation of mAbs produced by a recombinant Chinese hamster ovary (CHO) cell line were investigated. The following approaches were applied: (1) construct a small-scale (3-L) bioreactor model capable of maintaining different pCO2 levels while keeping other process parameters constant; (2) examine the effects of pCO2 and its interactions with other process parameters (e.g., manganese and substrate conservation) on mAb afucosylation; and (3) investigate the possible underlying mechanisms behind any observed effects of pCO2 and other process levels on mAb afucosylation. Mn was chosen because it is a cofactor for several glycosylation enzymes (Rouiller et al., (2014), Biotechnol Prog, 30(3), 571–583) and has been used to modulate glycosylation levels in CHO cell culture studies (Gramer et al., (2011), Biotechnol Bioeng, 108(7), 1591–1602; Surve et al., (2014), Biotechnol Prog., 31(2): 460–647), even though it had not previously shown any effect on non-fucosylation and was not known to be a cofactor for α1,6-fucosyltransferase (FUT8). Matrix preservation was studied because in large-scale operations, production matrix is ​​preserved in the bioreactor prior to inoculation and its effects on product quality attributes have not previously been reported.

[0656] 4.2 Materials and methods

[0657] 4.2.1 Cell culture

[0658] The same recombinant CHO cell line expressing immunoglobulin G1 (IgG1) subclass mAbs was used in all studies reported herein. The cells were thawed and expanded to inoculate production cultures in 3-L glass bioreactors (Applikon) as previously described (Yuk et al., (2015), Biotechnol Prog, 31 (1), 226-237). The set points for temperature, pH, and dissolved oxygen (DO) in the bioreactor were controlled by a Finesse Smart Controller with TruBio DeltaV (Thermo Fisher Scientific). The temperature, pH, and DO of all production cultures were maintained as follows: on the first day, at 37°C, 7.15, and 30% (air saturation); between the first and third days, at 34°C, 7.15, and 30%; and from the third day onwards, at 34°C, 7.00, and 30% DO. Three days after inoculation, concentrated nutrient feed was added to the production culture at 1:7 (v / v).

[0659] The following parameters were studied: manganese supplementation, L-fucose supplementation, substrate preservation, pCO2 levels, and osmotic pressure (using sorbitol and NaCl as osmotic pressure titrators). The basal substrate used for the inoculation series and production cultures contained a nominal amount of Mn and did not contain L-fucose. Supplemented Mn and / or L-fucose were added immediately after inoculation of the production culture to achieve the target day 0 concentration, as described in the study. Matrix preservation was performed by incubating the basal substrate (for the N-1 inoculation series and production culture) in separate 3-L bioreactors at 37°C for 36 hours with air sparging (10 sccm), stirring (75 rpm), and one-sided (CO2 only) pH control. Osmotic pressure was adjusted by adding a stock solution of NaCl (100 g / L) or sorbitol (182 g / L).

[0660] 4.2.2 High and low pCO2 model configuration

[0661] To achieve different pCO2 characteristics, the bioreactor configuration was modified to generate high pCO2 models ( Fig. 20A ) and low pCO2 models ( Fig. 20B In both models, pH was controlled by sparging CO2 through an open tube (5 mm inner diameter) to lower pH and adding Na2CO3 to increase pH as previously described (Hsu et al., (2012), Cytotechnology 64(6):667–678).

[0662] For the high pCO2 model ( Fig. 20A ), the bioreactor working volume was ≥1.9 L. DO was controlled by supplying air / O2 through a micro-jet device (15 μm pore size). The DO controller was set to use air to control DO, with a minimum output of 2 sccm; after the air output reached 12 sccm, DO control was switched to O2, with a minimum output of 2 sccm.

[0663] For the low pCO2 model ( Fig. 20B ), the bioreactor working volume was ≤1.5 L. DO was controlled by sparging air / O2 through the same open tube used for CO2. The DO controller was set to use air output with a minimum sparge of 10 sccm and a maximum sparge of 50 sccm. After the output reached 50 sccm, the O2 sparge was increased and the air sparge was reduced. When the O2 sparge reached 50 sccm, the air was turned off and the O2 output was increased as needed to a maximum of 250 sccm.

[0664] 4.2.3 Cell culture analysis

[0665] Packed cell volume (PCV), viable cell concentration, culture viability, pH, DO, pCO2, glucose, lactate, osmotic pressure, Na+, ammonium, mAb product titer, glycosylation variants, charge variants, size variants, and Mn concentration (by inductively coupled plasma mass spectrometry) were measured as previously described (Hsu et al., (2012), Cytotechnology 64(6):667–678; Yuk et al., (2015), Biotechnol Prog, 31(1), 226-237). The structures of N-linked glycosylation species are described in detail in Thomann et al. (2016) and are described in detail in Fig.28A Non-fucosylation was normalized to G0F and defined as:

[0666]

[0667] 4.2.4 Intracellular pH analysis

[0668] Intracellular pH (pHi) was measured using SNARF-4F 5-(and-6)-carboxylic acid, acetoxymethyl acetate (SNARF-4F) (Molecular Probes; Cat# S23921, Thermo Fisher Scientific). SNARF-4F is a fluorinated derivative of carboxyl SNARF-1 and has a pKa value of approximately 6.4. The pHi measurement and calculation method is based on Reynolds et al. (Reynolds et al., (1996), Cytometry, 25, 349-357) and deZengotita et al. (deZengotita et al., (2002), Biotechnol Bioeng, 77(44), 369-380).

[0669] In preparation for pHi measurements, fresh matrix was pre-equilibrated to the desired conditions for at least 6 hours. For the pCO2 titration case, the matrix was equilibrated in a TAP ambr 15 system (Sartorius Stedim Biotech) controlled at 37°C and 600 rpm agitation while sparging CO2 to achieve the desired pCO2 level. Next, the pH was adjusted to 7.0 using 0.5 M Na2CO3 and the osmolarity was adjusted to approximately 400 mOsm / kg using 100 g / L NaCl. For the osmolarity titration case, the matrix osmolarity was adjusted using 100 g / L NaCl and placed in an incubator controlled at 37°C, 5% CO2 and 50 rpm agitation (2.5 cm orbit) overnight. The cells were pelleted (500,000 cells, 200 g, 2 minutes) and washed twice in phosphate buffered saline (PBS). The pellet was quickly resuspended in fresh matrix pre-equilibrated to the desired conditions and SNARF-4F (final concentration 1.5 μg / mL) was added. The cell-dye mixture was incubated under the same conditions as the pre-equilibrated matrix (30 min). pHi was measured immediately using an Attune NxT flow cytometer (Thermo Fisher Scientific) with a 488 nm laser and detection at 585 nm and 640 nm. Extracellular pH, pCO2, osmolarity, and Na were measured using the Nova Bioprofile FLEX + A pH calibration curve was generated using cells stained in buffers of known pH in the presence of nigericin (Sigma Cat# N7143, Sigma-Aldrich) as previously described (Salvi et al., (2002), AAPS PharmSci, 4(4), 1-8).

[0670] 4.2.5 Proteomic analysis

[0671] For proteomic analysis by mass spectrometry (MS), cells from production cultures at day 7 and day 12 were pelleted (10 million cells, 200 g, 2 minutes), washed twice with PBS, snap frozen on dry ice, and stored at -80°C until analysis. Proteins were extracted from each sample, digested into peptides, and labeled with tandem mass tags (TMT) as previously described (Vildhede et al., (2018), Drug Metab Dispos, 46(5), 692–696), and analyzed using an Orbitrap Lumos mass spectrometer (Thermo Scientific) using the SPS-MS3 method (McAlister et al., (2014), Anal Chem, 84(16), 7150–7158).

[0672] MS / MS spectra were assigned using the MASCOT search algorithm to search all entries for C. cristae (Chinese hamster) in UniProt (downloaded in June 2016). A search of all tryptic peptides (2 incomplete cleavages) was performed and a 50 ppm precursor tolerance was used to limit the number of candidate peptides, while a tolerance of 0.8 Da was used to match MS / MS data collected in the ion trap. Static modifications included TMT (+229.16293) and cysteine ​​alkylation (57.0215) at the N-terminal end of the peptide and lysine residues, while variable modifications included methionine oxidation (15.9949) and TMT labeling of tyrosine (229.1629). Peptide spectrum matches were filtered to a 2% false discovery rate using a target-decoy approach scored with a linear discriminant analysis algorithm before filtering to a 2% false discovery rate at the protein level as previously described (Kirkpatrick et al., (2013), PNAS, 110(48), 19462-19431).

[0673] Quantitative values ​​were extracted and corrected for isotopic impurities using Mojave (Zhuang et al., (2013), SciSignal, 6(271), 1-11). Additionally, quantitative events with precursor purity <0.7 (±0.25 Da) or total intensity <50,000 were discarded before the quantitative values ​​were normalized and converted to "relative abundance" values ​​using a custom script in R. Relative abundance values ​​for each protein were calculated by dividing the sample intensity by the total intensity of the protein and then normalizing the result to 100. After data normalization, principal component analysis (PCA) was performed using a custom script in R. To determine pathway enrichment in the data set, UniProt identifiers were converted to homologous mouse, rat, or human identifiers and processed by ingenuity pathway analysis (IPA; QIAGEN Inc.) as described previously (Kramer et al., (2014), Bioinformatics, 30(4), 523–530).

[0674] 4.3 Results and Discussion

[0675] 4.3.1 Low and high pCO2

[0676] In order to develop a small-scale bioreactor model capable of maintaining different pCO levels, the CO stripping rate in the 3-L bioreactor was adjusted (Figure 20). Although the NaHCO concentration in the substrate can be adjusted to change the pCO level in the bioreactor culture (Goudar et al., (2006), Biotechnol Bioeng, 96(6), 1107-1117; Zhu et al., (2005), Biotechnol Prog, 21(1), 70-77), the bioreactor gas sparge rate was adjusted instead because this is an effective method to adjust the CO stripping (and thus the pCO level) while keeping agitation and the vessel aspect ratio constant.

[0677] In order to achieve low gas injection rates while maintaining sufficient k L aTo support the metabolic needs of cells for oxygen, a porous micro-jet device is used for DO control in the high pCO2 model. The micro-jet device produces a small bubble size, thereby increasing the total gas-liquid surface area interface. In order to increase the residence time of CO2 and further reduce CO2 stripping (Matsunaga et al. (2009), Journal of Bioscience and Bioengineering, 107(4), 419-424), the high pCO2 model is operated at a higher working volume (≥1.9 L). The low pCO2 model uses an open tube sparger and a lower working volume (≤1.5 L) to increase CO2 stripping. These differences in bioreactor configuration lead to the required pCO2 level separation between the two models ( Fig. 21B ). Although high pCO2 has been shown to affect hybridoma cell growth, viability and antibody production (deZengotita et al., (1998), Cytotechnology, 28, 219-227), no negative effects on these attributes and product quality were observed for the CHO cell line used in this study (Figure 29). This is believed to be the first report demonstrating that pCO2 levels in CHO cultures can be regulated without controlling the NaHCO3 concentration in the culture medium in the same small bioreactor.

[0678] 4.3.2 Effects of pCO2 levels, matrix preservation, and supplemented Mn on non-fucosylation

[0679] Using high and low pCO2 bioreactor models, the effects of pCO2 levels, substrate preservation, and supplemental Mn on non-fucosylation, as well as potential interactions, were examined in a fully factorial design of experiments (DOE). Consistent with the established effects of Mn on galactosylation (Gramer et al., (2011), Biotechnol Bioeng, 108(7), 1591-1602), non-galactosylated species (GOF) were reduced in the context of supplemented Mn (Figure 21). In the low pCO2 background, non-fucosylation was about 1% higher in the presence of Mn supplementation (Figure 2A). This result was unexpected because Mn is not known to affect fucosylation. For example, while several other divalent cations significantly inhibit FUT8 activity, Mn does not (Kaminska et al., (1998), Glycoconjuagte Journal, 15, 783-788). In the presence of supplemented Mn, non-fucosylation was about 2% higher in the presence of matrix preservation or high pCO2 (Figure 2B). Fig.21A ). In the presence of supplemented Mn, matrix preservation, and high pCO2, non-fucosylation was highest (about 4%), suggesting an interaction between these three factors.

[0680] This observed interaction could be confirmed at multiple Mn supplementation levels using both high and low pCO2 models (Figure 22). In the high pCO2 model, the increase in non-fucosylation was more pronounced in the presence of matrix preservation at all Mn levels ( Fig.22A ). This example demonstrates the effects and interactions of Mn, matrix conservation, and pCO2 levels in regulating afucosylation. Thus, Mn, matrix conservation, and pCO2 levels can each regulate afucosylation on their own, but when they act together, their effects on afucosylation are amplified.

[0681] 4.3.3 Elimination of high pCO2, osmotic pressure and Na + Effects on non-fucosylation

[0682] Higher culture osmolarity and Na were observed in the high pCO2 model compared to the low pCO2 model. + ( Figures 22C-22D ). This can be explained by the fact that CO2 in solution forms H + and HCO3 - , as shown in the simplified balanced equation (Equation 1):

[0683]

[0684] In a pH-controlled environment, base (Na2CO3 in this study) is added to the bioreactor to neutralize H+, thereby pushing the equilibrium to the right of reaction equation 1 and increasing the culture osmotic pressure and HCO3 - The concentration of Na2CO3 can also be increased by adding Na2CO3. + These findings raise the question of whether the increased mAb afucosylation is caused by high pCO2, osmotic pressure, or Na + Caused by the problem.

[0685] Attempts to disentangle the effects of high pCO2 and osmotic pressure on afucosylation have resulted in conflicting results in the literature. Observations range from minimal effects of increasing pCO2 and / or osmotic pressure on afucosylation (Kimura et al., (1997), Biotechnol Prog, 13, 311-317; Schmelzer et al., (2002), Biotechnol Prog, 18, 346-353) to decreased afucosylation with increasing osmotic pressure (Konno et al., (2012), Cytotechnology, 64, 249-265). In these previous studies, production cultures were started under high pCO2 and / or osmotic pressure; this does not represent typical conditions in bioreactors, where pCO2 and osmotic pressure are initially low and then increase over the course of production cultures (Hsu et al., (2012), Cytotechnology 64(6): 667–678).

[0686] To distinguish pCO2 from osmotic pressure and Na + To examine the effects of this in an environment more representative of typical CHO bioreactor cultures, the osmolarity in the low pCO2 model was titrated with either NaCl or sorbitol to match the time course osmolarity profile of production cultures in the high pCO2 model operated at 1.9 L (peak osmolarity ~450 mOsm / kg) and 2.2 L (peak osmolarity ~550 mOsm / kg) (Figure 23). For all cases, afucosylation increased with increasing osmolarity. Afucosylation was similar between the NaCl titrated (low pCO2) and high pCO2 cases at both target peak osmolarity levels, whereas afucosylation was ~1% lower in the sorbitol titrated (low pCO2). These results suggest that the increase in afucosylation observed in the high pCO2 model is likely due to the Na2CO3 addition for pH control. + It is the elevated concentration, not the high pCO2 or osmolarity alone.

[0687] 4.3.4 Changes in intracellular pH: high pCO2 and high osmotic pressure / Na +

[0688] To study high pCO2 and / or Na + Mechanisms behind the greater increase in afucosylation in the background when subjecting recombinant CHO cell lines to different pCO2 and osmolarity / Na + When the pH is level, measure its i CO2 can diffuse across the cell membrane (Endeward et al., (2014), Frontiers in Physiology, 4, 1-21), encountering the equilibrium described in reaction equation 1 inside the cell, thereby lowering the pH i In addition, Na from alkali addition + (Na2CO3)t can control the extracellular pH by Na + / H + exchange (Orlowski et al., (1997), J Biol Chem, 272(36), 22373-22376) and / or Na + -dependent Cl - / HCO3 - Exchange (Reusch et al., (1995), American Physiological Society, C147-C153) affects pH i pH i Changes in pH can affect enzyme expression (Bumke et al., (2003), Proteomics, 3(5), 675-688). In addition, each enzyme has a pH range for optimal activity. Therefore, pH i changes (due to high pCO2 and / or Na + ) may affect the expression and / or activity of enzymes involved in fucosylation.

[0689] To test this hypothesis, the pH of the recombinant CHO cell line was measured at different pCO2 and osmolarity (osmolarity was titrated using NaCl). i pH i As pCO2 levels increase, they decrease ( Fig.24A ), and as the osmotic pressure / Na + increases with the increase of Fig. 24B ). These data show the pCO2 and osmotic pressure / Na + Both can affect pH i, which is consistent with previous findings (deZengotita et al., (2002)., Biotechnol Bioeng, 77(44), 369-380; Reusch et al., (1995), American Physiological Society, C147-C153).

[0690] 4.3.5 Whole proteomic analysis

[0691] Untargeted proteomics was performed to reveal Mn, matrix conservation, and high pCO2 / Na + Potential mechanisms behind the effects on afucosylation. Production cultures were subjected to four different conditions ( Fig.25A ). PCA( Fig.26B ) shows a clear separation of samples by day (7 vs. 12 days) and by treatment (i.e., cell culture condition). IPA showed that pathways related to glucose and amino acid metabolism (glycolysis, gluconeogenesis, methionine degradation, and cysteine ​​biosynthesis) were upregulated in the presence of Mn, matrix preservation, and high pCO2 ( Fig.26C Among the glycolytic enzymes, fructose bisphosphate aldolase of case iv showed the highest upregulation of differential expression relative to case i ( Fig.26D ). + The increase in pH i and phosphofructokinase activity, the rate-limiting enzyme in the glycolytic pathway that converts fructose-6-phosphate (Fru-6-P) to fructose-1,6-bisphosphate (Fidelman et al., (1982), Am J Physiol, 242(1), C87-93). Increased phosphofructokinase activity increases the conversion of Fru-6-P to fructose-1,6-bisphosphate, thereby reducing the level of Fru-6-P and upregulating the expression of fructose-bisphosphate aldolase. Since Fru-6-P is a precursor of GDP-mannose, it is an upstream precursor of GDP-fucose in the de novo synthesis pathway ( Fig.27A ), so a decrease in Fru-6-P would reduce the supply of GDP-mannose and GDP-fucose and thus increase afucosylation.

[0692] 4.3.6 GDP-fucose synthesis pathway: proteomic analysis and L-fucose supplementation

[0693] To evaluate the possibility that GDP-fucose was affected in the cell culture conditions that resulted in higher mAb non-fucosylation (i.e., cases ii-iv), the differential expression of key enzymes in the de novo and salvage GDP-fucose synthesis pathways was examined ( Fig.27A). The downregulation of GMD and L-fucokinase was positively correlated with the level of afucosylation: the expression of GMD and L-fucokinase was lowest in the culture treatment with the highest afucosylation (case iv) ( Fig.27B ). No consistent changes in FX were observed for cases ii-iv relative to case i.

[0694] In previous studies, knockout of GMD or FX in CHO cell lines reduced GDP-fucose and increased afucosylation (Kanda et al., (2007), J Biotechnol, 130(30), 300-310; Louie et al., (2016), Biotechnol Bioeng, 114(3), 632-644) and afucosylation could be reduced by L-fucose supplementation to utilize the salvage pathway to synthesize GDP-fucose (Louie et al., (2016), Biotechnol Bioeng, 114(3), 632-644). Based on the proteomic observations here and knockout studies elsewhere, it can be hypothesized that the higher afucosylation with higher pCO2 and / or supplemented Mn is at least partially due to GDP-fucose limitation. To test this hypothesis, a full factorial DOE ( Fig.27C In previous titration studies, the effect of L-fucose on afucosylation reached saturation after approximately 0.3 g / L, so supplementation with L-fucose at 1 g / L was chosen for this evaluation. Supplementation with L-fucose under conditions known to generate high afucosylation restored afucosylation levels to lower levels without affecting G0F ( Fig.31 In conclusion, both proteomics and L-fucose supplementation results confirmed that GDP-fucose limitation resulted in relatively high mAb afucosylation for the combination of high pCO2, matrix preservation and supplemented Mn.

[0695] 4.3.7 Proteomic analysis: FUT8

[0696] Considering the key role of FUT8 in non-fucosylation, the differential expression of FUT8 in four culture treatment cases i-iv was analyzed ( Fig.25A ). Relative to case i, for cases ii-iv, FUT8 was downregulated and negatively correlated with non-fucosylation ( Fig.28B ). This is believed to be the first study to demonstrate downregulation of FUT8 in CHO cells by high pCO2, matrix preservation, and supplemented Mn.

[0697] 4.3.8 Proteomic analysis: other glycosylation enzymes, pH i , Golgi pH

[0698] and Golgi apparatus Mn concentration

[0699] Differential expression of glycosylases upstream (Man I, GnTII) and downstream (GalT3, GalT4, and GalT7) of FUT8 was assessed to determine if there were any additional bottlenecks in this part of the glycosylation pathway (Hossler et al., (2009), Glycobiology, 19(9), 936-949; Kremkow et al., (2018), Metabol Eng, 47, 134-142). Man I and GnTII showed little variation among the test cases and culture days, while GalT3, GalT4, and GalT7 expression levels did not vary consistently ( Fig.28B Even if these glycosylation enzymes do not show differential expression, proteomics cannot determine changes in enzyme activity. The activity of these enzymes may be affected by pH. i Or the effect of changes in intracellular Mn content, because Mn is a cofactor for some substances (Rouiller et al., (2014), Biotechnol Prog, 30(3), 571-583; Gramer et al., (2011), Biotechnol Bioeng, 108(7), 1591-1602). Proteomic data support these theories, as described below.

[0700] NHE1, a protein involved in pH i Regulated Na + / H + exchanger (Orlowski et al., (1997), J Biol Chem, 272(36), 22373-22376) and GPR89, a Golgi pH regulator (Maeda et al., (2008), Nature Cell Biology, 10(10), 1135-1145) and is associated with non-fucosylation and pCO2 / Na + Positively correlated ( Fig.28B ). These observations suggest that pH i and Golgi pH is affected by pCO2 / Na + The effect of pH i The results were consistent (Figure 24).

[0701] ATP2A1, an ATP-dependent transporter for Mn entry into the Golgi apparatus (Baelen et al., (2004), Biochimica et Biophysica Acta, 1742(1-3), 103-112), was upregulated and correlated with non-fucosylation and pCO2 / Na + Positively correlated ( Fig.28BGPP130, a Golgi protein whose degradation is completely dependent on intracellular Mn levels (Mukhopadhyay et al., (2010), Molecular Biology of the Cell, 21, 1282-1292; Masuda et al., (2013), Synapse, 67(5), 205-215; Venkat et al., (2017), Molecular Biology of the Cell, 28, 2569-2578) is downregulated and correlated with non-fucosylation and pCO2 / Na + Negatively correlated ( Fig.28B ). These results suggest that the intracellular Mn level was highest in case iv relative to the other cases. Higher intracellular Mn levels may increase the activity of GnT and GalT, favoring the overall flux to non-fucosylated glycoforms. Therefore, enhanced Mn transport and intracellular Mn levels under culture conditions of high pCO2 / Na+, supplemented Mn, and matrix preservation may contribute to higher non-fucosylation.

[0702] It will be understood that the foregoing is only illustrative of the principles of the present disclosure and that various modifications may be made by those skilled in the art without departing from the scope and spirit of the disclosure. For example, but not by way of limitation, the volume of the bioreactor used in the examples can be between about 1 L and about 20,000 L (e.g., about 1 L, about 1.5 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L, about 3000 L, about 4000 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10,000 L, about 11,000 L, about 12,000 L, about 13,000 L, about 14,000 L, about 15,000 L, about 16,000 L, about 17,000 L, about 18,000 L, about 19,000 L, or about 20,000 L). Additionally, the bioreactor configuration can be modified to adjust the level of pCO2, substrate retention duration, osmotic pressure, Na+, Mn, temperature, pH, fucose, galactose, or a combination thereof.

[0703] Example 5: Matrix storage to modulate glycosylation

[0704] A matrix storage experiment was performed for Antibody VI to evaluate changes in glycosylation (e.g., non-fucosylation and galactosylation) with increasing matrix storage time at elevated temperatures in a bioreactor. Prior to use in inoculating cultures, media for production and expansion cultures were stored at 38°C for 0, 36, 48, and 72 hours prior to production in a bioreactor with air sparging, agitation, and pH control. Figures 33A-33BThe effect of substrate storage time on glycosylation at high temperature (38°C) is shown. The correlation between substrate storage time and G0 is shown in Fig.33A The correlation between matrix storage time and non-fucosylation (eg, normalized G0-F) is shown in Fig.33B In addition, non-fucosylation increased with the storage time of the matrix. Fig.33B As shown, when medium preservation was applied to the cell culture medium, the level of normalized G0-F showed a significant medium preservation time-dependent increase. ( Fig.33A )

[0705] Matrix storage experiments were performed for Antibody III to evaluate changes in afucosylation. To evaluate the cumulative effect of matrix storage on afucosylation of Antibody III, four cases were tested in duplicate bioreactors. Production (N) and / or inoculation series (N-1) matrix was stored at elevated temperature (approximately 37°C) for 48 hours. Control cases represented the use of production and inoculation series matrix that was not stored at elevated temperature prior to use to inoculate the bioreactor. Fig.33C As shown, the results demonstrate that both N and N-1 matrices increase afucosylation on their own (expressed here as G0-F%). The greatest increase in afucosylation was observed when N matrix preservation was used in combination with N-1 matrix preservation, demonstrating the cumulative effect of matrix preservation on afucosylation.

[0706] Matrix storage and Mn supplementation experiments were performed for Antibody III to evaluate changes in afucosylation. To evaluate the discrete and combined / synergistic effects of matrix storage and Mn supplementation on afucosylation of Antibody III, four cases were tested in duplicate bioreactors. The production matrix was stored at elevated temperature (approximately 37°C) for 48 hours with / without supplementation with 250 nM Mn. The control case represents the use of production matrix that was not stored at elevated temperature prior to use to inoculate the bioreactor and lacked Mn supplementation. For the conditions tested, as Fig.33D As shown, matrix preservation showed a greater effect on non-fucosylation (expressed as G0-F%) than Mn supplementation. The greatest increase in non-fucosylation was observed when matrix preservation was combined with Mn supplementation. In particular, the greatest increase in G0-F% was observed when 48 h matrix preservation was combined with 250 nM Mn supplementation ( Fig.33D ). Exposure to 48 hours of matrix storage increased the level of G0-F%. When 250 nM Mn was supplemented to the matrix exposed to matrix storage, the level of G0-F% increased.

[0707] An increase in normalized G0-F with matrix storage time (36 h storage time) was also observed at 37°C, as shown in Fig.21AAn interaction between substrate storage time, pCO2, and Mn was also observed. The unexpected synergistic interaction resulted in an increase in normalized G0-F that was greater than the sum of the increases in normalized G0-F observed for each individual factor ( Fig.21A ).

[0708] It will be understood that the foregoing is only illustrative of the principles of the present disclosure and that various modifications may be made by those skilled in the art without departing from the scope and spirit of the disclosure. For example, but not by way of limitation, the volume of the bioreactor used in the examples can be between about 1 L and about 20,000 L (e.g., about 1 L, about 1.5 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L, about 3000 L, about 4000 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10,000 L, about 11,000 L, about 12,000 L, about 13,000 L, about 14,000 L, about 15,000 L, about 16,000 L, about 17,000 L, about 18,000 L, about 19,000 L, or about 20,000 L). Additionally, the bioreactor and its operation can be modified to adjust the level of pCO2, substrate retention duration, culture duration, osmotic pressure, Na+, Mn, culture temperature, fucose, galactose, or a combination thereof.

[0709] Example 6: Galactose addition to modulate glycosylation

[0710] Three studies were conducted to evaluate the addition of galactose, Mn, and combinations thereof on the glycosylation of Antibody VI. The results of Studies 1, 2, and 3 are shown in Figures 34-36. Galactose or Mn, or a combination thereof, can be adjusted to target a specific distribution of glycosylation (e.g., G0 and normalized G0-F). Increases in galactose levels and / or Mn levels resulted in lower non-galactosylation (G0) and higher non-fucosylation (normalized G0-F). Additionally, at lower levels of galactose, G0 was more sensitive to changes in Mn levels than at higher levels of galactose. Similarly, at lower levels of Mn, G0 was more sensitive to changes in galactose levels than at higher levels of Mn. In particular, an unexpected synergistic decrease in G0% was observed when different concentrations of galactose were added together with Mn supplementation ( Fig.34A and 35A Each galactose and Mn supplementation reduced the level of G0 in a dose-dependent manner. However, when galactose and Mn were both added to the culture medium, the level of G0 was significantly and synergistically reduced. When galactose and Mn were both added to the culture medium, the level of G0-F showed relatively little change ( Fig.34B and 35B ).

[0711] Example 7: Fucose supplementation and culture temperature to regulate glycosylation

[0712] 7.1 Introduction

[0713] This example summarizes the effect of fucose supplementation on glycosylation. Higher fucose supplementation levels and / or earlier fucose supplementation resulted in a greater reduction in non-fucosylation (e.g., G0-F). An interaction with culture temperature was observed, with a greater effect on non-fucosylation observed at lower culture temperatures.

[0714] 7.2 Evaluation of fucose concentration

[0715] The effect of fucose addition on glycosylation was evaluated in three antibody VI studies. Figures 37A-37B The effect of fucose concentration on afucosylation (eg, G0-F) and galactosylation (eg, G0) is shown. An increase in fucose levels results in higher afucosylation (normalized to G0-F).

[0716] 7.3 Evaluation of the timing of fucose addition

[0717] The effect of the timing of fucose addition on glycosylation was evaluated using Antibody VI at two different fucose levels. Fucose was added to the production culture as a post-inoculation supplement at five different time points. Figures 38A-38B The effect of the timing of fucose addition on non-fucosylation (e.g., G0-F) and galactosylation (e.g., G0) is shown. A greater reduction in G0-F was observed with earlier fucose addition. G0 was not affected by the timing of fucose addition.

[0718] 7.4 Evaluation of fucose supplementation and its interaction with temperature

[0719] To evaluate the effects of fucose concentration and temperature as well as the interaction between fucose and temperature on glycosylation, a central multiplex design study was performed with Antibody VI. Fucose was added as a post-inoculation supplement on day 0 of the production culture. Figures 39A-39B The effects of fucose and temperature on non-fucosylation (e.g., G0-F) and galactosylation (e.g., G0) are shown. Increasing fucose concentration and decreasing temperature resulted in lower levels of non-fucosylation (e.g., G0-F). An unexpected interaction between fucose and temperature was observed, where the effect of fucose on non-fucosylation (e.g., G0-F) was greater at lower temperatures than at higher temperatures. Figures 39A-39B As shown, more fucose is required to achieve the same G0-F level at high temperatures. Decreasing temperature leads to higher G0 levels.

Claims

1. A method for regulating the glycosylation pattern of a glycoprotein of interest in cell culture, comprising: The following parameters are adjusted in the cell culture medium and / or in the cell culture environment, either alone or in any combination: a. Under high partial pressure CO2 (pCO2) conditions, the Mn concentration ranges from about 1 nM to about 20000 nM; b. Under low pCO2 conditions, the Mn concentration is from about 1 nM to about 30000 nM; c. pCO2 from about 10 mmHg to about 250 mmHg; d. The cell culture medium is stored for a duration of from about 0 hours to about 120 hours before inoculation at a temperature of about 25°C to 39°C; e. cell culture duration from about 0 days to about 150 days; f. Na+ concentration from about 0 mM to about 300 mM; g. an osmotic pressure concentration of from about 250 mOsm / kg to about 550 mOsm / kg; h. a galactose concentration from about 0 mM to about 60 mM; i. a fucose concentration from about 0 mM to about 60 mM; and j. The culture temperature is from about 29°C to about 39°C.

2. A method for preparing a cell culture medium, a feed matrix, a hydrolysate or an additive, the method comprising adjusting one or more of the following steps: a. Mn concentration in high partial pressure CO2 (pCO2) culture ranges from about 1 nM to about 20,000 nM; b. Mn concentration in low pCO2 culture from about 1 nM to about 30000 nM; c. the pCO2 is from about 10 mmHg to about 250 mmHg; d. the cell culture medium is stored for a duration of from about 0 hours to about 120 hours before inoculation; e. cell culture duration from about 0 days to about 150 days; f. Na+ concentration from about 0 mM to about 300 mM; g. an osmotic pressure concentration of from about 250 mOsm / kg to about 550 mOsm / kg; h. a galactose concentration from about 0 mM to about 60 mM; i. a fucose concentration from about 0 mM to about 60 mM; and j. The culture temperature is from about 29°C to about 39°C; The cell culture medium, feed matrix, hydrolysate or additive modulates the glycosylation pattern of the glycoprotein of interest.

3. A cell culture composition comprising: a. A host cell engineered to express a glycoprotein of interest; and b. A cell culture and / or a cell culture medium which is adjusted to target one or more predetermined parameters selected from: i. Mn concentration in high partial pressure CO2 (pCO2) culture ranges from about 1 nM to about 20,000 nM; ii. Mn concentrations in low pCO2 cultures range from about 1 nM to about 30,000 nM; iii. the pCO2 is from about 10 mmHg to about 250 mmHg; iv. the cell culture medium is stored for a duration of from about 0 hours to about 120 hours before inoculation; v. cell culture duration from about 0 days to about 150 days; vi. Na+ concentration from about 0 mM to about 300 mM; vii. an osmotic pressure concentration of from about 250 mOsm / kg to about 550 mOsm / kg; viii. galactose concentration from about 0 mM to about 60 mM; ix. a fucose concentration of from about 0 mM to about 60 mM; and x. The culture temperature is from about 29°C to about 39°C.

4. A method for producing a glycoprotein of interest in cell culture, comprising: a. subjecting a cell culture medium suitable for culturing eukaryotic cells to a method according to any one of claims 1 to 75, b. inoculating conditioned cell culture medium with the eukaryotic cells expressing the recombinant protein; c. culturing the eukaryotic cells to allow the recombinant protein to be expressed.

5. A method for regulating the glycosylation of a target glycoprotein, the method comprising: a. measuring the cell culture medium to determine whether the manganese concentration of the cell culture medium falls within the target range; as well as b. culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium falling within the target range; Wherein glycosylation of the glycoprotein of interest is modulated compared to glycosylation of the glycoprotein of interest expressed by the host cell in culture medium falling outside of the target range of manganese concentration.

6. A cell culture composition comprising: a. a cell culture medium which is assayed to determine whether the manganese concentration of the cell culture medium is within a target range; and b. Host cells engineered to express the glycoprotein of interest.

7. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a. a cell culture medium which is assayed to determine whether the manganese concentration of the cell culture medium is within a target range; b. a host cell engineered to express a glycoprotein of interest; and c. the target glycoprotein.

8. A method for regulating the glycosylation of a target glycoprotein, the method comprising: a. Under high CO2 conditions, supplementing the cell culture medium used when culturing host cells expressing the target glycoprotein with between about 10 nM and about 2000 nM manganese; or b. supplementing the cell culture medium used in culturing host cells expressing the target glycoprotein with between about 10 nM and about 3000 nM manganese under low CO2 conditions; Wherein the glycosylation of the glycoprotein of interest is modulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium that has not been so supplemented.

9. A cell culture composition comprising: a. Cell culture medium supplemented with: i. between about 10 nM and about 2000 nM manganese under high CO2 conditions; or ii. between about 10 nM and about 3000 nM manganese under low CO2 conditions; and b. Host cells engineered to express the glycoprotein of interest.

10. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a. Cell culture medium supplemented with manganese, i. wherein the culture is supplemented with: between about 10 nM and about 2000 nM manganese under high CO2 conditions; or ii. between about 10 nM and about 3000 nM manganese under low CO2 conditions; b. a host cell engineered to express the glycoprotein of interest; and c. the target glycoprotein.

11. A method for regulating the glycosylation of a target glycoprotein, the method comprising: a. exposing the cell culture medium having a target pH of 6.30 to 7.25 to a high temperature, short time (HTST) heat treatment; as well as b. culturing a host cell expressing the target glycoprotein in the cell culture medium; Wherein the glycosylation of the target glycoprotein is modulated compared to the glycosylation of the target glycoprotein expressed by the host cell in a culture medium having a target pH value greater than 7.25 before the HTST heat treatment.

12. A cell culture composition comprising: a. a cell culture medium exposed to HTST heat treatment having a target pH of about 6.30 to about 7.25; and b. Host cells engineered to express the glycoprotein of interest.

13. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a. a cell culture medium exposed to HTST heat treatment having a target pH of about 6.30 to about 7.25; b. a host cell engineered to express a glycoprotein of interest; and c. the target glycoprotein.

14. A method for regulating the glycosylation of a target glycoprotein, the method comprising: a. culturing a host cell expressing the target glycoprotein in a cell culture medium, wherein: i. exposing the cell culture to high pCO2, ii. said cell culture is exposed to a prolonged matrix storage time, and / or iii. said cell culture comprises an increased Na+ concentration; Wherein the glycosylation of the glycoprotein of interest is modulated compared to the fucosylation of a preparation of the glycoprotein of interest expressed by the host cell in a medium exposed to low pCO2, shortened substrate storage time and / or reduced Na+ concentration.

15. A cell culture composition comprising: a. Cell culture medium comprising high pCO2, extended substrate storage time and / or increased Na+ concentration; and b. Host cells engineered to express the glycoprotein of interest.

16. A composition comprising a glycoprotein of interest, wherein the preparation comprises: a. Cell culture medium comprising high pCO2, extended substrate storage time and / or increased Na+ concentration; b. a host cell engineered to express a glycoprotein of interest; and c. the target glycoprotein.

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