Cell culture strategies for modulating protein glycosylation

Abstract

To provide a method for modulating a glycosylation pattern of protein.SOLUTION: A method for modulating a glycosylation pattern of glycoprotein of interest in a cell culture includes modulating, for example, the following parameters, either alone or in any combination, in a cell culture medium, and / or, in a cell culture environment: a. a Mn concentration of approximately 1nM to approximately 20000nM in a high partial pressure CO2(pCO2) condition; b. a Mn concentration of approximately 1nM to approximately 30000nM in a low pCO2 condition; c. a pCO2 of approximately 10 mmHg to approximately 250 mmHg; d. a pre-inoculation cell culture media hold duration of approximately 0 hours to approximately 120 hours at a temperature of approximately 25°C to 39°C.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 717,751, filed Aug. 10, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] The subject matter disclosed herein 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, and cell culture and glycoprotein compositions prepared using such media and / or strategies. [Background technology]

[0003] N-linked glycosylation can affect the physiochemical 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 for a 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 several studies, mAbs with increased terminal galactosylation, which represents the addition of terminal galactose residues to N-acetylglucosamine (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). Thus, optimal and consistent levels of galactosylation may be highly desirable for mAb production with CDC as a mechanism of action. Other studies have shown that mAbs with reduced core fucosylation, which represents 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). Thus, an optimal and consistent level of fucosylation deficiency (i.e., lack of core fucose on N-linked glycans) can be highly desirable for mAb products with ADCC as a mechanism of action. Strategies for modulating mAb glycosylation (eg, galactosylation and / or fucosylation) during cell culture processes generally fall into one of four categories:(1) genetic manipulation 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) modifying 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. al.,(2017), mAbs,9(4),715-734); and (4) fine-tuning of culture process parameters (Konno et al.,(2012), Cytotechnology,64,249-265). While many cell culture process parameters are known to affect galactosylation (Hossler et al.,(2009), Glycobiology,19(9),936-949), fewer have been identified to modulate fucosylation. Summary of the Invention

[0004] The subject matter disclosed herein relates to modulating 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 modulating glycosylation to achieve or maintain a desired glycoprotein glycosylation pattern (e.g., galactosylation and / or fucosylation pattern). Methods by which glycosylation can be modulated in accordance with the present disclosure include, but are not limited to, the following: (1) For example, by Mn concentration analysis of the raw material, Mn supplementation to and / or during cell culture medium, and / or

[0005] (2) adjusting the cell culture medium and / or cell culture manganese (Mn) concentration with respect to minimizing Mn loss from the cell culture by establishing a lowered pH target or set point for medium pH adjustment prior to high temperature short time (HTST) heat treatment of the medium; 2 , medium retention time, culture period, culture temperature and osmolality / Na + and (3) adjusting the cell culture medium and / or cell culture galactose and / or fucose concentrations. The presently disclosed subject matter is also directed to cell cultures and glycoprotein compositions prepared when such process parameters are adjusted as described herein.

[0006] In certain embodiments, the present disclosure provides a method for modulating the glycosylation pattern of a glycoprotein of interest in a cell culture, comprising adjusting the following parameters in the cell culture medium and / or in the cell culture environment: high partial pressure CO 2 (pCO 2 ) conditions of Mn concentration of about 1 nM to about 20,000 nM; low pCO 2 Mn concentration of about 1 nM to about 30,000 nM; pCO2 of about 10 mmHg to about 250 mmHg 2 a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25°C to 39°C; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C, either alone or in any combination.

[0007] In some embodiments, the cell culture environment is in a bioreactor with or without cells. 2 The conditions are approximately 10 to approximately 100 mmHg and high pCO 2 The conditions are about 20 to about 250 mmHg.

[0008] In one embodiment, the pCO2 The conditioning period encompasses at least the first half of the cell culture period.

[0009] In some embodiments, the glycoprotein of interest is a recombinant protein. In some 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 (dock-and-lock trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody). In some embodiments, the antibody is a chimeric antibody, a humanized antibody or a human antibody. In some embodiments, the antibody is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is ocrelizumab. In certain embodiments, the antibody or antibody fragment exhibits a %G0-F (percent fucosylation-deficient 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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%.

[0010] In certain embodiments, glycosylation is modulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated galactosylation deficient G0)), or to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0), or to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0), or to achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F).

[0011] In one embodiment, modulating the glycosylation pattern of a glycoprotein of interest comprises reducing pCO 2 Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 2 The conditions include adjusting an Mn concentration of about 1 nM to about 20,000 nM, as well as the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours, at a temperature of about 25° C. to 39° C.; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29° C. to about 39° C.

[0012] In one embodiment, modulating the glycosylation pattern of a glycoprotein of interest comprises reducing pCO 2 Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 2 The conditions include adjusting a Mn concentration of about 1 nM to about 20,000 nM, and the following parameters in the cell culture medium and / or cell culture environment: a pre-seeding cell culture medium holding time of about 0 hours to about 120 hours, at a temperature of about 25° C. to 39° C.; and a cell culture period of about 0 days to about 150 days.

[0013] In one embodiment, modulating the glycosylation pattern of a glycoprotein of interest comprises reducing pCO 2 Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 2 The conditions include adjusting the Mn concentration from about 1 nM to about 20,000 nM, and the following parameters in the cell culture medium and / or cell culture environment: a galactose concentration from about 0 mM to about 60 mM; and / or a fucose concentration from about 0 mM to about 60 mM.

[0014] In certain embodiments, modulating the glycosylation pattern of a glycoprotein of interest comprises adjusting the pre-seeding cell culture medium holding period and any of the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: high partial pressure CO 2 (pCO 2 ) conditions of Mn concentration of about 1 nM to about 20,000 nM; low pCO 2 Mn concentration of about 1 nM to about 30,000 nM; pCO2 of about 10 mmHg to about 250 mmHg 2 a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolarity of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C, wherein the cell culture medium retention period is about 0 hours to about 120 hours at a temperature of about 25°C to 39°C.

[0015] In one embodiment, the step of modulating the glycosylation pattern of a glycoprotein of interest can be achieved by adjusting the pre-seeding cell culture medium holding period and the following parameters in the cell culture medium and / or in the cell culture environment: high partial pressure CO 2 (pCO 2 ) conditions, such as Mn concentrations of about 1 nM to about 20,000 nM, or low pCO 2 Mn concentration of about 1 nM to about 30,000 nM; pCO2 of about 10 mmHg to about 250 mmHg 2 and adjusting the cell culture period from about 0 days to about 150 days, wherein the cell culture medium holding period is from about 0 hours to about 120 hours at a temperature of from about 25° C. to about 39° C.

[0016] In one embodiment, the step of modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture duration and any of the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: Na+ concentration from about 0 mM to about 300 mM; osmolarity 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., and the cell culture duration is from about 0 days to about 150 days.

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

[0018] In one embodiment, the step of modulating the glycosylation pattern of the glycoprotein of interest comprises adjusting the Na concentration and pCO2 concentration from about 10 mmHg to about 250 mmHg. 2 This includes adjusting the

[0019] In certain embodiments, the step of modulating the glycosylation pattern of the glycoprotein of interest comprises adjusting the osmolarity and any of the following parameters, alone or in any combination, in the cell culture medium and / or cell culture environment: a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29° C. to about 39° C., with an osmolarity of about 250 mOsm / kg to about 550 mOsm / kg.

[0020] In one embodiment, modulating the glycosylation pattern of a glycoprotein of interest comprises reducing pCO 2Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 2 The conditions include adjusting the Mn concentration from about 1 nM to about 20,000 nM, adjusting the Na+ concentration from about 0 mM to about 300 mM, and adjusting the pre-seeding cell culture medium retention period from about 0 hours to about 120 hours.

[0021] In one embodiment, the modulation of the glycosylation pattern of the glycoprotein of interest comprises an osmolality of about 250 mOsm / kg to about 550 mOsm / kg and a pCO2 of about 10 mmHg to about 250 mmHg. 2 This includes adjusting the

[0022] In one embodiment, modulating the glycosylation pattern of the glycoprotein of interest comprises adjusting 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.

[0023] In one embodiment, the modulation of the glycosylation pattern of the glycoprotein of interest is achieved by administering a pCO 2 and adjusting the fucose concentration from about 0 mM to about 60 mM.

[0024] In certain embodiments, modulating the glycosylation pattern of a 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.

[0025] In one embodiment, the modulation of the glycosylation pattern of the glycoprotein of interest is achieved by pCO 2 Concentration and any of the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: high partial pressure CO 2 (pCO 2 ) conditions of Mn concentration of about 1 nM to about 20,000 nM; low pCO 2a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25° C. to about 39° C.; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29° C. to about 39° C., 2 The concentration is about 10 mmHg to about 250 mmHg.

[0026] In one embodiment, the Mn concentration is 2 Approximately 1 nM to approximately 20,000 nM in culture; high pCO 2 In culture, about 1nM to about 10000nM, about 1nM to about 5000nM, about 1nM to about 4000nM, about 1nM to about 3000nM, about 1nM to about 2000nM, about 1nM to about 1000nM; high pCO 2 In culture, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 20 nM to about 2000 nM, about 20 nM M ~ about 3000nM, about 20nM - about 10000nM, about 20nM - about 20,000nM, about 20nM - about 300nM, about 30nM - about 110nM.

[0027] In one embodiment, the Mn concentration is 2 Approximately 1 nM to approximately 30,000 nM in culture; low pCO 2 In culture, about 1nM to about 20000nM; about 1nM to about 10000nM, about 1nM to about 5000nM, about 1nM to about 4000nM, about 1nM to about 3000nM , about 1nM to about 2000nM, about 1nM to about 1000nM; about 1nM to about 500nM, about 1nM to about 100nM, about 1nM to about 50nM, about 1nM to about 20nM , about 20 nM to about 100 nM, about 20 nM to about 300 nM, about 20 nM to about 500 nM, about 20 nM to about 1000 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 5000 nM, about 20 nM to about 10,000 nM, about 20 nM to about 20,000 nM, or about 30 nM to about 110 nM.

[0028] In one embodiment, adjusting the Mn concentration includes determining the Mn content in a cell culture feedstock and selecting a feedstock lot for adjusting the Mn concentration.

[0029] In some embodiments, adjusting the Mn concentration includes (i) adjusting the cell culture medium or a material in contact with the cell culture to adjust the Mn concentration; or (ii) considering the concentration of leached Mn in the cell culture medium or during the cell culture; or a combination of (i) and (ii). In some embodiments, the leached Mn is generated by contacting the cell culture and / or cell culture medium with (i) a filter; (ii) a medium preparation, holding or culture vessel; or (iii) a combination of (i) and (ii). In some embodiments, the filter includes, but is not limited to, a depth filter, a column, a membrane, and a disk. In some embodiments, the filter material includes, but is not limited to, diatomaceous earth, hollow fiber, or resin.

[0030] In certain embodiments, the cell culture medium is a basal medium, a reconstituted medium, a feed medium, a hydrolysate, a supplement, a serum or an additive.

[0031] In certain embodiments, the cell culture medium is replenished during the production stage of the cell culture.

[0032] In certain embodiments, the cell culture medium is replenished prior to the production stage of the cell culture.

[0033] In certain embodiments, the cell culture medium contains one or more of Mn, fucose, galactose and / or Na+, and supplementation is based on a predefined schedule or criteria.

[0034] In certain embodiments, one or more of Mn, fucose, galactose and Na+ are supplemented as a bolus, as intermittent supplementation, as continuous supplementation, as semi-continuous supplementation, as feedback loop based supplementation, or as a combination of one or more of these.

[0035] In certain embodiments, the cell culture medium consists essentially of one or more of: i) Mn; ii) fucose; iii) galactose; and / or iv) Na+.

[0036] In certain embodiments, adjusting the Mn concentration includes utilizing 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.

[0037] In one embodiment, the modulation of the glycosylation pattern of the glycoprotein of interest is achieved by pCO 2 This includes adjusting the

[0038] In one embodiment, the cell culture or cell culture medium is in a bioreactor and pCO 2 is achieved by adjusting the bioreactor working volume; the bioreactor gassing strategy; the bioreactor agitation strategy; the bioreactor medium exchange strategy, the bioreactor perfusion strategy, the bioreactor feeding strategy, or any combination thereof.

[0039] In one embodiment, the pCO 2 Regulation is high pCO 2 In one embodiment, the pCO 2 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.

[0040] In one embodiment, the pCO 2 Adjustment is at low pCO 2 In one embodiment, 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.

[0041] In one embodiment, the pCO 2 The regulation occurs at day 0 of culture.

[0042] In one embodiment, the pCO 2 The conditioning occurs for approximately the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0043] In one embodiment, the pCO 2 Regulation occurs for approximately the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0044] In certain embodiments, modulating the glycosylation pattern of the glycoprotein of interest comprises modulating a pre-seeding cell culture medium holding period, wherein the pre-seeding cell culture medium holding period 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.

[0045] In certain embodiments, the temperature of the medium during pre-seeding cell culture medium holding 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.

[0046] In certain embodiments, modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture period, wherein the cell culture period 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.

[0047] In certain embodiments, modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the Na+ concentration, wherein the Na+ concentration is from about 0 mM to about 300 mM; from about 20 mM to about 20 mM; from about 30 mM to about 150 mM; or from about 40 mM to about 130 mM.

[0048] In one embodiment, the regulation of the Na+ concentration is 2 CO 3 , NaHCO 3 The method includes supplementing the cell culture with Na compounds, including but not limited to NaOH, NaCl, or combinations thereof.

[0049] In some embodiments, adjusting the glycosylation pattern of the glycoprotein of interest comprises adjusting the osmolarity, the osmolarity being 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. In some embodiments, adjusting the osmolarity comprises supplementing the cell culture with an osmolarity-adjusting medium component. In some embodiments, the osmolarity-adjusting medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof. In some embodiments, the osmolarity-adjusting medium component is supplemented as a bolus, as an intermittent supplement, as a continuous supplement, as a semi-continuous supplement, as a feedback loop-based supplement, or one or more combinations thereof.

[0050] In certain embodiments, modulating 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.

[0051] In certain embodiments, modulating 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.

[0052] In certain embodiments, modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture temperature, wherein the cell culture temperature is from about 29°C to about 39°C, from about 30°C to about 39°C, from about 31°C to about 38°C, or from about 34°C to about 38°C.

[0053] In certain embodiments, the cell culture temperature is adjusted during the production stage of the cell culture.

[0054] In certain embodiments, the cell culture temperature is adjusted prior to the production stage of the cell culture.

[0055] In certain embodiments, the cell culture temperature is adjusted based on a predefined schedule or criteria.

[0056] In some embodiments, the cell culture comprises eukaryotic cells. In some embodiments, the eukaryotic cells are insect, avian, fungal, plant or mammalian cells. In some embodiments, the fungal cells are yeast, Pichia or any filamentous fungal cells. In some embodiments, the yeast cells are S. cerevisiae cells. In some embodiments, the mammalian cells are CHO cells.

[0057] In certain embodiments, the cell culture is in a bioreactor, including but not limited to, a single-use technology (SUT) bag or bioreactor; a WAVE bioreactor; a stainless steel bioreactor; a flask; a tube and a chamber.

[0058] In certain embodiments, the volume of the cell culture is 1 mL to 35,000 L. In certain embodiments, the volume of the cell culture is 1 mL to 10 ml, 1 mL to 50 ml, 1 mL to 100 ml, 1 mL to 200 ml, 1 mL to 300 ml, 1 mL to 500 ml, 1 mL to 1000 ml, 1 mL to 2000 ml, 1 mL to 3000 ml, 1 mL to 4000 ml, 1 mL to 5000 ml, 1 mL to 1 L, 1 mL to 2 L, 1 mL to 3 L, 1 mL to 4 L, 1 mL to 5 L, 1 mL to 6 L, 1 mL to 10 L, 1 mL to 20 L, 1 mL ~30L, 1mL~40L, 1mL~50L, 1mL~60L, 1mL~70L, 1mL~100L, 1mL~200L, 1mL~300L, 1mL~400L, 1mL~500L, 1mL~1000L, 1mL~2000L, 1mL~3000L, 1mL~4000L, 1mL~5000L, 1mL~10,000L, 1mL~20,000L, 1mL~30,000L, 1mL~30,000L, and 1mL~35,000L.

[0059] In certain embodiments, the present disclosure provides a method for preparing a cell culture medium, feed medium, hydrolysate, or additive, comprising administering to a subject a culture medium, feed medium, or additive comprising: 2 (pCO 2 ) Mn concentration in the culture; low pCO of about 1 nM to about 30,000 nM 2 Mn concentration in culture; pCO of about 10 mmHg to about 250 mmHg 2 the cell culture medium, feed medium, hydrolysate or additive modulates the glycosylation pattern of a glycoprotein of interest, comprising one or more steps of adjusting a Na+ concentration from about 0 mM to about 300 mM; an osmolality 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.

[0060] In certain embodiments, these methods involve administering a pCO2 concentration of about 10 mmHg to about 250 mmHg. 2, including adjusting the Na concentration from about 0 mM to about 300 mM and the pre-seeding cell culture medium holding period from about 0 hours to about 120 hours.

[0061] In certain embodiments, these methods involve administering to a subject a subject a Mn concentration of about 1 nM to about 30,000 nM, a pCO 2 and adjusting the Na+ concentration from about 0 mM to about 300 mM.

[0062] In certain embodiments, these methods involve administering to a subject a subject a Mn concentration of about 1 nM to about 30,000 nM, a pCO 2 , including adjusting the Na concentration from about 0 mM to about 300 mM and the pre-seeding cell culture medium retention period from about 0 hours to about 72 hours.

[0063] In certain embodiments, these methods involve administering a pCO2 concentration of about 10 mmHg to about 250 mmHg. 2 and adjusting the Na+ concentration from about 0 mM to about 300 mM.

[0064] In certain embodiments, these methods include administering to a patient a physiological saline solution containing an osmolality of about 250 mOsm / kg to about 550 mOsm / kg and a pCO2 of about 10 mmHg to about 250 mmHg. 2 This includes adjusting the

[0065] In certain embodiments, these methods involve administering a pCO2 concentration of about 10 mmHg to about 250 mmHg. 2 , adjusting the Mn concentration from about 1 nM to about 30,000 nM, the cell culture period from about 0 days to about 150 days, and the pre-seeding cell culture medium retention period from about 0 hours to about 120 hours.

[0066] In certain embodiments, these methods involve adjusting the Mn concentration from about 1 nM to about 30,000 nM and the galactose concentration from about 0 mM to about 60 mM.

[0067] In certain embodiments, these methods involve adjusting the fucose concentration from about 0 mM to about 60 mM and the Mn concentration from about 1 nM to about 30,000 nM.

[0068] In certain embodiments, these methods involve administering to a patient a fucose concentration of about 0 mM to about 60 mM and a pCO2 concentration of about 10 mmHg to about 250 mmHg. 2 This includes adjusting the

[0069] In certain embodiments, these methods involve the use of a fucose concentration of about 0 mM to about 60 mM, an Mn concentration of about 1 nM to about 30,000 nM, and a pCO2 concentration of about 10 mmHg to about 250 mmHg. 2 This includes adjusting the

[0070] In certain embodiments, these methods include adjusting the fucose concentration from about 0 mM to about 60 mM, and the cell culture temperature is from about 29°C to about 39°C.

[0071] In certain embodiments, these methods involve adjusting the fucose concentration from about 0 mM to about 60 mM and the cell culture period from about 0 days to about 150 days.

[0072] In one embodiment, the Mn concentration is 2 Approximately 1 nM to approximately 20,000 nM in culture; high pCO 2 Approximately 1 nM to approximately 1000 nM in culture; high pCO 2 About 20 nM to about 300 nM in culture; or high pCO 2 About 30 nM to about 110 nM in culture.

[0073] In one embodiment, the Mn concentration is 2 Approximately 1 nM to approximately 30,000 nM in culture; low pCO 2 Approximately 1 nM to approximately 3000 nM in culture; low pCO 2 About 20 nM to about 300 nM in culture; or low pCO 2 About 30 nM to about 110 nM in culture.

[0074] In one embodiment, adjusting the Mn concentration includes determining the Mn content in a cell culture feedstock and selecting a feedstock lot for adjusting the Mn concentration.

[0075] In certain embodiments, modulating the Mn concentration includes: i) adjusting the cell culture medium or a material in contact with the cell culture to modulate the Mn concentration; or (ii) taking into account the concentration of Mn leached into the cell culture medium or during the cell culture; or a combination of (i) and (ii).

[0076] In some embodiments, the leached Mn is produced by contacting the cell culture and / or cell culture medium with (i) a filter; (ii) a medium preparation, holding or culture vessel; or (iii) a combination of (i) and (ii). In some embodiments, the filter includes, but is not limited to, a depth filter, a column, a membrane and a disk. In some embodiments, the filter material includes, but is not limited to, diatomaceous earth, hollow fiber or resin.

[0077] In certain embodiments, adjusting the Mn concentration comprises utilizing 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.

[0078] In one embodiment, the pCO 2 In one embodiment, the cell culture medium is in a bioreactor and pCO 2 The regulation of pCO is accomplished by adjusting the bioreactor working volume; the bioreactor gas sparging strategy; the bioreactor agitation strategy; the bioreactor feeding strategy, the bioreactor perfusion strategy, the bioreactor medium exchange strategy, or any combination thereof. In one embodiment, 2 Regulation is high pCO 2 In one embodiment, the pCO 2 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.

[0079] In one embodiment, the pCO2 Adjustment is at low pCO 2 In one embodiment, the pCO 2 In one embodiment, the pCO 2 The modulation occurs at day 0 of culture. 2 The modulation occurs for about the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days. In one embodiment, the pCO 2 The regulation occurs for approximately the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0080] In some embodiments, the pre-seeding cell culture medium retention period 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 some embodiments, the temperature of the medium during pre-seeding cell culture medium retention 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.

[0081] In certain embodiments, the cell culture period is from about 0 to about 150 days, from about 0 to about 15 days, from about 0 to about 12 days, from about 0 to about 7 days, or from about 0 to about 5 days.

[0082] In one embodiment, 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. 2 CO 3 , NaHCO 3 The method includes supplementing the cell culture with Na compounds, including but not limited to NaOH, NaCl, or combinations thereof.

[0083] In some embodiments, the osmolarity 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. In some embodiments, adjusting the osmolarity comprises supplementing the cell culture with an osmotically adjusted medium component. In some embodiments, the osmotically adjusted medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

[0084] In certain embodiments, the galactose concentration is from about 0 mM to about 60 mM or from about 0 mM to about 50 mM.

[0085] 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.

[0086] 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.

[0087] In some embodiments, the present disclosure is directed to a eukaryotic cell fermentation process for the production of recombinant proteins. In some embodiments, the recombinant proteins are antibodies or antibody fragments, scFvs (single chain variable fragments), BsDbs (bispecific diabodies), scBsDbs (single chain bispecific diabodies), scBsTaFvs (single chain bispecific tandem variable domains), DNL-(Fab)3 (dock-and-lock trivalent Fabs), sdAbs (single domain antibodies) and BssdAbs (bispecific single domain antibodies). In some embodiments, the antibodies are chimeric, humanized or human antibodies. In some embodiments, the antibodies are anti-CD20 antibodies. In some embodiments, the anti-CD20 antibodies are ocrelizumab. In some embodiments, the antibody or antibody fragment exhibits a %G0-F (percent fucosylation-deficient glycoprotein) of about 0% to about 20%; about 1% to about 15%; about 1% to about 10%; or about 1% to about 8%; 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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%. In some embodiments, the eukaryotic cell is an insect, avian, fungal, plant or mammalian cell. In some embodiments, the fungal cell is a yeast, Pichia or any filamentous fungal cell. In some embodiments, the yeast cell is a S. cerevisiae cell. In some embodiments, the mammalian cell is a CHO cell.

[0088] In one embodiment, the present disclosure provides a method for the treatment of a glycoprotein comprising administering to a host cell engineered to express a glycoprotein of interest, the method comprising administering to a host cell an amount of high partial pressure CO at about 1 nM to about 20,000 nM. 2 (pCO 2 ) Mn concentration in the culture; low pCO of about 1 nM to about 30,000 nM 2 Mn concentration in culture; pCO of about 10 mmHg to about 250 mmHg 2the cell culture medium being adjusted to target one or more predetermined parameters selected from: a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C.

[0089] In one embodiment, the Mn concentration is from about 1 nM to about 30,000 nM, and the pre-seeding cell culture medium retention period is from about 0 hours to about 120 hours.

[0090] In one embodiment, the pCO 2 The pressure is about 10 mmHg to about 250 mmHg, the Na + concentration is about 0 mM to about 300 mM, and the cell culture medium retention period before seeding is about 0 hours to about 120 hours.

[0091] In one embodiment, the Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 is about 10 mmHg to about 250 mmHg, and the Na + concentration is about 0 mM to about 300 mM.

[0092] In one embodiment, the Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 is about 10 mmHg to about 250 mmHg, the Na + concentration is about 0 mM to about 300 mM, and the pre-seeding cell culture medium retention period is about 0 hours to about 120 hours.

[0093] In one embodiment, the pCO 2 is about 10 mmHg to about 250 mmHg, and the Na + concentration is about 0 mM to about 300 mM.

[0094] In one embodiment, the osmolality is about 250 mOsm / kg to about 550 mOsm / kg, and the pCO 2 is about 10 mmHg to about 250 mmHg.

[0095] In one embodiment, the pCO 2 is about 10 mmHg to about 250 mmHg, the Mn concentration is about 1 nM to about 30,000 nM, the cell culture period is about 0 days to about 150 days, and the cell culture medium retention period before seeding is about 0 hours to about 120 hours.

[0096] In one embodiment, the Mn concentration is about 1 nM to about 30,000 nM, and the galactose concentration is about 0 mM to about 60 mM.In one embodiment, the fucose concentration is about 0 mM to about 60 mM, and the Mn concentration is about 1 nM to about 30,000 nM.

[0097] In one embodiment, the fucose concentration is about 0 mM to about 60 mM, and the pCO 2 In one embodiment, the fucose concentration is about 0 mM to about 60 mM, the Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 is about 10 mmHg to about 250 mmHg. In one embodiment, the fucose concentration is about 0 mM to about 60 mM, and the cell culture temperature is about 29° C. to about 39° C. In one embodiment, the fucose concentration is about 0 mM to about 60 mM, and the cell culture period is about 0 to about 150 days.

[0098] In certain embodiments, the present disclosure is directed to a method of producing a glycoprotein of interest in a cell culture, the method comprising providing a cell culture medium suitable for culturing eukaryotic cells, seeding the conditioned cell culture medium with eukaryotic cells expressing the recombinant protein, and culturing the eukaryotic cells such that the recombinant protein is expressed, according to any one of the embodiments disclosed herein.

[0099] In certain embodiments, the present disclosure is directed to a method of modulating glycosylation of a glycoprotein of interest comprising assaying a cell culture medium to determine whether the manganese concentration of the cell culture medium falls within a targeted range, and culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium that falls within the targeted range, wherein glycosylation of the glycoprotein of interest is modulated compared to glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium that falls outside the targeted range of manganese concentrations.

[0100] In certain embodiments, the present disclosure is directed to a composition comprising a glycoprotein of interest, the composition comprising a cell culture medium, the preparation of which comprises a cell culture medium assayed to determine whether the manganese concentration of the cell culture medium falls within a targeted range, a host cell engineered to express the glycoprotein of interest, and the glycoprotein of interest.

[0101] In an embodiment, the present disclosure provides a method for modulating glycosylation of a glycoprotein of interest, comprising administering high CO to a cell culture medium used in culturing a host cell expressing the glycoprotein of interest. 2 Supplementing the cell culture medium used in culturing the host cells expressing the glycoprotein of interest with about 10 nM to about 2000 nM manganese under conditions of low CO 2 The present invention is directed to a method comprising supplementing a culture medium containing about 10 nM to about 3000 nM manganese under conditions, wherein glycosylation of a glycoprotein of interest is modulated compared to glycosylation of the glycoprotein of interest expressed by a host cell in a culture medium that has not been so supplemented.

[0102] In one embodiment, the present disclosure provides a cell culture composition comprising: 2 Approximately 10 nM to approximately 2000 nM manganese under conditions of low CO 2The present invention is directed to a cell culture composition comprising a cell culture medium supplemented with about 10 nM to about 3000 nM manganese under conditions, and a host cell engineered to express a glycoprotein of interest.

[0103] In an embodiment, the present disclosure provides a composition comprising a glycoprotein of interest, the preparation being a cell culture medium supplemented with manganese, the culture being in an environment of elevated CO 2 Under conditions of about 10 nM to about 2000 nM manganese or low CO 2 The present invention is directed to a composition comprising a cell culture medium supplemented with about 10 nM to about 3000 nM manganese under conditions, a host cell engineered to express a glycoprotein of interest, and the glycoprotein of interest.

[0104] In some embodiments, the method of modulating glycosylation of a glycoprotein of interest includes assaying a cell culture medium and / or cell culture to determine whether the manganese concentration of the cell culture medium and / or cell culture falls within a targeted range, and culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium that falls within the targeted range. In some embodiments, 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 and / or cell culture that falls outside the targeted range of manganese concentration. In some embodiments, the manganese concentration target range is 20 nM to 200 nM. In non-limiting embodiments, the manganese concentration target range is about 30 nM to about 110 nM.

[0105] In some embodiments, the disclosed glycoprotein of interest is an antibody. The antibody can be a chimeric antibody, a humanized antibody, or a human antibody. In a non-limiting embodiment, the antibody is ocrelizumab.

[0106] In certain embodiments, the disclosed host cells are mammalian cells. The host cells can be Chinese Hamster Ovary (CHO) cells.

[0107] In some embodiments, the disclosed cell culture media assays include assaying a component of the cell culture media for manganese concentration. In some embodiments, the disclosed cell culture media assays include assaying a component of the cell culture media for manganese concentration. The cell culture media component is a hydrolysate or serum. The cell culture media component can also be a complex mixture of multiple components.

[0108] In some embodiments, glycosylation is modulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated, galactosylation-deficient G0)). In some embodiments, glycosylation is modulated to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0).

[0109] In some embodiments, glycosylation is modulated to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0-F). In some embodiments, glycosylation is modulated to achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F).

[0110] In certain embodiments, the subject matter disclosed herein is directed to a cell culture composition comprising a cell culture medium and / or cell culture assayed to determine whether the manganese concentration of the cell culture medium and / or cell culture falls within a targeted range, and a host cell engineered to express a glycoprotein of interest. In certain embodiments, the manganese concentration is regulated through the selection or avoidance of raw materials in contact with the culture medium and / or cell culture that can leach manganese (e.g., depth and / or media filters, media preparation and / or holding vessels, and bioreactors). In certain embodiments, the cell culture composition further comprises a glycoprotein of interest. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric, humanized, or human antibody. In certain embodiments, the antibody is ocrelizumab. In certain embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0111] In some embodiments, the subject matter disclosed herein is directed to a preparation comprising a glycoprotein of interest, the preparation comprising a cell culture medium assayed to determine whether the manganese concentration of the cell culture medium falls within a targeted range, a host cell engineered to express the glycoprotein of interest, and the glycoprotein of interest. In some embodiments, the manganese concentration is controlled through the selection of a raw material that contains manganese at a desired level. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0112] In certain embodiments, the presently disclosed subject matter provides a method for modulating glycosylation of a glycoprotein of interest, comprising administering high CO to a cell culture medium used in culturing a host cell expressing the glycoprotein of interest.2 Supplementing the cell culture medium used in culturing the host cells expressing the glycoprotein of interest with about 10 nM to about 2000 nM manganese under conditions of low CO 2 The present invention is directed to a method comprising supplementing about 10 nM to about 3000 nM manganese under conditions, wherein glycosylation of a glycoprotein of interest is modulated compared to glycosylation of a glycoprotein of interest expressed by a host cell in a culture medium not so supplemented. In an embodiment, manganese is supplemented directly to the cell culture. In an embodiment, the glycoprotein of interest is an antibody. In an embodiment, the antibody is a chimeric, humanized or human antibody. In an embodiment, the antibody is ocrelizumab. In an embodiment, the host cell is a mammalian cell, e.g., a CHO cell. In an embodiment, glycosylation is modulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated G0)). In an embodiment, glycosylation is modulated to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0).

[0113] In certain embodiments, the presently disclosed subject matter provides a cell culture composition comprising: 2 Approximately 10 nM to approximately 2000 nM manganese under conditions of low CO 2 The present invention is directed to a cell culture composition comprising a cell culture medium and / or cell culture supplemented with about 10 nM to about 3000 nM manganese under conditions and a host cell engineered to express a glycoprotein of interest. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0114] In certain embodiments, the presently disclosed subject matter provides a composition comprising a glycoprotein of interest, the preparation being a cell culture medium supplemented with manganese, the culture being in an environment of elevated CO 2 Under conditions of about 10 nM to about 2000 nM manganese or low CO 2 The present invention is directed to a composition comprising a cell culture medium supplemented with about 10 nM to about 3000 nM manganese under conditions, a host cell engineered to express a glycoprotein of interest, and a glycoprotein of interest. In some embodiments, the manganese is supplemented by using a source (e.g., depth and / or media filters, media preparation and / or holding vessels, and bioreactors) that leach manganese while in contact with the culture medium and / or cell culture. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0115] In certain embodiments, the subject matter disclosed herein is directed to a method of modulating glycosylation of a glycoprotein of interest, comprising exposing a cell culture medium comprising a pH target 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 modulated compared to the glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium having a pre-HTST heat treatment pH target of greater than pH 7.25. In certain embodiments, the glycoprotein of interest is an antibody. In certain embodiments, the antibody is a chimeric, humanized or 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 modulated to achieve increased G0-F (fucosylation-deficient G0) while decreasing G0 (fucosylated G0).

[0116] In some embodiments, the subject matter disclosed herein is directed to a method of modulating Mn levels in a cell culture medium and / or cell culture, comprising using 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 a glycoprotein of interest in the cell culture medium. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric, humanized or human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell. In some embodiments, glycosylation is modulated to achieve increased G0-F (fucosylation-deficient G0) while decreasing G0 (fucosylated G0).

[0117] In some embodiments, the subject matter disclosed herein is directed to a cell culture composition comprising a cell culture medium comprising 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. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0118] In some embodiments, the subject matter disclosed herein is directed to a cell culture composition comprising a cell culture medium comprising a pH target of about 6.3 to about 7.3 prior to exposure to HTST heat treatment, and a host cell engineered to express a glycoprotein of interest. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric, humanized or human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0119] In some embodiments, the subject matter disclosed herein is directed to a composition comprising a glycoprotein of interest, the preparation comprising a cell culture medium comprising a pH target of about 6.10 to about 7.25 exposed to HTST heat treatment, a host cell engineered to express the glycoprotein of interest, and the glycoprotein of interest. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0120] In certain embodiments, the subject matter disclosed herein is directed to a composition comprising a glycoprotein of interest, wherein the preparation comprises a cell culture medium comprising 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.

[0121] In certain embodiments, the presently disclosed subject matter provides a method of modulating glycosylation of a glycoprotein of interest, 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 no supplementation), and is at or above high or low pCO 2 and / or the cell culture is exposed to extended or shortened medium retention times and / or culture duration, the culture is maintained at a higher or lower culture temperature, maintained at a higher or lower osmolality, and / or the cell culture contains an increased or decreased Na+ concentration and / or any combination thereof, and glycosylation of the glycoprotein of interest is enhanced by a low pCO 2The present invention is directed to a method of producing a glycoprotein of interest in a cell culture medium exposed to a shortened culture medium retention time and / or a reduced Na+ concentration, wherein the glycoprotein of interest is modulated relative to the fucosylation and / or galactosylation of a preparation of the glycoprotein of interest expressed by the host cell in the cell culture medium. In an embodiment, the glycoprotein of interest is an antibody. In an embodiment, the antibody is a chimeric, humanized or human antibody. In an embodiment, the antibody is ocrelizumab. In an embodiment, the host cell is a mammalian cell, e.g., a CHO cell. In an embodiment, glycosylation is modulated to achieve increased G0-F (fucosylation-deficient G0), while increasing G0 (fucosylated G0), while decreasing G0 (fucosylated G0) or reduced G0-F (fucosylation-deficient G0). In an embodiment, glycosylation is modulated to achieve increased or reduced fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0). In certain embodiments, glycosylation is modulated to achieve increased or decreased galactosylation deficiency (eg, G0) without affecting fucosylation deficiency (eg, G0-F).

[0122] In certain embodiments, the presently disclosed subject matter provides a cell culture composition, comprising manganese, galactose and / or fucose supplementation (or no supplementation), high or low pCO 2The present invention is directed to a cell culture composition comprising a cell culture medium and / or cell culture comprising a higher or lower culture temperature, a higher or lower osmolality, and / or an increased or decreased Na+ concentration, and / or any combination thereof, and a host cell engineered to express a glycoprotein of interest. In some embodiments, the cell culture composition further comprises a glycoprotein of interest. In some embodiments, the glycoprotein of interest is an antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is ocrelizumab. In some embodiments, the host cell is a mammalian cell, e.g., a CHO cell.

[0123] In certain embodiments, the subject matter disclosed herein is a composition comprising a glycoprotein of interest, the preparation being administered with or without manganese, galactose and / or fucose supplementation, high or low pCO 2 The present invention is directed to a composition comprising a cell culture medium and / or cell culture comprising a higher or lower culture temperature, a higher or lower osmolality, and / or an increased or decreased Na+ concentration, and / or any combination thereof, a host cell engineered to express a glycoprotein of interest, and a glycoprotein of interest. In one embodiment, the glycoprotein of interest is an antibody. In one embodiment, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the antibody is ocrelizumab. In one embodiment, the host cell is a mammalian cell, e.g., a CHO cell. [Brief description of the drawings]

[0124] [Figure 1]FIG. 1 illustrates that variation in Mn levels at day 0 of production cultures correlates with variation in galactosylation-deficient, %G0 (fucosylated G0, bottom) and fucosylation-deficient, normalized %G0-F (fucosylation-deficient, top) antibody species. [Diagram 2] 2A and 2B illustrate the effect of manganese concentration on day 0 on galactosylation and fucosylation of mAb products during ocrelizumab cell culture. A plot of G0 versus Mn concentration (nM) on day 0 is shown in FIG. 2A. A plot of normalized G0-F versus Mn concentration (nM) on day 0 is shown in FIG. 2B. [Diagram 3] 3A and 3B illustrate the effect of supplemented Mn on galactosylation and fucosylation of mAb products during ocrelizumab cell culture at day 0. A plot of G0 versus supplemented Mn concentration (nM) is shown in FIG. 3A. A plot of G0-F versus supplemented Mn concentration (nM) is shown in FIG. 3B. [Figure 4] Figures 4A and 4B illustrate the effect of Mn concentration on day 0 on galactosylation and fucosylation of mAb products in the ocrelizumab cell culture process at various levels of cell culture scale. A plot of normalized G0-F against Mn concentration (nM) on day 0 is shown in Figure 4A. A plot of G0 against Mn concentration (nM) on day 0 is shown in Figure 4B. The 2L scale-dependent factor represents the use of a high pCO2 environment in the bioreactor. [Diagram 5] Figures 5A and 5B illustrate the effect of supplemented Mn on galactosylation and fucosylation of mAb products in the ocrelizumab cell culture process at various levels of cell culture scale. A plot of normalized G0-F against supplemented Mn concentration (nM) is shown in Figure 5A. A plot of G0 against supplemented Mn concentration (nM) is shown in Figure 5B. The 2L scale-dependent factor represents the use of a high pCO2 environment in the bioreactor. [Figure 6]Figures 6A and 6B illustrate the effect of supplemented Mn on the Antibody I cell culture process. A plot showing that Mn supplementation increases total fucosylation deficiency (G0) is shown in Figure 6A. A plot showing that Mn supplementation reduces galactosylation deficiency (%G0F) is shown in Figure 6B. [Figure 7] Figure 7 illustrates the effect of supplemented Mn on the Antibody II cell culture process. Mn supplementation increases %G0-F (top) and decreases %G0 (bottom). [Figure 8] Figures 8A and 8B illustrate the effect of supplemented Mn on the antibody III cell culture process. A plot showing that Mn supplementation increases %G0-F is shown in Figure 8A. A plot showing that Mn supplementation decreases %G0 is shown in Figure 8B. [Figure 9] Figures 9A and 9B illustrate the effect of supplemented Mn on the antibody IV cell culture process. A plot showing that Mn supplementation increases %G0-F is shown in Figure 9A. A plot showing that Mn supplementation decreases %G0 is shown in Figure 9B. [Figure 10] Figures 10A and 10B illustrate the effect of supplemented Mn on an antibody V cell culture process. A plot showing that Mn supplementation increases %G0-F is shown in Figure 10A. A plot showing that Mn supplementation decreases %G0 is shown in Figure 10B. [Figure 11] FIG. 11 illustrates the effect of Mn addition timing on glycosylation (top: %G0-F and bottom: %G0). [Figure 12] 12A-12B illustrate the effect of Mn addition timing during production culture on glycosylation (FIG. 12A) and normalized G0-F (FIG. 12B). [Figure 13] FIG. 13 illustrates exemplary typical and atypical high temperature short time (HTST) pressure (top) and flow rate profiles (bottom) observed during ocrelizumab HTST thermal treatment. [Figure 14]FIG. 14 illustrates the change in turbidity (left) and Mn loss (right) versus pre-HTST pH adjustment of media during the Sand Bath HTST screen. [Figure 15] Figure 15 illustrates the impact of pre-HTST pH adjustment on 2L cell culture performance. Key performance indicators are shown: % final viability (top), IVPCV (middle) and final titer (bottom). [Figure 16] Figure 16 illustrates the effect of pre-HTST pH adjustment on 2L cell culture performance. Charge-related variants (% mildly protected acidic region (top), % major IE-HPLC (middle) and % basic region (bottom)) are shown. [Figure 17] Figure 17 illustrates the impact of pre-HTST pH adjustment on 2L cell culture performance. Size-related variants (% HMWS (top), main peak SE-HPLC (middle) and % Fab (bottom)) are shown. [Figure 18] Figure 18 illustrates the effect of pre-HTST pH adjustment on glycans from the 2L bioreactor. %G0, %G0-F, %normalized G0-F, %G2+NANA, %Man5, %G1 / G1', %G2 are shown. [Figure 19]Figures 19A-19H illustrate the effect of pH adjustment target on media before HTST heat treatment with antibody III. A schematic showing the design of the experiment is illustrated in Figure 19A. Variability charts of manganese concentration versus media pH target before HTST heat treatment are illustrated in Figure 19B. Variability charts of final viability (top) and IVPCV (bottom) are illustrated in Figure 19C. Variability charts of 13-day (left) and 14-day (right) titers are illustrated in Figure 19D. Variability charts of 13-day (left) and 14-day %G0-F (right) are illustrated in Figure 19E. Variability charts of 13-day (left) and 14-day %G0 (right) are illustrated in Figure 19F. Variability charts of 13-day (left column) and 14-day size variants (right column) are illustrated in Figure 19G, where size-related variants include %HMWS, %Main Peak, and %LMWS. Variability charts for 13 day (left column) and 14 day charge related variants (right column) are illustrated in FIG. 19H, where charge related variants include % lightly protected acidic region, % main peak IE-HPLC and % basic region. [Figure 20] Figures 20A and 20B illustrate schematic diagrams of an exemplary bioreactor. A high partial pressure of carbon dioxide (pCO2) model (top) and a plot showing an aeration strategy to maintain constant dissolved oxygen in the high pCO2 model (bottom) are illustrated in Figure 20A. A low pCO2 model and a plot showing an aeration strategy to maintain constant dissolved oxygen in the low pCO2 model (bottom) are illustrated in Figure 20B. [Figure 21] Figures 21A and 21B illustrate the effect of the pCO2 model, medium retention and Mn supplementation and their combinations on fucosylation loss of mAbs (calculated as normalized G0-F) at time of harvest (12 days) and the pCO2 profiles for the cultures. A plot showing that Mn levels at day 0 are approximately 5-fold higher in Mn-supplemented cultures compared to unsupplemented cultures is shown in Figure 21A. A plot showing the pCO2 profiles for cultures maintained in the low and high pCO2 models is shown in Figure 21B. [Figure 22]Figures 22A-22D illustrate the effect of pCO2 and media retention on CHO cells. A plot showing the loss of fucosylation (calculated as normalized G0-F) of mAb at time of harvest (day 12) with increasing levels of Mn supplementation (at day 0) is shown in Figure 22A. A plot showing the pCO2 profile during cell culture is shown in Figure 22B. A plot showing the osmolality profile during call culture is shown in Figure 22C. A plot showing the Na+ profile during cell culture is shown in Figure 22D. [Diagram 23] Figures 23A-23D illustrate the effect of osmolarity, pCO2 model and type of osmolarity titrant on CHO cells. A plot showing fucosylation loss (calculated as normalized G0-F) of mAb at time of harvest (day 12) is shown in Figure 23A. A plot showing Na+ profile during cell culture is shown in Figure 23B. A plot showing pCO2 profile during cell culture is shown in Figure 23C. A plot showing osmolarity profile during call culture is shown in Figure 23D. [Figure 24] Figures 24A and 24B illustrate the effect of pCO2 and osmolality on intracellular pH (pHi) measured in CHO cells. Plots showing different pCO2 levels while maintaining similar osmolality (406-413 mOsm / kg) and Na+ (83-87 mM) levels are shown in Figure 24A. Plots showing different osmolality levels (using NaCl; 46-152 mM Na+ as the osmolality titrant) while maintaining similar pCO2 levels (23-28 mmHg) are shown in Figure 24B. [Diagram 25] Figures 25A and 25B illustrate the effect of different culture conditions and culture duration on fucosylation loss (calculated as normalized G0-F) of mAbs produced in 3-L bioreactors. A chart showing the differences in culture conditions is illustrated in Figure 25A. A plot showing fucosylation loss levels at 7 days and at harvest (12 days) is illustrated in Figure 25B. [Figure 26]Figures 26A-26D illustrate the global proteome analysis. A schematic showing the experimental design and work flow is shown in Figure 26A. Plots showing principal component analysis (PCA) separated samples by day (PC1) and cell culture treatment (PC2) are shown in Figure 26B. Ingenuity pathway analysis (IPA) of the canonical pathways for all cases is shown in Figure 26C. Expression of glycolytic enzymes for each treatment and day compared to case is shown in Figure 26D. [Figure 27] Figures 27A-27C illustrate the results evaluating the possibility that GDP-fucose is affected in cell culture conditions that resulted in higher mAb fucosylation deficiency. The de novo and salvage pathways for the synthesis of GDP-fucose are illustrated in Figure 27A. A heat map of key enzymes in the GDP-fucose synthesis pathway is illustrated in Figure 27B. A plot showing the effect of L-fucose supplementation (day 0) on fucosylation deficiency levels (calculated as normalized G0-F) at the time of harvest (day 12) is illustrated in Figure 27C. [Figure 28] Figures 28A and 28B illustrate proteomic analysis to determine differential expression of key proteins in the glycosylation pathway under different culture conditions in 3-L bioreactors. A description of the four cases (i-iv) tested in 3-L bioreactors and the resulting fucosylation deficiency levels are provided in Figure 25A. A diagram of the glycosylation pathway in the Golgi showing only the glycosylation variant (Man5-G2) associated with fucosylation deficiency is illustrated in Figure 28A. 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 7 and 12 days of production culture are illustrated in Figure 28B. [Figure 29]Figures 29A-29E illustrate the performance of recombinant CHO cells cultured in 3-L bioreactors using high and low pCO2 models. Growth as represented by packed cell volume (PCV) is shown in Figure 29A. A plot representing recombinant CHO cell viability is shown in Figure 29B. A plot representing mAb titer is shown in Figure 29C. A plot representing charge variants (12 days) is shown in Figure 29D. A plot representing size variants (12 days) is shown in Figure 29E. HWMS represents high molecular weight species and LWMS represents low molecular weight species. [Diagram 30] Figure 30 illustrates the effect of the pCO2 model, media retention and Mn supplementation on G0 of mAbs at the time of harvest (day 12). Plots show the effect of each factor alone and in combination with the other factors on G0. [Diagram 31] Figure 31 illustrates the effect of L-fucose addition (day 0) and manganese addition (day 0) on G0 of mAb produced in a 3-L bioreactor at the time of harvest (day 12). The plot shows the effect on G0 of fucose and manganese supplementation alone and their combined effect. [Diagram 32] FIG. 32 illustrates the variation in manganese content in PP3 and GEM. [Diagram 33] Figures 33A-33D illustrate the effect of media retention, Mn supplementation, and combinations thereof on G0 and G0-F. Figures 33A-33B illustrate the effect of media retention time at elevated temperature (38°C) on galactosylation-deficient, G0 (Figure 34A) and fucosylation-deficient, normalized G0-F (Figure 33B). Figure 33C illustrates the cumulative effect of media retention on fucosylation-deficient (%G0-F) on antibody III. Figure 33D illustrates the effect of media retention, Mn supplementation, and combinations thereof on fucosylation-deficient (%G0-F) on antibody III. [Diagram 34] 34A-34B illustrate the effect of galactose and supplemented Mn and their interactions on galactosylation-deficient, G0 (FIG. 34A) and fucosylation-deficient, normalized G0-F (FIG. 34B) from study 1. [Diagram 35] 35A-35B illustrate the effects of galactose and Mn and their interactions on galactosylation-deficient, G0 (FIG. 35A) and fucosylation-deficient, normalized G0-F (FIG. 35B) from study 2. [Diagram 36] 36A-36B illustrate the effect of galactose on galactosylation-deficient, G0 (FIG. 36A) and fucosylation-deficient, normalized G0-F (FIG. 36B) from study 3. [Figure 37] Figures 37A-37B illustrate the effect of fucose supplementation on fucosylation-deficient, G0-F (Figure 37A) and galactosylation-deficient, G0 (Figure 37B). [Figure 38] Figures 38A-38B illustrate the effect of timing of fucose addition on fucosylation-deficient, G0-F (Figure 38A) and galactosylation-deficient, G0 (Figure 38B). [Figure 39] 39A-39B illustrate the effect of fucose concentration and temperature and their interactions on fucosylation-deficient, G0-F (FIG. 39A) and galactosylation-deficient, G0 (FIG. 39B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0125] The subject matter disclosed herein relates to modulating the glycosylation (e.g., galactosylation and / or fucosylation) of a recombinant glycoprotein of interest, e.g., a mAb, such that the recombinant glycoprotein of interest falls within a desired quality attribute range. For example, and not intended to be limiting, the subject matter disclosed herein is applicable to modifying the glycosylation profile of a mAb to fall within a narrower band of quality attribute ranges than is achieved using conventional cell culture media, media preparation strategies, and / or cell culture strategies. Methods by which glycosylation can be modulated according to the present disclosure include, but are not limited to, (1) establishing a lowered pH set point for media pH adjustment, e.g., prior to Mn concentration analysis of raw materials, adjusting manganese (Mn) concentration of cell culture media for Mn supplementation during cell culture, and / or high temperature, short time (HTST) heat treatment of the media; and (2) adjusting process parameters, e.g., pCO2, during cell culture. 2 , medium retention time and osmolality / Na + The presently disclosed subject matter is also directed to cell cultures and glycoprotein compositions prepared when such process parameters are adjusted as described herein.

[0126] For clarity of disclosure, and not intended to be limiting, the detailed description is divided into the following subsections.

[0127] 1.Definition 2. Adjusting the raw material to control glycosylation 3. Manganese supplementation to regulate glycosylation 4. Modified pH target of the medium prior to high temperature, short time (HTST) treatment to regulate glycosylation by minimizing manganese loss during HTST. 5. pCO to regulate glycosylation 2 , manganese, medium retention, culture period, culture temperature and osmolality / Na + 6. Galactose to regulate glycosylation 7. Fucose and incubation temperature for regulating glycosylation and combinations thereof 8. Cell Culture and Glycoprotein Composition

[0128] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Certain methods and materials are described below, In carrying out or testing the subject matter disclosed herein, methods and materials similar or equivalent to those described herein can be used.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0129] As used herein, the terms "comprise(s) / include(s)," "having / has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or constructs. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0130] For the indication of numerical ranges herein, each intervening number with the same precision is expressly assumed. For example, for the range 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are assumed, and for the range 6.0 to 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 assumed.

[0131] As used herein, the term "about" or "approximately" means within an acceptable error range of a value determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limits of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, as practiced in the art. Alternatively, "about" can mean within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0132] The term "replenishment" is used in the broadest sense to encompass a variety of types, techniques, or methods for adding target molecules, materials, objects, or combinations thereof. Instantaneous, fully continuous, semi-continuous, intermittent, time-based, and feedback loop-based additions are examples of supplementation.

[0133] As used herein, the term "modulate" is used to denote an increase or decrease in a respective attribute.

[0134] The term "antibody" is used herein in the broadest sense and encompasses a variety of 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.

[0135] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen that the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 linear antibodies; single-chain antibody molecules (eg, scFv); and multispecific antibodies formed from antibody fragments.

[0136] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (V H and L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) To confer antigen-binding specificity, a single V H or V L In addition, an antibody that binds to a specific antigen may have a V domain that is sufficient to H or V L Using the domains, respectively, the complementary V L or V H Domains may also be isolated by screening libraries (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0137] The term "homologous sequence" as used herein refers to sequences that share significant sequence similarity when 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 compare sequences and calculate the statistical significance of matches based on factors such as sequence length, sequence identity and similarity, and the presence and length of sequence mismatches and gaps.Homologous sequences can refer to both DNA and protein sequences.

[0138] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.) and other modifications known in the art are also included in this definition. The terms "polypeptide" and "protein" as used herein specifically encompass antibodies.

[0139] The term "glycoprotein" refers to a polypeptide or protein bound to at least one carbohydrate moiety, e.g., a polysaccharide or oligosaccharide, that is attached to the protein via an oxygen- or nitrogen-containing side chain of an amino acid residue, e.g., a serine or threonine residue ("O-linked") or an asparagine residue ("N-linked"). The term "glycan" refers to a polysaccharide or oligosaccharide, e.g., a polymer composed of monosaccharides. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched.

[0140] As used herein, "glycosylation pattern" and "glycosylation profile" of a recombinant glycoprotein of interest refer to various physical characteristics of the poly- or oligosaccharides of the glycoprotein, such as, for example, the amount and quality of the various monosaccharides present, the degree of branching and / or attachment (e.g., N-linked or O-linked).

[0141] "Fucosylation" refers to the degree and distribution of fucose residues on polysaccharides and oligosaccharides, such as N-glycans, O-glycans and glycolipids. "Afucosylation-deficient" refers to the absence of fucose residues on polysaccharides and oligosaccharides. G0 glycans refer to glycans that lack terminal galactose residues. The art has identified two different nomenclatures for identifying fucosylated / fucosylated-deficient G0 glycans. (1) "G0-F" (i.e., "G0 minus" F) is used to represent fucosylated G0 glycans and "G0" is used to represent fucosylated G0 glycans; and

[0142] (2) "G0" is used to represent fucosylation-deficient G0 glycans, and "G0F" is used to represent fucosylated G0 glycans.

[0143] Identifying which convention is used in a particular context involves analyzing the use of G0 and the use of either G0-F or G0F. Therapeutic glycoproteins, such as antibodies or Fc fusion proteins, with non-fucosylated or "fucosylated-deficient" N-glycans exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) due to enhanced FcγRIIIa binding ability without detectable changes in complement-dependent cytotoxicity (CDC) or antigen binding ability. In some situations, such as cancer treatment, non-fucosylated or "fucosylated-deficient" antibodies are desirable because they can achieve therapeutic efficacy at low doses while inducing high cytotoxicity against tumor cells and triggering high effector function in NK cells through enhanced interaction with FcγRIIIa. In other situations, 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. The term "% fucose" or "% fucosylation deficiency" as used herein refers to the percentage of non-fucosylated N-glycans present on the recombinant glycoprotein of interest. Higher % fucose deficiency or % fucosylation deficiency represents a higher number of non-fucosylated N-glycans, and lower % fucose deficiency or % fucosylation deficiency represents a higher number of fucosylated N-glycans. Fucosylation deficiency is sometimes expressed as % normalized G0-F and is calculated by: TIFF2025037936000002.tif16170

[0144] The term "galactosylation" as used in the context of the present invention refers to the addition of galactose units to the oligosaccharide chains on a glycoprotein. The term "galactosylation-deficient" refers to the absence of galactose units on the oligosaccharide chains on a glycoprotein. The term "galactosylated" antibody as used herein refers to an antibody whose N-linked glycan contains at least one galactose residue (e.g., G1 and G2 glycans). The term "galactosylation-deficient" antibody as used herein refers to an antibody whose N-linked glycan lacks galactose residues (e.g., G0 and G0F glycans).

[0145] The term "expression" as used herein refers to transcription and / or translation. In some embodiments, the level of transcription of a desired product can be determined based on the amount of corresponding mRNA present. For example, the mRNA transcribed from a sequence of interest can be quantified by PCR or by Northern hybridization. In some embodiments, the protein encoded by a sequence of interest can be quantified by various methods, for example, by ELISA, by assaying the biological activity of the protein, or by utilizing an assay independent of such activity, such as Western blotting or radioimmunoassay, that uses an antibody that recognizes and binds to the protein.

[0146] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which it is introduced. In certain embodiments, vectors direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0147] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII, and FcR expression on hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0148] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, for example as described in Gazzano-Santoro et al., J.Immunol.Methods.202:163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding capacity are described, for example, in U.S. Pat. No. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al. J.Immunol.164:4178-4184 (2000).

[0149] "Culturing" cells refers to contacting the cells with cell culture medium under conditions suitable for cell survival and / or growth and / or proliferation. Cell culture can be performed under a variety of conditions, including but not limited to batch, fed-batch, continuous, and perfusion processes. Cell culture duration can vary depending on the process. For example, and not intended to be limiting, fed-batch processes can be performed for fewer days, e.g., 0-20 days, whereas a typical perfusion process can be performed for up to 150 days or longer.

[0150] By "batch culture" is meant a culture in which all components for cell culture, including cells and all culture nutrients, are supplied to the culture vessel at the beginning of the culture process.

[0151] As used herein, the term "fed-batch cell culture" refers to a batch culture in which cells and culture medium are initially fed to a culture vessel, additional culture nutrients are fed to the culture continuously or in discrete increments during the culture process, and cells and / or product are or are not periodically harvested prior to the end of the culture.

[0152] The term "bioreactor agitation strategy" refers to the agitation rate and / or physical manipulation of the culture and / or culture medium in a bioreactor.

[0153] The term "bioreactor medium exchange strategy" refers to any process in which a change of the medium in contact with the cells of a bioreactor and / or culture occurs, including, but not limited to, a process in which cells are spun down from a cell culture sample taken from a bioreactor and resuspended in new medium that may differ from the original cell culture medium used to initially grow the cells.

[0154] A "perfusion culture" is a culture in which cells are maintained in culture, e.g., by filtration, encapsulation, attachment to microcarriers, etc., and culture medium is continuously or intermittently introduced and removed from the culture vessel.

[0155] "Cultivation vessel", "culture vessel" and "bioreactor" refer to a vessel used to culture cells. Cultivation vessels can be of any size as long as they are useful for culturing cells. In certain embodiments, a bioreactor for use in the methods disclosed herein is a stainless steel vessel. In certain embodiments, a bioreactor for use in the methods disclosed herein is a rocker bag. In certain embodiments, a bioreactor for use in the methods disclosed herein is a disposable bioreactor.

[0156] The terms "culture medium" and "cell culture medium" refer to a nutrient source used to grow or maintain cells. As will be understood by those skilled in the art, a nutrient source may contain components required by cells for growth and / or survival or may contain components that support cell growth and / or survival. Cell culture medium also refers to any liquid supernatant for growing or maintaining cells. Media components refer to any components that can be added to a cell culture or cell culture medium at any culture stage, at any time, or in any form. Media components also refer to components obtained from raw materials for cell culture media. Vitamins, essential or non-essential amino acids, and trace elements are examples of media components. It should be understood that throughout this specification "medium" and "media" are used interchangeably.

[0157] A "chemically defined cell culture medium" or "CDM" is a medium having a specified composition that is free of animal-derived or undefined products, such as animal serum and peptones. As will be appreciated by those skilled in the art, CDM can be used in the process of polypeptide production, where cells are contacted with the CDM and secrete the polypeptide into the CDM. Thus, it is understood that a composition can contain CDM and a polypeptide product, and that the presence of a polypeptide product does not make the CDM chemically undefined.

[0158] "Chemically undefined cell culture medium" refers to a medium whose chemical composition cannot be specified and may contain one or more animal-derived or undefined products, such as animal serum and peptones. As will be understood by those skilled in the art, chemically undefined cell culture media may contain animal-derived products as a nutrient source.

[0159] "Medium retention" refers to the practice of cell culture whereby cell culture medium is retained in a culture vessel (e.g., bioreactor, disposable bag) or a vessel used for medium preparation or medium storage (e.g., stainless steel tank, disposable vessel) prior to use in culturing cells. In cell culture operations, the culture medium can be warmed and kept at or near the culture temperature prior to using the medium to seed cells in a bioreactor. "Medium retention period" or "medium retention time" refers to the extent of time that the medium is held (e.g., at a temperature above ambient) prior to using the medium to seed cells in a bioreactor. It is understood that "medium retention", "medium retention period" and "medium retention time" are used interchangeably throughout this specification.

[0160] "HTST" stands for "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 can form, HTST equipment can become fouled, and media components can fall out of solution. Adjustments to certain culture media parameters can be made to reduce or inhibit the formation of precipitates in the media from the HTST treatment.

[0161] As used herein, "low pCO 2 The term describes operation in a relatively narrow carbon dioxide range, with the upper limit of CO2 being "high pCO 2 " Lower than the upper limit of CO2 used in operation. Low pCO 2can be about 10 mmHg to about 100 mmHg, about 10 mmHg 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 broader carbon dioxide range, with the upper limit of pCO2 being lower than the lower pCO2. 2 pCO used in operation 2 Higher than the upper limit of pCO 2 The pCO2 concentration as described herein may be about 20 to about 250 mmHg, about 20 mmHg to about 200 mmHg, about 20 mmHg to about 150 mmHg, or about 30 mmHg to about 150 mmHg. 2 The adjustment can occur for at least the first half of the cell culture period. For example, and not intended to be limiting, for a 20 day culture, pCO 2 Adjustments can occur over at least the first 10 days. Depending on the duration of cell culture, pCO 2 Regulation may also vary as a result.

[0162] 2. Adjusting the raw material to control glycosylation It is known that multiple cell culture factors have the potential to affect glycosylation of glycoproteins, e.g., mAbs. These factors include process parameters and media components, such as galactose and trace metals, among others. Variations in the levels of individual media components can be introduced during the mAb cell culture process through the use of complex raw materials. For example, but not intended to be limiting, cell culture media, e.g., basal or feed media (and their respective components, e.g., hydrolysates or various types of serum), can exhibit lot-to-lot variations that can affect mAb glycosylation. Thus, in certain embodiments, the present disclosure is directed to compositions and methods aimed at reducing the variability of cell culture media to modulate the glycosylation (e.g., galactosylation and / or fucosylation) of mAbs. For example, but not intended to be limiting, Mn supplementation can be achieved by using media components that contain Mn as an impurity or raw materials (e.g., depth filters, stainless steel or glass vessels) that can release Mn into the cell culture media or cell culture.

[0163] In certain embodiments, the present disclosure is directed to strategies for screening cell culture media and / or individual components thereof to modulate mAb glycosylation (e.g., galactosylation and / or fucosylation). In non-limiting embodiments, cell culture media can be screened for specific target amounts of individual components, e.g., Mn concentration, galactose concentration. For example, and not intended to be limiting, cell culture media can be screened and selected based on Mn concentration target ranges 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 (media outside such target ranges are not utilized for cell culture of mAbs). In certain embodiments, cell culture media can be further supplemented with 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) of galactose. In certain embodiments, the cell culture medium can be supplemented with up to about 6 g / L of galactose.

[0164] Certain embodiments described herein relate to modulating glycosylation (e.g., fucosylation deficiency and / or galactosylation) by screening cell culture media and / or cell cultures based on the disclosed Mn concentration target ranges to achieve or maintain a desired glycoprotein glycosylation pattern. In certain non-limiting embodiments, the desired glycoprotein glycosylation pattern can be modulation of fucosylation deficiency and / or galactosylation of glycoproteins. For example, and not by way of limitation, the target range of fucosylation deficiency G0 (%G0-F or %normalized G0-F) can be about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%. The %G0-F or %normalizedG0-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 glycoprotein glycosylation pattern can be a decrease or increase in fucosylation of the glycoprotein. For example, and not by way of limitation, the target range of fucosylated galactosylation-deficient G0 (%G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85% or about 60% to about 80%. The %G0 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%.

[0165] In certain embodiments, the desired glycoprotein glycosylation pattern can be a combination of fucosylation deficiency and galactosylation modulation of the glycoprotein. For example, and not by way of limitation, the target range of %G0-F or %normalizedG0-F can be 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 can be about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%. The %G0-F or %normalizedG0-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% and the %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0166] 3. Manganese supplementation to regulate glycosylation As described above, the Mn concentration of the cell culture medium can affect glycosylation, e.g., mAb galactosylation and / or fucosylation. As also described herein, the Mn concentration of the cell culture can affect glycosylation, e.g., mAb galactosylation and / or fucosylation. Thus, in certain embodiments, mAb glycosylation can be regulated not only by adjusting 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, increasing the Mn concentration can increase fucosylation deficiency (by increasing the level of G0-F, a fucosylation-deficient form of G0) and / or increase galactosylation (resulting in a reduction in G0, galactosylation-deficient and fucosylated glycan species).

[0167] As outlined in Figures 5-10, increasing Mn concentration in cell culture medium and / or cell culture can generally result in increased G0-F (fucosylation-deficient G0) and decreased G0 (fucosylated G0), however, identifying trends across six different mAbs, the extent to which fucosylation-deficient (and thus G0-F species) are increased and galactosylation-deficient (and thus G0 species) are decreased, can be refined by establishing specific Mn supplementation action 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 fall outside the desired product quality specifications. In certain embodiments, Mn is supplemented to achieve a selected range in 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 within the disclosed ranges). In certain embodiments, the concentration of Mn supplementation can be about 20 nM to about 300 nM. In non-limiting embodiments, the concentration of Mn supplementation can be about 30 nM to about 110 nM. Mn supplementation can be used in combination with high CO 2 In certain embodiments, including those occurring in cell culture medium exposed to Mn, 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, such concentrations have the unexpected ability to increase fucosylation deficiencies (and thus G0-F glycans) and decrease galactosylation deficiencies (and thus G0 glycans) while not causing the resulting mAb to fall outside the desired product quality specifications.

[0168] In certain embodiments, the timing of Mn supplementation of the culture can also affect glycosylation (e.g., galactosylation and fucosylation deficiencies). Mn supplementation can be added during the pre-production expansion culture stage and / or during the production culture stage. In certain embodiments, Mn supplementation can result from leaching of Mn from the cell culture medium and / or materials in contact with the cell culture (e.g., depth or media filters, culture vessels, media holding vessels). In a non-limiting embodiment, Mn supplementation can be achieved by using a depth filter containing diatomaceous earth, which leaches Mn and other trace metals during the filtering media preparation process, thereby supplementing the culture.

[0169] In certain embodiments, mAbs can be harvested after Mn supplementation. For example, and not by way of limitation, mAbs can be harvested at about day 2 to about day 25 of culture (e.g., about day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of cell culture). In non-limiting embodiments, mAbs can be harvested at about day 7 to about day 15 of cell culture. In some embodiments, mAbs can be harvested at about day 5 to about day 20 of cell culture).

[0170] In certain embodiments, the media compositions and cell culture processes disclosed herein may be modified with or without the addition of the following: fucose, ammonia, sodium, uridine, N-acetylglucosamine, N-acetylgalactosamine, cadmium, lipoic acid, V 2+ , Cr 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Ca 2+ , Mg 2+ and kifunensine.

[0171] 4. Modified pH target of the medium before high temperature short time (HTST) treatment to regulate glycosylation As disclosed herein, cell culture medium concentration of Mn can modulate glycosylation of glycoproteins, e.g., mAbs. Thus, the present disclosure, in certain embodiments, is directed to a method of modulating cell culture medium Mn concentration to modulate glycosylation, e.g., galactosylation and / or fucosylation of mAbs. The present disclosure, in certain embodiments, is also directed to a method of modulating cell culture Mn concentration to modulate glycosylation, e.g., galactosylation and / or fucosylation of mAbs. For example, and not by way of limitation, the present disclosure notes that performing HTST treatment of a medium with a pre-HTST medium pH adjustment target of greater than about 7.0 can result in a decrease in cell culture medium Mn concentration after HTST treatment. Thus, in certain embodiments, the present disclosure is directed to performing HTST using a medium adjusted to a pH target of less than about 7.25 (e.g., about 6.1 to about 7.2). In certain embodiments, the present disclosure is directed to performing HTST using a medium prepared to a pH target of less than about 7.3 (e.g., about 6.1 to about 7.3). In certain embodiments, the pH target for a medium prepared for HTST 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.

[0172] 5. pCO2, manganese, medium retention, culture duration, culture temperature and Na+ / osmolality to regulate glycosylation As disclosed herein, the pCO 2 Adjusting the medium holding time, culture time, culture temperature, manganese concentration, osmolality / Na+ concentration and / or combinations thereof can result in modulation of fucosylated and / or fucosyl-deficient G0 glycans of glycoproteins, e.g., mAbs, cultured in such media. In certain embodiments, the present disclosure provides a method for the preparation of pCO 2The present invention is directed to a method of cell culture that utilizes a medium or cell culture in which the medium, manganese concentration, medium retention time, culture period, culture temperature, Na+ concentration, osmolality or combinations thereof have been adjusted as outlined herein.

[0173] 5.1 Media retention As described herein, medium retention time at a particular temperature or temperature range can affect glycosylation (e.g., galactosylation and / or fucosylation deficiency). In certain embodiments, the elevated medium retention temperature can be 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. In certain embodiments, medium retention time at a particular 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 maintained at a temperature of about 25°C to about 39°C for a period of about 0 hours to about 72 hours to modulate glycosylation (e.g., fucosylation deficiency and / or galactosylation). In certain embodiments, the cell culture medium so maintained is utilized in the production culture, the growth culture, or both.

[0174] Certain embodiments described herein relate to modulating glycosylation (e.g., fucosylation deficiency and / or galactosylation) by applying the disclosed medium retention times at specific temperatures or temperature ranges to achieve or maintain a desired glycoprotein glycosylation pattern. In certain embodiments, the desired glycoprotein glycosylation pattern can be modulation of fucosylation deficiency of glycoproteins. For example, and not by way of limitation, the target range of fucosylation deficiency G0 (%G0-F or %normalized G0-F) can be 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 %normalizedG0-F can be modulated 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 modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range of galactosylation deficiency (e.g., %G0) can be about 40% to about 90%, about 50% to about 90%, about 55% to about 85% or about 60% to about 80%. In certain embodiments, %G0 can be modulated 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 fucosylation deficiency and modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range for %G0-F or %normalizedG0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 55% to about 85%, or about 60% to about 80%.In certain embodiments, %G0-F or %normalizedG0-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%, and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0175] 5.2 Partial pressure of carbon dioxide (pCO 2 ) In certain embodiments, the present disclosure provides a method for controlling the partial pressure of carbon dioxide (pCO ) in cell culture to modulate mAb glycosylation (e.g., galactosylation and / or fucosylation). 2 In a non-limiting embodiment, the present invention is directed to a strategy for adjusting pCO 2 The level of pCO2 can be between 0mmHg and 250mmHg. 2 The model was designed to maintain a pCO2 concentration of approximately 0 to 250 mmHg, approximately 20 to 250 mmHg, approximately 20 to 200 mmHg, approximately 20 to 150 mmHg, or approximately 30 to 150 mmHg for the majority of the incubation period, beginning on day 0. 2 It can have a range of low pCO 2 The model was designed to maintain a pCO2 concentration of approximately 10mmHg to approximately 100mmHg, approximately 10mmHg to approximately 80mmHg, approximately 10mmHg to approximately 70mmHg, or approximately 10mmHg to approximately 60mmHg for the majority of the culture period, beginning on day 0. 2 Certain embodiments described herein may have a pCO2 concentration within a target range to achieve or maintain a desired glycoprotein glycosylation pattern. 2The present invention relates to modulating glycosylation by adjusting the level of . For example, in certain embodiments, the desired glycoprotein glycosylation pattern can be modulation of fucosylation deficiency of glycoproteins. In certain embodiments, the target range of fucosylation deficiency G0 (%G0-F or %normalized G0-F) can be 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 modulated 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 modulation of galactosylation of glycoproteins. For example, and not by way of limitation, the target range of galactosylation deficiency (%G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, %G0 can be modulated 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 fucosylation deficiency and modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range for %G0-F or %normalizedG0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%.In certain embodiments, %G0-F or %normalizedG0-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%, and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0176] 5.3 Sodium (Na+) concentration In certain embodiments, the present disclosure is directed to strategies for adjusting the concentration of sodium (Na+) in cell culture medium and / or cell cultures to modulate mAb glycosylation (e.g., galactosylation and / or fucosylation). For example, and not by way of limitation, cell culture medium may be supplemented with sodium (Na+) to achieve a Na+ concentration target range 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. 2 CO 3 , NaHCO 3 , NaCl, NaOH and / or Na+ compounds (e.g., for pH adjustment), or combinations thereof.

[0177] Certain embodiments described herein relate to modulating glycosylation by adjusting the Na+ concentration in cell culture medium and / or cell culture to a specified target range to achieve or maintain a desired glycoprotein glycosylation pattern. In certain embodiments, the desired glycoprotein glycosylation pattern can be modulation of fucosylation deficiency of glycoproteins. For example, and not by way of limitation, the target range of fucosylation deficiency (%G0-F or %normalizedG0-F) can be about 0% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 8%. The %G0-F or %normalizedG0-F can be adjusted to 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 some embodiments, the desired glycoprotein glycosylation pattern can be the modulation of galactosylation of glycoproteins. For example, and not by way of limitation, the target range of galactosylation deficiency (%G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. %G0 can be modulated 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 some embodiments, the desired glycoprotein glycosylation pattern can be a combination of fucosylation deficiency and modulation of galactosylation of glycoproteins. For example, and not by way of limitation, the target range for %G0-F or %normalized G0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 50% to about 85% or about 60%.In certain embodiments, %G0-F or %normalizedG0-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%, and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0178] 5.4 Osmolarity In certain embodiments, the present disclosure is directed to strategies for adjusting the osmolality of culture media to modulate mAb glycosylation (e.g., galactosylation and / or fucosylation). For example, and not by way of limitation, the osmolality of cell culture media can be adjusted by adding sorbitol, KCl, osmoprotectants (e.g., betaine) and / or NaCl to achieve an osmolality 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.

[0179] Certain embodiments described herein relate to modulating glycosylation by adjusting the osmolality level of the culture medium and / or cell culture to a target range to achieve or maintain a desired glycoprotein glycosylation pattern. In certain embodiments, the desired glycoprotein glycosylation pattern can be modulation of fucosylation deficiency of glycoproteins. For example, and not by way of limitation, the target range of fucosylation deficiency (%G0-F or %normalizedG0-F) can be 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 %normalizedG0-F can be adjusted to 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 some embodiments, the desired glycoprotein glycosylation pattern can be the modulation of galactosylation of glycoproteins. For example, and not by way of limitation, the target range of galactosylation deficiency (%G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In some embodiments, %G0 can be modulated 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 some embodiments, the desired glycoprotein glycosylation pattern can be a combination of fucosylation deficiency and modulation of galactosylation of glycoproteins. For example, and not by way of limitation, the target range for %G0-F or %normalizedG0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%.In certain embodiments, %G0-F or %normalizedG0-F or %normalizedG0-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% and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0180] 5.5 Manganese supplementation As described above in sections 2 and 3, the Mn concentration 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 is It can be adjusted by adjusting the amount of Mn present in the cell culture medium raw material, as described above, as well as by supplementing the cell culture medium and / or cell culture with Mn, to achieve a particular Mn concentration target or target range as outlined in this section, including in combination with one or more other parameters. In certain embodiments, the concentration of Mn supplement is selected to achieve a final target concentration or concentration range of less than 10 μM (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 μM, including concentrations falling within the disclosed ranges).

[0181] Certain embodiments described herein relate to modulating glycosylation by supplementing cell culture medium and / or cell culture with the disclosed concentrations of Mn to achieve or maintain a desired glycoprotein glycosylation pattern. In a non-limiting embodiment, the desired glycoprotein glycosylation pattern can be modulation of fucosylation deficiency of glycoproteins. For example, and not by way of limitation, the target range of fucosylation deficiency G0 (%G0-F or %normalized G0-F) can be 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 modulated 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 some embodiments, the desired glycoprotein glycosylation pattern can be modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range of galactosylation deficiency (%G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In some embodiments, %G0 can be modulated 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%.

[0182] In certain embodiments, the desired glycoprotein glycosylation pattern achieved by Mn supplementation can be a combination of modulation of glycoprotein fucosylation deficiency and modulation of fucosylation. For example, and not by way of limitation, the target range of %G0-F or %normalizedG0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, %G0-F or %normalizedG0-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%, and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0183] 5.6 pCO 2 , medium holding, culture period, supplemented Mn, osmolality and Na+ concentration combinations In certain embodiments, a combination of the disclosed techniques for modulating glycosylation (e.g., fucosylation deficiency and / or galactosylation) of a glycoprotein can be used. For example, and not by way of limitation, pCO2 can be used to achieve or maintain a desired glycoprotein glycosylation pattern. 2 Combinations of the disclosed conditions of medium maintenance, culture duration, supplemented Mn, osmolality and / or Na+ concentration can be utilized in cell culture medium and / or cell cultures. In non-limiting embodiments, supplemented Mn (e.g., about 1 nM to about 30,000 nM), pCO 2The disclosed medium retention times (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.) in combination with the culture level (e.g., about 0 mmHg to about 250 mmHg), culture period (e.g., about 0 days to about 25 days), Na concentration (e.g., about 0 mM to 250 mM) and osmolality (e.g., about 250 mOsm / kg to about 600 mOsm / kg) can be applied to the culture medium.

[0184] In one embodiment, the pCO 2 The disclosed combinations of conditions of medium maintenance, culture duration, supplemented Mn, osmolality and Na+ concentration can induce combinations or synergistic effects on the fucosylation loss and / or galactosylation profile of glycoproteins. For example, but not by way of limitation, pCO 2 When the disclosed combinations of conditions of medium maintenance, culture duration, supplemented Mn, osmolality and Na+ concentration are utilized, synergistic modulation (e.g., increase or decrease) of %G0-F of glycoproteins can occur. Additionally, in non-limiting embodiments, pCO 2 When the disclosed combinations of conditions of medium maintenance, culture duration, supplemented Mn, osmolality and Na+ concentration are utilized, synergistic modulation (e.g., increase or decrease) of %GO of glycoproteins can occur. Additionally, in certain embodiments, pCO 2 When the disclosed combinations of conditions of medium retention, culture duration, supplemented Mn, osmolality and Na+ concentration are utilized, synergistic regulation of %G0-F can occur.

[0185] Certain embodiments described herein provide for the administration of disclosed conditions (e.g., pCO 2The present invention relates to modulating glycosylation by modifying a combination of factors such as: concentration of fucosylation, medium retention, culture duration, supplemented Mn, osmolality, and Na+ concentration. In certain non-limiting embodiments, the desired glycoprotein glycosylation pattern can be an increase or decrease in fucosylation deficiency of the glycoprotein. For example, and not by way of limitation, the target range of fucosylation deficiency G0 (%G0-F or %normalized G0-F) can be 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 to 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 some embodiments, the desired glycoprotein glycosylation pattern can be the modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range of galactosylation (e.g., %G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In some embodiments, %G0 can be modulated 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 some embodiments, the desired glycoprotein glycosylation pattern can be a combination of fucosylation deficiency and modulation of galactosylation of the glycoprotein. In certain embodiments, the target range for %G0-F or %normalized G0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 50% to about 85% or about 60% to about 80%.In certain embodiments, %G0-F or %normalizedG0-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%, and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0186] 6. Galactose to regulate glycosylation 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 fucosylation deficiency). Thus, mAb glycosylation can be modulated by supplementing galactose, Mn, or a combination thereof. For example, and not by way of limitation, the concentration of galactose can be about 10 g / L total (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 about 100 mM total. For example, and not by way of limitation, the concentration of galactose can be 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. The 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 μM. Target concentration ranges for galactose and Mn can include concentrations that fall within the ranges described. Non-limiting examples of galactose and Mn addition can include addition to the production culture and / or growth culture up to the production culture stage.

[0187] Certain embodiments described herein relate to modulating glycosylation by supplementing the culture medium with the disclosed concentrations of galactose, with / without the disclosed concentrations of Mn, to achieve or maintain a desired glycoprotein glycosylation pattern. In non-limiting embodiments, the desired glycoprotein glycosylation pattern can be modulation of fucosylation deficiency of glycoproteins. For example, and not by way of limitation, the target range of fucosylation deficiency G0 (%G0-F or %normalized G0-F) can be 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 %normalizedG0-F can be modulated 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 modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range of galactosylation deficiency (%G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85% or about 60% to about 80%. In certain embodiments, %G0 can be modulated 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 fucosylation deficiency and modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range for %G0-F or %normalizedG0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%.In certain embodiments, %G0-F or %normalizedG0-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%, and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0188] 7. Fucose and incubation temperature for regulating glycosylation and combinations thereof In certain embodiments, the addition of L-fucose (fucose) to cell culture medium and / or cell cultures can also affect glycosylation (e.g., galactosylation and fucosylation deficiencies). Thus, in certain embodiments, mAb glycosylation can be modulated by supplementing the cell culture medium with fucose. The addition of fucose results in modulation of fucosylation deficiencies (e.g., G0-F), and the degree to which the fucosylation deficiency is modulated can be refined by the fucose concentration and / or timing of fucose addition. In certain non-limiting embodiments, the concentration of fucose added can be about 0 g / L to 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 can be about 100 mM total. For example, and not by way of limitation, the concentration of fucose can be 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, the timing of fucose addition can be from about 0 days after inoculation of the production culture to 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) with different fucose concentrations (e.g., about 0.1 g / L, about 0.5 g / L, including concentrations falling within the disclosed ranges). In certain embodiments, fucose addition at a level in the range of about 0 g / L to about 1 g / L can be performed 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 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. In one embodiment, fucose supplementation at a level of about 0 g / L to about 1 g / L or about 0 mM to about 60 mM results in low pCO 2 or high pCO 2 This can be done in combination with Mn supplementation at background levels of about 0 nM to 20000 nM. Non-limiting examples of fucose addition include addition to the production culture and / or to the growth culture up to the production culture stage.

[0189] Certain embodiments described herein provide for the administration of disclosed conditions (e.g., pCO2, pCO3, pCO4, pCO5, pCO6, pCO7, pCO8, pCO9, pCO10, pCO11, pCO20, pCO30, pCO40, pCO50, pCO60, pCO12, pCO13, pCO24, pCO15, pCO25, pCO30, pCO40, pCO16, pCO17, pCO25, pCO30, pCO40, pCO18, pCO25, pCO30, pCO40, pCO19, pCO26, pCO30, pCO40, pCO5 ...8, pCO26, pCO30, pCO40, pCO50, pCO19, pCO26, pCO30, p 2The present invention relates to modulating glycosylation by supplementing fucose at the disclosed concentrations under conditions of increased fucose concentration (e.g., supplemented Mn). In a non-limiting embodiment, a desired glycoprotein glycosylation pattern that can be achieved by increasing the fucose concentration is modulation (e.g., increase or decrease) of fucosylation deficiency of a glycoprotein. For example, and not by way of limitation, the target range of fucosylation deficiency G0 (%G0-F or %normalized G0-F) can be 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 modulated 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 a modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range of fucosylated G0 (%G0) can be about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%. In certain embodiments, %G0 can be modulated 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 modulation of fucosylation deficiency and modulation of galactosylation of the glycoprotein. For example, and not by way of limitation, the target range for %G0-F or %normalizedG0-F can be 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 about 40% to about 90%, about 50% to about 90%, about 50% to about 85%, or about 60% to about 80%.In certain embodiments, %G0-F or %normalizedG0-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%, and %G0 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%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 50%.

[0190] 8. Cell Culture and Glycoprotein Composition In certain embodiments, the present disclosure relates to compositions of glycoproteins, e.g., mAbs, obtained through the use of the cell culture strategies outlined herein. Such compositions can include specific cell culture compositions defined by the properties of the cell culture medium, the host cell, and the glycoprotein being expressed. For example, and not by way of limitation, the compositions of the present disclosure are directed to compositions of glycoproteins of interest, e.g., mAbs, that exhibit a particular glycosylation profile, e.g., a particular amount of fucosylated and / or galactosylated G0 glycans. In certain embodiments, the compositions of the present disclosure are directed to compositions of cell culture medium that contain or are supplemented to contain an advantageous Mn concentration. In certain embodiments, the present disclosure can be directed to the combination of such cell culture medium with such glycoproteins of interest that exhibit a particular glycosylation profile.

[0191] In certain embodiments, the present disclosure relates to compositions of glycoproteins, e.g., mAbs, obtained by screening and selecting cell culture media for compatibility with specific action goals or by adjusting for other cell culture media component variations. For example, and not by way of limitation, the present disclosure is directed to mAb compositions, where the compositions are obtained from cell cultures where the cell culture media Mn concentration falls within the range of 30 nM to 110 nM, and where media outside of such range of Mn concentrations are not utilized in connection with cell cultures producing mAbs. The present disclosure is also directed to mAb compositions, where the compositions are obtained from cell cultures where the cell culture Mn concentration falls within the range of 30 nM to 110 nM, and where cell cultures outside of such range of Mn concentrations are not utilized in connection with cell cultures producing mAbs. In certain embodiments, the present disclosure is directed to compositions of cell culture media where the Mn concentration of the raw material has been screened and / or selected. In certain embodiments, the present disclosure can be directed to the combination of such cell culture media with such glycoproteins of interest exhibiting a particular glycosylation profile.

[0192] In an embodiment, the present disclosure relates to a composition of glycoprotein, e.g., mAb, exhibiting a particular glycosylation profile, e.g., a particular amount of fucosylated and / or galactosylated G0 glycans, obtained by adjusting the concentration of Mn in a cell culture medium through performing a HTST treatment of the medium with a pre-HTST pH adjustment target of less than about 7.3 or less than about 7.0. For example, and not by way of limitation, the pre-HTST pH adjustment target for the medium can 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 an embodiment, the composition of the present disclosure is directed to a composition of cell culture medium in which a HTST treatment step has been performed with a pre-HTST pH adjustment target as disclosed herein. In an embodiment, the present disclosure can be directed to a combination of such a cell culture medium and such a glycoprotein of interest exhibiting a particular glycosylation profile.

[0193] In one embodiment, the present disclosure provides a method for determining pCO during a cell culture process, as outlined herein. 2 The present disclosure relates to compositions of glycoproteins, e.g., mAbs, that exhibit a particular glycosylation profile, e.g., a particular amount of fucosylated and / or galactosylated G0 glycans, obtained by adjusting the medium holding time, culture period, culture temperature, manganese, galactose, fucose and / or osmolarity / Na+ concentration, or a combination thereof. In certain embodiments, the compositions of the present disclosure are 2 The present disclosure is directed to compositions of cell culture media in which the medium holding period, culture period, culture temperature, manganese, galactose, fucose and / or osmolarity / Na+ concentration or combinations thereof are adjusted as outlined herein. In certain embodiments, the present disclosure may be directed to combinations of such cell culture media with such glycoproteins of interest exhibiting specific glycosylation profiles.

[0194] In certain embodiments, various bioreactor configurations can be used for the composition of the cell culture media. For example, and not by way of limitation, the volume of the bioreactor can be from about 1 L to 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 a non-limiting embodiment, the bioreactor configuration is 2 The medium can be altered to adjust holding time, osmolarity, Na+, Mn, temperature, pH, levels of fucose, galactose or combinations thereof.

[0195] In addition to the various embodiments depicted and claimed, the subject matter of the present disclosure is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the features presented herein may be combined with each other in other ways within the scope of the subject matter of the present disclosure, such that the subject matter of the present disclosure includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure is presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject matter of the present disclosure to the disclosed embodiments.

[0196] It is obvious to those skilled in the art that various modifications and variations can be made to the compositions and methods of the subject matter of the present disclosure without departing from the spirit or scope of the subject matter of the present disclosure.Therefore, it is intended that the subject matter of the present disclosure 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.

[0197] Embodiments of the present disclosure The following are non-limiting embodiments of the present disclosure.

[0198] 1. A method for modulating the glycosylation pattern of a glycoprotein of interest in a cell culture, comprising the steps of: increasing or decreasing the glycosylation pattern of a glycoprotein of interest in a cell culture medium and / or in the cell culture environment, alone or in any combination, by increasing or decreasing the glycosylation pattern of a glycoprotein of interest in a cell culture medium and / or in the cell culture environment, alone or in any combination, by increasing or decreasing the glycosylation pattern of a glycoprotein of interest in a cell culture medium and / or in the cell culture environment, alone or in any combination, by decreasing ... 2 (pCO 2 ) conditions of Mn concentration of about 1 nM to about 20,000 nM; low pCO 2 Mn concentration of about 1 nM to about 30,000 nM; pCO2 of about 10 mmHg to about 250 mmHg 2 a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25°C to 39°C; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C.

[0199] 2. The method of embodiment 1, wherein said cell culture environment is in a bioreactor with or without cells.

[0200] 3. Low pCO 2 The condition is about 10 to about 100 mmHg, and the high pCO 2 3. The method of embodiment 1 or embodiment 2, wherein the conditions are about 20 to about 250 mmHg.

[0201] 4. pCO 2 4. The method of embodiment 3, wherein the conditioning period encompasses at least the first half of the cell culture period.

[0202] 5. The method of any one of embodiments 1 to 4, wherein the glycoprotein of interest is a recombinant protein.

[0203] 6. The method according to any one of the preceding claims, 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 domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

[0204] 7. The method of any one of embodiments 1 to 6, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0205] 8. The method of any one of embodiments 1 to 7, wherein the antibody is an anti-CD20 antibody.

[0206] 9. The method of any one of embodiments 1-8, wherein the anti-CD20 antibody is ocrelizumab.

[0207] 10. The method of any one of embodiments 6 to 8, wherein the antibody or antibody fragment exhibits a %G0-F (percent fucosylation-deficient 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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%.

[0208] 11. The method of any one of embodiments 6 to 9, in which glycosylation is modulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated galactosylation-deficient G0)), or to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0), or to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0), or to achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F).

[0209] 12. The step of modulating the glycosylation pattern of a glycoprotein of interest is carried out by using a low pCO 2 Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 2 The method according to any one of embodiments 1 to 11, comprising adjusting under the conditions a Mn concentration of about 1 nM to about 20,000 nM, and the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours, at a temperature of about 25° C. to 39° C.; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolarity of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29° C. to about 39° C.

[0210] 13. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises the step of: 2 Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 213. The method of embodiment 12, comprising adjusting the Mn concentration under conditions of about 1 nM to about 20,000 nM, and the following parameters in the cell culture medium and / or cell culture environment: a pre-seeding cell culture medium holding time of about 0 hours to about 120 hours, at a temperature of about 25° C. to 39° C.; and a cell culture period of about 0 days to about 150 days.

[0211] 14. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises the step of: 2 Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 2 13. The method of embodiment 12, comprising adjusting the Mn concentration under the conditions from about 1 nM to about 20,000 nM, and the following parameters in the cell culture medium and / or cell culture environment: a galactose concentration from about 0 mM to about 60 mM; and / or a fucose concentration from about 0 mM to about 60 mM.

[0212] 15. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises adjusting the pre-seeding cell culture medium holding period and any of the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: high partial pressure CO 2 (pCO 2 ) conditions of Mn concentration of about 1 nM to about 20,000 nM; low pCO 2 Mn concentration of about 1 nM to about 30,000 nM; pCO2 of about 10 mmHg to about 250 mmHg 2 15. The method according to any one of embodiments 1 to 14, comprising adjusting a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolarity of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C, wherein the cell culture medium retention period is about 0 hours to about 120 hours at a temperature of about 25°C to 39°C.

[0213] 16. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises adjusting the pre-seeding cell culture medium holding period and the following parameters in the cell culture medium and / or in the cell culture environment: high partial pressure CO 2 (pCO 2 ) conditions of about 1 nM to about 20,000 nM Mn concentration or low pCO 2 Mn concentration of about 1 nM to about 30,000 nM; pCO2 of about 10 mmHg to about 250 mmHg 2 and controlling the cell culture period from about 0 days to about 150 days, 16. The method according to embodiment 15, wherein the cell culture medium holding period is from about 0 hours to about 120 hours at a temperature of from about 25° C. to 39° C.

[0214] 17. The method of any one of embodiments 12 to 15, wherein the glycoprotein of interest is an antibody or an antibody fragment thereof.

[0215] 18. The method of embodiment 17, wherein the antibody or antibody fragment thereof is an anti-CD20 antibody.

[0216] 19. The method of embodiment 18, wherein the anti-CD20 antibody is ocrelizumab.

[0217] 20. The method according to any one of embodiments 1 to 19, wherein the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises adjusting the cell culture period and any of the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: Na+ concentration of about 0 mM to about 300 mM; osmolarity of about 250 mOsm / kg to about 550 mOsm / kg; galactose concentration of about 0 mM to about 60 mM; fucose concentration of about 0 mM to about 60 mM; and culture temperature of about 29°C to about 39°C, and the cell culture period is about 0 days to about 150 days.

[0218] 21. The method according to any one of embodiments 1 to 20, wherein the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises adjusting a Na+ concentration of about 0 nM to about 300 nM and any of the following parameters, alone or in any combination, in the cell culture medium and / or in the cell culture environment: an osmolarity of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C, wherein the Na+ concentration is about 0 mM to about 300 mM.

[0219] 22. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises administering to the subject a Na concentration and a pCO2 concentration of about 10 mmHg to about 250 mmHg. 2 The method of any one of embodiments 1 to 21, comprising regulating

[0220] 23. The method according to any one of embodiments 1 to 22, wherein the step of adjusting the glycosylation pattern of the glycoprotein of interest comprises adjusting the osmolarity and any of the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C, wherein the osmolarity is about 250 mOsm / kg to about 550 mOsm / kg.

[0221] 24. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises the step of: 2 Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO 2 2. The method of embodiment 1, comprising adjusting the Mn concentration under the conditions from about 1 nM to about 20,000 nM, adjusting the Na+ concentration from about 0 mM to about 300 mM, and adjusting the pre-seeding cell culture medium retention period from about 0 hours to about 120 hours.

[0222] 25. The modulation of the glycosylation pattern of the glycoprotein of interest comprises an osmolality of about 250 mOsm / kg to about 550 mOsm / kg and a pCO2 of about 10 mmHg to about 250 mmHg. 2 2. The method of embodiment 1, comprising regulating

[0223] 26. The method of embodiment 1, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating 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.

[0224] 27. The modulation of the glycosylation pattern of the glycoprotein of interest comprises a pCO2 concentration of about 10 mmHg to about 250 mmHg. 2 and adjusting the fucose concentration to about 0 mM to about 60 mM.

[0225] 28. The method of embodiment 1, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating a fucose concentration of about 0 mM to about 60 mM and a culture temperature of about 29°C to about 39°C.

[0226] 29. The modulation of the glycosylation pattern of the glycoprotein of interest is pCO 2 Concentration and any of the following parameters in the cell culture medium and / or cell culture environment, alone or in any combination: high partial pressure CO 2 (pCO 2 ) conditions of Mn concentration of about 1 nM to about 20,000 nM; low pCO 2 a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25° C. to about 39° C.; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29° C. to about 39° C., 22. The method of embodiment 1, wherein the concentration is from about 10 mmHg to about 250 mmHg.

[0227] 30. The Mn concentration is high pCO 2 Approximately 1 nM to approximately 20,000 nM in culture; high pCO 2 In culture, about 1nM to about 10000nM, about 1nM to about 5000nM, about 1nM to about 4000nM, about 1nM to about 3000nM, about 1nM to about 2000nM, about 1nM to about 1000nM; high pCO 2 The method according to any one of embodiments 1 to 29, wherein the concentration in the culture is about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 10000 nM, about 20 nM to about 20,000 nM, about 20 nM to about 300 nM, or about 30 nM to about 110 nM.

[0228] 31. The Mn concentration is low pCO 2 Approximately 1 nM to approximately 30,000 nM in culture; low pCO 2 In culture, about 1nM to about 20000nM; about 1nM to about 10000nM, about 1nM to about 5000nM, about 1nM to about 4000nM, about 1nM to about 3000nM, about 1nM ~about 2000nM, about 1nM to about 1000nM; about 1nM to about 500nM, about 1nM to about 100nM, about 1nM to about 50nM, about 1nM to about 20nM, about 20nM to about 100 The method according to any one of embodiments 1 to 30, wherein the MgO is about 20 nM, about 20 nM to about 300 nM, about 20 nM to about 500 nM, about 20 nM to about 1000 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 5000 nM, about 20 nM to about 10000 nM, about 20 nM to about 20000 nM, or about 30 nM to about 110 nM.

[0229] 32. The method of any one of the preceding claims, wherein adjusting the Mn concentration comprises determining the Mn content in a cell culture feedstock and selecting a feedstock lot for adjusting the Mn concentration.

[0230] 33. The method of any one of embodiments 1-32, wherein adjusting the Mn concentration comprises: (i) adjusting the cell culture medium or a material in contact with the cell culture, to adjust the Mn concentration; or (ii) taking into account the concentration of Mn leached into the cell culture medium or during the cell culture; or a combination of (i) and (ii).

[0231] 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 medium preparation, holding or culture vessel; or (iii) a combination of (i) and (ii).

[0232] 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.

[0233] 36. The method of embodiment 34, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resin.

[0234] 37. The method of any one of embodiments 1 to 36, wherein the cell culture medium is a basal medium, a reconstituted medium, a feed medium, a hydrolysate, a supplement, a serum or an additive.

[0235] 38. The method according to any one of the preceding embodiments, wherein the cell culture medium is replenished during the production stage of the cell culture.

[0236] 39. The method according to any one of the preceding embodiments, wherein the cell culture medium is replenished prior to the production stage of the cell culture.

[0237] 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 the supplementation is based on a predefined schedule or criteria.

[0238] 41. The method of any one of embodiments 1-40, wherein one or more of Mn, fucose, galactose and Na+ are replenished as a bolus, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop-based replenishment, or as a combination of one or more thereof.

[0239] 42. The method of any one of the preceding embodiments, wherein the cell culture medium consists essentially of one or more of: i) Mn; ii) fucose; iii) galactose; and / or iv) Na+.

[0240] 43. The method of any one of embodiments 1-42, wherein the adjusting the Mn concentration comprises utilizing 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.

[0241] 44. The modulation of the glycosylation pattern of the glycoprotein of interest is 2 The method of any one of embodiments 1 to 43, comprising regulating

[0242] 45. The cell culture or the cell culture medium is in a bioreactor and pCO 2 The method according to any one of embodiments 1 to 44, wherein the adjustment is achieved by adjusting a bioreactor working volume; a bioreactor gas sparging strategy; a bioreactor agitation strategy; a bioreactor medium exchange strategy, a bioreactor perfusion strategy, a bioreactor feeding strategy, or any combination thereof.

[0243] 46. ​​The pCO 2 Regulation, high pCO 2 45. The method of embodiment 44, comprising establishing the culture.

[0244] 47. pCO 247. The method of embodiment 46, wherein the blood pressure 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.

[0245] 48. The pCO 2 Regulation, low pCO 2 The method according to any one of the preceding embodiments, comprising establishing a culture.

[0246] 49. The pCO 2 49. The method of embodiment 48, wherein the blood pressure is from about 10 mmHg to about 100 mmHg, from 10 mmHg to about 80 mmHg, from about 20 mmHg to about 70 mmHg, or from about 30 mmHg to about 60 mmHg.

[0247] 50. pCO 2 50. The method of any one of embodiments 1-49, wherein the modulation occurs on day 0 of culture.

[0248] 51. The pCO 2 51. The method of embodiment 50, wherein the modulation occurs for approximately the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0249] 52. The pCO 2 51. The method of embodiment 50, wherein the modulation occurs for approximately the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0250] 53. The method of embodiment 52, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the pre-seeding cell culture medium holding period, wherein the pre-seeding cell culture medium holding period 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.

[0251] 54. The method according to embodiment 53, wherein the temperature of the medium during the pre-seeding cell culture medium holding is from about 25°C to about 39°C, from about 30°C to about 39°C, from about 35°C to about 39°C, or from about 36°C to about 39°C.

[0252] 55. The method according to any one of embodiments 1 to 54, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture period, wherein the cell culture period 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.

[0253] 56. The method of any one of embodiments 1 to 55, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the Na+ concentration, wherein the Na+ concentration is from about 0 mM to about 300 mM; from about 20 mM to about 20 mM; from about 30 mM to about 150 mM; or from about 40 mM to about 130 mM.

[0254] 57. The regulation of the Na+ concentration is Na 2 CO 3 , NaHCO 3 57. The method of any one of the preceding embodiments, comprising supplementing the cell culture with a Na compound, including but not limited to, NaOH, NaCl, or a combination thereof.

[0255] 58. The method of any one of embodiments 1 to 57, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the osmolality, wherein the osmolality is from about 250 mOsm / kg to about 550 mOsm / kg; from about 300 mOsm / kg to about 450 mOsm / kg; or from about 325 mOsm / kg to about 425 mOsm / kg.

[0256] 59. The method of any one of embodiments 1 to 58, wherein adjusting the osmolarity comprises supplementing the cell culture with an osmolarity-adjusting medium component.

[0257] 60. The method of embodiment 59, wherein the osmotically regulated medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

[0258] 61. The method of embodiment 59, wherein the osmolarity-adjusting medium components are replenished as a bolus, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop-based replenishment, or as a combination of one or more of these.

[0259] 62. The method of any one of embodiments 1 to 61, wherein modulating 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.

[0260] 63. The method of any one of embodiments 1 to 62, wherein modulating 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.

[0261] 64. The method of any one of embodiments 1 to 63, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture temperature, and 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.

[0262] 65. The method of embodiment 64, wherein the cell culture temperature is adjusted during the production stage of the cell culture.

[0263] 66. The method of any one of the preceding embodiments, wherein the cell culture temperature is adjusted prior to the production stage of the cell culture.

[0264] 67. The method of any one of embodiments 1-66, wherein the cell culture temperature is adjusted based on a predefined schedule or criteria.

[0265] 68. The method of any one of embodiments 1 to 67, wherein the cell culture comprises eukaryotic cells.

[0266] 69. The method of embodiment 68, wherein the eukaryotic cell is an insect, avian, fungal, plant or mammalian cell.

[0267] 70. The method of embodiment 69, wherein the fungal cell is a yeast, Pichia or any filamentous fungal cell.

[0268] 71. The method of embodiment 70, wherein the yeast cell is a S. cerevisiae cell.

[0269] 72. The method of embodiment 69, wherein the mammalian cell is a CHO cell.

[0270] 73. The method of any one of embodiments 1 to 72, wherein the cell culture is in a bioreactor, including, but not limited to, a disposable technology (SUT) bag or bioreactor; a WAVE bioreactor; a stainless steel bioreactor; a flask; a tube and a chamber.

[0271] 74. The method of any one of embodiments 1 to 73, wherein the volume of the cell culture is from 1 mL to 35,000 L.

[0272] 75. The volume of the cell culture is 1mL~10ml, 1mL~50ml, 1mL~100ml, 1mL~200ml, 1mL~300ml, 1mL~500ml, 1mL~1000ml, 1mL~2000ml, 1mL~ 3000ml, 1mL~4000ml, 1mL~5000ml, 1mL~1L, 1mL~2L, 1mL~3L, 1mL~4L, 1mL~5L, 1mL~6L, 1mL~10L, 1mL~20L, 1mL~30L, 1mL~ 40L, 1mL to 50L, 1mL to 60L, 1mL to 70L, 1mL to 100L, 1mL to 200L, 1mL to 300L, 1mL to 400L, 1mL to 500L, 1mL to 1000L, 1mL to 2000L, 1mL to 3000L, 1mL to 4000L, 1mL to 5000L, 1mL to 10,000L, 1mL to 20,000L, 1mL to 30,000L, 1mL to 30,000L, or 1mL to 35,000L.

[0273] 76. Use of the method according to any one of embodiments 1 to 75 to obtain a glycoprotein of interest exhibiting a %G0-F (percent fucosylation-deficient 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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%.

[0274] 77. The use of the method according to any one of embodiments 1 to 75, wherein glycosylation is modulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated galactosylation-deficient G0)), or to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0), or to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0), or to achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F).

[0275] 78. A method for preparing a cell culture medium, feed medium, hydrolysate or additive, comprising administering to a subject a cell culture medium, feed medium, hydrolysate or additive at a high partial pressure of about 1 nM to about 20,000 nM CO. 2 (pCO 2 ) Mn concentration in the culture; low pCO of about 1 nM to about 30,000 nM 2 Mn concentration in culture; pCO of about 10 mmHg to about 250 mmHg 2 a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C, wherein the cell culture medium, feed medium, hydrolysate or additive modulates the glycosylation pattern of a glycoprotein of interest.

[0276] 79. The method of embodiment 78, wherein the glycoprotein of interest is an antibody or an antibody fragment.

[0277] 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%.

[0278] 81. The method of embodiment 79, wherein the glycosylation of the antibody or pre-antibody fragment is modulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated galactosylation-deficient G0)), or to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0), or to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0), or to achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F).

[0279] 82. The method according to any one of embodiments 78 to 81, comprising adjusting the Mn concentration from about 1 nM to about 30000 nM and the pre-seeding cell culture medium retention time from about 0 hours to about 120 hours.

[0280] 83. The pCO2 of about 10 mmHg to about 250 mmHg 2 83. The method of any one of embodiments 78-82, comprising adjusting the Na+ concentration from about 0 mM to about 300 mM and the pre-seeding cell culture medium retention time from about 0 hours to about 120 hours.

[0281] 84. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 and adjusting the Na+ concentration to about 0 mM to about 300 mM.

[0282] 85. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 85. The method of any one of embodiments 78-84, comprising adjusting the Na+ concentration from about 0 mM to about 300 mM and the pre-seeding cell culture medium retention time from about 0 hours to about 72 hours.

[0283] 86. The pCO2 of about 10 mmHg to about 250 mmHg 2 and adjusting the Na+ concentration to about 0 mM to about 300 mM.

[0284] 87. The osmolarity of about 250 mOsm / kg to about 550 mOsm / kg and the pCO2 of about 10 mmHg to about 250 mmHg. 2 The method of any one of embodiments 78 to 86, comprising regulating

[0285] 88. The pCO2 of about 10 mmHg to about 250 mmHg 2 88. The method of any one of embodiments 78-87, comprising adjusting the Mn concentration from about 1 nM to about 30,000 nM, the cell culture period from about 0 days to about 150 days, and the pre-seeding cell culture medium retention period from 0 hours to about 120 hours.

[0286] 89. The method according to any one of embodiments 78 to 88, comprising adjusting the Mn concentration from about 1 nM to about 30,000 nM and the galactose concentration from about 0 mM to about 60 mM.

[0287] 90. The method according to any one of embodiments 78 to 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.

[0288] 91. The fucose concentration is about 0 mM to about 60 mM and the pCO 2The method of any one of embodiments 78 to 90, comprising regulating

[0289] 92. The fucose concentration is about 0 mM to about 60 mM, the Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 The method of any one of embodiments 78 to 91, comprising regulating

[0290] 93. The method according to any one of embodiments 78 to 92, comprising adjusting the fucose concentration from about 0 mM to about 60 mM, and wherein the cell culture temperature is from about 29°C to about 39°C.

[0291] 94. The method according to any one of embodiments 78 to 93, comprising adjusting the fucose concentration from about 0 mM to about 60 mM and the cell culture period from about 0 days to about 150 days.

[0292] 95. The Mn concentration is high pCO 2 Approximately 1 nM to approximately 20,000 nM in culture; high pCO 2 Approximately 1 nM to approximately 1000 nM in culture; high pCO 2 About 20 nM to about 300 nM in culture; or high pCO 2 The method of any one of embodiments 78 to 94, wherein the concentration is from about 30 nM to about 110 nM in culture.

[0293] 96. The Mn concentration is low pCO 2 Approximately 1 nM to approximately 30,000 nM in culture; low pCO 2 Approximately 1 nM to approximately 3000 nM in culture; low pCO 2 About 20 nM to about 300 nM in culture; or low pCO 2 The method of any one of embodiments 78 to 95, wherein the concentration is about 30 nM to about 110 nM in culture.

[0294] 97. The method of embodiment 95 or embodiment 96, wherein adjusting the Mn concentration comprises determining the Mn content in a cell culture feedstock and selecting a feedstock lot for adjusting the Mn concentration.

[0295] 98. The method of embodiment 95 or embodiment 96, wherein adjusting the Mn concentration comprises: i) adjusting the cell culture medium or a material in contact with the cell culture, to adjust the Mn concentration; or (ii) taking into account the concentration of Mn leached into the cell culture medium or during the cell culture; or a combination of (i) and (ii).

[0296] 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 medium preparation, holding or culture vessel; or (iii) a combination of (i) and (ii).

[0297] 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.

[0298] 101. The method of embodiment 99, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resin.

[0299] 102. The method of embodiment 95 or embodiment 96, wherein the adjustment of the Mn concentration comprises utilizing 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.

[0300] 103. The pCO 2 The method of any one of embodiments 78 to 102, wherein the expression level of the IL-1 gene is modulated.

[0301] 104. The cell culture medium is in a bioreactor, and pCO 2 The method of embodiment 103, wherein adjusting is accomplished by adjusting a bioreactor working volume; a bioreactor gas sparging strategy; a bioreactor agitation strategy; a bioreactor feeding strategy; a bioreactor perfusion strategy; a bioreactor medium exchange strategy, or any combination thereof.

[0302] 105. The pCO2 Regulation, high pCO 2 104. The method of embodiment 103, comprising establishing the culture.

[0303] 106. The pCO 2 106. The method of embodiment 105, wherein the blood pressure 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.

[0304] 107. The pCO 2 Regulation, low pCO 2 104. The method of embodiment 103, comprising establishing the culture.

[0305] 108. The pCO 2 108. The method of embodiment 107, wherein the blood pressure is from about 10 mmHg to about 100 mmHg, from 10 mmHg to about 80 mmHg, from about 20 mmHg to about 70 mmHg, or from about 30 mmHg to about 60 mmHg.

[0306] 109. The pCO 2 104. The method of embodiment 103, wherein the modulation occurs on day 0 of the culture.

[0307] 110. The pCO 2 The method of embodiment 103, wherein the modulation occurs for approximately the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0308] 111. The pCO 2 104. The method of embodiment 103, wherein the modulation occurs for approximately the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0309] 112. The method according to any one of embodiments 78 to 111, wherein the pre-seeding cell culture medium retention period 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.

[0310] 113. The method according to embodiment 112, wherein the temperature of the medium during the pre-seeding cell culture medium holding is from about 25°C to about 39°C, from about 30°C to about 39°C, from about 35°C to about 39°C, or from about 36°C to about 39°C.

[0311] 114. The method according to any one of embodiments 78 to 113, wherein the cell culture period 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.

[0312] 115. The method according to any one of embodiments 78 to 114, 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.

[0313] 116. The regulation of the Na+ concentration is 2 CO 3 , NaHCO 3 116. The method of embodiment 115, comprising supplementing said cell culture with Na compounds, including but not limited to, NaOH, NaCl, or combinations thereof.

[0314] 117. The method of any one of embodiments 78 to 116, wherein the osmolality is from about 250 mOsm / kg to about 550 mOsm / kg, from about 300 mOsm / kg to about 450 mOsm / kg, or from about 325 mOsm / kg to about 425 mOsm / kg.

[0315] 118. The method of embodiment 117, wherein adjusting the osmolarity comprises supplementing the cell culture with an osmotically adjusted medium component.

[0316] 119. The method of embodiment 118, wherein the osmotically adjusted medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

[0317] 120. The method of any one of embodiments 787 to 119, wherein the galactose concentration is from about 0 mM to about 60 mM or from about 0 mM to about 50 mM.

[0318] 121. The method according to any one of embodiments 78 to 119, 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.

[0319] 122. The method according to any one of embodiments 78 to 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.

[0320] 123. Use of a medium according to any one of embodiments 78 to 121 in a eukaryotic cell fermentation process for the production of recombinant proteins.

[0321] 124. Use of a medium 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 domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

[0322] 125. Use of the medium according to embodiment 124, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0323] 126. Use of the medium according to embodiment 124, wherein the antibody is an anti-CD20 antibody.

[0324] 127. Use of the medium according to embodiment 124, wherein the anti-CD20 antibody is ocrelizumab.

[0325] 128. Use of a medium according to embodiment 124, wherein the antibody or antibody fragment exhibits a %G0-F (percent fucosylation-deficient glycoprotein) of about 0% to about 20%; about 1% to about 15%; about 1% to about 10%; or about 1% to about 8%; a standardized %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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%.

[0326] 129. The use of a medium according to embodiment 124, wherein the eukaryotic cells are insect, avian, fungal, plant or mammalian cells.

[0327] 130. The use of a medium according to embodiment 129, wherein the fungal cell is a yeast, Pichia or any filamentous fungal cell.

[0328] 131. The use of a medium according to embodiment 130, wherein the yeast cells are S. cerevisiae cells.

[0329] 132. The use of the medium according to embodiment 129, wherein the mammalian cells are CHO cells.

[0330] 133. The use of a medium according to any one of embodiments 123 to 132, wherein the cell culture is in a bioreactor, including but not limited to a disposable technology (SUT) bag or bioreactor; a WAVE bioreactor; a stainless steel bioreactor; a flask; a tube and a chamber.

[0331] 134. Use of a medium according to any one of embodiments 123 to 133, wherein the volume of the cell culture is between 1 mL and 35,000 L.

[0332] 135. The volume of the cell culture is 1mL~10ml, 1mL~50ml, 1mL~100ml, 1mL~200ml, 1mL~300ml, 1mL~500ml, 1mL~1000ml, 1mL~2000ml, 1mL~ 3000ml, 1mL~4000ml, 1mL~5000ml, 1mL~1L, 1mL~2L, 1mL~3L, 1mL~4L, 1mL~5L, 1mL~6L, 1mL~10L, 1mL~20L, 1mL~30L, 1mL~40 Use of a medium according to embodiment 134, wherein the medium is in a volumetric capacity of 1 mL, 1 mL to 50 L, 1 mL to 60 L, 1 mL to 70 L, 1 mL to 100 L, 1 mL to 200 L, 1 mL to 300 L, 1 mL to 400 L, 1 mL to 500 L, 1 mL to 1000 L, 1 mL to 2000 L, 1 mL to 3000 L, 1 mL to 4000 L, 1 mL to 5000 L, 1 mL to 10,000 L, 1 mL to 20,000 L, 1 mL to 30,000 L, 1 mL to 30,000 L, or 1 mL to 35,000 L.

[0333] 136. Host cells engineered to express a glycoprotein of interest and a high partial pressure of CO at about 1 nM to about 20,000 nM. 2 (pCO 2 ) Mn concentration in the culture; low pCO of about 1 nM to about 30,000 nM 2 Mn concentration in culture; pCO of about 10 mmHg to about 250 mmHg 2 a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours; a cell culture period of about 0 days to about 150 days; a Na+ concentration of about 0 mM to about 300 mM; an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; a galactose concentration of about 0 mM to about 60 mM; a fucose concentration of about 0 mM to about 60 mM; and a culture temperature of about 29°C to about 39°C.

[0334] 137. The composition of embodiment 136, wherein the cell culture environment is in a bioreactor.

[0335] 138. The composition according to any one of embodiments 136 to 137, wherein the glycoprotein of interest is an antibody or an antibody fragment.

[0336] 139. The composition of embodiment 138, wherein the antibody or antibody fragment exhibits a %G0-F (percent fucosylation-deficient 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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%.

[0337] 140. The composition of embodiment 138, wherein the glycosylation of the antibody or antibody fragment is modulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated galactosylation-deficient G0)), or to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0), or to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0), or to achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F).

[0338] 141. The composition of embodiment 136, wherein the Mn concentration is from about 1 nM to about 30,000 nM and the pre-seeding cell culture medium retention time is from about 0 hours to about 120 hours.

[0339] 142. The pCO 2 is from about 0 mM to about 300 mM, and the pre-seeding cell culture medium holding period is from about 0 hours to about 120 hours.

[0340] 143. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2is from about 10 mmHg to about 250 mmHg and the Na+ concentration is from about 0 mM to about 300 mM.

[0341] 144. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 is from about 10 mmHg to about 250 mmHg, said Na+ concentration is from about 0 mM to about 300 mM, and said pre-seeding cell culture medium holding period is from about 0 hours to about 120 hours.

[0342] 145. pCO 2 is from about 10 mmHg to about 250 mmHg and the Na+ concentration is from about 0 mM to about 300 mM.

[0343] 146. The osmolality is about 250 mOsm / kg to about 550 mOsm / kg, and the pCO 2 The composition of embodiment 136, wherein the blood flow is from about 10 mmHg to about 250 mmHg.

[0344] 147. pCO 2 137. The composition of embodiment 136, wherein the Mn concentration is from about 1 nM to about 30,000 nM, the cell culture period is from about 0 days to about 150 days, and the pre-seeding cell culture medium retention period is from about 0 hours to about 120 hours.

[0345] 148. The composition of embodiment 136, wherein the Mn concentration is from about 1 nM to about 30,000 nM and the galactose concentration is from about 0 mM to about 60 mM.

[0346] 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 30,000 nM.

[0347] 150. The fucose concentration is about 0 mM to about 60 mM, and the pCO 2 The composition of embodiment 136, wherein the blood flow is from about 10 mmHg to about 250 mmHg.

[0348] 151. The fucose concentration is about 0 mM to about 60 mM, the Mn concentration is about 1 nM to about 30,000 nM, and the pCO 2 The composition of embodiment 136, wherein the blood flow is from about 10 mmHg to about 250 mmHg.

[0349] 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 from about 29 °C to about 39 °C.

[0350] 153. The composition according to embodiment 136, wherein the fucose concentration is from about 0 mM to about 60 mM and the cell culture period is from about 0 days to about 150 days.

[0351] 154.Mn concentration, high pCO 2 Approximately 1 nM to approximately 20,000 nM in culture; high pCO 2 In culture, about 1nM to about 10000nM, about 1nM to about 5000nM, about 1nM to about 4000nM, about 1nM to about 3000nM, about 1nM to about 2000nM, about 1nM to about 1000nM; high pCO 2 The composition according to any one of embodiments 136 to 153, wherein the concentration in the culture is about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 10000 nM, about 20 nM to about 20,000 nM, about 20 nM to about 300 nM, or about 30 nM to about 110 nM.

[0352] 155. The Mn concentration is low pCO 2 Approximately 1 nM to approximately 30,000 nM in culture; low pCO 2In culture, about 1nM to about 20000nM; about 1nM to about 10000nM, about 1nM to about 5000nM, about 1nM to about 4000nM, about 1nM to about 3000nM, about 1nM About 2000nM, about 1nM to about 1000nM; about 1nM to about 500nM, about 1nM to about 100nM, about 1nM to about 50nM, about 1nM to about 20nM, about 20nM to about 100n The method of any one of embodiments 136 to 153, wherein the M is about 20 nM to about 300 nM, about 20 nM to about 500 nM, about 20 nM to about 1000 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 5000 nM, about 20 nM to about 10,000 nM, about 20 nM to about 20,000 nM, or about 30 nM to about 110 nM.

[0353] 156. The composition of embodiment 154 or embodiment 155, wherein adjusting the Mn concentration comprises determining the Mn content in a cell culture raw material and selecting a raw material lot to adjust the Mn concentration.

[0354] 157. The composition of embodiment 154 or embodiment 155, wherein adjusting the Mn concentration comprises: (i) adjusting the cell culture medium or a material in contact with the cell culture to adjust the Mn concentration; or (ii) taking into account the concentration of Mn leached into the cell culture medium or during the cell culture; or a combination of (i) and (ii).

[0355] 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 medium preparation, holding or culture vessel; or (iii) a combination of (i) and (ii).

[0356] 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.

[0357] 160. The composition of embodiment 158, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resins.

[0358] 161. The composition of embodiment 154 or embodiment 155, wherein Mn is supplemented as a component of the cell culture medium.

[0359] 162. The composition of embodiment 161, wherein the cell culture medium is a feed medium, a hydrolysate or an additive.

[0360] 163. The composition of embodiment 162, wherein the feed medium, the hydrolysate or the additive comprises Mn.

[0361] 164. The composition of embodiment 162, wherein the feed medium or the additive consists essentially of Mn.

[0362] 165. The composition of embodiment 154 or embodiment 155, wherein the Mn is replenished during the production stage of the cell culture.

[0363] 166. The composition of embodiment 154 or embodiment 155, wherein the Mn is supplemented prior to the production stage of the cell culture.

[0364] 167. The composition of embodiment 154 or embodiment 155, wherein the Mn is replenished based on a predefined schedule or criteria.

[0365] 168. The composition of embodiment 154 or embodiment 155, wherein the Mn is supplemented as a bolus dose, as intermittent supplementation, as continuous supplementation, as semi-continuous supplementation, as feedback loop-based supplementation, or a combination of one or more of these.

[0366] 169. The composition of embodiment 154 or embodiment 155, wherein adjusting the Mn concentration comprises utilizing 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.

[0367] 170. The modulation of the glycosylation pattern of the glycoprotein of interest is 2 The composition of any one of embodiments 136 to 153, comprising regulating

[0368] 171. The cell culture or cell culture medium is in a bioreactor, and pCO 2 The composition of embodiment 170, wherein the adjustment is achieved by adjusting a bioreactor working volume; a bioreactor gas sparging strategy; a bioreactor agitation strategy; a bioreactor feeding strategy; a bioreactor perfusion strategy; a bioreactor medium exchange strategy, or any combination thereof.

[0369] 172. The pCO 2 Regulation, high pCO 2 The composition of embodiment 170, comprising establishing the culture.

[0370] 173. The pCO 2 The composition of embodiment 172, wherein the blood flow rate is from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 150 mmHg, or from about 30 mmHg to about 150 mmHg.

[0371] 174. pCO 2 Regulation, low pCO 2 The composition of embodiment 170, comprising establishing the culture.

[0372] 175. pCO 2 The composition of embodiment 174, wherein the blood flow rate is from about 10 mmHg to about 100 mmHg, from 10 mmHg to about 80 mmHg, from about 20 mmHg to about 70 mmHg, or from about 30 mmHg to about 60 mmHg.

[0373] 176. pCO 2 The composition of embodiment 170, wherein the modulation occurs on day 0 of culture.

[0374] 177. pCO 2The composition of embodiment 170, wherein the modulation occurs for approximately the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0375] 178. pCO 2 The composition of embodiment 170, wherein the modulation occurs for approximately the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

[0376] 179. The composition of any one of embodiments 1136 to 153, wherein the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the pre-seeding cell culture medium retention period, wherein the pre-seeding cell culture medium retention period 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.

[0377] 180. The composition of embodiment 179, wherein the temperature of the medium during the pre-seeding cell culture medium holding 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.

[0378] 181. The composition of any one of embodiments 136 to 153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture period, and the cell culture period 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.

[0379] 182. The composition of any one of embodiments 136 to 153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating 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.

[0380] 183. The regulation of the Na+ concentration is 2 CO 3 , NaHCO 3183. The composition of embodiment 182, comprising supplementing said cell culture with a Na compound, including but not limited to, NaOH, NaCl, or a combination thereof.

[0381] 184. The composition of embodiment 182, wherein Na+ is replenished during the production stage of the cell culture.

[0382] 185. The composition of embodiment 182, wherein the Na+ is replenished prior to the production stage of the cell culture.

[0383] 186. The composition of embodiment 182, wherein the Na+ is replenished based on a predefined schedule or criteria.

[0384] 187. The composition of embodiment 182, wherein the Na+ is replenished as a bolus, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop-based replenishment, or a combination of one or more of these.

[0385] 188. The composition of any one of embodiments 136 to 153, wherein the modulation of the glycosylation pattern of the glycoprotein of interest comprises modulating the osmolality, wherein the osmolality is from about 250 mOsm / kg to about 550 mOsm / kg; from about 300 mOsm / kg to about 450 mOsm / kg; or from about 325 mOsm / kg to about 425 mOsm / kg.

[0386] 189. The composition of embodiment 188, wherein adjusting the osmolarity comprises supplementing the cell culture with an osmolarity-adjusting medium component.

[0387] 190. The composition of embodiment 189, wherein the osmoregulatory medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

[0388] 191. The composition of embodiment 189, wherein the osmolarity-adjusting medium components are replenished during the production stage of the cell culture.

[0389] 192. The composition of embodiment 189, wherein the osmolarity-adjusting medium components are replenished prior to the production stage of the cell culture.

[0390] 193. The composition of embodiment 189, wherein the osmotically regulated medium components are replenished based on a predefined schedule or criteria.

[0391] 194. The composition of embodiment 189, wherein the osmolarity-adjusting medium components are replenished as a bolus, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop-based replenishment, or a combination of one or more of these.

[0392] 195. The composition of any one of embodiments 136 to 153, wherein modulating 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.

[0393] 196. The composition of embodiment 195, wherein galactose is supplemented as a component of the cell culture medium.

[0394] 197. The composition of embodiment 196, wherein the cell culture medium is a feed medium, a hydrolysate or an additive.

[0395] 198. The composition of embodiment 197, wherein the feed medium, the hydrolysate or the additive comprises galactose.

[0396] 199. The composition of embodiment 197, wherein the feed medium or the additive consists essentially of galactose.

[0397] 200. The composition of embodiment 196, wherein the galactose is supplemented during the production stage of the cell culture.

[0398] 201. The composition of embodiment 196, wherein the galactose is supplemented prior to the production stage of the cell culture.

[0399] 202. The composition of embodiment 196, wherein the galactose is supplemented based on a predefined schedule or criteria.

[0400] 203. The composition of embodiment 196, wherein the galactose is supplemented as a bolus dose, as intermittent supplementation, as continuous supplementation, as semi-continuous supplementation, as feedback loop-based supplementation, or a combination of one or more of these.

[0401] 204. The composition of any one of embodiments 136 to 153, wherein modulating 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.

[0402] 205. The composition of embodiment 204, wherein fucose is supplemented as a component of the cell culture medium.

[0403] 206. The composition of embodiment 205, wherein the cell culture medium is a feed medium, a hydrolysate or an additive.

[0404] 207. The composition of embodiment 206, wherein the feed medium, the hydrolysate or the additive comprises fucose.

[0405] 208. The composition of embodiment 206, wherein the feed medium or the additive consists essentially of fucose.

[0406] 209. The composition of embodiment 205, wherein the fucose is replenished during the production stage of the cell culture.

[0407] 210. The composition of embodiment 205, wherein the fucose is supplemented prior to the production stage of the cell culture.

[0408] 211. The composition of embodiment 205, wherein the fucose is replenished based on a predefined schedule or criteria.

[0409] 212. The composition of embodiment 205, wherein the fucose is replenished as a bolus dose, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop-based replenishment, or a combination of one or more of these.

[0410] 213. The composition of any one of embodiments 136 to 153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture temperature, and 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.

[0411] 214. The composition of embodiment 213, wherein the cell culture temperature is regulated during the production stage of the cell culture.

[0412] 215. The composition of embodiment 213, wherein the cell culture temperature is adjusted prior to the production stage of the cell culture.

[0413] 216. The composition of embodiment 213, wherein the cell culture temperature is adjusted based on a predefined schedule or criteria.

[0414] 217. The composition of any one of embodiments 136 to 153, wherein the cell culture comprises eukaryotic cells.

[0415] 218. The composition of embodiment 217, wherein the eukaryotic cell is a fungal cell or a mammalian cell.

[0416] 219. The composition of embodiment 218, wherein the fungal cell is a yeast cell.

[0417] 220. The composition of embodiment 219, wherein the yeast cell is a S. cerevisiae cell.

[0418] 221. The composition of embodiment 218, wherein the mammalian cell is a CHO cell.

[0419] 222. The composition of any one of embodiments 136 to 221, wherein the cell culture is in a bioreactor, including but not limited to a single-use technology (SUT) bag or bioreactor; a WAVE bioreactor; a stainless steel bioreactor; a flask; a tube and a chamber.

[0420] 223. The composition of any one of embodiments 136 to 222, wherein the volume of the cell culture is from 1 mL to 35,000 L.

[0421] 224. A method for producing a glycoprotein of interest in cell culture, comprising providing a cell culture medium suitable for culturing eukaryotic cells, the method according to any one of embodiments 1 to 75, seeding the conditioned cell culture medium with eukaryotic cells expressing the recombinant protein, and culturing the eukaryotic cells so that the recombinant protein is expressed.

[0422] 225. A method for producing a glycoprotein of interest in a cell culture according to embodiment 224, wherein the cell culture is in a bioreactor.

[0423] 226. Low pCO 2 The condition is about 10 to about 100 mmHg, and the high pCO 2 225. A method for producing a glycoprotein of interest in a cell culture as described in embodiment 224, wherein the conditions are about 20 to about 250 mmHg.

[0424] 227.pCO 24. The method for producing a glycoprotein of interest in a cell culture as described in embodiment 3, wherein the period of conditioning encompasses at least the first half of the cell culture period.

[0425] 228. The method of any of embodiments 224-227, wherein the glycoprotein of interest is a recombinant protein.

[0426] 229. The 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 domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

[0427] 230. The method of embodiment 229, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0428] 231. The method of embodiment 229, wherein the antibody is an anti-CD20 antibody.

[0429] 232. The method of embodiment 231, wherein the anti-CD20 antibody is ocrelizumab.

[0430] 233. The method of any one of embodiments 224 to 232, wherein the antibody or antibody fragment exhibits a %G0-F (percent fucosylation-deficient 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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85% or about 60% to about 80%.

[0431] 234. The method of any one of embodiments 224 to 233, wherein the glycosylation is regulated to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated galactosylation-deficient G0)), or to achieve decreased fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0), or to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0), or to achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F).

[0432] 235. A method for modulating glycosylation of a glycoprotein of interest, comprising assaying a cell culture medium to determine whether the manganese concentration of the cell culture medium is within a targeted range; culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium that is within the targeted range, wherein glycosylation of the glycoprotein of interest is modulated compared to glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium that is outside the targeted range of manganese concentrations.

[0433] 236. The method of embodiment 235, wherein the glycoprotein of interest is an antibody.

[0434] 237. The method of embodiment 236, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0435] 238. The method of any one of embodiments 236-237, wherein the antibody is an anti-CD20 antibody.

[0436] 239. The method of any one of embodiments 297-298, wherein the anti-CD20 antibody is ocrelizumab.

[0437] 240. The method of embodiment 235, wherein the host cell is a mammalian cell.

[0438] 241. The method of any one of embodiments 239-240, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

[0439] 242. The method of embodiment 235, wherein the target range of manganese concentration is from about 30 nM to about 110 nM.

[0440] 243. The method of embodiment 235, wherein assaying the cell culture medium comprises assaying the manganese concentration of a component of the cell culture medium.

[0441] 244. The method of embodiment 243, wherein the component of the cell culture medium is a hydrolysate or serum.

[0442] 245. The method according to any one of embodiments 235 to 244, wherein glycosylation is modulated to achieve increased G0-F (fucosylation-deficient G0) while decreasing G0 (fucosylated G0).

[0443] 246. A cell culture composition comprising: a cell culture medium that has been assayed to determine whether the manganese concentration of the cell culture medium is within a targeted range; and a host cell engineered to express a glycoprotein of interest.

[0444] 247. The cell culture composition of embodiment 246, wherein the composition further comprises the glycoprotein of interest.

[0445] 248. The cell culture composition of embodiment 247, wherein the glycoprotein is an antibody.

[0446] 249. The cell culture composition of embodiment 248, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0447] 250. The cell culture composition of any one of embodiments 248 to 249, wherein the antibody is an anti-CD20 antibody.

[0448] 251. The cell culture composition of any one of embodiments 248 to 249, wherein the anti-CD20 antibody is ocrelizumab.

[0449] 252. The cell culture composition of embodiment 246, wherein the host cell is a mammalian cell.

[0450] 253. The cell culture composition of any one of embodiments 252, wherein the host cells are CHO cells.

[0451] 254. The cell culture composition of embodiment 246, wherein the target range of manganese concentration is from about 30 nM to about 110 nM.

[0452] 255. A composition comprising a glycoprotein of interest, the preparation comprising cell culture medium that has been assayed to determine whether the manganese concentration of the cell culture medium is within a targeted range, a host cell engineered to express the glycoprotein of interest, and the glycoprotein of interest.

[0453] 256. The composition described in embodiment 255, wherein the glycoprotein is an antibody.

[0454] 257. The composition of embodiment 256, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0455] 258. The cell culture composition of any one of embodiments 256 to 257, wherein the antibody is an anti-CD20 antibody.

[0456] 259. The cell culture composition of any one of embodiments 256 to 257, wherein the anti-CD20 antibody is ocrelizumab.

[0457] 260. The cell culture composition of embodiment 255, wherein the host cell is a mammalian cell.

[0458] 261. The cell culture composition of embodiment 260, wherein the host cells are CHO cells.

[0459] 262. A method for modulating glycosylation of a glycoprotein of interest, comprising: injecting high CO into a cell culture medium used in culturing a host cell expressing the glycoprotein of interest. 2 Supplementing the cell culture medium used in culturing the host cells expressing the glycoprotein of interest with about 10 nM to about 2000 nM manganese under conditions of low CO 2 The method comprises supplementing a culture medium with about 10 nM to about 3000 nM manganese under conditions, wherein glycosylation of a glycoprotein of interest is modulated compared to glycosylation of a glycoprotein of interest expressed by a host cell in a culture medium not so supplemented.

[0460] 263. The method of embodiment 262, wherein the glycoprotein of interest is an antibody.

[0461] 264. The method of embodiment 263, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0462] 265. The method of any one of embodiments 263-264, wherein the antibody is ocrelizumab.

[0463] 266. The method of embodiment 262, wherein the host cell is a mammalian cell.

[0464] 267. The method of embodiment 266, wherein the host cell is a CHO cell.

[0465] 268. The method according to any one of embodiments 262 to 267, wherein glycosylation is modulated to achieve increased G0-F (fucosylation-deficient G0) while decreasing G0 (fucosylated G0).

[0466] 269. A cell culture composition comprising:2 Approximately 10 nM to approximately 2000 nM manganese under conditions of low CO 2 1. A cell culture composition comprising: a cell culture medium supplemented with about 10 nM to about 3000 nM manganese under conditions; and a host cell engineered to express a glycoprotein of interest.

[0467] 270. The cell culture composition of embodiment 269, wherein the composition further comprises a glycoprotein of interest.

[0468] 271. The cell culture composition of embodiment 269, wherein the glycoprotein is an antibody.

[0469] 272. The cell culture composition of embodiment 271, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0470] 273. The cell culture composition of any one of embodiments 271-272, wherein the antibody is ocrelizumab.

[0471] 274. The cell culture composition of embodiment 269, wherein the host cell is a mammalian cell.

[0472] 275. The cell culture composition of embodiment 274, wherein the host cells are CHO cells.

[0473] 276. A composition comprising a glycoprotein of interest, the preparation being a cell culture medium supplemented with manganese, the culture being exposed to high CO 2 Under conditions of about 10 nM to about 2000 nM manganese or low CO 2 1. A composition comprising: a cell culture medium supplemented with about 10 nM to about 3000 nM manganese under conditions; a host cell engineered to express a glycoprotein of interest; and the glycoprotein of interest.

[0474] 277. The composition described in embodiment 276, wherein the glycoprotein is an antibody.

[0475] 278. The composition of embodiment 277, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0476] 279. The cell culture composition of any one of embodiments 276 to 277, wherein the antibody is ocrelizumab.

[0477] 280. The cell culture composition of embodiment 276, wherein the host cell is a mammalian cell.

[0478] 281. The cell culture composition of embodiment 280, wherein the host cells are CHO cells.

[0479] 282. A method for modulating glycosylation of a glycoprotein of interest, comprising exposing a cell culture medium having a pH target of 6.30 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 glycosylation of the glycoprotein of interest is modulated compared to glycosylation of the glycoprotein of interest expressed by said host cell in a culture medium having a pre-HTST heat treatment pH target of greater than pH 7.25.

[0480] 283. The method of embodiment 282, wherein the glycoprotein of interest is an antibody.

[0481] 284. The method of embodiment 283, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0482] 285. The method of any one of embodiments 283-284, wherein the antibody is ocrelizumab.

[0483] 286. The method of embodiment 282, wherein the host cell is a mammalian cell.

[0484] 287. The method of embodiment 286, wherein the host cell is a CHO cell.

[0485] 288. The method according to any one of embodiments 282 to 287, wherein glycosylation is modulated to achieve increased G0-F (fucosylation-deficient G0) while decreasing G0 (fucosylated G0).

[0486] 289. A cell culture composition comprising a cell culture medium comprising a pH target of about 6.30 to about 7.25 that has been exposed to HTST heat treatment, and a host cell engineered to express a glycoprotein of interest.

[0487] 290. The cell culture composition of embodiment 289, wherein the composition further comprises a glycoprotein of interest.

[0488] 291. The cell culture composition of embodiment 290, wherein the glycoprotein is an antibody.

[0489] 292. The cell culture composition of embodiment 291, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0490] 293. The cell culture composition of any one of embodiments 291-292, wherein the antibody is ocrelizumab.

[0491] 294. The cell culture composition of embodiment 293, wherein the host cell is a mammalian cell.

[0492] 295. The cell culture composition of embodiment 294, wherein the host cells are CHO cells.

[0493] 296. A composition comprising a glycoprotein of interest, the preparation comprising a cell culture medium comprising a pH target of about 6.30 to about 7.25 that has been exposed to HTST heat treatment, a host cell engineered to express the glycoprotein of interest, and the glycoprotein of interest.

[0494] 297. The composition of embodiment 296, wherein the glycoprotein is an antibody.

[0495] 298. The composition of embodiment 297, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0496] 299. The cell culture composition of any one of embodiments 297 to 298, wherein the antibody is ocrelizumab.

[0497] 300. The cell culture composition of embodiment 296, wherein the host cell is a mammalian cell.

[0498] 301. The cell culture composition of embodiment 300, wherein the host cells are CHO cells.

[0499] 302. A method for modulating glycosylation of a glycoprotein of interest, comprising culturing a host cell expressing the glycoprotein of interest in a cell culture medium, wherein the cell culture is exposed to high pCO2, the cell culture is exposed to an extended medium retention time, and / or the cell culture contains an increased Na+ concentration; and wherein glycosylation of the glycoprotein of interest is modulated compared to fucosylation of a preparation of the glycoprotein of interest expressed by the host cell in a cell culture medium exposed to low pCO2, a shortened medium retention time, and / or a reduced Na+ concentration.

[0500] 303. The method of embodiment 302, wherein the glycoprotein of interest is an antibody.

[0501] 304. The method of embodiment 303, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0502] 305. The method of any one of embodiments 303-304, wherein the antibody is ocrelizumab.

[0503] 306. The method of embodiment 302, wherein the host cell is a mammalian cell.

[0504] 307. The method of embodiment 306, wherein the host cell is a CHO cell.

[0505] 308. The method according to any one of embodiments 302 to 307, wherein glycosylation is modulated to achieve increased G0-F (fucosylation-deficient G0) while decreasing G0 (fucosylated G0).

[0506] 309. A cell culture composition comprising a cell culture medium comprising high pCO2, extended medium retention time and / or increased Na+ concentration; and a host cell engineered to express a glycoprotein of interest.

[0507] 310. The cell culture composition of embodiment 309, wherein the composition further comprises a glycoprotein of interest.

[0508] 311. The cell culture composition of embodiment 310, wherein the glycoprotein is an antibody.

[0509] 312. The cell culture composition of embodiment 311, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0510] 313. The cell culture composition of any one of embodiments 311-312, wherein the antibody is ocrelizumab.

[0511] 314. The cell culture composition of embodiment 309, wherein the host cell is a mammalian cell.

[0512] 315. The cell culture composition of any one of embodiments 314, wherein the host cells are CHO cells.

[0513] 316. A composition comprising a glycoprotein of interest, the preparation comprising a cell culture medium comprising high pCO2, extended medium retention time and / or increased Na+ concentration; a host cell engineered to express the glycoprotein of interest; and the glycoprotein of interest.

[0514] 317. The composition of embodiment 316, wherein the glycoprotein is an antibody.

[0515] 318. The composition of embodiment 317, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0516] 319. The cell culture composition of any one of embodiments 317 to 318, wherein the antibody is ocrelizumab.

[0517] 320. The cell culture composition of embodiment 316, wherein the host cell is a mammalian cell.

[0518] 321. The cell culture composition of embodiment 320, wherein the host cells are CHO cells. EXAMPLES

[0519] The following examples are merely illustrative of the subject matter disclosed herein and should not be construed as limiting in any way.

[0520] Example 1: Adjustment of raw materials to control glycosylation Multiple cell culture factors are known to have the potential to affect glycosylation of monoclonal antibody therapeutics. These factors include process parameters, media treatments and media components such as galactose and trace metals. Variations in the levels of individual media components can be introduced during the mAb cell culture process through the use of complex raw materials such as Proteose Peptone No. 3 (PP3) and Genentech Essential Media (GEM) powder. These sources of variation in raw materials can result in significant differences in Mn concentrations at the beginning of the production culture (i.e., day 0), as outlined in Table 1. [Table 1]

[0521] Variation in Mn levels at day 0 of the production culture correlates with variation in %G0 (fucosylated G0) and %G0-F (fucosylation-deficient) antibody species (Figure 1). In Figure 1, sets 1 and 2 were performed using medium depth filtration through Mn leaching from the depth filter. Medium depth filtration can contribute significant additional Mn to the production culture medium composition. Day 0 Mn levels were measured by ICP-MS. Set 3 predicted day 0 Mn levels are based on the formula G0=109.57487-8.3488683*ln(Mn), obtained using data from sets 1-2 and 4-6. Fucosylation-deficient is represented by the normalized G0-F=100*[G0-F] / (G0+[G0-F]).

[0522] To improve control over Mn contribution from raw materials, one or both of the following strategies can be implemented: (a) test and select PP3 and GEM powders within the specified Mn range prior to use, and (b) adjust day 0 Mn levels after production culture seeding to within an established acceptable range (e.g., 30 nM-110 nM).

[0523] PP3 and GEM powders are selected based on the Mn ranges in Table 2 and Figure 32. These ranges were established based on the Day 0 Mn in the 2016 v1.0 batch and historical data from 36 lots of PP3 and 34 lots of GEM powder, taking into account losses during media preparation and processing. [Table 2]

[0524] To further ensure process consistency and adjustment of G0, normalized G0-F and CDC values ​​within specifications, action limits can be adjusted to a narrower range of day 0 post-seeding Mn levels of 30-110 nM with either <30 nM or >110 nM.

[0525] Example 2 Manganese supplementation to regulate glycosylation 2.1 Introduction This example summarizes the effect of manganese supplementation on ocrelizumab and other antibodies. With increasing manganese supplementation, an increase in fucosylation-deficient (G0-F) and a decrease in fucosylated (G0) (galactosylation-deficient) species were observed.

[0526] 2.2 Evaluation of manganese supplementation with ocrelizumab Manganese supplementation experiments were performed on ocrelizumab. Manganese (Mn) concentrations in the test cases were adjusted by post-inoculation addition to the ocrelizumab production cultures. The concentrations of manganese tested in these studies are listed in Table 3. The manganese concentrations listed correspond to the amount of additional manganese added to the cultures on a post-inoculation volume basis and do not reflect the total manganese concentration at day 0 due to the presence of manganese in the control process medium. Manganese additions were made using filtered sterile solutions of 0.05 mM and 0.5 mM manganese sulfate monohydrate and added through a septum. Controls and replicates of some test cases were included in each study. [Table 3] a The manganese concentrations evaluated correspond to manganese added during the ocrelizumab production culture process. Low levels of manganese are present in the control medium; b The 0.05 mM manganese sulfate solution used in these cases

[0527] Figure 2 shows the correlation between day 0 manganese concentration and fucosylation-deficient (normalized G0-F) and galactosylation-deficient (G0) in cell cultures. Figure 3 shows the impact of manganese supplementation on ocrelizumab fucosylation-deficient (normalized G0-F) and fucosylated galactosylation-deficient (G0) species. As the concentration of manganese increases, ocrelizumab G0-F increases and G0 decreases. Figures 2 and 3 show the same trends regarding the impact of manganese on fucosylation-deficient and galactosylation-deficient across bioreactor scales, demonstrating that the findings at the small scale (2L) can be extended. Notably, an increase in normalized G0-F and a decrease in G0 were observed in Mn-supplemented cultures compared to unsupplemented cultures (no Mn addition) at both 2L and 12kL bioreactor scales.

[0528] 2.2 Manganese supplementation and pCO for ocrelizumab 2 Evaluation Scale-dependent factor model (high pCO2 for 36 h at 37 °C) 2 Manganese titrations at 0, 50, 100, 150, 250, 350, 500, 750, 1000 and 2000 nM manganese were performed in both the 2L model and the standard 2L model. Media for this study were high temperature short time (HTST) heat treated with the following HTST conditions: 102°C, 10 second hold, 15 psig back pressure and cooled to 37°C after HTST. All other conditions and parameters were run at target conditions (i.e., with the same set points).

[0529] The results for fucosylation-deficient (normalized G0-F) and fucosylated galactosylation-deficient (g0) are shown in Figures 4 and 5. 2 In the presence of low pCO levels, the standard 2L model 2A larger effect was observed for standardized G0-F and G0 compared to G0-F. The trends in this study are consistent with the manganese titration study (see 2.2 above). As shown in Figures 2-5, bioreactors with various volumes (e.g., standard 2 L, 2 L scale-dependent factor, and 12,000 L) can be used for the cell culture medium composition. The cell culture medium conditions can be adjusted based on the volume of the bioreactor. For example, the cell culture medium composition used in the 2 L bioreactor can be scaled up for use in a 15,000 L bioreactor. Additionally, the cell culture medium composition used in the 15,000 L bioreactor can be scaled down for use in a 2 L bioreactor. The volume of the bioreactor can be from about 1 L to 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).

[0530] 2.3 Evaluation of manganese concentration for antibody I A full factorial experimental design (2 × 2 × 3) was designed investigating three factors: 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 goal of the 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. Results for G0-F (fucosylation-deficient) and G0 (G0F) are shown in Figure 6. Consistent with other antibodies, an increase in fucosylation-deficient and a decrease in G0 were observed with increasing manganese concentration. The results were consistent across all cell ages and iron concentrations, indicating that the effect of manganese is independent of the other tested parameters. [Table 4]

[0531] 2.4 Evaluation of manganese supplementation for antibody II The study design consisted of a full factorial experimental design combining three two-level variables: copper supplementation level, manganese supplementation level, and zinc supplementation level (Table 5). All proposed process conditions (containing copper, manganese, and zinc supplemented at target levels) were tested in duplicate. The results of this experiment are shown in Figure 7. Manganese had the largest effect estimates for both G0 and G0-F. The trends in G0 and G0-F with increasing manganese levels are consistent with the other antibodies. [Table 5]

[0532] 2.5 Evaluation of Manganese Supplementation for Antibody III A manganese titration study was performed on Antibody III. 0.5 mM manganese sulfate stock solution was added after production seeding, calculated based on the final working volume. The concentrations added in addition to the actual measured manganese from the test cases are shown in Table 6. As shown in Case 1, there is traces of manganese in the cell culture medium without additional manganese added to the production culture. While some variability exists in the measured manganese levels, in general, levels measured using inductively coupled plasma mass spectrometry confirmed that the correct amount of manganese stock solution was added to each bioreactor after production seeding. All control runs were performed within expectations. Further manganese supplementation had no effect on growth and titer. As expected, with increasing Mn, fucosylated (G0) (galactosylation deficient) decreased and fucosylation deficient (G0-F) increased. Results are shown in Figure 8. [Table 6]

[0533] 2.6 Evaluation of manganese supplementation for Antibody IV and Antibody V Antibody IV and Antibody V were evaluated in combination with zinc, manganese, iron and copper using a full factorial experimental design to determine the effect of zinc, manganese, iron and copper on cell culture process performance and product quality of Antibody IV and Antibody V. Table 7 shows the design of these studies. The results for Antibody IV are shown in FIG. 9 and the results for Antibody V are shown in FIG. 10. Figures 9 and 10 show the actual measured manganese. Due to the presence of manganese in the basal medium, this is different compared to the supplemented amount of manganese listed in Table 7. As the manganese concentration increases, G0-F (fucosylation deficient) increases and G0 (fucosylated galactosylation deficient) decreases for both Antibody IV and Antibody V. This effect is independent of zinc, iron and copper concentrations. [Table 7]

[0534] 2.7 Evaluation of the timing of manganese supplementation for antibody VI To evaluate the effect of different timing of addition on glycosylation, Mn addition timing experiments were performed on antibody VI. In the first study (Figure 11), cultures were supplemented with 500 nM manganese (during the previous growth passage or on day 0 or day 3 of the production culture) or not. In the second study (Figure 12), cultures were supplemented with 80 nM manganese (on day 0 or daily during the production culture) or not. Regardless of the timing of Mn supplementation, addition of Mn to cell cultures decreased G0 (fucosylated galactosylation deficient) and increased normalized G0-F (fucosylation deficient).

[0535] It will be understood that the foregoing is merely illustrative of the principles of the disclosure and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the disclosure. For example, and not by way of limitation, the volume of the bioreactor used in this example can be from about 1 L to 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 are pCO 2 The medium may be altered to adjust the levels of: medium holding time, culture duration, osmolarity, Na+, Mn, culture temperature, fucose, galactose, or combinations thereof.

[0536] [Example 3] Pre-high temperature short time (HTST) heat treatment pH target 3.1 Introduction This example summarizes the selection of and supporting experimental results for pH adjustment targets for medium prior to high temperature, short time (HTST) heat treatment for ocrelizumab (rhuMAb 2H7) production medium preparation. A lower pre-HTST heat treatment medium pH target can reduce medium turbidity, associated precipitate formation, HTST heat transfer surface fouling, and filter clogging during HTST operations (see, e.g., U.S. Patent No. 9,493,744).

[0537] Trends of increasing pressure measured before the cooler and decreasing flow rates were observed for several media preparations (Figure 13). These trends were found to correlate with increasing manganese (Mn) losses across the HTST and filtration operations (Table 9). The increase in HTST pressure (before the cooler) and the decrease in HTST flow rates in these examples can be attributed to turbidity / precipitate formation and subsequent pressure increase on the media filter (increased filter clogging). To mitigate the variability of this atypical HTST profile in ocrelizumab manufacturing runs and the decrease in Mn losses across HTST and filtration, a lower pre-HTST pH media adjustment target along with a post-HTST pH adjustment was evaluated and implemented for ocrelizumab production media. [Table 9]

[0538] All manganese measurements were performed using an inductively coupled plasma mass spectrometry (ICP-MS) assay method.

[0539] 3.2 Evaluation of manganese losses from HTST heat treatment and filtration with different pH adjustment targets An initial screening study with a benchtop Sand Bath HTST heat treatment method was used to evaluate the turbidity change and manganese loss after heat treatment over a wide range of pH targets. Ocrelizumab production culture medium was used for this study. The Sand Bath HTST heat treatment method represents the worst case conditions for HTST heat treatment due to the extended heat treatment time required to reach 102°C compared to the HTST production skid. The turbidity change before and after heat treatment and manganese loss over heat treatment / filtration are shown in Figure 14. Below the pH adjustment target of 7.00 for the production medium before HTST, no significant increase in turbidity or decrease in Mn loss was observed. At pH adjustment targets of 7.00 or higher for the production medium before HTST, no greater increase in turbidity and Mn loss was observed. This indicates that adjusting the medium pH to a target below 7.00 prior to HTST heat treatment can be expected to reduce Mn loss over the production HTST heat treatment and filtration operations.

[0540] A preliminary scale HTST study was conducted to evaluate three pre-HTST pH adjustment targets of about 6.30 (non-pH adjusted medium), 6.70, and 7.10. Ocrelizumab production culture medium was used for this study. Mn measurements and Mn losses throughout HTST and filtration are shown in Table 10. Consistent with the Sand Bath HTST heat treatment study, the about 6.30 and 6.70 pre-HTST pH cases demonstrated less Mn loss throughout HTST and filtration compared to the pH 7.10 case, indicating that a lower pre-HTST pH target may help reduce Mn losses observed throughout the production HTST and filtration operations. [Table 10]

[0541] 3.3 Effect of Pre-HTST pH Adjustment on 2L Cell Culture Performance and Product Quality Production medium processed in the preliminary scale HTST study was used in two 2L experiments. After HTST heat treatment and before filtration, the production medium was adjusted to a final pH target of 7.10 + / - 0.10. This pH adjustment step would occur after HTST and filtration and target 7.15 + / - 0.10. A control was included in each experiment using medium prepared with the same Proteose Peptone 3 (PP3) and Genentech Basal Medium (GEM) powder 2 raw material lots, without HTST heat treatment. N-1 and N medium retention at 36 hours and higher pCO 2 A select run was performed using a scale-dependent 2L model with a modified scattering strategy to generate levels.

[0542] The following results from these studies are compared to a 2L control run performed during process characterization and validation (PC / PV) as well as a historical manufacturing run. 2L control and manufacturing product quality data are derived from affinity pools on each AO assay. Figure 15 shows the KPIs. Figures 16-18 show product quality. KPIs, charge related variants, size related variants and glycans showed no significant impact from varying the pre-HTST pH target between approximately 6.30, 6.70 and 7.10. These studies indicate that cell culture performance and product quality will not be affected by varying the pre-HTST pH target between 6.30-7.10 with a post-HTST pH adjustment of 7.10+ / -0.10.

[0543] 3.4 Effect of pH adjustment on osmolarity in ocrelizumab production medium The osmolality changes from sodium carbonate addition for pH adjustment are shown in Tables 11 and 12 for the sand bath HTST and initial pre-scale HTST studies. Both the osmolality before HTST heat treatment at a pH of 6.90 and the final osmolality after the final pH adjustment to 7.10 were observed to be within the current production medium target osmolality. [Table 11] [Table 12]

[0544] 3.5 Ocrelizumab Production Medium Pre-HTST pH and Osmolality Targets and Target Range Recommendations Based on the results summarized in this example, the recommended ocrelizumab production medium pre-HTST pH adjustment target is 6.90+ / -0.10 or 6.70+ / -0.10 with an osmolality warning range of 320-350 mOsm / kg. Results of the small-scale study 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 production 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 HTST heat treatment, filtration and weighing of the 12,000 L bioreactor, the ocrelizumab production medium will require pH adjustment to a final target pH of 7.15+ / -0.10. At the time of the final pH adjustment in the 12,000 L bioreactor, an osmolality check should be performed to ensure the medium is within the target osmolality of 340 + / - 20 mOsm / kg prior to inoculation with the production culture.

[0545] 3.6 Use of Lower Pre-HTST pH Set Points for Additional mAbs During the preparation of production basal media for further mAb examples, antibody III experienced atypical HTST performance, as observed for ocrelizumab (Figure 13). To mitigate future HTST performance issues and to prevent manganese loss due to atypical HTST performance, media pH before HTST heat treatment and filtration was evaluated for further mAbs. Media was prepared to pH targets of 7.1, 6.6 and 6.1. Each media preparation was split for use with and without HTST heat treatment. Figure 19B shows manganese results from media preparations. Blue symbols indicate manganese levels in HTST heat treated media. There is a clear trend of increased media pH target and decreased manganese after HTST heat treatment. This indicates that at higher media pH, higher manganese loss is observed over HTST heat treatment and filtration. This result is consistent with the results performed for ocrelizumab.

[0546] A 2L study was conducted to evaluate the impact of lower media pH before HTST heat treatment and filtration on cell culture performance and product quality. KPI results are shown in Figures 19C and 19D. Product quality results are shown in Figures 19E-19H. This study shows that cell culture performance and product quality for the additional mAb would not be affected by changing the pre-HTST pH target from 6.10 to 7.10.

[0547] Based on the results of this study, the pre-HTST heat treatment and filtration medium pH target for additional mAbs was reduced to 6.6.

[0548] [Example 4] pCO2, manganese, medium retention, osmolarity and Na+ to regulate fucosylation 4.1 Introduction pCO 2 Among the cell culture process parameters that have unknown effects on glycosylation (e.g., galactosylation and / or fucosylation defects), the partial pressure of carbon dioxide in the culture medium (pCO2 ) is of great interest and therefore has similar pCO 2 Maintaining the pCO profile is a challenge often encountered during process scale-up. Previous studies have shown that 2 have shown that levels of riboflavin can 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). These studies demonstrated that pCO 2 did not demonstrate an effect of pCO 2 The profiles are not representative of the pCO2 encountered in large-scale bioreactor cultures.

[0549] To better understand and establish robust regulation of fucosylation deficiency across bioreactor scale, we performed pCO2 deficiency assays on fucosylation deficiency of mAbs produced by recombinant Chinese Hamster Ovary (CHO) cell lines. 2 The effects of pCO2 on the oxidative stress response and its possible interactions with other process measures were investigated. The following approach was applied: (1) Different levels of pCO2 were administered while holding other process parameters constant. 2 (2) Construct a small-scale (3-L) bioreactor model capable of maintaining pCO 2and other process parameters, such as manganese and its interaction with medium retention, on mAb fucosylation deficiency, and (3) the effect of pCO 2 and other process measures to investigate possible mechanisms behind any observed effects. Mn was chosen because it is a cofactor for multiple 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 has not previously been shown to have any effect on fucosylation defects and is not known to be a cofactor for α1,6-fucosyltransferase (FUT8). Medium retention was studied because production medium was retained in the bioreactor prior to seeding in large-scale operations and its impact on product quality attributes has not been previously reported.

[0550] 4.2 Materials and Methods 4.2.1 Cell culture The same recombinant CHO cell line expressing mAbs of the immunoglobulin G1 (IgG1) subclass was used in all studies reported herein. Cells were thawed and grown to inoculate production cultures in 3-L glass bioreactors (Applikon) as previously described (Yuk et al., (2015), Biotechnol Prog, 31(1), 226-237). Set points for temperature, pH and dissolved oxygen (DO) in the bioreactor were regulated by a Finesse SmartController (Thermo Fisher Scientific) with a TruBio DeltaV. Temperature, pH and DO for all production cultures were maintained at 37°C, 7.15 and 30% (of air saturation) on day 1, 34°C, 7.15 and 30% between days 1 and 3, and 34°C, 7.00 and 30% DO from day 3 onwards. Three days after inoculation, concentrated nutrient feed was added to the production culture at 1:7 (v / v).

[0551] The following parameters were examined: Mn supplementation, L-fucose supplementation, medium retention, pCO 2 The basal medium used for the inoculum train and production cultures contained nominal amounts of Mn and no L-fucose. Supplemental Mn and / or L-fucose were added immediately after inoculation of the production cultures to achieve the target day 0 concentrations as described for this study. Air sparging (10 sccm), agitation (75 rpm) and unilateral (CO 2 Medium retention was performed by maintaining the basal medium (used for the N-1 inoculum train and production culture) in separate 3-L bioreactors for 36 h at 37° C. with pH adjustment (only). Osmolarity was adjusted by adding stock solutions of NaCl (100 g / L) or sorbitol (182 g / L). 4.2.2 High and low pCO 2 Model Configuration

[0552] Different pCO2 To achieve this profile, the bioreactor configuration was modified to achieve a high pCO 2 Model (Figure 20A) and low pCO 2 A model (Figure 20B) was created. In both models, CO was introduced through an open pipe (5 mm inner diameter) to reduce the pH. 2 The pH was adjusted by spraying with 100% NaCl and by adding Na2CO3 to increase the pH as previously described (Hsu et al., (2012), Cytotechnology 64(6):667-678).

[0553] High pCO 2 For the model (Figure 20A), the working volume of the bioreactor was ≥ 1.9 L. DO was measured by air / O2 through a small sparger (15 μm pore size). 2 The DO regulator was set to use air to regulate DO with a minimum output of 2 sccm, and after the air output reached 12 sccm, the DO regulator was switched to O with a minimum output of 2 sccm. 2 Switched to.

[0554] low pCO 2 For the model (Figure 20B), the working volume of the bioreactor was 1.5 L or less. 2 Air / O through the same open pipe used for 2 DO was adjusted by sparging with O. The DO regulator settings used an air output with a minimum sparge of 10 sccm and a maximum sparge of 50 sccm. After the air output reached 50 sccm, O 2 Sparging increased and air sparging decreased. 2 When the sparge reaches 50 sccm, turn off the air and increase the O2, if necessary, up to a maximum of 250 sccm. 2 Output increased.

[0555] 4.2.3 Cell culture analysis Packed cell volume (PCV), viable cell concentration, culture viability, pH, DO, pCO 2, glucose, lactate, osmolality, Na+, ammonium, mAb product titer, glycosylation variants, charge variants, size variants and Mn concentrations (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 glycosylated species are detailed in Thomann et al. (2016) and illustrated in Figure 28A. Fucosylation defects were normalized to G0F and defined as follows: TIFF2025037936000014.tif16170

[0556] 4.2.4 Intracellular pH analysis SNARF-4F 5-(and -6-) carboxylic acid, acetoxymethyl ester acetate (SNARF-4F) (Molecular Probes; Cat #S23921, Thermo Fisher Scientific) was used to measure intracellular pH (pHi). SNARF-4F is a fluorinated derivative of carboxy SNARF-1 and has a pKa value of approximately 6.4. The pHi measurement and calculation methods were 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).

[0557] To prepare for pH measurements, fresh media was pre-equilibrated to the desired conditions for a minimum of 6 hours. 2 For titration cases, the desired pCO 2 CO to reach the level 2The medium was equilibrated in a TAP ambr 15 system (Sartorius Stedim Biotech) controlled at 37°C and 600 rpm agitation with sparging. The pH was then adjusted to 7.0 with 0.5 M Na2CO3 and the osmolarity was adjusted to approximately 400 mOsm / kg with 100 g / L NaCl. For osmolarity titration cases, the medium osmolarity was adjusted with 100 g / L NaCl and incubated at 37°C, 5% CO 2 The cells were placed overnight in an incubator conditioned with 50 rpm agitation (2.5 cm orbit) and 500 µg / mL. The cells were pelleted (500,000 cells, 200 g, 2 min) and washed twice in phosphate-buffered saline (PBS). The pellet was quickly resuspended in fresh medium pre-equilibrated to the desired conditions and SNARF-4F was added (1.5 µg / mL final concentration). The cell-dye mixture was incubated (30 min) under the same conditions as the pre-equilibrated medium. pHi was immediately measured 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, and β-acetylglucosamine were measured using a Nova Bioprofile FLEX. 2 , osmolality and Na + A pH calibration curve was generated using cells stained in known pH buffers in the presence of nigericin (Sigma Cat #N7143, Sigma-Aldrich) as previously described (Salvi et al., (2002), AAPS PharmSci, 4(4), 1-8).

[0558] 4.2.5 Proteomic analysis For proteomic analysis by mass spectrometry (MS), cells from day 7 and day 12 production cultures were sedimented (10 million cells, 200 g, 2 min), washed twice with PBS, flash frozen on dry ice, and stored at -80 °C until analysis. Proteins from each sample were extracted, digested into peptides, labeled with tandem mass tags (TMTs), and analyzed by SPS-MS3 methodology using an Orbitrap Lumos mass spectrophotometer (Thermo Scientific) as previously described (Vildhede et al., (2018), Drug Metab Dispos, 46(5), 692-696) (McAlister et al., (2014), Anal Chem, 84(16), 7150-7158).

[0559] Assignment of MS / MS spectra was performed using the MASCOT search algorithm to search against all entries for Cricketturus griseus (Chinese hamster) in UniProt (downloaded in June 2016). A search of tryptic peptides (two residual cleavages) was performed and a precursor tolerance of 50 ppm was used to limit the number of candidate peptides while a 0.8 Da error tolerance was used to match the MS / MS data collected in the ion trap. Static modifications included TMT on the N-terminus of the peptide and lysine residues (+229.16293) as well as cysteine ​​alkylation (57.0215), whereas variable modifications included methionine oxidation (15.9949) and TMT labeling of tyrosine (229.1629). Peptide spectral matches were filtered to a 2% false discovery rate using a target-decoy approach that was scored using a linear discriminant analysis algorithm before being filtered to a 2% false discovery rate at the protein level as previously described (Kirkpatrick et al., (2013), PNAS, 110(48), 19462-19431).

[0560] Quantitative values ​​were extracted and corrected for isotopic impurities using Mojave (Zhuang et al., (2013), Sci Signal, 6(271), 1-11). Furthermore, quantitative events with a precursor purity less than 0.7 (±0.25 Da) or a total intensity less than 50,000 were discarded before quantitative values ​​were normalized and converted to "relative abundance" values ​​using custom scripts in R. Relative abundance values ​​were calculated for each protein by dividing the sample intensity by the total intensity for that protein and then normalizing the result to 100. After data normalization, principal component analysis (PCA) was performed using custom scripts in R. To determine pathway enrichment within the dataset, UniProt identifiers were converted to homologous mouse, rat or human identifiers and processed by Ingenuity Pathway Analysis (IPA; QIAGEN Inc.) as previously described (Kramer et al., (2014), Bioinformatics, 30(4), 523-530).

[0561] 4.3 Results and Discussion 4.3.1 Low and high pCO 2 Different pCO 2 To develop a small-scale bioreactor model capable of maintaining CO levels in a 3-L bioreactor, 2 The stripping rate was controlled (Figure 20). pCO 2 To change the concentration of NaHCO in the medium, 3 The concentration can be adjusted (Goudar et al., (2006), Biotechnol Bioeng, 96(6), 1107-1117; Zhu et al., (2005), Biotechnol Prog, 21(1), 70-77), while keeping the stirring and vessel aspect ratio constant. 2 Stripping (and therefore pCO 2 Instead, the bioreactor gas sparge rate was controlled because this is an effective way to regulate the concentration of acetic acid (CO2) and the amount of CO2 released.

[0562] Sufficient k to support cellular metabolic demand for oxygen L To achieve low gas sparging rates while maintaining a high pCO 2 A fritted microsparger for DO regulation in the model was used. The small sparger results in a small bubble size and therefore increases the total gas-liquid surface area interface. 2 Increase the residence time of CO 2 To further reduce stripping (Matsunaga et al., (2009), Journal of Bioscience and Bioengineering, 107(4), 419-424), higher working volumes (>1.9 L) and higher pCO 2 The model was run. Low pCO 2 The model is CO 2 To increase stripping, an open pipe sparger and a lower working volume (1.5 L or less) were used. These differences in bioreactor geometry resulted in a significant difference in pCO between the two models. 2 This resulted in the desired separation of pCO levels (Figure 21B). 2 Even though NaHCO has been shown to affect hybridoma cell growth, viability and antibody production (deZengotita et al., (1998), Cytotechnology, 28, 219-227), no negative effects were observed on these characteristics and product quality for the CHO cell line used in this study (Figure 29). This is because NaHCO 3 pCO2 concentration was not manipulated for CHO cultures in the same small-scale bioreactor. 2 This is believed to be the first report to demonstrate control of levels.

[0563] 4.3.2 pCO for fucosylation deficiency 2 Levels, medium retention and effects of supplemented Mn High and low pCO 2Using a bioreactor model, we evaluated the effect of pCO on fucosylation deficiency in a full factorial design of experiments (DOE). 2 The effects of level, medium retention and supplemental Mn and potential interactions between these were examined. Consistent with the established effect of Mn on galactosylation (Gramer et al., (2011), Biotechnol Bioeng, 108(7), 1591-1602), the galactosylation-deficient species (G0F) was reduced in the supplemental Mn background (Figure 21). 2 With Mn supplementation in the background, the fucosylation defect was about 1% higher (Figure 1). This result is unexpected, since Mn is not known to have any effect on fucosylation. For example, several other divalent cations significantly inhibited FUT8 activity, but Mn did not (Kaminska et al., (1998), Glycoconjuagte Journal, 15, 783-788). The fucosylation defect was not observed in cultured cells in the presence of supplemented Mn or in cultured cells in the presence of high pCO 2 The fucosylation defect was due to supplemental Mn, medium retention and high pCO 2 was highest in the presence of (approximately 4%), indicating an interaction between these three factors.

[0564] This observed interaction was consistent with high and low pCO 2 Using both models, multiple levels of Mn supplementation were confirmed (Figure 22). An increase in fucosylation deficiency was observed at all Mn levels, with high pCO2 retention in the medium. 2 This example demonstrates the role of Mn, media retention, and pCO in regulating fucosylation defects. 2 The effects of Mn, media retention and pCO 2 Each level alone can regulate fucosylation defects, but when they act in combination their effect on fucosylation defects is magnified. 4.3.3 High pCO for fucosylation deficiencies 2 , osmolality and Na + Remove confounding factors from the effect of

[0565] low pCO 2 Higher pCO compared to the model 2 In the model, higher culture osmolality and Na + was observed (Figures 22C-22D). This is due to the fact that the CO in the solution is 2 is equilibrated to H + and HCO 3 - This can be explained by the fact that CO 2 +H 2 O ⇔ H + +HCO 3 - formula 1

[0566] In a pH-controlled environment, a base (Na in this study) is needed to neutralize H+. 2 CO 3 ) is added to the bioreactor, which shifts the equilibrium to the right side of Equation 1, decreasing the osmolality and HCO 3 - Increases Na concentration (deZengotita et al., (2002), Biotechnol Bioeng, 77(44), 369-380). + Levels are also Na 2 CO 3 These observations suggest that increased fucosylation of mAb increases the pCO 2 , osmolality or Na + This raises the question of whether the

[0567] The effect of high osmolarity on fucosylation defects 2 Attempts to eliminate the effect of increasing pCO 2and / or osmolarity range from minimal effect on fucosylation defects (Kimura et al., (1997), Biotechnol Prog, 13, 311-317; Schmelzer et al., (2002), Biotechnol Prog, 18, 346-353) to a decrease in fucosylation defects with increasing osmolarity (Konno et al., (2012), Cytotechnology, 64, 249-265). In these previous studies, production cultures were incubated with high pCO 2 and / or osmolality, which was initiated by pCO 2 and is not representative of typical conditions in bioreactors where osmolarity is initially low and then increases during production culture (Hsu et al., (2012), Cytotechnology 64(6):667-678).

[0568] In an environment more representative of a typical CHO bioreactor culture, pCO 2 The effect of osmolality and Na + To distinguish between the effects of 2 To match the time course osmolality profile of the production culture in the model, low pCO 2 Osmolarity was titrated in the model (Figure 23). In all cases, fucosylation defects increased with increasing osmolarity. At both target maximum osmolarity levels, NaCl titration (low pCO 2 ) cases and high pCO 2 Fucosylation defects were similar among cases, whereas fucosylation defects were not associated with sorbitol titration (low pCO 2 ) cases were approximately 1% lower. These results suggest that high pCO 2 The increased fucosylation defect observed in the model was due to high pCO 2 or Na used for pH adjustment rather than osmolality alone. 2 CO 3 Na from the addition of + This is likely due to increased concentrations.

[0569] 4.3.4 Intracellular pH changes: high pCO 2 and hyperosmolarity / Na + High pCO 2 and / or Na + To investigate the mechanism behind the increase in greater fucosylation defects in the context of 2 and osmolality / Na + pH when subjected to levels i was measured against a recombinant CHO cell line. 2 diffuses across the cell membrane (Endeward et al., (2014), Frontiers in Physiology, 4, 1-21) and reaches an equilibrium state inside the cell, as described in Equation 1, and therefore pH i In addition, the addition of base (Na 2 O) to regulate extracellular pH can be used to reduce the 2 CO 3 ) can affect pH through 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). i Changes in pH can affect enzyme expression (Bumke et al., (2003), Proteomics, 3(5), 675-688). Furthermore, each enzyme has a pH range for optimal activity. Therefore, (high pCO 2 and / or Na + (by pH) i A shift in can affect the expression and / or activity of enzymes involved in fucosylation.

[0570] To test this hypothesis, we used different levels of pCO 2 and osmolarity (using NaCl to titrate osmolarity) for recombinant CHO cell lines, pH i The pH was measured. ipCO 2 It decreased with increasing levels (Figure 24A), and the osmolality / Na + (Figure 24B). These data indicate that pCO 2 and osmolality / Na + Both are pH i This is consistent with previous findings (deZengotita et al., (2002)., Biotechnol Bioeng, 77(44), 369-380; Reusch et al., (1995), American Physiological Society, C147-C153).

[0571] 4.3.5 Global Proteomics Analysis Mn, medium retention and high pCO for fucosylation deficiency 2 / Na + To clarify the mechanisms behind the effects of Mn, untargeted proteomics was performed. Production cultures were subjected to four different conditions expected to affect fucosylation defects to various degrees (Figure 25A). PCA (Figure 26B) showed a clear separation of samples by day (7 vs. 12) and treatment (i.e., cell culture conditions). IPA demonstrated that pathways related to glucose and amino acid metabolism (glycolysis, gluconeogenesis, methionine degradation and cysteine ​​biosynthesis) were significantly affected by Mn, medium retention and high pCO 2 (Figure 26C). Among the glycolytic enzymes, fructose bisphosphate aldolase showed the highest upregulation of differential expression for case iv compared to case i (Figure 26D). + The increase in pH iand can increase the activity of phosphofructokinase, the rate-limiting enzyme in the glycolytic pathway that converts fructose-6-phosphate (Fru-6-P) to fructose 1,6-diphosphate (Fidelman et al., (1982), Am J Physiol, 242(1), C87-93). Enhanced activity of phosphofructokinase can increase the conversion of Fru-6-P to fructose 1,6-diphosphate, thereby decreasing the level of Fru-6-P and upregulating the expression of fructose diphosphate aldolase. Since Fru-6-P is a precursor to GDP-mannose, which is the upstream precursor to GDP-fucose in the de novo synthesis pathway (Figure 27A), a decrease in Fru-6-P would decrease the supply of GDP-mannose and GDP-fucose, thus increasing the fucosylation defect.

[0572] 4.3.6 GDP-fucose synthesis pathway: proteomic analysis and L-fucose supplementation To assess whether GDP-fucose may have been affected in cell culture conditions that produced higher mAb fucosylation defects (i.e., cases ii-iv), differential expression of key enzymes in the de novo and salvage GDP-fucose synthesis pathways was examined (Figure 27A). Downregulation of GMD and L-fucose kinase positively correlated with fucosylation defect levels, with GMD and L-fucose kinase expression being lowest for the culture treatment with the highest fucosylation defect (case iv) (Figure 27B). Consistent changes in FX were observed for cases ii-iv compared to case i.

[0573] Previous studies have shown that knockout of GMD or FX in CHO cell lines reduces GDP-fucose and increases fucosylation deficiency (Kanda et al., (2007), J Biotechnol, 130(30), 300-310; Louie et al., (2016), Biotechnol Bioeng, 114(3), 632-644), and the fucosylation deficiency can 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). In light of the observations obtained from the proteomics studies here and knockout studies elsewhere, it can be hypothesized that the increase in fucosylation deficiency due to high pCO2 and / or supplemental Mn is at least in part due to the limitation of GDP-fucose. To examine this hypothesis, we measured the pCO2-induced fucosylation deficiency and / or the fucosylation deficiency due to the fucosylation deficiency. 2 A full factorial experimental design was performed testing supplemented Mn and supplemented L-fucose (Figure 27C). The effect of L-fucose on fucosylation deficiency saturated after approximately 0.3 g / L in previous titration studies, so 1 g / L L-fucose supplementation was chosen for this evaluation. Supplementing with L-fucose in conditions known to generate high fucosylation deficiency restored the fucosylation deficiency to lower levels without affecting G0F (Figure 31). Taken together, both the proteomic and L-fucose supplementation results indicate that GDP-fucose limitation is a key factor in the regulation of fucosylation in the presence of high pCO 2 , contributing to the relatively high mAb fucosylation defect observed for the combination of medium-retained and supplemented Mn.

[0574] 4.3.7 Proteomic Analysis: FUT8 Considering the key role of FUT8 in fucosylation defects, we analyzed the differential expression of FUT8 in the four culture treatment cases i-iv (Figure 25A). FUT8 was downregulated and negatively correlated with fucosylation defects for cases ii-iv compared to case i (Figure 28B). This suggests that high pCO 2This is believed to be the first study to demonstrate downregulation of FUT8 by medium-maintained and supplemented Mn in CHO cells.

[0575] 4.3.8 Proteomic Analysis: Other Glycosylation Enzymes, pH i , Golgi pH and Golgi Mn concentration To determine whether any additional bottlenecks exist in this segment of the glycosylation pathway, differential expression of glycosylation enzymes upstream (Man I, GnTII) and downstream (GalT3, GalT4 and GalT7) of FUT8 was assessed (Hossler et al., (2009), Glycobiology, 19(9), 936-949; Kremkow et al, (2018), Metabol Eng, 47, 134-142). There was minimal change for Man I and GnTII and inconsistent change for GalT3, GalT4 and GalT7 expression levels across the test cases and culture days (Figure 28B). Even though these glycosylation enzymes did not show differential expression, proteomics cannot ascertain changes in enzyme activity. Since Mn is a cofactor for several enzymes, the activity of these enzymes may be affected by pH i or by changes in intracellular Mn content (Rouiller et al., (2014), Biotechnol Prog, 30(3), 571-583; Gramer et al., (2011), Biotechnol Bioeng, 108(7), 1591-1602). As discussed below, proteomic data support these theories.

[0576] NHE1, pH i Na is involved in regulation + / 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), were upregulated, and fucosylation defects and pCO 2 / Na + These observations were positively correlated with pCO 2 / Na + By pH i and Golgi pH is affected, i This is consistent with the findings of (Figure 24).

[0577] ATP2A1, the ATP-dependent transporter of Mn into the Golgi (Baelen et al., (2004), Biochimica et Biophysica Acta, 1742(1-3), 103-112), is upregulated and associated with fucosylation defects and pCO 2 / Na + GPP130, a Golgi protein whose degradation depends exclusively 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), was downregulated and correlated with defective fucosylation and pCO2 expression. 2 / Na + The results showed that the intracellular Mn level was highest in case iv compared to the other cases. Higher intracellular Mn levels may increase the activity of GnTs and GalTs to favor the overall flow toward fucosylation-deficient glycoforms. Thus, increased Mn transport and intracellular Mn levels may be associated with high pCO 2 In culture conditions of / Na+, supplemented Mn and medium retention, this may contribute to the trend towards higher fucosylation deficiency.

[0578] It will be understood that the foregoing is merely illustrative of the principles of the disclosure and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the disclosure. For example, and not by way of limitation, the volume of the bioreactor used in this example can be from about 1 L to 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 addition, the geometry of the bioreactor is 2 The medium can be altered to adjust holding time, osmolarity, Na+, Mn, temperature, pH, levels of fucose, galactose or combinations thereof.

[0579] [Example 5] Media retention to regulate glycosylation Media retention experiments were performed on antibody VI to evaluate glycosylation changes (e.g., fucosylation deficiency and galactosylation) with increasing media retention time at elevated temperature in the bioreactor. Media for production culture and pre-production growth culture were maintained in the bioreactor for 0, 36, 48 and 72 hours with air sparging, agitation and pH adjustment before being used to inoculate the cultures. Figures 33A-33B show the effect of media retention time at elevated temperature (38°C) on glycosylation. The correlation between media retention time and G0 is shown in Figure 33A. The correlation between media retention time and fucosylation deficiency (e.g., normalized G0-F) is shown in Figure 33B. Furthermore, fucosylation deficiency increased with increasing media retention time. As shown in Figure 33B, the levels of normalized G0-F showed a significant media retention time-dependent increase when media retention was applied to cell culture media. (Figure 33A).

[0580] To evaluate the change in fucosylation deficiency, media retention experiments were performed on antibody III. To evaluate the cumulative effect of media retention on fucosylation deficiency of antibody III, four cases were tested in duplicate bioreactors. Production (N) and / or inoculum train (N-1) media were held at elevated temperature (approximately 37° C.) for 48 hours. The control case represents the use of production and inoculum train media that were not held at elevated temperature prior to use in seeding the bioreactor. As shown in FIG. 33C, the results demonstrate that both N and N-1 media retention alone increase fucosylation deficiency (represented here by %G0-F). The greatest increase in fucosylation deficiency was observed when N media retention was used in combination with N-1 media retention, thereby demonstrating the cumulative effect of media retention on fucosylation deficiency.

[0581] To evaluate the change in fucosylation deficiency, media retention and Mn supplementation experiments were performed on antibody III. To evaluate the individual and combined / synergistic effects of media retention and Mn supplementation on the fucosylation deficiency of antibody III, four cases were tested in duplicate bioreactors. The production medium was held at elevated temperature (about 37° C.) for 48 hours with / without 250 nM Mn supplementation. The control case represents the use of production medium that was not held at elevated temperature prior to use in seeding the bioreactor and the absence of Mn supplementation. For the conditions tested, media retention showed a greater impact on fucosylation deficiency (represented by %G0-F) than Mn supplementation, as shown in FIG. 33D. The greatest increase in fucosylation deficiency was observed when media retention was used in combination with Mn supplementation. Notably, the greatest increase in %G0-F was observed when 48 hour media retention was used in combination with 250 nM Mn supplementation (FIG. 33D). Exposure to medium retention for 48 hours increased the levels of %G0-F. The levels of %G0-F increased when 250 nM Mn was supplemented in the medium exposed to medium retention.

[0582] As shown in FIG. 21A, a normalized increase in G0-F with culture retention time (36-hour retention time) was also observed at 37°C. Culture retention time, pCO 2 An interaction between α,β- and Mn was also observed, resulting in an increase in normalized G0-F that was greater than the sum of the normalized G0-F increases observed for each single element (Figure 21A).

[0583] It will be understood that the foregoing is merely illustrative of the principles of the disclosure and that various modifications can be made by those skilled in the art without departing from the scope and spirit of the disclosure. For example, and not by way of limitation, the volume of the bioreactor used in this example can be from about 1 L to 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 are pCO 2 The medium may be altered to adjust the levels of: medium holding time, culture duration, osmolarity, Na+, Mn, culture temperature, fucose, galactose, or combinations thereof.

[0584] [Example 6] Addition of galactose to regulate glycosylation Three studies were performed to evaluate the addition of galactose, Mn, and combinations thereof on glycosylation of antibody VI. Results from studies 1, 2, and 3 are shown in Figures 34-36. Galactose or Mn, or combinations thereof, can be adjusted to target specific distributions of glycosylation (e.g., G0 and normalized G0-F). Increasing galactose and / or Mn levels resulted in lower galactosylation deficiency (G0) and higher fucosylation deficiency (normalized G0-F). Furthermore, at lower levels of galactose, G0 was more sensitive to changes in Mn levels compared to higher levels of galactose. Similarly, at lower levels of Mn, G0 was more sensitive to changes in galactose levels compared to higher levels of Mn. Notably, an unexpected synergistic decrease in %G0 was observed when different concentrations of galactose were added in combination with Mn supplementation (Figures 34A and 35A). Each galactose and Mn supplementation dose-dependently decreased the level of G0. However, when galactose and Mn were added together in the culture medium, the level of G0 was significantly and synergistically decreased. When both galactose and Mn were added together in the culture medium, the level of G0-F showed a relatively lower change (Figures 34B and 35B).

[0585] [Example 7] Fucose supplementation and culture temperature to regulate glycosylation 7.1 Introduction This example summarizes the effect of fucose supplementation on glycosylation. The higher the level of fucose supplementation and / or the earlier the fucose supplementation, the greater the reduction in fucosylation defects (e.g., G0-F). An interaction with culture temperature was observed, with a greater effect on fucosylation defects observed at lower culture temperatures.

[0586] 7.2 Evaluation of fucose concentration In three antibody VI studies, the effect of fucose addition on glycosylation was evaluated. Figures 378A-378B show the effect of fucose concentration on fucosylation deficiency (e.g., G0-F) and galactosylation (e.g., G0). Increasing fucose levels resulted in higher fucosylation deficiency (normalized G0-F).

[0587] 7.3 Evaluation of the timing of fucose addition Antibody VI was used to evaluate the effect of fucose addition timing on glycosylation at two different fucose levels. Fucose was added as a post-seeding addition to the production culture at five different time points. Figures 38A-38B show the effect of fucose addition timing on fucosylation deficiency (e.g., G0-F) and galactosylation (e.g., G0). The earlier the fucose addition, the greater the reduction in G0-F observed. G0 was not affected by the timing of fucose addition.

[0588] 7.4 Evaluation of fucose supplementation and its interaction with temperature A central composite design study was performed with antibody VI to evaluate the effect of fucose concentration and temperature, and the interaction of fucose and temperature, on glycosylation. Fucose was added as a post-seeding addition on day 0 of the production culture. Figures 39A-39B show the effect of fucose and temperature on fucosylation deficiency (e.g., G0-F) and galactosylation (e.g., G0). Increasing fucose concentration and decreasing temperature resulted in lower fucosylation deficiency (e.g., G0-F) levels. An unexpected interaction between fucose and temperature was observed,

[0589] Fucose has a greater effect on fucosylation defects (e.g., G0-F) at lower temperatures than at higher temperatures. As shown in Figures 39A-39B, more fucose was required at elevated temperatures to reach the same G0-F levels. Decreasing the temperature resulted in higher G0 levels.

Claims

1. 1. A method for modulating the glycosylation pattern of a glycoprotein of interest in a cell culture, comprising: The following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: a. High partial pressure CO ２ (pCO ２ ) an Mn concentration of about 1 nM to about 20,000 nM under the conditions; b. low pCO ２ a Mn concentration in the condition of about 1 nM to about 30,000 nM; c. A pCO of about 10 mmHg to about 250 mmHg ２ ; d. a pre-inoculation cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25° C. to 39° C.; e. cell culture duration from about 0 days to about 150 days; f. a Na+ concentration of about 0 mM to about 300 mM; g. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; h. a galactose concentration of about 0 mM to about 60 mM; i. a fucose concentration of about 0 mM to about 60 mM; and j. an incubation temperature of about 29° C. to about 39° C.; The method includes adjusting the

2. 10. The method of claim 1, wherein the cell culture environment is in a bioreactor with or without cells.

3. The low pCO ２ The condition is about 10 to about 100 mmHg, and the high pCO ２ The method of claim 1 or claim 2, wherein the conditions are from about 20 to about 250 mmHg.

4. pCO ２ The method of claim 3 , wherein the conditioning period encompasses at least the first half of the cell culture period.

5. The method according to any one of claims 1 to 4, wherein the glycoprotein of interest is a recombinant protein.

6. The method according to any one of claims 1 to 5, 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 domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

7. The method of any one of claims 1 to 6, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

8. The method of any one of claims 1 to 7, wherein the antibody is an anti-CD20 antibody.

9. The method of any one of claims 1 to 8, wherein the anti-CD20 antibody is ocrelizumab.

10. The antibody or antibody fragment, i) %G0-F (percent fucosylated glycoproteins) 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 ii) a %G0 (percent galactosylation deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%; The method according to claims 6 to 8, wherein

11. The glycosylation a. to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated, galactosylation-deficient G0)); or b. to achieve reduced fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0); or c. to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0); or d. To achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F); The method according to claims 6 to 9, wherein the expression level is regulated.

12. The step of modulating the glycosylation pattern of a glycoprotein of interest comprises the steps of: ２ Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO ２ Under conditions comprising a Mn concentration of about 1 nM to about 20,000 nM and the following parameters, alone or in any combination, in the cell culture medium and / or in the cell culture environment: a. a pre-inoculation cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25° C. to 39° C.; b. A cell culture period of about 0 days to about 150 days; c. a Na+ concentration of about 0 mM to about 300 mM; d. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; e. a galactose concentration of about 0 mM to about 60 mM; f. a fucose concentration of about 0 mM to about 60 mM; and g. an incubation temperature of about 29° C. to about 39° C.; The method according to any one of claims 1 to 11, comprising regulating

13. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises the step of: ２ Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO ２ Under conditions of about 1 nM to about 20,000 nM Mn concentration and the following parameters in the cell culture medium and / or cell culture environment: a. a pre-inoculation cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25° C. to 39° C.; and b. A cell culture period of about 0 days to about 150 days; The method of claim 12, comprising regulating

14. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises the step of: ２ Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO ２ Under conditions of about 1 nM to about 20,000 nM Mn concentration and the following parameters in the cell culture medium and / or cell culture environment: a. a galactose concentration of about 0 mM to about 60 mM; and / or b. a fucose concentration of about 0 mM to about 60 mM; The method of claim 12, comprising regulating

15. The step of modulating the glycosylation pattern of said glycoprotein of interest comprises adjusting the pre-seeding cell culture medium holding period and the following parameters, alone or in any combination, in the cell culture medium and / or in the cell culture environment: a. High partial pressure CO ２ (pCO ２ ) an Mn concentration of about 1 nM to about 20,000 nM under the conditions; b. low pCO ２ a Mn concentration in the condition of about 1 nM to about 30,000 nM; c. A pCO of about 10 mmHg to about 250 mmHg ２ ; d. A cell culture period of about 0 days to about 150 days; e. a Na+ concentration of about 0 mM to about 300 mM; f. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; g. a galactose concentration of about 0 mM to about 60 mM; h. a fucose concentration of about 0 mM to about 60 mM; and i. an incubation temperature of about 29° C. to about 39° C.; [0033] 15. The method of any one of claims 1 to 14, wherein the cell culture medium holding period is from about 0 hours to about 120 hours at a temperature of from about 25°C to 39°C.

16. The step of modulating the glycosylation pattern of said glycoprotein of interest comprises adjusting the pre-seeding cell culture medium holding period and the following parameters in the cell culture medium and / or in the cell culture environment: a. High partial pressure CO ２ (pCO ２ ) at a Mn concentration of about 1 nM to about 20,000 nM or low pCO ２ a Mn concentration in the condition of about 1 nM to about 30,000 nM; b. A pCO of about 10 mmHg to about 250 mmHg ２ and c. A cell culture period of about 0 days to about 150 days; [0033] 16. The method of claim 15, wherein the cell culture medium holding period is from about 0 hours to about 120 hours at a temperature of from about 25° C. to 39° C.

17. The method according to any one of claims 12 to 15, wherein the glycoprotein of interest is an antibody or an antibody fragment thereof.

18. The method of claim 17, wherein the antibody or antibody fragment thereof is an anti-CD20 antibody.

19. 19. The method of claim 18, wherein the anti-CD20 antibody is ocrelizumab.

20. The step of modulating the glycosylation pattern of said glycoprotein of interest comprises adjusting the cell culture duration and any of the following parameters, alone or in any combination, in the cell culture medium and / or in the cell culture environment: a. a Na+ concentration of about 0 mM to about 300 mM; b. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; c. a galactose concentration of about 0 mM to about 60 mM; d. a fucose concentration of about 0 mM to about 60 mM; and e. an incubation temperature of about 29° C. to about 39° C.; [0033] The method according to any one of claims 1 to 19, wherein the cell culture period is from about 0 days to about 150 days.

21. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises adjusting a Na+ concentration from about 0 nM to about 300 nM and any of the following parameters, alone or in any combination, in the cell culture medium and / or in the cell culture environment: a. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; b. a galactose concentration of about 0 mM to about 60 mM; c. a fucose concentration of about 0 mM to about 60 mM; and d. an incubation temperature of about 29° C. to about 39° C.; [0033] 21. The method of any one of claims 1 to 20, wherein the Na+ concentration is from about 0 mM to about 300 mM.

22. The step of modulating the glycosylation pattern of the glycoprotein of interest comprises adjusting the Na concentration and pCO2 concentration from about 10 mmHg to about 250 mmHg. ２ The method according to any one of claims 1 to 21, comprising regulating

23. The step of modulating the glycosylation pattern of said glycoprotein of interest comprises adjusting the osmolarity and any of the following parameters in the cell culture medium and / or in the cell culture environment, alone or in any combination: a. a galactose concentration of about 0 mM to about 60 mM; b. a fucose concentration of about 0 mM to about 60 mM; and c. an incubation temperature of about 29° C. to about 39° C.; [0033] 23. The method of any one of claims 1 to 22, wherein the osmolality is from about 250 mOsm / kg to about 550 mOsm / kg.

24. modulating the glycosylation pattern of the glycoprotein of interest, a. Low pCO ２ Adjusting the Mn concentration to about 1 nM to about 30,000 nM under conditions of high pCO ２ adjusting the Mn concentration to about 1 nM to about 20,000 nM under the conditions; b. adjusting the Na+ concentration from about 0 mM to about 300 mM; and c) adjusting the pre-seeding cell culture medium holding period from about 0 hours to about 120 hours.

25. The modulation of the glycosylation pattern of the glycoprotein of interest comprises maintaining an osmolality of about 250 mOsm / kg to about 550 mOsm / kg and a pCO2 of about 10 mmHg to about 250 mmHg. ２ The method of claim 1 , comprising regulating

26. said modulation of the glycosylation pattern of said glycoprotein of interest comprising: a. an incubation temperature of about 29° C. to about 39° C.; and b. a galactose concentration of about 0 mM to about 60 mM; and / or a fucose concentration of about 0 mM to about 60 mM; The method of claim 1 , comprising regulating

27. The modulation of the glycosylation pattern of the glycoprotein of interest comprises a pCO ２ and adjusting the fucose concentration from about 0 mM to about 60 mM.

28. 2. The method of claim 1, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating a fucose concentration of about 0 mM to about 60 mM and a culture temperature of about 29°C to about 39°C.

29. The modulation of the glycosylation pattern of the glycoprotein of interest is ２ Concentration and any of the following parameters, alone or in any combination, in the cell culture medium and / or in the cell culture environment: a. High partial pressure CO ２ (pCO ２ ) an Mn concentration of about 1 nM to about 20,000 nM under the conditions; b. low pCO ２ a Mn concentration in the condition of about 1 nM to about 30,000 nM; c. a pre-inoculation cell culture medium holding period of about 0 hours to about 120 hours at a temperature of about 25° C. to 39° C.; d. A cell culture period of about 0 days to about 150 days; e. a Na+ concentration of about 0 mM to about 300 mM; f. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; g. a galactose concentration of about 0 mM to about 60 mM; h. a fucose concentration of about 0 mM to about 60 mM; and i. an incubation temperature of about 29° C. to about 39° C.; [0033] The pCO ２ 10. The method of claim 1, wherein the concentration is from about 10 mmHg to about 250 mmHg.

30. The Mn concentration is high pCO ２ About 1 nM to about 20,000 nM in culture; high pCO ２ In culture, about 1 nM to about 10000 nM, about 1 nM to about 5000 nM, about 1 nM to about 4000 nM, about 1 nM to about 3000 nM, about 1 nM to about 2000 nM, about 1 nM to about 1000 nM; high pCO ２ 30. The method of any one of claims 1 to 29, wherein the concentration in the culture is about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 10,000 nM, about 20 nM to about 20,000 nM, about 20 nM to about 300 nM, or about 30 nM to about 110 nM.

31. The Mn concentration is low pCO ２ about 1 nM to about 30,000 nM in culture; ２ In culture, about 1 nM to about 20,000 nM; about 1 nM to about 10,000 nM, about 1 nM to about 5,000 nM, about 1 nM to about 4,000 nM, about 1 nM to about 3,000 nM, about 1 nM ~about 2000nM, about 1nM to about 1000nM; about 1nM to about 500nM, about 1nM to about 100nM, about 1nM to about 50nM, about 1nM to about 20nM, about 20nM to about 10 31. The method of any one of claims 1 to 30, wherein the agonist is 0 nM, about 20 nM to about 300 nM, about 20 nM to about 500 nM, about 20 nM to about 1000 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 5000 nM, about 20 nM to about 10000 nM, about 20 nM to about 20000 nM, or about 30 nM to about 110 nM.

32. 32. The method of any one of claims 1 to 31, wherein adjusting the Mn concentration comprises determining the Mn content in a cell culture feedstock and selecting a feedstock lot for adjusting the Mn concentration.

33. 33. The method of any one of claims 1 to 32, wherein adjusting the Mn concentration comprises: (i) adjusting the cell culture medium or a material in contact with the cell culture, to adjust the Mn concentration; or (ii) taking into account the concentration of Mn leached into the cell culture medium or during the cell culture; or a combination of (i) and (ii).

34. 34. The method of claim 33, wherein the leached Mn is produced by contacting a cell culture and / or cell culture medium with: (i) a filter; (ii) a media preparation, holding or culture vessel; or (iii) a combination of (i) and (ii).

35. 35. The method of claim 34, wherein the filter includes, but is not limited to, a depth filter, a column, a membrane, and a disk.

36. 35. The method of claim 34, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resin.

37. 37. The method of any one of claims 1 to 36, wherein the cell culture medium is a basal medium, a reconstituted medium, a feed medium, a hydrolysate, a supplement, a serum or an additive.

38. 38. The method of any one of claims 1 to 37, wherein the cell culture medium is replenished during the production stage of the cell culture.

39. 39. The method of any one of claims 1 to 38, wherein the cell culture medium is replenished prior to the production stage of the cell culture.

40. 40. The method of any one of claims 1 to 39, wherein the cell culture medium comprises one or more of Mn, fucose, galactose and / or Na+, and the supplementation is based on a predefined schedule or criteria.

41. 41. The method of any one of claims 1-40, wherein one or more of Mn, fucose, galactose and Na+ are supplemented as a bolus, as intermittent supplementation, as continuous supplementation, as semi-continuous supplementation, as feedback loop based supplementation, or as a combination of one or more of these.

42. 42. The method of any one of claims 1-41, wherein the cell culture medium consists essentially of one or more of: i) Mn; ii) fucose; iii) galactose; and / or iv) Na+.

43. 43. The method of any one of claims 1-42, wherein the adjusting the Mn concentration comprises utilizing 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.

44. The modulation of the glycosylation pattern of the glycoprotein of interest is ２ The method of any one of claims 1 to 43, comprising regulating

45. The cell culture or the cell culture medium is in a bioreactor, and ２ is achieved by adjusting the bioreactor working volume; the bioreactor gassing strategy; the bioreactor agitation strategy; the bioreactor medium exchange strategy, the bioreactor perfusion strategy, the bioreactor feeding strategy or any combination thereof.

46. The pCO ２ Regulation of high pCO ２ 45. The method of claim 44, comprising establishing the culture.

47. The pCO ２ 47. The method of claim 46, wherein the blood flow is from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 150 mmHg, or from about 30 mmHg to about 250 mmHg.

48. The pCO ２ Regulation of low pCO ２ 48. The method of any one of claims 1 to 47, comprising establishing a culture.

49. The pCO ２ 49. The method of claim 48, wherein the blood flow is from about 10 mmHg to about 100 mmHg, from 10 mmHg to about 80 mmHg, from about 20 mmHg to about 70 mmHg, or from about 30 mmHg to about 60 mmHg.

50. The pCO ２ 50. The method of any one of claims 1 to 49, wherein the modulation occurs on day 0 of culture.

51. The pCO ２ 51. The method of claim 50, wherein the modulation occurs for approximately the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

52. The pCO ２ 51. The method of claim 50, wherein the modulation occurs for about the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

53. 53. The method of claim 52, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the pre-seeding cell culture medium holding period, wherein the pre-seeding cell culture medium holding period 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.

54. 54. The method of claim 53, wherein the temperature of the medium during the pre-seeding cell culture medium holding is from about 25°C to about 39°C, from about 30°C to about 39°C, from about 35°C to about 39°C, or from about 36°C to about 39°C.

55. 55. The method of any one of claims 1 to 54, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture duration, wherein the cell culture duration 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.

56. 56. The method of any one of claims 1-55, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the Na+ concentration, wherein the Na+ concentration is from about 0 mM to about 300 mM; from about 20 mM to about 20 mM; from about 30 mM to about 150 mM; or from about 40 mM to about 130 mM.

57. The adjustment of the Na+ concentration is ２ CO ３ , NaHCO ３ 57. The method of any one of claims 1 to 56, comprising supplementing the cell culture with Na compounds, including but not limited to, NaOH, NaCl or combinations thereof.

58. 58. The method of any one of claims 1 to 57, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the osmolality, wherein the osmolality is from about 250 mOsm / kg to about 550 mOsm / kg; from about 300 mOsm / kg to about 450 mOsm / kg; or from about 325 mOsm / kg to about 425 mOsm / kg.

59. 59. The method of any one of claims 1 to 58, wherein adjusting the osmolarity comprises supplementing the cell culture with an osmolarity-adjusting medium component.

60. 60. The method of claim 59, wherein the osmotically regulated medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

61. 60. The method of claim 59, wherein the osmolarity-adjusting media components are replenished as a bolus, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop based replenishment, or as a combination of one or more of these.

62. 62. The method of any one of claims 1 to 61, wherein modulating 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.

63. 63. The method of any one of claims 1 to 62, wherein modulating 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.

64. 64. The method of any one of claims 1 to 63, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture temperature, wherein the cell culture temperature is from about 29°C to about 39°C, from about 30°C to about 39°C, from about 31°C to about 38°C, or from about 34°C to about 38°C.

65. 65. The method of claim 64, wherein the cell culture temperature is adjusted during the production stage of the cell culture.

66. 66. The method of any one of claims 1 to 65, wherein the cell culture temperature is adjusted prior to the production stage of the cell culture.

67. 67. The method of any one of claims 1 to 66, wherein the cell culture temperature is adjusted based on a predefined schedule or criteria.

68. 68. The method of any one of claims 1 to 67, wherein the cell culture comprises eukaryotic cells.

69. 69. The method of claim 68, wherein the eukaryotic cell is an insect, avian, fungal, plant or mammalian cell.

70. 70. The method of claim 69, wherein the fungal cell is a yeast, Pichia or any filamentous fungal cell.

71. 71. The method of claim 70, wherein the yeast cell is a S. cerevisiae cell.

72. 70. The method of claim 69, wherein the mammalian cell is a CHO cell.

73. 73. The method of any one of claims 1-72, wherein the cell culture is in a bioreactor, including but not limited to a single-use technology (SUT) bag or bioreactor; a WAVE bioreactor; a stainless steel bioreactor; a flask; a tube and a chamber.

74. 74. The method of any one of claims 1 to 73, wherein the volume of the cell culture is between 1 mL and 35,000 L.

75. The volume of the cell culture is 1 ml to 10 ml, 1 ml to 50 ml, 1 ml to 100 ml, 1 ml to 200 ml, 1 ml to 300 ml, 1 ml to 500 ml, 1 ml to 1000 ml, 1 ml to 2000 ml, 1 ml to 30 ml. 00ml, 1ml to 4000ml, 1ml to 5000ml, 1ml to 1l, 1ml to 2l, 1ml to 3l, 1ml to 4l, 1ml to 5l, 1ml to 6l, 1ml to 10l, 1ml to 20l, 1ml to 30l, 1ml to 4 75. The method of claim 74, wherein the volume is 0 L, 1 mL to 50 L, 1 mL to 60 L, 1 mL to 70 L, 1 mL to 100 L, 1 mL to 200 L, 1 mL to 300 L, 1 mL to 400 L, 1 mL to 500 L, 1 mL to 1000 L, 1 mL to 2000 L, 1 mL to 3000 L, 1 mL to 4000 L, 1 mL to 5000 L, 1 mL to 10,000 L, 1 mL to 20,000 L, 1 mL to 30,000 L, 1 mL to 30,000 L, or 1 mL to 35,000 L.

76. a. %G0-F (percent fucosylated glycoproteins) 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 b. %G0 (percent galactosylation deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%; Use of the method according to any one of claims 1 to 75 for obtaining a glycoprotein of interest which exhibits

77. The glycosylation a. to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated, galactosylation-deficient G0)); or b. to achieve reduced fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0); or c. to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0); or d. To achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F); The use of the method according to any one of claims 1 to 75, which is regulated.

78. 1. A method for preparing a cell culture medium, a feed medium, a hydrolysate or an additive, comprising the steps of: a. High partial pressure CO ２ (pCO ２ ) a Mn concentration in the culture of about 1 nM to about 20,000 nM; b. low pCO ２ A Mn concentration in the culture of about 1 nM to about 30,000 nM; c. A pCO of about 10 mmHg to about 250 mmHg ２ ; d. a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours; e. cell culture duration from about 0 days to about 150 days; f. a Na+ concentration of about 0 mM to about 300 mM; g. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; h. a galactose concentration of about 0 mM to about 60 mM; i. a fucose concentration of about 0 mM to about 60 mM; and j. an incubation temperature of about 29° C. to about 39° C.; and adjusting the amount of A method wherein said cell culture medium, feed medium, hydrolysate or additive modulates the glycosylation pattern of a glycoprotein of interest.

79. 79. The method of claim 78, wherein the glycoprotein of interest is an antibody or an antibody fragment.

80. 80. The method of claim 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%.

81. Glycosylation of the antibody or antibody fragment a. to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated, galactosylation-deficient G0)); or b. to achieve reduced fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0); or c. to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0); or d. To achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F); 80. The method of claim 79, wherein said

82. 82. The method of any one of claims 78-81, comprising adjusting the Mn concentration from about 1 nM to about 30000 nM and the pre-seeding cell culture medium holding time from about 0 hours to about 120 hours.

83. The pCO2 of about 10 mmHg to about 250 mmHg. ２ 83. The method of any one of claims 78-82, comprising adjusting the Na+ concentration from about 0 mM to about 300 mM and the pre-seeding cell culture medium holding time from about 0 hours to about 120 hours.

84. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO ２ and adjusting the Na+ concentration from about 0 mM to about 300 mM.

85. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO ２ 85. The method of any one of claims 78-84, comprising adjusting the Na+ concentration from about 0 mM to about 300 mM and the pre-seeding cell culture medium holding time from about 0 hours to about 72 hours.

86. The pCO2 of about 10 mmHg to about 250 mmHg. ２ and adjusting the Na+ concentration from about 0 mM to about 300 mM.

87. said osmolarity being between about 250 mOsm / kg and about 550 mOsm / kg and said pCO2 being between about 10 mmHg and about 250 mmHg. ２ The method of any one of claims 78 to 86, comprising regulating

88. The pCO2 of about 10 mmHg to about 250 mmHg. ２ 88. The method of any one of claims 78-87, comprising adjusting the Mn concentration from about 1 nM to about 30,000 nM, the cell culture period from about 0 days to about 150 days, and the pre-seeding cell culture medium holding period from about 0 hours to about 120 hours.

89. 89. The method of any one of claims 78 to 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.

90. 90. The method of any one of claims 78 to 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.

91. The fucose concentration is about 0 mM to about 60 mM and the pCO ２ The method of any one of claims 78 to 90, comprising regulating

92. The fucose concentration is about 0 mM to about 60 mM, the Mn concentration is about 1 nM to about 30,000 nM, and the pCO ２ The method of any one of claims 78 to 91, comprising regulating

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

94. 94. The method of any one of claims 78-93, comprising adjusting the fucose concentration from about 0 mM to about 60 mM and the cell culture period from about 0 days to about 150 days.

95. The Mn concentration is high pCO ２ About 1 nM to about 20,000 nM in culture; high pCO ２ About 1 nM to about 1000 nM in culture; high pCO ２ about 20 nM to about 300 nM in culture; or high pCO ２ The method of any one of claims 78 to 94, wherein the concentration is about 30 nM to about 110 nM in culture.

96. The Mn concentration is low pCO ２ About 1 nM to about 30,000 nM in culture; low pCO ２ About 1 nM to about 3000 nM in culture; low pCO ２ About 20 nM to about 300 nM in culture; or low pCO ２ The method of any one of claims 78 to 95, wherein the concentration is about 30 nM to about 110 nM in culture.

97. 97. The method of claim 95 or claim 96, wherein adjusting the Mn concentration comprises determining the Mn content in a cell culture feedstock and selecting a feedstock lot for adjusting the Mn concentration.

98. 97. The method of claim 95 or claim 96, wherein adjusting the Mn concentration comprises: i) adjusting the cell culture medium or a material in contact with the cell culture to adjust the Mn concentration; or (ii) taking into account the concentration of Mn leached into the cell culture medium or during cell culture; or a combination of (i) and (ii).

99. 99. The method of claim 98, wherein the leached Mn is produced by contacting the cell culture and / or cell culture medium with: (i) a filter; (ii) a media preparation, holding or culture vessel; or (iii) a combination of (i) and (ii).

100. 100. The method of claim 99, wherein the filter includes, but is not limited to, a depth filter, a column, a membrane, and a disk.

101. 100. The method of claim 99, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resin.

102. 97. The method of claim 95 or claim 96, wherein the adjusting the Mn concentration comprises utilizing 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.

103. The pCO ２ The method according to any one of claims 78 to 102, wherein the is regulated.

104. The cell culture medium is in a bioreactor and ２ The method of claim 103, wherein adjusting is accomplished by adjusting a bioreactor working volume; a bioreactor gas sparging strategy; a bioreactor agitation strategy; a bioreactor feeding strategy; a bioreactor perfusion strategy; a bioreactor medium exchange strategy or any combination thereof.

105. The pCO ２ Regulation of high pCO ２ 104. The method of claim 103, comprising establishing the culture.

106. The pCO ２ is from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 150 mmHg, or from about 30 mmHg to about 150 mmHg.

107. The pCO ２ Regulation of low pCO ２ 104. The method of claim 103, comprising establishing the culture.

108. The pCO ２ is from about 10 mmHg to about 100 mmHg, from 10 mmHg to about 80 mmHg, from about 20 mmHg to about 70 mmHg, or from about 30 mmHg to about 60 mmHg.

109. The pCO ２ 104. The method of claim 103, wherein the modulation occurs on day 0 of culture.

110. The pCO ２ 104. The method of claim 103, wherein the conditioning occurs for approximately the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

111. The pCO ２ 104. The method of claim 103, wherein the modulation occurs for about the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

112. 112. The method of any one of claims 78-111, wherein the pre-seeding cell culture medium holding period 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.

113. 113. The method of claim 112, wherein the temperature of the medium during the pre-seeding cell culture medium holding is from about 25°C to about 39°C, from about 30°C to about 39°C, from about 35°C to about 39°C, or from about 36°C to about 39°C.

114. 114. The method of any one of claims 78-113, wherein the cell culture period 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.

115. 115. The method of any one of claims 78-114, 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.

116. The adjustment of the Na+ concentration is ２ CO ３ , NaHCO ３ 116. The method of claim 115, comprising supplementing the cell culture with Na compounds, including but not limited to, NaOH, NaCl, or combinations thereof.

117. 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.

118. 118. The method of claim 117, wherein adjusting the osmolarity comprises supplementing the cell culture with an osmolarity-adjusting medium component.

119. 119. The method of claim 118, wherein the osmotically regulated medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

120. The method of any one of claims 787-119, wherein the galactose concentration is from about 0 mM to about 60 mM or from about 0 mM to about 50 mM.

121. 120. The method of any one of claims 78 to 119, wherein the fucose concentration is from about 0 mM to about 60 mM, from about 0 mM to about 40 mM, from about 0 mM to about 20 mM, or from about 0 mM to about 10 mM.

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

123. Use of a medium according to any one of claims 78 to 121 in a eukaryotic cell fermentation process for the production of recombinant proteins.

124. 124. Use of a medium according to claim 123, wherein said 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 (dock and lock trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

125. 125. Use of the medium according to claim 124, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

126. 125. Use of the medium described in claim 124, wherein the antibody is an anti-CD20 antibody.

127. 125. Use of the medium described in claim 124, wherein the anti-CD20 antibody is ocrelizumab.

128. 125. Use of a medium according to claim 124, wherein said antibody or said antibody fragment exhibits a %G0-F (percent fucosylation-deficient glycoprotein) of about 0% to about 20%; about 1% to about 15%; about 1% to about 10%; or about 1% to about 8%; 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 (percent galactosylation-deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%.

129. 125. Use of the medium according to claim 124, wherein the eukaryotic cells are insect, avian, fungal, plant or mammalian cells.

130. 130. Use of a medium according to claim 129, wherein the fungal cell is a yeast, a Pichia or any filamentous fungal cell.

131. 131. Use of the medium of claim 130, wherein the yeast cells are S. cerevisiae cells.

132. 130. The use of the medium of claim 129, wherein the mammalian cells are CHO cells.

133. 133. Use of the medium of any one of claims 123 to 132, wherein the cell culture is in a bioreactor, including but not limited to: a single-use technology (SUT) bag or bioreactor; a WAVE bioreactor; a stainless steel bioreactor; a flask; a tube and a chamber.

134. 134. The use of a medium according to any one of claims 123 to 133, wherein the volume of the cell culture is between 1 mL and 35,000 L.

135. The volume of the cell culture is 1 ml to 10 ml, 1 ml to 50 ml, 1 ml to 100 ml, 1 ml to 200 ml, 1 ml to 300 ml, 1 ml to 500 ml, 1 ml to 1000 ml, 1 ml to 2000 ml, 1 ml to 300 ml. 0ml, 1ml to 4000ml, 1ml to 5000ml, 1ml to 1l, 1ml to 2l, 1ml to 3l, 1ml to 4l, 1ml to 5l, 1ml to 6l, 1ml to 10l, 1ml to 20l, 1ml to 30l, 1ml to 40l , 1 mL to 50 L, 1 mL to 60 L, 1 mL to 70 L, 1 mL to 100 L, 1 mL to 200 L, 1 mL to 300 L, 1 mL to 400 L, 1 mL to 500 L, 1 mL to 1000 L, 1 mL to 2000 L, 1 mL to 3000 L, 1 mL to 4000 L, 1 mL to 5000 L, 1 mL to 10,000 L, 1 mL to 20,000 L, 1 mL to 30,000 L, 1 mL to 30,000 L, or 1 mL to 35,000 L. Use of the medium according to claim 134.

136. a. a host cell engineered to express a glycoprotein of interest; b. i. High partial pressure CO ２ (pCO ２ ) a Mn concentration in the culture of about 1 nM to about 20,000 nM; ii. Low pCO ２ A Mn concentration in the culture of about 1 nM to about 30,000 nM; iii. A pCO of about 10 mmHg to about 250 mmHg ２ ; iv. a pre-seeding cell culture medium holding period of about 0 hours to about 120 hours; v. A cell culture period of about 0 days to about 150 days; vi. a Na+ concentration of about 0 mM to about 300 mM; vii. an osmolality of about 250 mOsm / kg to about 550 mOsm / kg; viii. a galactose concentration of about 0 mM to about 60 mM; ix. a fucose concentration of about 0 mM to about 60 mM; and x. an incubation temperature of about 29°C to about 39°C; and a cell culture and / or cell culture medium adjusted to target one or more predetermined parameters selected from the group consisting of:

137. The composition of claim 136, wherein the cell culture environment is in a bioreactor.

138. The composition of any one of claims 136 to 137, wherein the glycoprotein of interest is an antibody or an antibody fragment.

139. The antibody or antibody fragment, a. %G0-F (percent fucosylated glycoproteins) 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 b. %G0 (percent galactosylation deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%; The composition of claim 138,

140. Glycosylation of the antibody or antibody fragment a. to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated, galactosylation-deficient G0)); or b. to achieve reduced fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0); or c. to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0); or d. To achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F); The composition of claim 138, which is regulated.

141. 137. The composition of claim 136, wherein the Mn concentration is from about 1 nM to about 30,000 nM and the pre-seeding cell culture medium holding period is from about 0 hours to about 120 hours.

142. The pCO ２ is from about 10 mmHg to about 250 mmHg, said Na+ concentration is from about 0 mM to about 300 mM, and said pre-seeding cell culture medium holding period is from about 0 hours to about 120 hours.

143. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO ２ is from about 10 mmHg to about 250 mmHg and the Na+ concentration is from about 0 mM to about 300 mM.

144. The Mn concentration is about 1 nM to about 30,000 nM, and the pCO ２ is from about 10 mmHg to about 250 mmHg, said Na+ concentration is from about 0 mM to about 300 mM, and said pre-seeding cell culture medium holding period is from about 0 hours to about 120 hours.

145. The pCO ２ is from about 10 mmHg to about 250 mmHg and the Na+ concentration is from about 0 mM to about 300 mM.

146. The osmolality is about 250 mOsm / kg to about 550 mOsm / kg, and the pCO ２ The composition of claim 136, wherein the pressure is from about 10 mmHg to about 250 mmHg.

147. The pCO ２ is from about 10 mmHg to about 250 mmHg, said Mn concentration is from about 1 nM to about 30,000 nM, said cell culture duration is from about 0 days to about 150 days, and said pre-seeding cell culture medium retention period is from about 0 hours to about 120 hours.

148. 137. The composition of claim 136, wherein the Mn concentration is from about 1 nM to about 30,000 nM and the galactose concentration is from about 0 mM to about 60 mM.

149. The composition of claim 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 30,000 nM.

150. The fucose concentration is about 0 mM to about 60 mM, and the pCO ２ The composition of claim 136, wherein the pressure is from about 10 mmHg to about 250 mmHg.

151. The fucose concentration is about 0 mM to about 60 mM, the Mn concentration is about 1 nM to about 30,000 nM, and the pCO ２ The composition of claim 136, wherein the pressure is from about 10 mmHg to about 250 mmHg.

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

153. 137. The composition of claim 136, wherein the fucose concentration is from about 0 mM to about 60 mM and the cell culture period is from about 0 days to about 150 days.

154. Mn concentration, high pCO ２ About 1 nM to about 20,000 nM in culture; high pCO ２ In culture, about 1 nM to about 10000 nM, about 1 nM to about 5000 nM, about 1 nM to about 4000 nM, about 1 nM to about 3000 nM, about 1 nM to about 2000 nM, about 1 nM to about 1000 nM; high pCO ２ The composition of any one of claims 136 to 153, wherein the composition is at about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 10,000 nM, about 20 nM to about 20,000 nM, about 20 nM to about 300 nM, or about 30 nM to about 110 nM in culture.

155. The Mn concentration is low pCO ２ About 1 nM to about 30,000 nM in culture; low pCO ２ In culture, about 1 nM to about 20,000 nM; about 1 nM to about 10,000 nM, about 1 nM to about 5,000 nM, about 1 nM to about 4,000 nM, about 1 nM to about 3,000 nM, about 1 nM to about 2000 nM, about 1 nM to about 1000 nM; about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 20 nM to about 100 nM 154. The method of any one of claims 136 to 153, wherein the M is about 20 nM to about 300 nM, about 20 nM to about 500 nM, about 20 nM to about 1000 nM, about 20 nM to about 2000 nM, about 20 nM to about 3000 nM, about 20 nM to about 5000 nM, about 20 nM to about 10000 nM, about 20 nM to about 20000 nM, or about 30 nM to about 110 nM.

156. 156. The composition of claim 154 or claim 155, wherein adjusting the Mn concentration comprises determining the Mn content in a cell culture feedstock and selecting a feedstock lot to adjust the Mn concentration.

157. The composition of claim 154 or claim 155, wherein adjusting the Mn concentration comprises: (i) adjusting the cell culture medium or a material in contact with the cell culture to adjust the Mn concentration; or (ii) taking into account the concentration of Mn leached into the cell culture medium or during cell culture; or a combination of (i) and (ii).

158. 158. The composition of claim 157, wherein the leached Mn is produced by contacting the cell culture and / or cell culture medium with: (i) a filter; (ii) a media preparation, holding or culture vessel; or (iii) a combination of (i) and (ii).

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

160. 159. The composition of claim 158, wherein the filter material includes, but is not limited to, diatomaceous earth, hollow fibers, or resin.

161. 156. The composition of claim 154 or claim 155, wherein Mn is supplemented as a component of a cell culture medium.

162. 162. The composition of claim 161, wherein the cell culture medium is a feed medium, a hydrolysate or an additive.

163. 163. The composition of claim 162, wherein the feed medium, the hydrolysate or the additive comprises Mn.

164. 163. The composition of claim 162, wherein the feed medium or the additive consists essentially of Mn.

165. 156. The composition of claim 154 or claim 155, wherein the Mn is replenished during the production stage of the cell culture.

166. 156. The composition of claim 154 or claim 155, wherein the Mn is supplemented prior to the production stage of the cell culture.

167. 156. The composition of claim 154 or claim 155, wherein the Mn is replenished based on a predefined schedule or criteria.

168. The composition of claim 154 or claim 155, wherein the Mn is supplemented as a bolus dose, as intermittent supplementation, as continuous supplementation, as semi-continuous supplementation, as feedback loop based supplementation, or as a combination of one or more of these.

169. 156. The composition of claim 154 or claim 155, wherein adjusting the Mn concentration comprises utilizing 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.

170. The modulation of the glycosylation pattern of the glycoprotein of interest is ２ The composition of any one of claims 136 to 153, comprising regulating:

171. The cell culture or cell culture medium is in a bioreactor, and ２ The composition of claim 170, wherein the adjustment is achieved by adjusting a bioreactor working volume; a bioreactor gas sparging strategy; a bioreactor agitation strategy; a bioreactor feeding strategy, a bioreactor perfusion strategy, a bioreactor medium exchange strategy, or any combination thereof.

172. The pCO ２ Regulation of high pCO ２ The composition of claim 170, further comprising establishing a culture.

173. The pCO ２ is from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 250 mmHg, from about 20 mmHg to about 150 mmHg, or from about 30 mmHg to about 150 mmHg.

174. The pCO ２ Regulation of low pCO ２ The composition of claim 170, further comprising establishing a culture.

175. The pCO ２ is from about 10 mmHg to about 100 mmHg, from 10 mmHg to about 80 mmHg, from about 20 mmHg to about 70 mmHg, or from about 30 mmHg to about 60 mmHg.

176. The pCO ２ The composition of claim 170, wherein the modulation occurs on day 0 of culture.

177. The pCO ２ 171. The composition of claim 170, wherein the modulation occurs for approximately the majority of the cell culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

178. The pCO ２ 171. The composition of claim 170, wherein the modulation occurs for approximately the majority of the production culture; for about the first 5 days; for about the first 7 days; or for about the first 10 days.

179. The composition of any one of claims 1136-153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the pre-seeding cell culture medium holding period, wherein the pre-seeding cell culture medium holding period 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.

180. 180. The composition of claim 179, wherein the temperature of the medium during the pre-seeding cell culture medium holding is from about 25°C to about 39°C, from about 30°C to about 39°C, from about 35°C to about 39°C, or from about 36°C to about 39°C.

181. The composition of any one of claims 136-153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture duration, wherein the cell culture duration 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.

182. The composition of any one of claims 136-153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating 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.

183. The adjustment of the Na+ concentration is ２ CO ３ , NaHCO ３ 183. The composition of claim 182, comprising supplementing the cell culture with a Na compound, including, but not limited to, NaOH, NaCl, or a combination thereof.

184. The composition of claim 182, wherein Na+ is replenished during the production stage of the cell culture.

185. The composition of claim 182, wherein Na+ is replenished prior to the production stage of the cell culture.

186. The composition of claim 182, wherein Na+ is replenished based on a predefined schedule or criteria.

187. The composition of claim 182, wherein Na+ is replenished as a bolus dose, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop based replenishment, or as a combination of one or more of these.

188. The composition of any one of claims 136-153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the osmolality, wherein the osmolality is from about 250 mOsm / kg to about 550 mOsm / kg; from about 300 mOsm / kg to about 450 mOsm / kg; or from about 325 mOsm / kg to about 425 mOsm / kg.

189. The composition of claim 188, wherein adjusting the osmolarity comprises supplementing the cell culture with an osmolarity-adjusting medium component.

190. 190. The composition of claim 189, wherein the osmotically regulated medium component is NaCl, KCl, sorbitol, an osmoprotectant, or a combination thereof.

191. 190. The composition of claim 189, wherein the osmolarity-adjusting medium components are replenished during the production stage of the cell culture.

192. 190. The composition of claim 189, wherein the osmolarity-adjusting media components are replenished prior to the production stage of the cell culture.

193. 190. The composition of claim 189, wherein the osmotically regulated media components are replenished based on a predefined schedule or criteria.

194. The composition of claim 189, wherein the osmolarity-adjusting media components are replenished as a bolus, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop-based replenishment, or as a combination of one or more of these.

195. The composition of any one of claims 136 to 153, wherein modulating 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.

196. 196. The composition of claim 195, wherein galactose is supplemented as a component of the cell culture medium.

197. 200. The composition of claim 196, wherein the cell culture medium is a feed medium, a hydrolysate or an additive.

198. 200. The composition of claim 197, wherein the feed medium, the hydrolysate or the additive comprises galactose.

199. 200. The composition of claim 197, wherein the feed medium or the additive consists essentially of galactose.

200. 200. The composition of claim 196, wherein the galactose is supplemented during the production stage of the cell culture.

201. 200. The composition of claim 196, wherein the galactose is supplemented prior to the production stage of the cell culture.

202. The composition of claim 196, wherein the galactose is supplemented based on a predefined schedule or criteria.

203. The composition of claim 196, wherein the galactose is supplemented as a bolus dose, as intermittent supplementation, as continuous supplementation, as semi-continuous supplementation, as feedback loop based supplementation, or as a combination of one or more of these.

204. The composition of any one of claims 136-153, wherein modulating 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; 0 mM to about 40 mM; from about 0 mM to about 20 mM; or from about 0 mM to about 10 mM.

205. The composition of claim 204, wherein fucose is supplemented as a component of a cell culture medium.

206. 206. The composition of claim 205, wherein the cell culture medium is a feed medium, a hydrolysate or an additive.

207. 207. The composition of claim 206, wherein the feed medium, the hydrolysate or the additive comprises fucose.

208. 207. The composition of claim 206, wherein the feed medium or the additive consists essentially of fucose.

209. The composition of claim 205, wherein the fucose is replenished during the production stage of the cell culture.

210. The composition of claim 205, wherein the fucose is supplemented prior to the production stage of the cell culture.

211. The composition of claim 205, wherein the fucose is replenished based on a predefined schedule or criteria.

212. The composition of claim 205, wherein the fucose is replenished as a bolus dose, as intermittent replenishment, as continuous replenishment, as semi-continuous replenishment, as feedback loop-based replenishment, or as a combination of one or more of these.

213. 154. The composition of any one of claims 136-153, wherein modulating the glycosylation pattern of the glycoprotein of interest comprises modulating the cell culture temperature, wherein the cell culture temperature is from about 29°C to about 39°C, from about 30°C to about 39°C, from about 31°C to about 38°C, or from about 34°C to about 38°C.

214. 214. The composition of claim 213, wherein the cell culture temperature is regulated during the production stage of the cell culture.

215. 214. The composition of claim 213, wherein the cell culture temperature is adjusted prior to the production stage of the cell culture.

216. The composition of claim 213, wherein the cell culture temperature is adjusted based on a predefined schedule or criteria.

217. The composition of any one of claims 136-153, wherein the cell culture comprises eukaryotic cells.

218. The composition of claim 217, wherein the eukaryotic cell is a fungal cell or a mammalian cell.

219. The composition of claim 218, wherein the fungal cell is a yeast cell.

220. 220. The composition of claim 219, wherein the yeast cell is a S. cerevisiae cell.

221. The composition of claim 218, wherein the mammalian cell is a CHO cell.

222. The composition of any one of claims 136-221, wherein the cell culture is in a bioreactor, including but not limited to a single-use technology (SUT) bag or bioreactor; a WAVE bioreactor; a stainless steel bioreactor; a flask; a tube and a chamber.

223. The composition of any one of claims 136 to 222, wherein the cell culture volume is from 1 mL to 35,000 L.

224. 1. A method for producing a glycoprotein of interest in cell culture, comprising: a. subjecting a cell culture medium suitable for culturing eukaryotic cells to the method of any one of claims 1 to 75; b. seeding the conditioned cell culture medium with eukaryotic cells expressing a recombinant protein; c. culturing the eukaryotic cells so that the recombinant protein is expressed; A method comprising:

225. 225. A method for producing a glycoprotein of interest in a cell culture as described in claim 224, wherein the cell culture is in a bioreactor.

226. The low pCO ２ The condition is about 10 to about 100 mmHg, and the high pCO ２ 225. The method for producing a glycoprotein of interest in cell culture according to claim 224, wherein the conditions are from about 20 to about 250 mmHg.

227. pCO ２ 4. The method for producing a glycoprotein of interest in a cell culture according to claim 3, wherein the period of conditioning comprises at least the first half of the cell culture period.

228. The method of any one of claims 224 to 227, wherein the glycoprotein of interest is a recombinant protein.

229. 229. The method of claim 228, wherein said 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 (dock-and-lock trivalent Fab), sdAb (single domain antibody) and BssdAb (bispecific single domain antibody).

230. The method of claim 229, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

231. The method of claim 229, wherein the antibody is an anti-CD20 antibody.

232. The method of claim 231, wherein the anti-CD20 antibody is ocrelizumab.

233. The antibody or antibody fragment, i) %G0-F (percent fucosylated glycoproteins) 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 ii) a %G0 (percent galactosylation deficient glycoprotein) of about 40% to about 90%; about 50% to about 90%; about 55% to about 85%; or about 60% to about 80%; The method according to any one of claims 224 to 232, wherein

234. The glycosylation a. to achieve increased fucosylation deficiency (e.g., G0-F (fucosylation-deficient G0)) while decreasing galactosylation deficiency (e.g., G0 (fucosylated, galactosylation-deficient G0)); or b. to achieve reduced fucosylation deficiency (e.g., G0-F) while increasing galactosylation deficiency (e.g., G0); or c. to achieve increased or decreased fucosylation deficiency (e.g., G0-F) without affecting galactosylation deficiency (e.g., G0); or d. To achieve increased or decreased galactosylation deficiency (e.g., G0) without affecting fucosylation deficiency (e.g., G0-F); The method of any one of claims 224 to 233, wherein the

235. 1. A method for modulating glycosylation of a glycoprotein of interest, comprising: a. assaying a cell culture medium to determine whether the manganese concentration of the cell culture medium falls within a targeted range; and b. culturing a host cell engineered to express the glycoprotein of interest in a cell culture medium that is within said targeted range; The method, wherein glycosylation of a glycoprotein of interest is regulated compared to glycosylation of the glycoprotein of interest expressed by a host cell in a culture medium falling outside the targeted range of manganese concentrations.

236. The method of claim 235, wherein the glycoprotein of interest is an antibody.

237. The method of claim 236, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

238. The method of any one of claims 236 to 237, wherein the antibody is an anti-CD20 antibody.

239. The method of any one of claims 297 to 298, wherein the anti-CD20 antibody is ocrelizumab.

240. 236. The method of claim 235, wherein the host cell is a mammalian cell.

241. 241. The method of any one of claims 239 to 240, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

242. 236. The method of claim 235, wherein the target range of manganese concentration is from about 30 nM to about 110 nM.

243. 236. The method of claim 235, wherein assaying the cell culture medium comprises assaying a manganese concentration of a component of the cell culture medium.

244. 244. The method of claim 243, wherein the component of the cell culture medium is a hydrolysate or serum.

245. 245. The method of any one of claims 235 to 244, wherein glycosylation is modulated to achieve increased G0-F (fucosylated G0) while decreasing G0 (fucosylated G0).

246. 1. A cell culture composition comprising: a. cell culture medium assayed to determine whether the manganese concentration of the cell culture medium falls within a targeted range; b. a host cell engineered to express a glycoprotein of interest; 1. A cell culture composition comprising:

247. The cell culture composition of claim 246, wherein the composition further comprises a glycoprotein of interest.

248. The cell culture composition of claim 247, wherein the glycoprotein is an antibody.

249. The cell culture composition of claim 248, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

250. The cell culture composition of any one of claims 248-249, wherein the antibody is an anti-CD20 antibody.

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

252. The cell culture composition of claim 246, wherein the host cell is a mammalian cell.

253. 253. The cell culture composition of any one of claims 252, wherein the host cell is a CHO cell.

254. The cell culture composition of claim 246, wherein the target range of manganese concentration is from about 30 nM to about 110 nM.

255. A composition comprising a glycoprotein of interest, the preparation comprising: a. cell culture medium assayed to determine whether the manganese concentration of the cell culture medium falls within a targeted range; b. a host cell engineered to express a glycoprotein of interest; c. a glycoprotein of interest; A composition comprising:

256. The composition of claim 255, wherein the glycoprotein is an antibody.

257. The composition of claim 256, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

258. The cell culture composition of any one of claims 256 to 257, wherein the antibody is an anti-CD20 antibody.

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

260. The cell culture composition of claim 255, wherein the host cell is a mammalian cell.

261. 261. The cell culture composition of claim 260, wherein the host cell is a CHO cell.

262. 1. A method for modulating glycosylation of a glycoprotein of interest, comprising: a. The cell culture medium used in culturing the host cells expressing the glycoprotein of interest is supplemented with high CO ２ supplementing manganese at about 10 nM to about 2000 nM under conditions; or b. The cell culture medium used in culturing the host cells expressing the glycoprotein of interest is provided with low CO ２ supplementing manganese at about 10 nM to about 3000 nM under conditions; The method, wherein glycosylation of the glycoprotein of interest is modulated relative to glycosylation of the glycoprotein of interest expressed by the host cell in a culture medium that has not been so supplemented.

263. The method of claim 262, wherein the glycoprotein of interest is an antibody.

264. The method of claim 263, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

265. The method of any one of claims 263 to 264, wherein the antibody is ocrelizumab.

266. 263. The method of claim 262, wherein the host cell is a mammalian cell.

267. The method of claim 266, wherein the host cell is a CHO cell.

268. 268. The method of any one of claims 262 to 267, wherein glycosylation is modulated to achieve increased G0-F (fucosylated G0) while decreasing G0 (fucosylated G0).

269. 1. A cell culture composition comprising: a. i. High CO ２ About 10 nM to about 2000 nM manganese under conditions, or ii. low CO ２ About 10 nM to about 3000 nM manganese under conditions and a cell culture medium supplemented with b. a host cell engineered to express a glycoprotein of interest; 1. A cell culture composition comprising:

270. The cell culture composition of claim 269, wherein the composition further comprises a glycoprotein of interest.

271. The cell culture composition of claim 269, wherein the glycoprotein is an antibody.

272. The cell culture composition of claim 271, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

273. 273. The cell culture composition of any one of claims 271-272, wherein the antibody is ocrelizumab.

274. 270. The cell culture composition of claim 269, wherein the host cell is a mammalian cell.

275. 275. The cell culture composition of claim 274, wherein the host cell is a CHO cell.

276. A composition comprising a glycoprotein of interest, the preparation comprising: a. A cell culture medium supplemented with manganese, comprising: i. The cultures are treated with high CO ２ About 10 nM to about 2000 nM manganese under conditions, or ii. low CO ２ cell culture medium supplemented with about 10 nM to about 3000 nM manganese under conditions; b. a host cell engineered to express a glycoprotein of interest; c. a glycoprotein of interest; A composition comprising:

277. The composition of claim 276, wherein the glycoprotein is an antibody.

278. The composition of claim 277, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

279. The cell culture composition of any one of claims 276-277, wherein the antibody is ocrelizumab.

280. The cell culture composition of claim 276, wherein the host cell is a mammalian cell.

281. The cell culture composition of claim 280, wherein the host cell is a CHO cell.

282. 1. A method for modulating glycosylation of a glycoprotein of interest, comprising: a. exposing a cell culture medium containing a pH target of 6.30 to 7.25 to a high temperature short time (HTST) heat treatment; and b. culturing a host cell expressing the glycoprotein of interest in a cell culture medium; The method, wherein glycosylation of the glycoprotein of interest is modulated relative to glycosylation of the glycoprotein of interest expressed by a host cell in a culture medium having a pre-HTST heat treatment pH target of greater than pH 7.

25.

283. The method of claim 282, wherein the glycoprotein of interest is an antibody.

284. The method of claim 283, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

285. The method of any one of claims 283 to 284, wherein the antibody is ocrelizumab.

286. 283. The method of claim 282, wherein the host cell is a mammalian cell.

287. 287. The method of claim 286, wherein the host cell is a CHO cell.

288. 288. The method of any one of claims 282 to 287, wherein glycosylation is modulated to achieve increased G0-F (fucosylation-deficient G0) while decreasing G0 (fucosylated G0).

289. 1. A cell culture composition comprising: a. a cell culture medium comprising a target pH of about 6.30 to about 7.25 that has been exposed to a HTST heat treatment; b. a host cell engineered to express a glycoprotein of interest; 1. A cell culture composition comprising:

290. The cell culture composition of claim 289, wherein the composition further comprises a glycoprotein of interest.

291. The cell culture composition of claim 290, wherein the glycoprotein is an antibody.

292. The cell culture composition of claim 291, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

293. 293. The cell culture composition of any one of claims 291-292, wherein the antibody is ocrelizumab.

294. 300. The cell culture composition of claim 293, wherein the host cell is a mammalian cell.

295. 300. The cell culture composition of claim 294, wherein the host cell is a CHO cell.

296. A composition comprising a glycoprotein of interest, the preparation comprising: a. a cell culture medium comprising a target pH of about 6.30 to about 7.25 that has been exposed to a HTST heat treatment; b. a host cell engineered to express a glycoprotein of interest; c. the glycoprotein of interest; A composition comprising:

297. The composition of claim 296, wherein the glycoprotein is an antibody.

298. The composition of claim 297, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

299. 300. The cell culture composition of any one of claims 297-298, wherein the antibody is ocrelizumab.

300. 300. The cell culture composition of claim 296, wherein the host cell is a mammalian cell.

301. The cell culture composition of claim 300, wherein the host cell is a CHO cell.

302. 1. A method for modulating glycosylation of a glycoprotein of interest, comprising: a. culturing a host cell expressing the glycoprotein of interest in a cell culture medium; i. the cell culture is exposed to elevated pCO2; ii. the cell culture is exposed to an extended medium retention time; and / or iii. the cell culture contains an increased Na+ concentration; The method, wherein glycosylation of the glycoprotein of interest is modulated compared to the fucosylation of a preparation of the glycoprotein of interest expressed by a host cell in a cell culture medium exposed to low pCO2, shortened culture medium retention time and / or reduced Na+ concentration.

303. The method of claim 302, wherein the glycoprotein of interest is an antibody.

304. The method of claim 303, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

305. The method of any one of claims 303 to 304, wherein the antibody is ocrelizumab.

306. The method of claim 302, wherein the host cell is a mammalian cell.

307. The method of claim 306, wherein the host cell is a CHO cell.

308. 308. The method of any one of claims 302 to 307, wherein glycosylation is modulated to achieve increased G0-F (fucosylated G0) while decreasing G0 (fucosylated G0).

309. 1. A cell culture composition comprising: a. cell culture medium containing high pCO2, extended medium retention time and / or increased Na+ concentration; b. a host cell engineered to express a glycoprotein of interest; 1. A cell culture composition comprising:

310. The cell culture composition of claim 309, wherein the composition further comprises a glycoprotein of interest.

311. The cell culture composition of claim 310, wherein the glycoprotein is an antibody.

312. The cell culture composition of claim 311, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

313. The cell culture composition of any one of claims 311-312, wherein the antibody is ocrelizumab.

314. The cell culture composition of claim 309, wherein the host cell is a mammalian cell.

315. 315. The cell culture composition of any one of claims 314, wherein the host cell is a CHO cell.

316. A composition comprising a glycoprotein of interest, the preparation comprising: a. cell culture medium containing high pCO2, extended medium retention time and / or increased Na+ concentration; b. a host cell engineered to express a glycoprotein of interest; c. a glycoprotein of interest; A composition comprising:

317. The composition of claim 316, wherein the glycoprotein is an antibody.

318. The composition of claim 317, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

319. The cell culture composition of any one of claims 317-318, wherein the antibody is ocrelizumab.

320. The cell culture composition of claim 316, wherein the host cell is a mammalian cell.

321. The cell culture composition of claim 320, wherein the host cell is a CHO cell.

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