Eukaryotic cell media

Abstract

This disclosure relates to eukaryotic cell culture media useful in the production of recombinant proteins from cultured cell lines. The eukaryotic cell culture media comprise integrated stress response inhibitor (ISRIB), and / or hypotaurine and / or increased level of iron, and / or suramin. The disclosure also provides methods using said eukaryotic cell culture media, e.g. for culturing mammalian cells with enhanced product titer, and / or growth, and / or viability and / or for producing a recombinant protein of comparable or better product quality by culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant protein in said media under conditions suitable for production of the polypeptide. Also provided are use of the eukaryotic cell culture media for the production of a recombinant protein from a cultured cell line comprising a polynucleotide encoding the recombinant protein.

Description

Docket No. P39212-WO-1Eukaryotic Cell MediaCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to United States Patent Application No. 63 / 725,938, filed November 27, 2024, and United States Patent Application No. 63 / 828,731, filed June 23, 2025, each of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] This invention relates to eukaryotic cell culture media useful in the production of recombinant proteins from cultured cell lines. The invention also relates to culturing methods using said eukaryotic cell culture media and uses of said eukaryotic cell culture media.BACKGROUND

[0003] Recombinant therapeutic proteins are predominantly produced using mammalian cells that have been transfected to express the therapeutic product. The primary goal of cell culture processes in biopharmaceutical manufacturing is to produce the recombinant protein at high titers with acceptable product quality. This can be challenging. For example, we have observed that when growth is increased, cell-specific productivity (Qp) can decrease such that the benefits of higher growth are not fully realized by the product titer at harvest. For example, we have observed that when growth is increased, the product quality can be compromised such that the levels of high molecular weight (HMW) species / forms and / or low molecular weight (LMW) species / forms in terms of size variants and the levels of acidic variants and / or basic variants in terms of charge variants can increase. In other words, high titers in biopharmaceutical manufacturing may suffer from poor product quality.

[0004] It is an aim of the present invention to provide new cell culture media that increase titer while providing the intended protein product at high product quality.Docket No. P39212-WO-1BRIEF SUMMARY OF THE DISCLOSURE

[0005] The invention provides cell cultures that improve titer, viability and / or product quality of recombinant proteins.

[0006] In accordance with a first aspect of the present invention, there is provided a eukaryotic cell culture medium comprising integrated stress response inhibitor (ISRIB).

[0007] In accordance with a second aspect of the present invention, there is provided a eukaryotic cell culture medium comprising hypotaurine. In accordance with a related aspect, there is provided a eukaryotic cell culture medium comprising hypotaurine and increased level of iron.

[0008] In accordance with a third aspect of the present invention, there is provided a eukaryotic cell culture medium comprising suramin.

[0009] Without wishing to be bound by theory, it is thought that the cell culture media of the invention give rise to an increase in titer of recombinant protein compared to known cell culture media without negatively impacting the product quality. In particular, the inventors have found that when growth is increased, cell-specific productivity (Qp) can decrease such that the benefits of higher growth are not fully realized by the product titer at harvest. It is thought that the lower Qp observed in production cultures (in presence of high growth or cell density) is due to bottlenecks in the ability of the cells to fully utilize oxidative phosphorylation (OxPhos) for energy production and lack of stimulus to transition from growth to production phase. Additionally, it is thought that endoplasmic reticulum (ER) stress is exacerbated in cells producing the more complex molecules (e.g., bispecifics) as opposed to standard recombinant monoclonal antibodies. Such ER stress can negatively impact not just Qp but also the product quality of the complex molecules (e.g, increase the extent of molecule self-aggregation, as manifested by increase in HMWFs). Thus, without wishing to be bound by theory, it is thought that the media components in the cell culture media of the invention increase titers and maintain desired product quality by improving access to OxPhos and lower ER stress.Docket No. P39212-WO-1

[0010] In a fourth aspect of the present invention, there is provided a method of culturing mammalian cells with enhanced growth and / or viability, comprising culturing the mammalian cells in a lx eukaryotic cell culture medium of the invention.

[0011] In a fifth aspect of the present invention, there is provided a method of producing a recombinant protein, comprising culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant protein under conditions suitable for production of the polypeptide in a lx eukaryotic cell culture medium of the invention.

[0012] In a sixth aspect of the present invention, there is provided a use of a eukaryotic cell culture medium of the invention for culturing a eukaryotic cell, optionally wherein the culturing provides enhanced growth and / or productivity compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0013] In a seventh aspect of the present invention, there is provided a use of a eukaryotic cell culture medium of the invention for culturing a eukaryotic cell, optionally wherein the culturing provides enhanced growth and / or productivity compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.

[0014] In an eighth aspect of the present invention, there is provided a use of a eukaryotic cell culture medium of the invention for the production of a recombinant protein from a cultured cell line comprising a polynucleotide encoding the recombinant protein, optionally wherein the titer of the recombinant protein is enhanced and / or the product quality of the recombinant protein is enhanced compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0015] In a ninth aspect of the present invention, there is provided a use of a eukaryotic cell culture medium of the invention for the production of a recombinant protein from a cultured cell line comprising a polynucleotide encoding the recombinant protein; optionally wherein the titer of the recombinant protein is enhanced and / or the product quality of the recombinant protein is enhanced compared to a said use of a similarDocket No. P39212-WO-1 eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin; and / or optionally wherein the culture viability is enhanced compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin, further optionally wherein the increase in culture viability is about 5-30% higher than with a similar eukaryotic cell culture medium that does not comprise ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0017] Figure 1 depicts the structure of Integrated Stress Response Inhibitor B (ISRIB; for e.g., CAS # 1597403-47-8). ISRIB attenuates the integrated stress response by promoting the assembly of active eIF2B, thereby increasing translation. Inclusion of ISRIB in cell culture medium increased titer 1.3x to 3. lx across 6 molecules tested (see FIG. 2, FIG. 3, and FIG 16).

[0018] Figure 2 demonstrates the improvement of titers and harvest viabilities with the inclusion of ISRIB in cell culture medium across three different recombinant protein molecules. Figure 2A shows the titer in presence of 2 pM ISRIB normalized to the control titer (i.e., without ISRIB), indicating a 1.3x, 1.5x, and 1.8x improvement for molecules 1, 2, and 3, respectively. Figure 2B shows the actual titer obtained for each protein molecule with and without ISRIB. Figure 2C shows the harvest viabilities of the cell lines used to generate the recombinant proteins. ISRIB improved the harvest viabilities for molecules 1, 2, and 3 by 16%, 10%, and 17%, respectively, compared in absolute terms to the control without ISRIB. With the exception of Molecule 1 which harvested Day 8, all other cultures harvested Day 12. The fed-batch production cell culture was conducted in a Sartorius AMBR250 High Throughput Bioreactor system utilizing three different molecules expressed in stable CHO cell lines.

[0019] Figure 3 demonstrates the improvement of titers with the inclusion of ISRIB in cell culture medium across three different recombinant protein molecules. Figure 3 ADocket No. P39212-WO-1 shows the harvest titer in the presence of 2 pM ISRIB normalized to the control titer (i.e., without ISRIB), indicating a 2. Ox, 3.2x, and 1.8x improvement for molecules 4, 5, and 6, respectively. Figure 3B shows the actual harvest titer obtained for the three protein molecules 4, 5, and 6 with and without ISRIB. Figure 3C shows the harvest viabilities of the cell lines used to generate the recombinant proteins. ISRIB improved the harvest viability for molecule 4 by 22% compared in absolute terms to the control without ISRIB. The production cell culture was conducted for 7 days in a shake flask (incubated with agitation at 37°C with 5% CO2) utilizing two different molecules expressed in a stable CHO cell line.

[0020] Figure 4 depicts the structure of hypotaurine (for e.g., CAS # 300-84- 5). Hypotaurine is an antioxidant that serves as a free radical scavenger.

[0021] Figure 5 shows the lack of impact on cell culture performance in the production of a monoclonal antibody in a 14 day standard fed batch process when hypotaurine alone is added to the cell culture medium. Figure 5A shows the viable cell density (VCD) profile (displayed in units of e5 cells / mL) obtained over the duration of the production culture. Figure 5B shows the titer profile obtained over the duration of the production culture. The results demonstrate that addition of hypotaurine (Diamond and Triangle markers) alone does not improve growth or titer when added to cell culture medium as compared to the control (Square and Circle markers). This lack of impact of hypotaurine addition alone on cell growth and monoclonal antibody production (titer) was also previously demonstrated (Vijayasankaran et al., 2018: “Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody” Biotechnology Progress 34(5): 1298-1307).

[0022] Figure 6 shows the positive impact of hypotaurine addition on cell culture performance — in terms of culture viability (Fig 6A), cumulative cell growth calculated as integrated viable cell concentration (IVCC; Fig 6B), and titer (Fig 6C).

[0023] Figure 7 shows hypotaurine has no notable impact on acidic variants (Fig 7A), but decreased basic variants in Cell Line 1 (Figure 7B) to improve main species for Cell Line 1 (Figure 7C).Docket No. P39212-WO-1

[0024] Figure 8 shows hypotaurine improved size variant profilesfor Molecule 8 produced by both Cell Lines 1 and 2. Figure 8A shows a decrease in product aggregates, represented by high molecular weight (HMW) forms, also known as High Molecular Weight Species (HMWS). Figure 8B shows decrease in product fragments for Molecule 8, represented by low molecular weight (LMW) forms, also known as Low Molecular Weight Species (LMWS). As illustrated in Figures 6-8, when hypotaurine was evaluated in combination with other culture parameters (pH shift, b vitamins, and manganese) in a design of experiments (DOE) study, the positive impact on cell culture performance (to boost cell viability, growth and productivity as shown in Figure 6A) and product quality (to decrease charge variants as shown in Figure 7 and size variants as shown in Figure 8) was observed via statistical modelling of the responses for two different cell lines expressing a different recombinant monoclonal antibody, Molecule 8. Cultures marked as -1 did not contain hypotaurine, while those marked as +1 were supplemented with 10 mM hypotaurine.

[0025] Figure 9 shows a comparison of the titer and harvest viability in four cell culture media: a control with lower levels of iron; a control with higher levels of iron; control with higher levels of iron and low hypotaurine concentration (1 mM); and control with higher levels of iron and high hypotaurine concentration (10 mM). Although iron is present in the control cultures (represented by “Control”), the addition of higher levels of iron (represented by “Control + Iron”) boosts the titer by 1.4x relative to the “Control,” as shown in Figure 9A. When the increased iron is combined with hypotaurine (represented by “Control + Iron w / Low Hypotaurine” and “Control + Iron w / High Hypotaurine”) to control product color, unexpectedly, titer (at day 12 harvest) increased beyond the “Control” and “Control + Iron” conditions. Titer was shown to increase in a dosedependent manner with hypotaurine, with low (1 mM) and high (10 mM) hypotaurine leading to 1.7x and 2. Ox titer improvements relative to the “Control” with neither increased iron nor hypotaurine. The viability is marginally decreased in the presence of elevated iron, and marginally increased when further supplemented with hypotaurine as shown in Figure 9B. The fed-batch 12-day production cell culture was conducted in a Sartorius AMBR250 High Throughput Bioreactor system utilizing a stable CHO cell line producing a recombinant bispecific antibody (molecule 1).Docket No. P39212-WO-1

[0026] Figure 10 depicts the improved growth observed using increased level of iron and increased level of iron + 1 mM hypotaurine during inoculum train culture stage (i.e., N-l, prior to production stage) with N-l perfusion. The cell growth improvement obtained is shown by the viable cell density (VCD) profiles represented in units of e6 cells / mL over the culture duration (in days). The VCD in the control culture without the additional iron or hypotaurine is represented by the square symbol. The culture with additional iron (i.e., higher levels of iron than the control culture) is represented by the circle symbol. The culture with the additional iron and hypotaurine is represented by the star symbol. The improved N-l growth supports the process intensification approach of inoculating N production cultures at high cell densities to maximize titers in a shortened culture duration (and hence increase productivity). The 7 day N-l perfusion culture was conducted in a Sartorius AMBR250 High Throughput Perfusion Bioreactor system utilizing a stable CHO cell line producing a recombinant bispecific antibody (molecule 1).

[0027] Figure 11 shows the effects of different antioxidants on titer and harvest viability compared to the control and the control combined with high iron. Five antioxidants (including hypotaurine) were tested in the presence of high levels of iron in fed-batch production cultures using a stable CHO cell line expressing a recombinant bispecific antibody (molecule 1). Figure 11 A shows that when glutathione and ascorbate were combined with high iron, there was no additional improvement beyond the 1.6x titer increase achieved by high iron alone, relative to the “Control” lacking high iron. Balcalein reduced performance when combined with iron. Bisglycinate improved performance when combined with iron (1.7x relative to the “Control” with no high iron), but not to the extent observed when hypotaurine was combined with iron (1.8x). Figure 1 IB shows that supplementing with baicalein or glutathione on top of high iron leads to worse harvest viabilities than supplementing with high iron alone. On the other hand, supplementing with ascorbate, hypotaurine, or bisglycinate in the high iron background improves harvest viabilities compared to supplementing with high iron alone. The fed-batch 12-day production cell culture was performed in a Sartorius AMBR15 High Throughput Bioreactor System utilizing a stable CHO cell line producing a recombinant bispecific antibody (molecule 1).

[0028] Figure 12 depicts the structure of Suramin (for e.g. CAS # 219-46-4).Docket No. P39212-WO-1

[0029] Figure 13 shows the impact of suramin on production cultures. Figure 13 A shows >10% improvement in titer for three molecules produced in stable CHO cell lines (Molecules 1, 2 and 4) in the presence of suramin. Cells expressing molecules 1 and 2 were cultured in 24-well-plate high throughput screening system with and without 35 pM suramin, and in the background without increased iron, ISRIB or hypotaurine. Cells expressing molecules 4, 5, and 6 were cultured in a Sartorius AMBR250 High Throughput Bioreactor system, in the presence of increased iron, 2 pM ISRIB and 10 mM hypotaurine, and with or without 35 pM suramin. Therefore, molecule 4 showed the additional benefit of -10% titer increase on top of the additional benefits conferred by increased iron, ISRIB and hypotaurine in the control cultures. Figure 13B shows a significant improvement in viable cell density (VCD in units of e6 cells / mL in upper panel) and cell viability (in units of % in lower panel) in molecules 4 (15% improvement in harvest viability) and 5 (17% improvement in harvest viability) when cell culture medium containing high iron, hypotaurine and ISRIB also contains suramin (Square markers) as compared to a control medium containing high iron, hypotaurine and ISRIB but without suramin (Triangle markers).

[0030] Figure 14 shows the impact of suramin addition on product quality and titer. Figure 14A shows the size distribution measured for Molecule 4 from harvest samples. The size distribution is divided amongst the high molecular weight species (HMWS, which represent protein aggregates), low molecular weight species (LMWS, which represent protein fragments), and main peak (which represents monomer). The addition of suramin recovers the monomer (i.e., main peak) expression when cultivated with high iron, hypotaurine and ISRIB, by decreasing the expression of the HMWS and LMWS. Figure 14B shows the impact of increased iron with ISRIB and hypotaurine addition in the presence or absence of suramin on titer and effective titer (which is represented by actual concentration of monomer) as compared to the control culture. Increased iron with ISRIB and hypotaurine significantly increased the titer over the control culture, and the further addition of suramin resulted in a further increase in titer. An overall increase in titer of the monomer species is also achieved upon suramin addition, and this concentration of the monomer is represented in Figure 14B by the term “Effective Titer”. Figure 14C shows the charge distribution measured for Molecule 4 from harvest samples. The chargeDocket No. P39212-WO-1 distribution is divided amongst acidic species, basic species and main peak (i.e., neutral species). The charge profile of molecule 4 cultured in media containing high iron, hypotaurine and ISRIB was improved with the addition of suramin — suramin restores the main peak to the levels observed in cell cultures using media without high iron, hypotaurine and ISRIB, by decreasing the acidic species. Suramin was tested using a stable CHO cell line expressing a recombinant bispecific antibody (Molecule 4) cultured over 12 days in fed-batch mode using a Sartorius AMBR250 High Throughput Bioreactor system.

[0031] Figure 15A depicts the structure of Ensulizole (for e.g. CAS#27503-81- 7). Figure 15B shows that when Ensulizole was cultured in a 24 well plate, high throughput screening system at 37°C with 5% CO2, improved titer for Molecule 1 and Molecule 2 were obtained in stable CHO Cell lines.

[0032] Figure 16A and 16B show a comparison of the harvest titer increase from a 3- factor, 2-level design of experiments (DoE) upon the addition of either 2 pM ISRIB, 10 mM Hypotaurine, and / or 350 pM Iron Citrate, each represented byThe control case, which received no additions, is denoted by “ — ”. The fed-batch 12-day production cell culture was conducted across two Sartorius Ambr 250 High Throughput Bioreactor systems utilizing a stable CHO cell line producing either a recombinant bispecific antibody (Molecule 1, Molecule 4) or a fragmented antigen-binding antibody (Molecule 5). The addition of Hypotaurine and Iron improves harvest titer by 80% when added together. Hypotaurine, Iron, and ISRIB can increase titer up to 40% when added separately at inoculation.

[0033] Figure 17A shows the actual titer vs. predicted titer values as normalized by the control “ - ” condition that was not provided ISRIB, Hypotaurine, or Iron Citrate.The regression model predicts that single-component additions of either 2 pM ISRIB, 10 mM Hypotaurine, or 350 pM Iron Citrate improve harvest titer by up to 40% and up to 80% when added in combination (R2 = 0.88). Figure 17B shows that Iron Citrate (represented by “XI”) is responsible for 45% of the improved titer effect, Hypotaurine (represented by “X2”) is responsible for 25% of the improved titer effect, the combined effect of Iron Citrate and Hypotaurine (represented by “X1*X2”) is responsible for 12% ofDocket No. P39212-WO-1 the improved titer effect, and ISRIB (represented by “X3”) is responsible for 12% of the improved titer effect.

[0034] Figures 18 A, 18B, and 18C show the influence of influence of either 2 pMISRIB (represented by10 mM Hypotaurine (represented byand / or 350 pM Iron Citrate (represented by “+ / -”) on harvest viability detected by trypan blue exclusion from a 3-factor, 2-level design of experiments (DoE). Upon the addition of 10 mM Hypotaurine without 350 pM Iron Citrate, a 4% increase in harvest viability is observed for Molecules 4 and 5, respectively. When 10 mM Hypotaurine is added in addition to 350 pM Iron Citrate, an increase in harvest viability anywhere from 5-13% is observed for Molecules 4 and 5, respectively.

[0035] Figure 19A, 19B, and 19C show the influence of influence of either 2 pM ISRIB (represented by10 mM Hypotaurine (represented byand / or 350 pM Iron Citrate (represented by “+ / -”) on the charge variant profile by ion exchange chromatography (Molecule 1, Molecule 4) and imaged capillary isoelectric focusing (Molecule Z) from a 3-factor, 2-level design of experiments (DoE). Upon the addition of 350 pM Iron Citrate the acidic charge variant peak sum may increase up to 20% for Molecule 1, 5% for Molecule 5, and 10% for Molecule 4. 10 mM Hypotaurine is able to decrease the acidic species 5% in the presence of 350 pM Iron Citrate. Also, upon the addition of 350 pM Iron Citrate, the main peak sum may decrease up to 20% for Molecule 1, 8% for Molecule 5, and 11% for Molecule 4. Upon the addition of 10 mM Hypotaurine without 350 pM Iron Citrate, a 2% increase in the basic peak sum is observed for Molecules 1 and 4, respectively.

[0036] Figure 20 shows the influence of 2 pM ISRIB, 10 mM Hypotaurine, and 350 pM Iron Citrate on the charge variant profile of Molecule 1 as assayed by ion exchange chromatography from a 3-factor, 2-level design of experiments (DoE). Upon the addition of 350 pM Iron Citrate, the acidic charge variant peak sum may increase up to 20% and the main peak sum may decrease up to 20%.10 mM Hypotaurine is able to decrease the acidic species approximately 5% in the presence of 350 pM Iron Citrate.

[0037] Figure 21 shows the influence of 2 pM ISRIB, 10 mM Hypotaurine, and 350 pM Iron Citrate on the charge variant profile of Molecule 5 as assayed by imagedDocket No. P39212-WO-1 capillary isoelectric focusing from a 3-factor, 2-level design of experiments (DoE). Upon the addition of 350 pM Iron Citrate, the acidic charge variant peak sum may increase up to 5% and the main peak sum may decrease up to 8%. 10 mM Hypotaurine is able to decrease the acidic species approximately 5% in the presence of 350 pM Iron Citrate.

[0038] Figure 22 shows the influence of 2 pM ISRIB, 10 mM Hypotaurine, and 350 pM Iron Citrate on the charge variant profile of Molecule 4 as assayed by ion exchange chromatography from a 3-factor, 2-level design of experiments (DoE). Upon the addition of 350 pM Iron Citrate, the acidic charge variant peak sum may increase up to 10% and the main peak sum may decrease up to 10%. The addition of 10 mM Hypotaurine may increase main peak by 4% and the basic peak by 3%. 10 mM Hypotaurine is able to decrease the acidic species approximately 5% in the presence of 350 pM Iron Citrate.

[0039] Figure 23 A, 23B, and 23C show the influence of influence of either 2 pM ISRIB (represented by “+ / -”), 10 mM Hypotaurine (represented by “+ / -”), and / or 350 pM Iron Citrate (represented by “+ / -”) on the size variant profile of Molecule 1 Molecule 5, and Molecule 4 from a 3-factor, 2-level design of experiments (DoE). LMWS were measured by capillary electrophoresis - sodium dodecyl sulfate while monomer and HMWS were measured by size exclusion chromatography. Upon the addition of 350 pM Iron Citrate the monomer species may decrease ~ 3% while HMWS may increase 2% for Molecule 4. Molecule 5 exhibits minimal effect.

[0040] Figure 24 shows the influence of 2 pM ISRIB, 10 mM Hypotaurine, and 350 pM Iron Citrate on the size variant profile of Molecule 1 as assayed by either capillary electrophoresis - sodium dodecyl sulfate (LMWS) or size exclusion chromatography (monomer species (i.e., main peak) & HMWS) from a 3-factor, 2-level design of experiments (DoE). Upon the addition of either iron citrate, hypotaurine, or ISRIB, monomer species may deviate by 3%.

[0041] Figure 25 shows the influence of 2 pM ISRIB, 10 mM Hypotaurine, and 350 pM Iron Citrate on the size variant profile of Molecule 5 as assayed by either capillary electrophoresis - sodium dodecyl sulfate (LMWS) or size exclusion chromatography (monomer species & HMWS) from a 3-factor, 2-level design of experiments (DoE). UponDocket No. P39212-WO-1 the addition of either iron citrate, hypotaurine, or ISRIB, monomer species may decrease by 1%.

[0042] Figure 26 shows the influence of 2 pM ISRIB, 10 mM Hypotaurine, and 350 pM Iron Citrate on the size variant profile of Molecule 4 as assayed by either capillary electrophoresis - sodium dodecyl sulfate (LMWS) or size exclusion chromatography (monomer species (Main Peak) & HMWS) from a 3 -factor, 2-level design of experiments (DoE). Upon the addition of iron citrate, monomer species may decrease by 3%.

[0043] Figure 27 shows a comparison of the harvest titer increase from a 3 x 2 factorial design of experiments (DoE) upon the addition of either 0, 1, or 10 mM Hypotaurine and either 150 or 350 pM Iron Citrate. The fed-batch 12-day production cell culture was conducted in a Sartorius Ambr 250 High Throughput Bioreactor system utilizing a stable CHO cell line producing a recombinant bispecific antibody (Molecule 9). Each cell culture was fortified with 2 pM ISRIB. When Hypotaurine is added at 10 mM, an additional ~ 1 g / L can be achieved. Both 150 and 350 pM Iron Citrate as well as 0 and 1 mM Hypotaurine have minimal influence on titer.

[0044] Figure 28 shows a comparison of the charge variant profile from a 3 x 2 factorial design of experiments (DoE) upon the addition of either 0, 1, or 10 mM Hypotaurine and either 150 or 350 pM Iron Citrate. The fed-batch 12-day production cell culture was conducted in a Sartorius Ambr 250 High Throughput Bioreactor system utilizing a stable CHO cell line producing a recombinant bispecific antibody (Molecule 9 ). Each cell culture was fortified with 2 pM ISRIB. When Hypotaurine is added at 10 mM with 150 pM Iron Citrate, a 15% shift from acidic peak % to main peak % is observed.

[0045] Figure 29 shows a comparison of the size variant profile from a 3 x 2 factorial design of experiments (DoE) upon the addition of either 0, 1, or 10 mM Hypotaurine and either 150 or 350 pM Iron Citrate. The fed-batch 12-day production cell culture was conducted in a Sartorius Ambr 250 High Throughput Bioreactor system utilizing a stable CHO cell line producing a recombinant bispecific antibody (Molecule 9 ). Each cell culture was fortified with 2 pM ISRIB. All conditions show negligible influence on size variants (note 350 pM Iron Citrate + 1 mM Hypotaurine condition is an outlier and removed from models).Docket No. P39212-WO-1DETAILED DESCRIPTION

[0046] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0047] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0048] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0049] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.Docket No. P39212-WO-1

[0050] For the avoidance of doubt, it is hereby stated that the information disclosed earlier in this specification under the heading “Background” is relevant to the invention and is to be read as part of the disclosure of the invention.

[0051] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.DEFINITIONS

[0052] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0053] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0054] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0055] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems orDocket No. P39212-WO-1 processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0056] The terms “cell culture medium” and “culture medium” refer to a nutrient solution used for growing mammalian cells that typically provides at least one component from one or more of the following categories:1) an energy source, usually in the form of a carbohydrate such as glucose;2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine;3) vitamins and / or other organic compounds required at low concentrations;4) free fatty acids; and5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

[0057] Such a culture medium may comprise a basal medium, which may be supplemented with additional components, e.g. via a nutrient fed or supplement. As used herein, references to a “similar eukaryotic cell culture medium” refers to an identical eukaryotic cell culture medium, but for the specified difference(s), for e.g., an identical eukaryotic cell culture medium but does not comprise ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0058] The nutrient solution can optionally be supplemented with one or more components from any of the following categories:1) hormones and other growth factors as, for example, insulin, transferrin, and epidermal growth factor;2) salts and buffers as, for example, calcium, magnesium, and phosphate;3) nucleosides and bases such as, for example, adenosine, thymidine, and hypoxanthine; and4) protein and tissue hydrolysates.Docket No. P39212-WO-1

[0059] Nutrient solutions, for example a nutrient of the invention (or of use in the invention), may also contain additional components or compounds, as disclosed elsewhere in the present application.

[0060] “Culturing” a cell refers to contacting a cell with a cell culture medium under conditions suitable to the survival and / or growth and / or proliferation of the cell. Nonlimiting examples of types of cell culture include batch culture, fed-batch culture, intensified culture, continuous culture and perfusion culture.

[0061] “Batch culture” refers to a culture in which all components for cell culturing (including the cells and all culture nutrients) are supplied to the culturing bioreactor at the start of the culturing process.

[0062] “Fed-batch culture,” as used herein refers to a batch culture wherein the cells and culture medium are supplied to the culturing bioreactor initially, and additional culture nutrients are fed, continuously or in discrete increments, to the culture during the culturing process, with or without periodic cell and / or product harvest before termination of culture.

[0063] “Intensified culture” as used herein refers to a culture wherein the production process is intensified to achieve higher productivities by, e.g., achieving higher titers in a given culture duration or achieving the same titers but in a shortened culture duration. The higher productivities can be achieved through higher cell mass, concentrated nutrient feeds, and / or perfusion (intermittent or continuous). For example, the high cell mass in production (N) can be achieved by using an intensified inoculum train process prior to production (i.e., at N-l stage) to reach high N-l cell densities, which can be achieved by perfusion and / or nutrient feeds in N-l bioreactor. The intensified culture in N stage may be conducted in fed-batch or perfusion modes, or a combination of both, including with intermittent perfusion (e.g., intermittent-perfusion fed-batch mode).

[0064] “Perfusion culture,” sometimes referred to as continuous culture, is a culture by which the cells are restrained in the culture by, e.g., filtration, encapsulation, anchoring to microcarriers, etc., and the culture medium is continuously, step-wise or intermittently introduced (or any combination of these) and removed from the culturing bioreactor.Docket No. P39212-WO-1

[0065] As used herein, the term “cell,” refers to animal cells, mammalian cells, cultured cells, host cells, recombinant cells and recombinant host cells. Such cells are generally cell lines obtained or derived from mammalian tissues which are able to grow and survive when placed in media containing appropriate nutrients and / or growth factors.

[0066] The term “cell line” as used herein includes reference to a culture of eukaryotic cells that can be propagated repeatedly. The eukaryotic cells of the cell line may be selected from any cell as defined herein.

[0067] The terms “host cell,” “host cell line” and “host cell culture” are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid can be subsequently introduced to create recombinant cells. These host cells may also have been modified (i.e., engineered) to alter or delete the expression of certain endogenous host cell products (e.g., endogenous virus-like particles or endogenous host cell proteins). Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny does not need to be completely identical in nucleic acid content to a parent cell, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The introduction of exogenous nucleic acid (e.g., by transfection) to these host cells would create recombinant cells that are derived from the original “host cell,” “host cell line” or “host cell line”. The terms “host cell,” “host cell line” and “host cell culture” may also refer to such recombinant cells and their progeny. The terms “recombinant cell”, “recombinant cell line” and “recombinant cell culture” are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid has been introduced to enable the expression of recombinant product of interest. The recombinant product expressed by such cells may be a recombinant protein, a recombinant viral particle, or a recombinant viral vector.

[0068] The term “mammalian host cell” or “mammalian cell” refers to cell lines derived from mammals that are capable of growth and survival when placed in either monolayer culture or in suspension culture in a medium containing the appropriate nutrients and growth factors. The necessary growth factors for a particular cell line areDocket No. P39212-WO-1 readily determined empirically without undue experimentation, as described for example in Mammalian Cell Culture (Mather, J. P. ed., Plenum Press, N.Y. 1984), and Barnes and Sato, (1980) Cell, 22:649. Typically, the cells are capable of expressing and secreting large quantities of a particular protein, e.g., glycoprotein, of interest into the culture medium. Examples of suitable mammalian host cells within the context of the present disclosure can include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dpl2.CHO cells (EP 307,247 published 15 Mar. 1989); CHO-K1 (ATCC, CCL-61); human embryonic kidney 293 cells (HEK293, Graham et al., The Journal of General Virology, 36(l):59-74, 1977), human cells, baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980); canine kidney cells (MDCK, ATCC CCL 34); ); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); mouse mammary tumor (MMT 060562, ATCC CCL51). In certain embodiments, the mammalian cells include Chinese hamster ovary cells (CHO), HEK293 cells, or human cells (for cell therapy). The cells may be CHO cells or HEK293 cells. The cells may be human cells (e.g. for cell therapy). The cells may be CHO cells. In certain embodiments, the Chinese hamster ovary (CHO) cells may comprise one or more of CHO KI, CHO S, DG44, DXB11, CHOZN GS- / -, ExpiCHO-S, Freestyle CHO-S, CHOK1SV and CHO / dhfr-. In certain embodiments, the cells comprise a polynucleotide that encodes a polypeptide. In another embodiment, the cell expresses the polypeptide transiently or expresses the polypeptide stably. In a further embodiment, the cells expressing the polypeptide stably comprises a polynucleotide that is integrated in the cellular genome of the cell at a targeted location. In another further embodiment, the cells expressing the polypeptide stably comprises a polynucleotide that is integrated in the cellular genome of the cell at a random location.

[0069] Growth phase” of the cell culture refers to the period of exponential cell growth (the log phase) where cells are generally rapidly dividing. The duration of time for which the cells are maintained at growth phase can vary based on the cell-type, the rate of growth of cells and / or the culture conditions, for example. In certain embodiments, during this phase, cells are cultured for a period of time, usually between 1-8 days, and under such conditions that cell growth is maximized. The determination of the growth cycle for the host cell can be determined for the particular host cell envisioned without undueDocket No. P39212-WO-1 experimentation. “Period of time and under such conditions that cell growth is maximized” and the like, refer to those culture conditions that, for a particular cell line, are determined to be optimal for cell growth and division. In certain embodiments, during the growth phase, cells are cultured in nutrient medium containing the necessary additives generally at about 30°-40°C in a humidified, controlled atmosphere, such that optimal growth is achieved for the particular cell line. In certain embodiments, during the growth phase, cells are fed additional nutrients to maintain the growth and / or the culture medium is perfused to add fresh nutrients while removing metabolic wastes. In certain embodiments, cells are maintained in the growth phase for a period of between about one and about eight days, between about one and about four days, usually between about two to about three days.

[0070] “Production phase” of the cell culture refers to the period of time during which cell growth is / has plateaued. The logarithmic cell growth typically decreases before or during this phase and protein production takes over. During the production phase, logarithmic cell growth has ended, and protein production is primary. During this period of time the medium is generally supplemented to support continued protein production and to achieve the desired glycoprotein product. Fed-batch and / or perfusion cell culture processes supplement the cell culture medium or provide fresh medium during this phase to achieve and / or maintain desired cell density, viability and / or recombinant protein product titer. A production phase can be conducted at large scale.

[0071] The term “production culture” refers to the final culture stage (N) for the cells, and it is typically used to produce the desired recombinant product (e.g., monoclonal antibody).

[0072] The term “inoculum train culture” refers to the stages of cell expansion leading to the production culture. In particular, the term “N-l” refers to the final inoculum train passage used to inoculate the production culture, i.e., N-l culture is used to seed the N culture. The N-l culture represents the penultimate culture passage during the cell culture process (starting from thaw of cell bank and extending all the way through to production culture).Docket No. P39212-WO-1

[0073] The term “activity” as used herein with respect to activity of a protein refers to any activity of a protein including, but not limited to, enzymatic activity, ligand binding, drug transport, ion transport, protein localization, receptor binding, and / or structural activity. Such activity can be modulated, e.g., reduced or eliminated, by reducing or eliminating the expression of the protein, thereby reducing or eliminating the presence of the protein. Such activity can also be modulated, e.g., reduced or eliminated, by altering the nucleic acid sequence encoding the protein such that the resulting modified protein exhibits reduced or eliminated activity relative to a wild type protein.

[0074] The term “expression” or “expresses” are used herein to refer to transcription and translation occurring within a host cell. The level of expression of a product gene in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the product gene that is produced by the cell. For example, mRNA transcribed from a product gene is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a product gene can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein.Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989). When reference is made to reduction and / or elimination of the expression of one or more endogenous products relative to the expression of the endogenous product(s) in an unmodified cell, such reductions and / or eliminations of expression encompass reductions and / or eliminations of the active endogenous product, notwithstanding the presence of mRNA encoding all or a portion of the endogenous product or the presence of endogenous product translated from such mRNA.

[0075] As used herein, “polypeptide” refers generally to peptides and proteins having more than about ten amino acids. The polypeptides can be homologous to the host cell, or preferably, can be exogenous, meaning that they are heterologous, i.e., foreign, to the host cell being utilized, such as a human protein produced by a Chinese hamster ovary cell, or a yeast polypeptide produced by a mammalian cell. In certain embodiments, mammalianDocket No. P39212-WO-1 polypeptides (polypeptides that were originally derived from a mammalian organism) are used, more preferably those which are directly secreted into the medium.

[0076] The term “protein” is meant to refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and / or quaternary structure. This is to distinguish from “peptides” or other small molecular weight drugs that do not have such structure. Typically, the protein herein will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD. Examples of proteins encompassed within the definition herein include host cell proteins as well as all mammalian proteins, in particular, therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and / or intrachain disulfide bonds.

[0077] The term “glycoprotein” refers to a protein which contains an oligosaccharide chain covalently attached to amino acid side-chains. The oligosaccharide(s) may be attached to the protein in a co-translational or post-translational modification, during a process known as glycosylation. Exemplary glycoproteins include antibodies, which typically have an N-linked oligosaccharide on each heavy chain.

[0078] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence, including multimers of such pairings), multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity. A therapeutic antibody is an antibody that may be used in the treatment of a disease.

[0079] An “antibody fragment,” “antigen-binding portion” of an antibody (or simply “antibody portion”) or “antigen-binding fragment” of an antibody, as used herein, refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For aDocket No. P39212-WO-1 review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0080] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0081] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. In certain embodiments, the antibody is of the IgGl isotype. In certain embodiments, the antibody is of the IgG2 isotype. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 8, a, y and p, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (K) and lambda (1), based on the amino acid sequence of its constant domain.

[0082] The term “titer” as used herein refers to the total amount of recombinantly expressed antibody produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of antibody per milliliter or liter of medium (mg / ml or mg / L). In certain embodiments, titer is expressed in grams of antibody per liter of medium (g / L). Titer can be expressed or assessed in terms of a relative measurement, such as a percentage increase in titer as compared obtaining the protein product under different culture conditions.

[0083] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody asDocket No. P39212-WO-1 being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies in accordance with the presently disclosed subject matter can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0084] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0085] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human complementarity determining regions (CDRs) and amino acid residues from human framework regions (FRs). In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non- human antibody, refers to an antibody that has undergone humanization.

[0086] As used herein, the term “recombinant protein” refers generally to peptides and proteins, including antibodies, that are encoded by a nucleic acid that is “heterologous,” i.e., foreign to the host cell being utilized, such as a nucleic acid encoding a human antibody that is introduced into a non-human host cell.

[0087] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence, including multimers of such pairings), multispecific antibodies (e.g., bispecific antibodies) and antibody fragments soDocket No. P39212-WO-1 long as they exhibit the desired antigen-binding activity. A therapeutic antibody is an antibody that may be used in the treatment of a disease.

[0088] An “antibody fragment,” “antigen-binding portion” of an antibody (or simply “antibody portion”) or “antigen-binding fragment” of an antibody, as used herein, refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0089] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0090] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. In certain embodiments, the antibody is of the IgGl isotype. In certain embodiments, the antibody is of the IgG2 isotype. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 8, a, y and p, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (K) and lambda (1), based on the amino acid sequence of its constant domain.

[0091] The term “titer” as used herein refers to the total amount of recombinantly expressed protein produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of grams of protein per liter of medium (g / L). In certain embodiments, titer is expressed in milligrams of protein per liter of medium (mg / L). Titer can be expressed or assessed in terms of a relative measurement, such as a percentage increase in titer as compared to obtaining the protein product under different culture conditions.Docket No. P39212-WO-1

[0092] The term “product quality of the recombinant protein” may be determined by analysis of the charge distribution of the recombinant protein and by analysis of the size distribution of the recombinant protein. The main peak (by charge distribution) corresponds to the recombinant polypeptide with the desired neutral charge; the acidic species typically have additional acid groups, such as sialic acid and / or a reduced number of amine moieties; and the basic species typically have additional amine moieties or a reduced number of acidic groups. The main peak / monomer (by size distribution) is the recombinant polypeptide monomer peak; while the non-monomer peaks identified by size distribution typically comprise low molecular weight species (LMWS) as protein fragments or high molecular weight species (HMWS) as protein aggregates (e.g., multimers). An improvement in product quality may be determined by where the main peak (by charge distribution) percentage or main peak (by size distribution) percentage for the recombinant protein produced using cell culture media of the invention is higher than the corresponding main peak(s) for a recombinant protein obtained using another different cell culture media (e.g., where the different cell culture media lacks one or more specific components of the cell culture media of the invention).

[0093] The term “main peak” in relation to a recombinant protein refers to the peak obtained for the monomeric target protein for size distribution, as measured by size exclusion chromatography or capillary electrophoresis sodium dodecyl-sulfate, and for charge state, when measured by charge separation methods such as ion exchange chromatography, capillary zone electrophoresis or imaged capillary isoelectric focusing. Examples of such analytical methods have been described in Hopp et al. 2009 (“Development of a high throughput protein A well-plate purification method for monoclonal antibodies” Biotechnology Progress 25(5): 1427-1432).

[0094] The following abbreviations are used herein:CHO Chinese Hamster OvaryDNA Deoxyribonucleic acid eccDNA extrachromosomal circular DNADocket No. P39212-WO-1 ecDNA extrachromosomal DNAER Endoplasmic ReticulumHMW High Molecular WeightHMWS High Molecular Weight Species (i.e., protein aggregates), also known as HMW formsIgG Immunoglobulin GISRIB Integrated Stress Response InhibitorIVCC Integrated Viable Cell ConcentrationLMW Low Molecular WeightLMWS Low Molecular Weight Species (i.e., protein fragments)N-l Final inoculum train passage used to inoculate production bioreactorProduction bioreactorOxidative PhosphorylationQp Cell-specific productivityUHPLC Ultra High Pressure Liquid ChromatographyVCD Viable Cell DensityCell Culture Media

[0095] It will be appreciated that the cell culture media disclosed herein include various different types of media. For example, the media may include: thaw, seed train, inoculum train medium (related to the passage(s) before going into production culture); production basal medium (such as medium used at start of production culture); feed / nutrient medium (such as medium fed during product culture and / or during inoculum train culture); and perfusion medium (i.e., perfused during production culture and / orDocket No. P39212-WO-1 during inoculum train culture); and the like. Unless required otherwise by the context, the medium of the disclosure or of the invention may be a thaw medium, a seed train medium, an inoculum train medium, a production basal medium, a feed / nutrient medium, a perfusion medium, or the like.

[0096] Where the medium comprises a basal medium, the basal medium may be any basal medium that is suitable for the relevant cell type, as would be known to the person of skill in the art. For example, media and feeds for CHO cell culture contain the necessary components for CHO cell cultures. These media and feeds are chemically defined, providing a consistent lot-to-lot defined composition. Examples of chemically-defined media have been described in patent literature, such as U.S. Patent Nos. 4,767,704;5,691,202; 6,048,728; 6,900,056; and 7,601,535; as well as in European Patent No. 1482031. Such media are considered derivatives of DMEM-F12 Media. DMEM-F12 (available from Thermo Fisher Scientific) comprises a composition as set out in Table 1.Table 1: DMEM-F12Docket No. P39212-WO-1Docket No. P39212-WO-1Docket No. P39212-WO-1

[0097] In a first aspect, the invention provides a eukaryotic cell culture medium comprising integrated stress response inhibitor (ISRIB).

[0098] ISRIB has the following chemical structure:

[0099] In embodiments, the ISRIB is adapted to be present at a level of from about 0.01 pM to about 200 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.01 pM to about 150 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.01 pM to about 100 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted toDocket No. P39212-WO-1 be present at a level of from about 0.01 pM to about 80 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.01 pM to about 60 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.01 pM to about 40 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.01 pM to about 20 pM when the medium is provided as a IX medium formulation.

[0100] It may be that the ISRIB is adapted to be present at a level of from about 0.025 pM to about 200 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.05 pM to about 200 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.06 pM to about 200 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.07 pM to about 200 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.08 pM to about 200 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.09 pM to about 200 pM when the medium is provided as a IX medium formulation.

[0101] It may be that the ISRIB is adapted to be present at a level of from about 0.025 pM to about 100 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.05 pM to about 60 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.08 pM to about 40 pM when the medium is provided as a IX medium formulation. It may be that the ISRIB is adapted to be present at a level of from about 0.1 pM to about 20 pM when the medium is provided as a IX medium formulation.

[0102] In embodiments, ISRIB may be present at a level of from about 0.01 pM to about 200 pM when the medium is provided as a IX medium formulation; optionally wherein the ISRIB is adapted to be present at a level of from about 0.1 pM to about 20Docket No. P39212-WO-1 pM when the medium is provided as a IX medium formulation; further optionally wherein the ISRIB is adapted to be present at a level of from about 2 pM to about 200 pM when the medium is provided as a IX medium formulation; further optionally wherein the ISRIB is adapted to be present at a level of from about 2 pM to about 20 pM when the medium is provided as a IX medium formulation.

[0103] In embodiments, the cell culture medium further comprises hypotaurine.

[0104] In embodiments, the cell culture medium further comprises hypotaurine and an increased level of iron. In embodiments, the iron may be in the form of Fe2+, Fe3+or a combination thereof. In embodiments, the iron may be or comprise chelated iron. For the avoidance of doubt, the term “increased iron” or “increased level of iron” may refer to a concentration of at least 100 pM of iron.

[0105] In the embodiments comprising hypotaurine or hypotaurine and an increased level of iron, the hypotaurine may be present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation.

[0106] It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 17.5 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 15 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 12.5 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 10 mM when the medium is provided as a IX medium formulation.

[0107] It may be that the hypotaurine may be present at a level of from about 0.2 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.3 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.4 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.5 mM to about 20 mM when the medium is provided as a IX mediumDocket No. P39212-WO-1 formulation. It may be that the hypotaurine may be present at a level of from about 0.6 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.7 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.8 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.9 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation.

[0108] In embodiments, the hypotaurine is present at a level of from about 0.2 mM to about 17.5 mM when the medium is provided as a IX medium formulation. In embodiments, the hypotaurine is present at a level of from about 0.5 mM to about 15 mM when the medium is provided as a IX medium formulation. In embodiments, the hypotaurine is present at a level of from about 0.7 mM to about 12.5 mM when the medium is provided as a IX medium formulation. In embodiments, the hypotaurine is present at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

[0109] In embodiments, the hypotaurine may be present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation; optionally at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation; further optionally at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

[0110] In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation.

[0111] In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 4500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 4000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 3500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted toDocket No. P39212-WO-1 be present at a level of from about 50 pM to about 3000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 2500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 2000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 1500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 750 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0112] In embodiments, the iron is adapted to be present at a level of from about 55 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 60 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 65 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 70 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 75 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 80 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 85 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 90 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 95 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a levelDocket No. P39212-WO-1 of from about 100 pM to about 5000 pM when the medium is provided as a IX medium formulation.

[0113] In embodiments, the iron is adapted to be present at a level of from about 60 pM to about 4000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 75 pM to about 2500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 90 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 95 pM to about 750 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 100 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0114] In embodiments, the iron may be adapted at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to about 350 pM; further optionally at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, or from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation.

[0115] In embodiments, the cell culture medium further comprises suramin.

[0116] In these embodiments, it may be that the suramin is adapted to be present at a level of from about 0.01 pM to about 1000 pM when the medium is provided as a IX medium formulation.

[0117] In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 900 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 800 pM when the medium is provided as a IX medium formulation. InDocket No. P39212-WO-1 embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 700 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 600 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0118] In embodiments, the suramin is adapted to be present at a level of from about 0.02 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.03 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.04 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.05 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.06 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.07 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.08 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.09 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.1 pM to about 1000 pM when the medium is provided as a IX medium formulation.

[0119] In embodiments, the suramin is adapted to be present at a level of from about 0.05 pM to about 800 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.1 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0120] In embodiments, the suramin may be adapted at a level of from about 0.01 pM to about 1000 pM, or from about 0.1 pM to about 500 pM when the medium is providedDocket No. P39212-WO-1 as a IX medium formulation; optionally at a level of from about 2 pM to about 400 pM, or from about 3.5 pM to about 400 pM, or from about 35 pM to about 400 pM when the medium is provided as a IX medium formulation; further optionally at a level of from about 3.5 pM to about 350 pM, or from about 35 pM to about 350 pM when the medium is provided as a IX medium formulation; further optionally at a level of from about from about 3.5 pM to about 35 pM when the medium is provided as a IX medium formulation.

[0121] In a second aspect of the invention, there is provided a eukaryotic cell culture medium comprising hypotaurine.

[0122] In embodiments, the eukaryotic cell culture medium comprises hypotaurine and an increased level of iron.

[0123] In embodiments, the iron may be in the form of Fe2+, Fe3+or a combination thereof. In embodiments, the iron may be chelated iron. For the avoidance of doubt, the term “increased iron” or “increased level of iron” may refer to a concentration of at least 100 pM of iron.

[0124] In embodiments, the hypotaurine may be present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation.

[0125] It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 17.5 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 15 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 12.5 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.1 mM to about 10 mM when the medium is provided as a IX medium formulation.

[0126] It may be that the hypotaurine may be present at a level of from about 0.2 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.3 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.4 mM to about 20 mM when the medium is provided asDocket No. P39212-WO-1 a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.5 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.6 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.7 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.8 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 0.9 mM to about 20 mM when the medium is provided as a IX medium formulation. It may be that the hypotaurine may be present at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation.

[0127] In embodiments, the hypotaurine is present at a level of from about 0.2 mM to about 17.5 mM when the medium is provided as a IX medium formulation. In embodiments, the hypotaurine is present at a level of from about 0.5 mM to about 15 mM when the medium is provided as a IX medium formulation. In embodiments, the hypotaurine is present at a level of from about 0.7 mM to about 12.5 mM when the medium is provided as a IX medium formulation. In embodiments, the hypotaurine is present at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

[0128] In embodiments, the hypotaurine may be present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation; optionally at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation; further optionally at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

[0129] In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation.

[0130] In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 4500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 4000 pM when the medium is provided as a IX medium formulation. In embodiments, theDocket No. P39212-WO-1 iron is adapted to be present at a level of from about 50 pM to about 3500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 3000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 2500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 2000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 1500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 750 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 50 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0131] In embodiments, the iron is adapted to be present at a level of from about 55 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 60 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 65 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 70 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 75 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 80 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 85 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 90 pM to about 5000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 95 pM to about 5000 pM when the medium is providedDocket No. P39212-WO-1 as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 100 pM to about 5000 pM when the medium is provided as a IX medium formulation.

[0132] In embodiments, the iron is adapted to be present at a level of from about 60 pM to about 4000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 75 pM to about 2500 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 90 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 95 pM to about 750 pM when the medium is provided as a IX medium formulation. In embodiments, the iron is adapted to be present at a level of from about 100 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0133] In embodiments, the iron may be adapted at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to about 350 pM; further optionally at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, or from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation.

[0134] In embodiments, the cell culture medium further comprises suramin.

[0135] In these embodiments, it may be that the suramin is adapted to be present at a level of from about 0.01 pM to about 1000 pM when the medium is provided as a IX medium formulation.

[0136] In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 900 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM toDocket No. P39212-WO-1 about 800 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 700 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 600 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0137] In embodiments, the suramin is adapted to be present at a level of from about 0.02 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.03 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.04 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.05 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.06 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.07 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.08 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.09 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.1 pM to about 1000 pM when the medium is provided as a IX medium formulation.

[0138] In embodiments, the suramin is adapted to be present at a level of from about 0.05 pM to about 800 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.1 pM to about 500 pM when the medium is provided as a IX medium formulation.Docket No. P39212-WO-1

[0139] In a third aspect of the invention, there is provided a eukaryotic cell culture medium comprising suramin.

[0140] In these embodiments, it may be that the suramin is adapted to be present at a level of from about 0.01 pM to about 1000 pM when the medium is provided as a IX medium formulation.

[0141] In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 900 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 800 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 700 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 600 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.01 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0142] In embodiments, the suramin is adapted to be present at a level of from about 0.02 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.03 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.04 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.05 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.06 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.07 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.08 pM to about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.09 pM toDocket No. P39212-WO-1 about 1000 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.1 pM to about 1000 pM when the medium is provided as a IX medium formulation.

[0143] In embodiments, the suramin is adapted to be present at a level of from about 0.05 pM to about 800 pM when the medium is provided as a IX medium formulation. In embodiments, the suramin is adapted to be present at a level of from about 0.1 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0144] The following embodiments may apply to the eukaryotic cell culture medium of any of the first, second or third aspects.

[0145] In embodiments, the eukaryotic cell culture medium further comprises ensulizole.

[0146] In embodiments, the ensulizole is adapted to be present at a level of from about 0.2 pM to about 500 pM when the medium is provided as a IX medium formulation.

[0147] In embodiments, the ensulizole is adapted to be present at a level of from about 0.2 pM to about 400 pM when the medium is provided as a IX medium formulation. In embodiments, the ensulizole is adapted to be present at a level of from about 0.2 pM to about 300 pM when the medium is provided as a IX medium formulation. In embodiments, the ensulizole is adapted to be present at a level of from about 0.2 pM to about 200 pM when the medium is provided as a IX medium formulation.

[0148] In embodiments, the ensulizole is adapted to be present at a level of from about 0.5 pM to about 500 pM when the medium is provided as a IX medium formulation. In embodiments, the ensulizole is adapted to be present at a level of from about 1 pM to about 500 pM when the medium is provided as a IX medium formulation. In embodiments, the ensulizole is adapted to be present at a level of from about 1.5 pM to about 500 pM when the medium is provided as a IX medium formulation. In embodiments, the ensulizole is adapted to be present at a level of from about 2 pM to about 500 pM when the medium is provided as a IX medium formulation.Docket No. P39212-WO-1

[0149] In embodiments, the ensulizole is adapted to be present at a level of from about 2 pM to about 200 pM when the medium is provided as a IX medium formulation.

[0150] In embodiments, the medium is selected from the group consisting of: a thaw medium, a seed train medium, an inoculum train medium, a production medium, a perfusion medium for inoculum train culture, a perfusion medium for production culture, a nutrient feed and a stock solution. For example, the medium may be a production medium (N), or an inoculum medium (such as the N-l inoculum medium). The medium may be a production medium (N). The medium may be an N-l inoculum medium.

[0151] In embodiments, the medium is capable of supporting high-density growth of mammalian cells in suspension cell culture. In embodiments, the medium is capable of supporting the expression of recombinant protein from the cells.

[0152] In embodiments, the eukaryotic cells are Chinese hamster ovary (CHO) cells, HEK293 cells, or human cells (for cell therapy) In embodiments, the eukaryotic cells are Chinese hamster ovary (CHO) cells. In embodiments, the CHO cells are adapted to produce recombinant protein.

[0153] In embodiments, the CHO cells are selected from CHO KI cells, CHO KI SV cells, DG44 cells, DUKXB-11 cells, CHOK1S cells, or CHO KIM cells, or their derivatives.

[0154] In embodiments, the polynucleotide that encodes the polypeptide is integrated in the cellular genome of the CHO cells. In other embodiments, the polynucleotide that encodes the polypeptide is randomly integrated in the cellular genome of the CHO cells

[0155] In embodiments, the medium is a IX medium formulation.

[0156] In embodiments, the medium is a concentrated or dry medium formulation. In embodiments, the medium is a concentrated medium formulation. In embodiments, the medium is a dry medium formulation. In these embodiments, the medium may be a 10X medium formulation. Alternatively, the medium may be a greater than 10X concentration medium formulation.Docket No. P39212-WO-1

[0157] In embodiments, the medium is a chemically defined medium.

[0158] In embodiments, at least one of the ISRIB, hypotaurine, increased level of iron, suramin, and ensulizole are added, individually or in combination, through supplementation, through nutrient feeds, by addition to basal media, or by addition to perfusion media.

[0159] In embodiments, at least one of the ISRIB, hypotaurine, increased level of iron, suramin, and ensulizole are added, individually or in combination, to a stock solution. In embodiments, the stock solution is used in the production culture.

[0160] In embodiments, at least one of the ISRIB, hypotaurine, increased level of iron, suramin, and ensulizole are added, individually or in combination, to the thaw, seed train or inoculum train medium.

[0161] In embodiments, at least one of the ISRIB, hypotaurine, increased level of iron, suramin, and ensulizole are added, individually or in combination, to the production medium.

[0162] In embodiments, at least one of the ISRIB, hypotaurine, increased level of iron, suramin, and ensulizole are added, individually or in combination, to the nutrient feed.

[0163] In embodiments, at least one of the ISRIB, hypotaurine, increased level of iron, suramin, and ensulizole are added, individually or in combination, to the perfusion medium.

[0164] In embodiments, use of hypotaurine is provided for enhancing growth and / or viability and / or productivity of a eukaryotic cell culture is provided, wherein the hypotaurine is present in the eukaryotic cell culture medium, wherein the eukaryotic cell culture medium comprises iron at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally wherein the iron is adapted to be present at a level of from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to aboutDocket No. P39212-WO-1350 pM; further optionally wherein the iron is adapted to be present at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, or from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation.

[0165] In embodiments, use of hypotaurine is provided for enhancing the titer and / or product quality of a recombinant protein product, wherein the recombinant protein product is produced by a eukaryotic cell culture, and wherein the hypotaurine is present in the eukaryotic cell culture medium, wherein the eukaryotic cell culture medium comprises iron at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally at a level of from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to about 350 pM; further optionally wherein the iron is adapted to be present at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, or from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation.

[0166] In embodiments, use of hypotaurine is provided, wherein the hypotaurine is present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation; optionally wherein the hypotaurine is present at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation; further optionally wherein the hypotaurine is present at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

[0167] In embodiments, use of ISRIB is provided for enhancing growth and / or viability and / or productivity of a eukaryotic cell culture is provided, wherein the ISRIB is present in the eukaryotic cell culture medium.Docket No. P39212-WO-1

[0168] In embodiments, use of ISRIB is provided for enhancing the titer and / or product quality of a recombinant protein product is provided, wherein the recombinant protein product is produced by a eukaryotic cell culture, and wherein the ISRIB is present in the eukaryotic cell culture medium.

[0169] In embodiments, use of suramin is provided for enhancing growth and / or viability and / or productivity of a eukaryotic cell culture is provided, wherein the suramin is present in the eukaryotic cell culture medium.

[0170] In embodiments, use of suramin is provided for enhancing the titer and / or product quality of a recombinant protein product, wherein the recombinant protein product is produced by a eukaryotic cell culture, and wherein the suramin is present in the eukaryotic cell culture medium.

[0171] In any of the above embodiments, use is provided wherein enhancing the growth and / or viability and / or productivity of the eukaryotic cell culture comprises increasing growth and / or viability and / or productivity of the eukaryotic cell culture compared to what would be obtained performing a comparative method with a similar cell culture medium not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.

[0172] In the above embodiment, use is provided wherein growth and / or viability is measured as viable cell density and / or final viability and / or viable cell count, optionally integrated viable cell count, optionally wherein the final viability and / or integrated viable cell count is at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% higher than that what would be obtained performing the comparative method.

[0173] In any of the above embodiments, use is provided wherein productivity is measured as the titer of a recombinant polypeptide expressed by the mammalian cells.

[0174] In any of the above embodiments, use is provided wherein the enhancing the product quality comprises: a higher percentage of the main peak (by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture that not comprising ISRIB and / or hypotaurine (andDocket No. P39212-WO-1 optionally increased level of iron) and / or suramin, optionally wherein the higher percentage of the main peak (by charge distribution) is about 10-20% higher than with the similar eukaryotic cell culture medium; and / or a lower percentage of the high molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin, optionally wherein the lower percentage in the high molecular weight species / forms is about 1-10% lower than with the similar eukaryotic cell culture medium; and / or a lower percentage of the low molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin, optionally wherein the lower percentage in the low molecular weight species / forms is about 1-10% lower than with the similar eukaryotic cell culture medium.

[0175] In any of the above embodiments, use is provided wherein enhancing the product titer comprises enhancing the titer of the recombinant polypeptide expressed by the mammalian cells compared to that what would be obtained performing a comparative method with a similar cell culture medium not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin optionally wherein enhancing the product titer comprises enhancing the titer of the recombinant polypeptide expressed by the mammalian cells by at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to that what would be obtained performing the comparative method.

[0176] In any of the above embodiments, use is provided wherein the eukaryotic cell culture is a CHO cell culture.

[0177] In any of the above embodiments, use is provided wherein the eukaryotic cell culture is a culture of a cell therapy product.Methods of CulturingDocket No. P39212-WO-1

[0178] In another aspect, the invention provides a method of culturing mammalian cells with enhanced growth and / or viability, comprising culturing the mammalian cells in a lx eukaryotic cell culture medium as provided herein.

[0179] The eukaryotic cell culture medium may be as defined herein. As used herein, references to a “similar eukaryotic cell culture medium” refers to an identical eukaryotic cell culture medium, but for the specified difference(s), for e.g., an identical eukaryotic cell culture medium but does not comprise ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0180] In embodiments, the culturing comprises culturing for a time period in the range of from about 3 to about 30 days. In embodiments, the culturing comprises culturing for a time period in the range of from about 4 to about 21 days. In embodiments, the culturing comprises culturing for a time period in the range of from about 6 to about 18 days. In embodiments, the culturing comprises culturing for a time period in the range of from about 7 to about 14 days.

[0181] In embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, or human cells (for cell therapy). In embodiments, the mammalian cells are CHO cells. In embodiments, the CHO cells are adapted to produce recombinant protein.

[0182] In embodiments, the CHO cells are selected from CHO KI cells, CHO KI SV cells, DG44 cells, DUKXB-11 cells, CHOK1S cells, or CHO KIM cells, or their derivatives.

[0183] In embodiments, the culturing is either a batch process, a standard fed-batch process, an intensified process or a perfusion process. In embodiments, the culturing is an intensified fed-batch process. In embodiments, the culturing is a perfusion process or an intermittent (semi-continuous) perfusion process.

[0184] In embodiments, the culturing is performed under batch culture conditions, fed-batch culture conditions, or perfusion culture conditions.

[0185] It may be that the culturing is performed under fed-batch culture conditions. The fed-batch culture conditions may be intensified fed-batch culture conditions.Docket No. P39212-WO-1

[0186] It may be that the culturing is performed under perfusion culture conditions. The perfusion culture conditions may be semi-continuous perfusion or continuous perfusion.Methods of Producing a Recombinant Protein

[0187] In another aspect, there is provided a method of producing a recombinant protein, comprising culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant protein under conditions suitable for production of the polypeptide in a lx eukaryotic cell culture medium as provided herein.

[0188] The eukaryotic cell culture medium may be as defined herein. As used herein, references to a “similar eukaryotic cell culture medium” refers to an identical eukaryotic cell culture medium, but for the specified difference(s), for e.g., an identical eukaryotic cell culture medium but does not comprise ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0189] In embodiments, the culturing is either a batch process, a standard fed-batch process, an intensified fed-batch process, or a perfusion process. In embodiments, the culturing is an intensified fed-batch process. In embodiments, the culturing is a perfusion process.

[0190] In embodiments, the polynucleotide that encodes the polypeptide is integrated in the cellular genome of the cells of the cell line at a targeted location. In alternative embodiments, the polynucleotide that encodes the polypeptide is randomly integrated in the cellular genome of the cells of the cell line.

[0191] In embodiments, the polynucleotide that encodes the polypeptide is an extrachromosomal polynucleotide. The extrachromosomal polynucleotide may comprise extrachromosomal DNA (ecDNA) and / or extrachromosomal circular DNA (eccDNA).

[0192] In embodiments, the polynucleotide that encodes the polypeptide is integrated into a chromosome of the cells of the cell line.

[0193] In embodiments, the recombinant polypeptide is an antibody, an antigen, an enzyme, a gene vector or a vaccine.Docket No. P39212-WO-1

[0194] In embodiments, the recombinant polypeptide is a therapeutic antibody.

[0195] In embodiments, the therapeutic antibody is selected from anti-HER receptor family antibodies (such as anti-HERl (EGFR), anti-HER2, anti-HER3 and anti-HER4); anti-CD protein antibodies (such as anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD25, anti-CD33, anti-CD34, anti-CD38, anti-CD52); anti- IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies, anti-CDl la antibodies; anti-CD 18 antibodies; anti-IgE antibodies; anti -Apo-2 receptor antibodies; anti-Tissue Factor (TF) antibodies; anti- cell adhesion molecules such as LFA-1, Mol, pl50,95, VLA- 4, ICAM-1, VCAM, anti-human ouP? integrin antibodies, anti-human avps integrin antibodies, anti-avP3 antibodies including either a or P or subunits thereof (e.g. anti- CD1 la, anti-CD18 or anti-CDl lb antibodies); anti-EGFR antibodies; anti-Fc receptor antibodies; anti-carcinoembryonic antigen (CEA) antibodies; anti-human renal cell carcinoma antibodies; anti-human colorectal tumor antibodies; anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma; antibodies directed against breast epithelial cells; antibodies that bind to colon carcinoma cells; anti-EpCAM antibodies; anti-GpIIb / IIIa antibodies; anti-RSV antibodies; anti-CMV antibodies; anti -HIV antibodies; anti -hepatitis antibodies; anti-CA 125 antibodies; antihuman 17-1A antibodies; and anti-human leukocyte antigen (HLA) antibodies, and anti- HLA DR antibodies, anti-growth factors such as vascular endothelial growth factor (anti- VEGF) or fragments; anti-IgE; anti-blood group antigens; anti-flk2 / flt3 receptor; and antiobesity (OB) receptor; anti-amyloid antibodies, anti-alpha-synuclein (e.g.: prasinezumab), anti-amyloid-beta, anti-growth hormone (GH), including human growth hormone (hGH) and bovine growth hormone (bGH); anti-growth hormone releasing factor; antiparathyroid hormone; anti-thyroid stimulating hormone; anti-lipoproteins; anti- a-1 - antitrypsin; anti-insulin A-chain; anti-insulin B-chain; anti-proinsulin; anti-follicle stimulating hormone; anti-calcitonin; anti-luteinizing hormone; anti-glucagon; anticlotting factors such as factor VIIIC, tissue factor or von Willebrands factor; anti-clotting factors such as Protein C; anti-atrial natriuretic factor; anti-lung surfactant; anti- a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; anti-tumor necrosis factor-a and -P; anti-enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); anti-human macrophageDocket No. P39212-WO-1 inflammatory protein (MIP-l-a); anti- a serum albumin such as human serum albumin (HSA); anti- mullerian-inhibiting substance; anti- relaxin A-chain; anti- relaxin B-chain; anti-prorelaxin; anti- mouse gonadotropin-associated peptide; anti-DNase; anti-inhibin; anti-activin; anti- receptors for hormones or growth factors; anti- protein A or D; anti- rheumatoid factors; anti- a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT -4, NT-5, or NT-6), or a nerve growth factor such as NGF-P; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); anti- transforming growth factor (TGF) such as TGF-a and TGF-P, including TGF-pi, TGF-P2, TGF-P3, TGF-P4, or TGF- P5; anti- insulin-like growth factor-I and -II (IGF-I and IGF-II); anti- des(l-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins (IGFBPs); anti- erythropoietin (EPO); anti- thrombopoietin (TPO); anti- osteoinductive factors; anti- immunotoxins; anti- a bone morphogenetic protein (BMP); anti- an interferon such as interferon-a, -P, and -y; anticolony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; anti- interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; anti- T-cell receptors; anti- surface membrane proteins; anti- decay accelerating factor (DAF); anti- a viral antigen such as, for example, a portion of the AIDS envelope; anti- transport proteins; anti- homing receptors; anti- addressins; anti- regulatory proteins; anti- immunoadhesins; and a therapeutic antibody directed against biologically active fragments or variants of any of the abovelisted polypeptides.

[0196] In embodiment, the therapeutic antibody is selected from AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranibizumab), RAPTIVA® (efalizumab), RITUXAN® (rituximab), ACTEMRA® (tocilizumab - anti-IL-6 receptor), XOLAIR® (omalizumab), OCREVUS® (ocrelizumab - anti-CD20 antibody), PERJETA® (pertuzumab - HER dimerization inhibitors (HDIs)), TECENTRIQ® (atezolizumab - anti- PD-L1 antibody), LUNSUMIO® or COLUMVI™ (mosunetuzumab or glofitamab - anti- CD20 X anti-CD3 bispecific antibody), VABYSMO® (faricimab - anti- VEGF-A X anti- angiopoietin-2 bispecific antibody), anti-CD79b antibody, anti-OX40 ligand, anti-oxidized LDL (oxLDL), anti-amyloid beta (e.g., trontinemab), anti-CD4 (MTRX1011A), anti- EGFL7 (EGF -like-domain 7), anti-IL13, Apomab (anti -DR5 -targeted pro-apoptotic receptor agonist (PARA), anti-BR3 (CD268, anti-BLyS receptor 3, anti-BAFF-R, (BAFFDocket No. P39212-WO-1Receptor), anti-TIGIT (anti-T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif domains) antibodies, astegolimab (anti- ST2, an IL-33 receptor), anti -beta 7 integrin subunit, anti- avps integrin antibodies, dacetuzumab (Anti-CD40), GAI 01 (obinutuzumab - anti-CD20 monoclonal antibody), MetMAb (onartuzumab - anti -MET receptor tyrosine kinase), cevostamab (anti-Fc receptor-homolog 5 (FcRH5) X anti-CD3 bispecific antibody), anti -neuropilin- 1 (NRP1), and rhuMAb IFN alpha.

[0197] In embodiments, when the recombinant polypeptide is an antibody, the antibody is a multispecific antibody or antigen-binding fragment thereof.

[0198] In embodiments, the antibody consists of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof. In embodiments, the antibody comprises a chimeric antibody, a human antibody or a humanized antibody. In embodiments, the antibody comprises a monoclonal antibody.

[0199] In embodiments, the cell line is a Chinese hamster ovary (CHO) cell line. In embodiments, the CHO cell line is selected from a CHO KI cell line, a CHO KI SV cell line, a DG44 cell line, a DUKXB-11 cell line, a CHOK1S cell line, a CHO KIM cell line, or their derivatives.Commercial Manufacturing

[0200] The cell culture media of the present disclosure may be employed in the production of a molecule of interest at manufacturing scale. “Manufacturing scale" production of therapeutic proteins, or other proteins, utilize cell cultures ranging from about 400 L to about 80,000 L, depending on the protein being produced and the need. Typically, such manufacturing scale production utilizes cell culture sizes from about 400 L to about 25,000 L. Within this range, specific cell culture sizes, such as about 4,000 L, about 6,000 L, about 8,000, about 10,000, about 12,000 L, about 14,000 L, about 16,000 L or about 25,000 L may be utilized.

[0201] The cell culture media of the present disclosure may be employed to support the high-density growth of mammalian cells in suspension cell culture, and / or to support the expression of recombinant protein from mammalian cells, and / or in the production ofDocket No. P39212-WO-1 large quantities of a molecule of interest in a shorter timeframe as compared to conventional media or media used previously in cell culture. In certain embodiments, the cell culture media of the present disclosure can be employed for improved quality of the molecule of interest as compared to conventional media or media used previously in cell culture. In certain embodiments, the cell culture media of the present disclosure can be used to enhance seed train stability by preventing chronic toxicity that can be caused by products that cause cell stress and clonal instability over time. In certain embodiments, the cell culture media of the present disclosure can be used for the optimal expression of cell culture products such as polypeptides, proteins, antibodies (monoclonal, bispecific, trispecific, multispecific, etc.), antibody fragments, etc.

[0202] In certain embodiments, the polypeptide of interest cultured in cell culture media of the present disclosure exhibits reduced coloration relative to a polypeptide expressed in conventional media or media used previously in cell culture. In certain embodiments, the polypeptide of interest exhibits a reduced negative charge in media of the present disclosure as compared to a polypeptide expressed in conventional media or media used previously in cell culture. In certain embodiments, the polypeptide of interest exhibits increased galactosylation and / or sialylation on N-glycans as compared to conventional media or media used previously in cell culture.

[0203] In certain embodiments, the media of the present disclosure can be used for cell culture process optimization and / or process development.

[0204] In certain embodiments, the cell culture media of the present disclosure can be employed to reduce levels of aggregates (HMWS) or fragments (LMWS) of a molecule of interest as compared to conventional media or media used previously in cell culture.

[0205] In certain embodiments, the cell culture media of the present disclosure can be used to achieve increased expression of a polypeptide (or polypeptides) of interest. For example, but not by way of limitation, the cell culture media of the present disclosure can achieve expression of standard and half antibodies at titers of at least 2 g / L, 2.5 g / L, 3 g / L,3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L,9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, or more, and expression of multispecific antibodies, e.g., bispecific antibodies, of at least 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5Docket No. P39212-WO-1 g / L, 5 g / L, 5.5 g / L, 6 g / L, or more. For example, but not by way of limitation, the cell culture media of the present disclosure can achieve bispecific content of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or more.

[0206] In certain embodiments, the cell culture media of the present disclosure may be used as an investigational tool. In certain embodiments, the cell culture media of the present disclosure maybe used as a diagnostic tool to map out the root causes of low protein expression for problematic molecules in various cells. In certain embodiments, the cell culture media of the present disclosure can be used to directly link an observed phenomenon or cellular behavior to the transgene expression in the cells. In certain embodiments, the cell culture media of the present disclosure can be exploited to identify and mitigate problems with respect to transgene(s) transcription and expression in cells.Manufacture of Antibodies using the Media of the present Disclosure

[0207] Therapeutic antibodies that can be produced in the media described in this disclosure include, without limitation, anti-HER receptor family antibodies (such as anti- HER1 (EGFR), anti-HER2, anti-HER3 and anti-HER4); anti-CD protein antibodies (such as anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti- CD25, anti-CD33, anti-CD34, anti-CD38, anti-CD52); anti-IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies, anti-CDl la antibodies; anti-CD 18 antibodies; anti-IgE antibodies; anti-Apo-2 receptor antibodies; anti-Tissue Factor (TF) antibodies; anti- cell adhesion molecules such as LFA-1, Mol, pl50,95, VLA-4, ICAM-1, VCAM, anti-human ouP? integrin antibodies, anti-human avps integrin antibodies, anti-avP3 antibodies including either a or P or subunits thereof (e.g. anti-CD 1 la, anti-CD 18 or anti-CD 1 lb antibodies); anti -EGFR antibodies; anti-Fc receptor antibodies; anti-carcinoembryonic antigen (CEA) antibodies; anti-human renal cell carcinoma antibodies; anti-human colorectal tumor antibodies; anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma; antibodies directed against breast epithelial cells; antibodies that bind to colon carcinoma cells; anti-EpCAM antibodies; anti-GpIIb / IIIa antibodies; anti-RSV antibodies; anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies; anti-CA 125 antibodies; anti-human 17-1A antibodies; and anti-human leukocyte antigen (HLA) antibodies, and anti-HLA DRDocket No. P39212-WO-1 antibodies; anti-growth factors such as vascular endothelial growth factor (anti-VEGF) or fragments; anti-IgE; anti-blood group antigens; anti-flk2 / flt3 receptor; and anti-obesity (OB) receptor. Other exemplary proteins to which therapeutic antibodies are designed include anti-amyloid antibodies, anti-alpha-synuclein (e.g. : prasinezumab), anti-amyloid- beta, anti-growth hormone (GH), including human growth hormone (hGH) and bovine growth hormone (bGH); growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; a-1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, tissue factor or von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; tumor necrosis factor-a and -P; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-l-a); serum albumin such as human serum albumin (EISA); mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin; activin; receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT -4, NT-5, or NT-6), or a nerve growth factor such as NGF-P; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-a and TGF-P, including TGF-pi, TGF-P2, TGF-P3, TGF-P4, or TGF- P5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins (IGFBPs); erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-a, -P, and -y; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor (DAF); a viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; and biologically active fragments or variants of any of the above-listed polypeptides. Many other antibodies and / or other proteins may be used in accordance with the instant disclosure, and the above lists are not meant to be limiting.Docket No. P39212-WO-1

[0208] Therapeutic antibodies of particular interest include those that are commercially available or are in clinical development such as: AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranibizumab), RAPTIVA® (efalizumab), RITUXAN® (rituximab), ACTEMRA® (tocilizumab - anti-IL-6 receptor), XOLAIR® (omalizumab), OCREVUS® (ocrelizumab - anti-CD20 antibody), PERJETA® (pertuzumab - HER dimerization inhibitors (HDIs)), TECENTRIQ® (atezolizumab - anti- PD-L1 antibody), LUNSUMIO® or COLUMVI™ (mosunetuzumab or glofitamab - anti- CD20 X anti-CD3 bispecific antibody), VABYSMO® (faricimab - anti- VEGF-A X anti- angiopoietin-2 bispecific antibody), anti-CD79b antibody, anti-OX40 ligand, anti-oxidized LDL (oxLDL), anti-amyloid beta (e.g., trontinemab), anti-CD4 (MTRX1011A), anti- EGFL7 (EGF -like-domain 7), anti-IL13, Apomab (anti -DR5 -targeted pro-apoptotic receptor agonist (PARA), anti-BR3 (CD268, anti-BLyS receptor 3, anti-BAFF-R, (BAFF Receptor), anti-TIGIT (anti-T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif domains) antibodies, astegolimab (anti- ST2, an IL-33 receptor), anti -beta 7 integrin subunit, anti- avps integrin antibodies, dacetuzumab (Anti-CD40), GAI 01 (obinutuzumab - anti-CD20 monoclonal antibody), MetMAb (onartuzumab - anti -MET receptor tyrosine kinase), cevostamab (anti-Fc receptor-homolog 5 (FcRH5) X anti-CD3 bispecific antibody), anti -neuropilin- 1 (NRP1), rhuMAb IFN alpha, etc. Many other antibodies and / or other proteins may be used in accordance with the instant disclosure, and the above lists are not meant to be limiting.Protein isolation, titer and quality

[0209] In embodiments, the method further comprises isolating the recombinant polypeptide. The isolating may be performed using any suitable method for the recombinant polypeptide, such as a method that uses a suitable affinity reagent. For example, when the recombinant protein is an IgG antibody or derivative thereof, the isolation may comprise use of a relevant affinity reagent such as Protein A, Protein G, Protein A / G, or Protein L. In embodiments, the method further comprises formulating the isolated recombinant polypeptide in a pharmaceutical composition, wherein the pharmaceutical composition comprises the recombinant polypeptide and a pharmaceutically acceptable excipient or carrier.Docket No. P39212-WO-1

[0210] In embodiments, the recombinant polypeptide is obtained at a higher titer. It may be that the higher titer is higher than the titer that would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0211] In embodiments, the lx eukaryotic cell culture medium comprises hypotaurine, thereby providing higher titer of the recombinant protein and / or an improved product quality.

[0212] In embodiments, the improved product quality comprises a lower percentage of the basic peak (by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium than not comprising hypotaurine. In embodiments, the lower percentage in the basic peak is about 1-10% lower than with a similar eukaryotic cell culture medium that does not comprise hypotaurine.

[0213] In embodiments, the improved product quality comprises a lower percentage of the acidic peak (by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising hypotaurine. In embodiments, the lower percentage in the acidic peak is about 1-10% lower than with a similar eukaryotic cell culture medium that does not comprise hypotaurine.

[0214] In embodiments, the lx eukaryotic cell culture medium comprises ISRIB and / or hypotaurine, thereby providing higher titer of the recombinant protein and / or an improved product quality.

[0215] In embodiments, the improved product quality comprises a lower percentage of the high molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising ISRIB and / or hypotaurine. In embodiments, the lower percentage in the high molecular weight species / forms is about 1- 10% lower than with a similar eukaryotic cell culture medium that does not comprise ISRIB and / or hypotaurine.Docket No. P39212-WO-1

[0216] In embodiments, the improved product quality comprises a lower percentage of the low molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising hypotaurine and / or ISRIB. In embodiments, the lower percentage in the low molecular weight species / forms is about 1- 10% lower than with a similar eukaryotic cell culture medium that does not comprise hypotaurine.

[0217] In embodiments, the lx eukaryotic cell culture medium comprises suramin, thereby providing the recombinant protein with an improved product quality.

[0218] In embodiments, the improved product quality comprises a higher percentage of the main peak (i.e., neutral species by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium that does not comprise suramin. In embodiments, the higher percentage in the main peak is about 10-20% higher than with a similar eukaryotic cell culture medium that does not comprise suramin.

[0219] In embodiments, the improved product quality comprises a higher percentage of the monomer (i.e., main peak by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium that does not comprise suramin. In embodiments, the higher percentage in the main peak is about 10-20% higher than with a similar eukaryotic cell culture medium that does not comprise suramin.Viability

[0220] In embodiments, the improved viability comprises a higher percentage of viable cells either throughout the cell culture or at harvest than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising hypotaurine or ISRIB. In embodiments, the higher percentage in the viability is about 5-30% higher than with a similar eukaryotic cell culture medium that does not comprise hypotaurine or ISRIB.UsesDocket No. P39212-WO-1

[0221] In another aspect of the invention, there is provided a use of a eukaryotic cell culture medium as provided herein for culturing a eukaryotic cell. It may be that the culturing provides enhanced growth and / or productivity compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0222] In another aspect of the invention, there is provided a use of a eukaryotic cell culture medium as provided herein for culturing a eukaryotic cell. It may be that the culturing provides enhanced growth and / or productivity compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally an increased level of iron) and / or suramin.

[0223] The culturing may be as further defined in relation to the methods disclosed herein.

[0224] In another aspect of the invention, there is provided a use of a eukaryotic cell culture medium as provided herein for the production of a recombinant protein from a cultured cell line comprising a polynucleotide encoding the recombinant protein. It may be that the titer of the recombinant protein is enhanced and / or the product quality of the recombinant protein is enhanced compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine and increased level of iron and / or suramin.

[0225] In another aspect of the invention, there is provided a use of a eukaryotic cell culture medium as provided herein for the production of a recombinant protein from a cultured cell line comprising a polynucleotide encoding the recombinant protein. It may be that the titer of the recombinant protein is enhanced and / or the product quality of the recombinant protein is enhanced compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin. It may be that the culture viability is enhanced compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin, further optionally wherein the increase in culture viability is about 5-30% higher than with a similar eukaryotic cell culture medium that does not comprise ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.Docket No. P39212-WO-1

[0226] In embodiments, the enhanced product quality comprises that an increased proportion of the recombinant protein is present as the main peak (by charge distribution).

[0227] In embodiments, the enhanced product quality comprises that an increased proportion of the recombinant protein is present as the monomer (by size distribution).

[0228] In embodiments, the polynucleotide that encodes the polypeptide is integrated in the cellular genome of the cells of the cell line at a targeted location. In alternative embodiments, the polynucleotide that encodes the polypeptide is randomly integrated in the cellular genome of the cells of the cell line.

[0229] In embodiments, the polynucleotide that encodes the polypeptide is an extrachromosomal polynucleotide. The extrachromosomal polynucleotide may comprise extrachromosomal DNA (ecDNA) and / or extrachromosomal circular DNA (eccDNA).

[0230] In embodiments, the polynucleotide that encodes the polypeptide is integrated into a chromosome of the cells of the cell line.

[0231] In embodiments, the recombinant polypeptide is an antibody, an antigen, an enzyme, a gene vector or a vaccine.

[0232] In embodiments, when the recombinant polypeptide is an antibody, the antibody is a multispecific antibody or antigen-binding fragment thereof.

[0233] In embodiments, the antibody consists of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof. In embodiments, the antibody comprises a chimeric antibody, a human antibody or a humanized antibody. In embodiments, the antibody comprises a monoclonal antibody.

[0234] In embodiments, the cell line is a Chinese hamster ovary cell (CHO) cell line, a HEK293 cell line, or a human cell (for cell therapy).

[0235] In embodiments, the cell line is a CHO cell line. In embodiments, the CHO cell line is selected from a CHO KI cell line, a CHO KI SV cell line, a DG44 cell line, a DUKXB-1 1 cell line, a CHOK1S cell line, a CHO KIM cell line, or their derivatives.Docket No. P39212-WO-1

[0236] In embodiments, the recombinant protein is a therapeutic antibody such as examples of which are disclosed herein above.

[0237] In embodiments, the media or feeds described here are used to culture a cell therapy product, such as an engineered T cell or other.EXAMPLES

[0238] The disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.Materials and Methods

[0239] CHO cell lines genetically engineered to secrete nine different recombinant humanized antibodies were used, including some antibodies having complex formats (e.g., bispecifics), as listed in Table 2 below. Cell lines were developed in a targeted integration host described in WO 2019 / 126634, hereby incorporated by reference. CHO cells were cultured in a proprietary DMEM / F12 based medium in shake flask vessels agitated at 150 rpm, and maintained in a 35-37°C, and 5-10% CO2 environment. Cells were passaged at a seeding density of -3-20 x 105cells / mL every 3-4 days.Table 2: Antibody and derivative molecule types produced by the CHO cell lines testedDocket No. P39212-WO-1Feb -Batch Production Culture

[0240] Fed-batch production cultures were performed in 24 well deep well plates, shake flask vessels, AMBR15 High Throughput Bioreactor System (Sartorius Stedim) or AMBR250 High Throughput Bioreactor System (Sartorius Stedim). Cells cultured in 24 well deep well plates used a 3 mL working volume and were inoculated at 2e6 cells per mL and grown for 7 days with a day 3 proprietary feed. Cells cultured in shake flasks were inoculated at 20e6 cells per mL and grown for 7 days with a day 1 and day 3 proprietary feed. AMBR250 bioreactors were inoculated at from a 7 day N-l perfusion culture . AMBR15 bioreactors were inoculated from either a 7 day N-l perfusion culture or from shake flask cultures. The AMBR15 and AMBR250 bioreactor units were maintained at a pH of 7.2 and dissolved oxygen (DO) at 40% of air saturation. AMBR cultures were fed on days 1, 3, 6 and 9. AMBR15 vessels were agitated at 1400 RPM and and 35°C whereas AMBR250 vessels were agitated at 477 RPM and 35°C.Cell Culture Medium

[0241] Cell culture medium for high throughput screening of components was prepared without additional iron, hypotaurine or ISRIB. Screened components wereDocket No. P39212-WO-1 supplemented to the control medium (a DMEM / F 12-based medium) at 0.1, lx and lOx of anticipated effective concentration for each component. Unless indicated otherwise, iron was supplemented as either just 50 pM ferrous sulfate, or with an additional 50-350 pM F erric c itrate, or just 50-350 pM ferric citrate without any ferrous sulfate. Hypotaurine was supplemented at 1 and 10 mM. ISRIB was supplemented at 2 pM, using a 2 mM stock solution in DMSO.

[0242] Forms of iron that can be used for eukaryotic cell culture include but are not limited to ferrous sulfate, ferric citrate, ferric ammonium citrate, ferric nitrate, ferric chloride, ferrous bisglycinate, and recombinant transferrin.Cell Viability Assessment

[0243] Cell viability was determined by Trypan blue dye exclusion using a Nova BioProfile FLEX Analyzer (Nova Biomedical), Vi-CELL XR (Beckman Coulter), or similar instrument.Titer Assessment

[0244] Titer was quantified by affinity chromatography (e.g. Protein A or Protein L high-performance liquid chromatography) or an immunoturbidometric method using the Cedex Bio HT Analyzer (Roche CustomBiotech) wherein the IgG reacts with a specific antiserum to form a precipitate, such that the change in absorbance can be measured photometrically and correlated to antibody concentration.Product Quality Assessment

[0245] Product quality was assessed using harvested cell culture material purified with either protein A or protein L affinity resin. Charge variants was assessed either by ion exchange chromatography or imaged capillary isoelectric focusing after the removal of the C-terminal lysine residues on the heavy chain by carboxypeptidase B. Size variants were assessed via size-exclusion chromatography by Ultra High Pressure Liquid Chromatography (SEC-UHPLC) to quantify monomer, high and low molecular weight forms of the product. In some cases, capillary electrophoresis-sodium dodecyl sulfate (CE- SDS) was also used to quantify the LMW forms. All protein product quality assays were developed in-house, and detailed protocols have been published (Hopp et al., 2009.Docket No. P39212-WO-1“Development of a high throughput protein A well-plate purification method for monoclonal antibodies” Biotechnology Progress 25(5): 1427-1432).Example 1: Cell culturing

[0246] Cell culture media was prepared via the addition of one or more of: 0.01 - 200 pM Integrated Stress Response Inhibitor B (ISRIB); 50 - 350 pM iron paired with 1-10 mM hypotaurine; and 2-400 pM Suramin to a basal medium.

[0247] It is thought that basal cell culture medium prepared with ISRIB achieves an increase in titer via the integrated stress response by activating eIF2B, thereby increasing translation. When tested individually, ISRIB resulted in titer increases in six different molecules representing bispecific, Fc Fusion and Fab IgGl therapeutics. The titer increase was observed in both bioreactors and shake flask production. Molecule 1 demonstrated a 1.3x increase in titer, Molecule 2 demonstrated a 1.5x increase in titer and Molecule 3 demonstrated a 1.8x increase in titer when cultured using an intensified fed batch process in a Sartorius AMBR250 High Throughput system. Molecule 4 demonstrated a 2. Ox increase in titer, Molecule 5 demonstrated a 3.2x increase in titer, and Molecule 6 demonstrated a 1.8x increase in titer using an intensified fed batch process in shake flask.

[0248] An increase in iron from 50 pM to 100 pM increased titer by30%. Hypotaurine has been demonstrated to protect antibody therapeutics from discoloration via oxidation (Vijayasankaran et al., 2018. “Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody” Biotechnology Progress 34(5): 1298-1307). Given that increased iron can result in increased oxidation, hypotaurine was paired with these conditions to reduce the risk of discoloration and control acidic variants increase due to iron. It was previously shown that hypotaurine does not increase growth or titer when included alone in cell culture medium for a given recombinant monoclonal antibody (Vijayasankaran et al., 2018. “Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody” Biotechnology Progress 34(5): 1298-1307) and demonstrated again with a different recombinant monoclonal antibody (M olecule 7) in a fed batch production process.Docket No. P39212-WO-1

[0249] Surprisingly, when assessed in combination with other cell culture process parameters (pH shift, B vitamins, and manganese), the use of hypotaurine improved cell culture performance (in terms of cell viability, growth, titer) and product quality (in terms of charge and size variants) for two different cell lines expressing a recombinant monoclonal antibody (Molecule 8). The surprising effects of hypotaurine were shown by statistical modelling of the responses from a design of experiments (DOE) study involving presence (+1) or absence (-1) of hypotaurine in the production cell culture.

[0250] Surprisingly, when paired with an increase in iron concentration, the inventors found that the use of hypotaurine synergistically improves titer. Without wishing to be bound by theory, it is thought that, in the absence of an antioxidant, the full potential of an iron increase is not achieved due to the secondary damage caused by iron through reactive oxygen species generation. Incorporation of hypotaurine as a Reactive Oxygen Species (ROS) scavenger leads to improved performance in the presence of increased iron. Similar synergistic effects were achieved using other antioxidants in place of hypotaurine, i.e. bisglycinate. However, other antioxidants did not show the benefits displayed by hypotaurine or bisglycinate, which makes this discovery all the more surprising.

[0251] Molecule 1, a bispecific IgGl therapeutic, demonstrated an increase in titer from 4 g / L to 6 g / L when iron was increased 2- fold. When paired with 1 mM hypotaurine, titer was improved to 7.5 g / L and further improved to 8.2 g / L when hypotaurine was increased to 10 mM. Increase in titer is driven from an increase in growth during N-l, thereby enabling a higher initial cell density to be achieved for inoculation of the N production culture. This is also consistent with the observed modest increase in viability at the increased level of iron, with viability further improved with hypotaurine.

[0252] Utilizing a 24 well plate high throughput screening process of potential components, suramin was tested and found to have a 10% increase in titer in both Molecules 1 and 2. Similar results were observed using ensulizole, which provided improved titer in both Molecules 1 and 2.Docket No. P39212-WO-1

[0253] Suramin demonstrated a similar increase in titer when cultured in AMBR250 production bioreactors, where Molecule 4 was found to have a 10% increase in titer. Molecules 4 and 5 demonstrated improved viability when suramin was present.

[0254] The titer advantage of adding more iron, hypotaurine and / or ISRIB to medium comes with a shift in the charge variant and aggregation profile. For example, the monomer species for molecule 4 decreased by ~2% when increased iron, hypotaurine and ISRIB were applied. Effective titer, defined as the titer from the bioreactor multiplied by the percentage of monomer species, does not reach its full potential due to a reduction in the main peak (in terms of size distribution). The presence of suramin when cultured with iron, hypotaurine and ISRIB rescues main peak (i.e., monomer) expression, resulting in an increase in effective titer. Additionally, cultures containing increased iron, hypotaurine and ISRIB result in increased acidic variants. The presence of suramin reduces acidic variants to control levels as demonstrated with molecule 4 and increases the percentage of the main species (i.e., neutral main peak).

Claims

1. Docket No. P39212-WO-1WHAT IS CLAIMED IS:

1. A eukaryotic cell culture medium comprising integrated stress response inhibitor (ISRIB).

2. The eukaryotic cell culture medium of claim 1, wherein the ISRIB is adapted to be present at a level of from about 0.01 pM to about 200 pM when the medium is provided as a IX medium formulation; optionally wherein the ISRIB is adapted to be present at a level of from about 0.1 pM to about 20 pM when the medium is provided as a IX medium formulation; further optionally wherein the ISRIB is adapted to be present at a level of from about 2 pM to about 200 pM when the medium is provided as a IX medium formulation; further optionally wherein the ISRIB is adapted to be present at a level of from about 2 pM to about 20 pM when the medium is provided as a IX medium formulation.

3. The eukaryotic cell culture medium of claim 1 or claim 2, further comprising hypotaurine.

4. The eukaryotic cell culture medium of claim 3, wherein the hypotaurine is present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation; optionally wherein the hypotaurine is present at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation; further optionally wherein the hypotaurine is present at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

5. The eukaryotic cell culture medium of claim 3 or claim 4, further comprising an increased level of iron.

6. The eukaryotic cell culture medium of claim 5, wherein the iron is adapted to be present at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally wherein the iron is adapted to be present at a level of from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to about 350 pM; further optionally wherein the iron is adapted to be present at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, orDocket No. P39212-WO-1 from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation.

7. The eukaryotic cell culture medium of any of claims 1 to 6, further comprising suramin.

8. The eukaryotic cell culture medium of claim 7, wherein the suramin is adapted to be present at a level of from about 0.01 pM to about 1000 pM, or from about 0.1 pM to about 500 pM when the medium is provided as a IX medium formulation; optionally wherein the suramin is adapted to be present at a level of from about 2 pM to about 400 pM, or from about 3.5 pM to about 400 pM, or from about 35 pM to about 400 pM when the medium is provided as a IX medium formulation; further optionally wherein the suramin is adapted to be present at a level of from about 3.5 pM to about 350 pM, or from about 35 pM to about 350 pM when the medium is provided as a IX medium formulation; further optionally wherein the suramin is adapted to be present at a level of from about from about 3.5 pM to about 35 pM when the medium is provided as a IX medium formulation.

9. A eukaryotic cell culture medium comprising hypotaurine.

10. The eukaryotic cell culture medium of claim 9, wherein the hypotaurine is present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation; optionally wherein the hypotaurine is present at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation; further optionally wherein the hypotaurine is present at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

11. The eukaryotic cell culture medium of claim 9 or claim 10, further comprising an increased level of iron.

12. The eukaryotic cell culture medium of claim 11, wherein the iron is adapted to be present at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally wherein the iron is adapted to beDocket No. P39212-WO-1 present at a level of from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to about 350 pM; further optionally wherein the iron is adapted to be present at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, or from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation..

13. The eukaryotic cell culture medium of any of claims 9 to 12, further comprising suramin.

14. The eukaryotic cell culture medium of claim 13, wherein the suramin is adapted to be present at a level of from about 0.01 pM to about 1000 pM when the medium is provided as a IX medium formulation; optionally wherein the suramin is adapted to be present at a level of from about 0.1 pM to about 500 pM when the medium is provided as a IX medium formulation.

15. A eukaryotic cell culture medium comprising suramin.

16. The eukaryotic cell culture medium of claim 15, wherein the suramin is adapted to be present at a level of from about 0.01 pM to about 1000 pM when the medium is provided as a IX medium formulation; optionally wherein the suramin is adapted to be present at a level of from about 0.1 pM to about 500 pM when the medium is provided as a IX medium formulation.

17. The eukaryotic cell culture medium of any preceding claim, further comprising ensulizole.

18. The eukaryotic cell culture medium of claim 17, wherein the ensulizole is adapted to be present at a level of from about 0.2 pM to about 500 pM when the medium is provided as a IX medium formulation; optionally wherein the ensulizole is adapted to be present at a level of from about 2 pM to about 200 pM when the medium is provided as a IX medium formulation.

19. The eukaryotic cell culture medium of any preceding claim, wherein the medium is selected from the group consisting of: a thaw medium, a seed train medium, anDocket No. P39212-WO-1 inoculum train medium, a production medium, a perfusion medium for inoculum train culture, a perfusion medium for production culture, a nutrient feed and a stock solution.

20. The eukaryotic cell culture medium of any preceding claim, wherein the medium is capable of supporting high-density growth of mammalian cells in suspension cell culture, and / or wherein the medium is capable of supporting the expression of recombinant protein from the cells.

21. The eukaryotic cell culture medium of claim 20, wherein the cells are Chinese hamster ovary (CHO) cells; optionally wherein the CHO cells are adapted to produce recombinant protein; optionally wherein the CHO cells comprise a polynucleotide encoding a recombinant polypeptide.

22. The eukaryotic cell culture medium of claim 21, wherein the CHO cells are selected from CHO KI cells, CHO KI SV cells, DG44 cells, DUKXB-11 cells, CHOK1S cells, or CHO KIM cells, or their derivatives.

23. The eukaryotic cell culture medium of claim 21 or claim 22, wherein the polynucleotide that encodes the polypeptide is integrated in the cellular genome of the CHO cells.

24. The eukaryotic cell culture medium of claim 21 or claim 22, wherein the polynucleotide that encodes the polypeptide is randomly integrated in the cellular genome of the CHO cells.

25. The eukaryotic cell culture medium of any preceding claim, wherein the medium is a IX medium formulation.

26. The eukaryotic cell culture medium of any of claims 1 to 24, wherein the medium is a concentrated or dry medium formulation.

27. The eukaryotic cell culture medium of claim 26, wherein the medium is a 1 OX medium formulation.

28. The eukaryotic cell culture medium of claim 26, wherein the medium is a greater than 10X concentration medium formulation.Docket No. P39212-WO-129. The eukaryotic cell culture medium of any of claims 1 to 25, wherein the medium is a chemically defined medium.

30. The eukaryotic cell culture medium of any of claims 1 to 25 and 29, wherein the ISRIB, hypotaurine, increased level of iron, suramin, or ensulizole are added, individually or in combination, through supplementation, through nutrient feeds, by addition to basal media, or by addition to perfusion media.

31. A method of culturing mammalian cells with enhanced growth and / or viability, and / or productivity comprising culturing the mammalian cells in a lx eukaryotic cell culture medium of any of claims 1 to 25, 29 or 30.

32. The method of claim 31, wherein the culturing comprises culturing for a time period in the range of from about 3 to about 30 days, optionally in the range of from about 6 to about 18 days.

33. A method of producing a recombinant protein, comprising culturing a eukaryotic cell line that comprises a polynucleotide encoding the recombinant protein under conditions suitable for production of the polypeptide in a lx eukaryotic cell culture medium of any of claims 1 to 25, 29 or 30.

34. The method of claim 33, wherein the culturing process is either a batch, a standard fed-batch, an intensified, or a perfusion.

35. The method of claim 33, wherein the polynucleotide that encodes the polypeptide is integrated in the cellular genome of the cells of the cell line at a targeted location.

36. The method of claim 33, wherein the polynucleotide that encodes the polypeptide is randomly integrated in the cellular genome of the cells of the cell line.

37. The method of any of claims 33-36, wherein the polynucleotide that encodes the polypeptide is an extrachromosomal polynucleotide.

38. The method of any of claims 33-37, wherein the polynucleotide that encodes the polypeptide is integrated into a chromosome of the cells of the cell line.

39. The method of any of claims 33-38, wherein the recombinant polypeptide is an antibody, an antigen, an enzyme, a gene vector or a vaccine.Docket No. P39212-WO-140. The method of claim 39, wherein the antibody is a multispecific antibody (including bispecific antibody) or antigen-binding fragment thereof.

41. The method of claim 39 or claim 40, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence or antigen-binding fragments thereof.

42. The method of any of claims 33-41, wherein the antibody comprises a chimeric antibody, a human antibody or a humanized antibody.

43. The method of any of claims 33-42, wherein the antibody comprises a monoclonal antibody.

44. The method of any of claims 33-43, further comprising isolating the recombinant polypeptide.

45. The method of any of claims 33-44, wherein the recombinant polypeptide is obtained at a higher titer; optionally wherein the higher titer is higher than the titer that would be obtained performing a comparative method with a similar eukaryotic cell culture not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.

46. The method of any of claims 33-45, wherein the lx eukaryotic cell culture medium comprises hypotaurine, thereby providing higher titer of the recombinant protein.

47. The method of any of claims 33-46, wherein the lx eukaryotic cell culture medium comprises hypotaurine, thereby providing the recombinant protein with an improved product quality; optionally wherein the improved product quality comprises a lower percentage of the acidic peak (by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium that does not comprise hypotaurine; further optionally wherein the lower percentage in the acidic peak is about 1-20% lower than with a similar eukaryotic cell culture medium that does not comprise hypotaurine.

48. The method of any of claims 33-46, wherein the lx eukaryotic cell culture medium comprises hypotaurine, thereby providing the recombinant protein with an improvedDocket No. P39212-WO-1 product quality; optionally wherein the improved product quality comprises a lower percentage of the basic peak (by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium that does not comprise hypotaurine; further optionally wherein the lower percentage in the basic peak is about 1-10% lower than with a similar eukaryotic cell culture medium that does not comprise hypotaurine.

49. The method of any of claims 33-46, wherein the lx eukaryotic cell culture medium comprises ISRIB and / or hypotaurine, thereby providing the recombinant protein with an improved product quality; optionally wherein the improved product quality comprises a lower percentage of the high molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising ISRIB and / or hypotaurine; further optionally wherein the lower percentage in the high molecular weight species / forms is about 1-10% lower than with a similar eukaryotic cell culture medium that does not comprise ISRIB and / or hypotaurine.

50. The method of any of claim 33-46, wherein the lx eukaryotic cell culture medium comprises ISRIB and / or hypotaurine, thereby providing the recombinant protein with an improved product quality; optionally wherein the improved product quality comprises a lower percentage of the low molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising ISRIB and / or hypotaurine; further optionally wherein the lower percentage in the low molecular weight species / forms is about 1-10% lower than with a similar eukaryotic cell culture medium that does not comprise ISRIB and / or hypotaurine.

51. The method of any of claims 31-50, wherein the lx eukaryotic cell culture medium comprises suramin, thereby providing the recombinant protein with an improved product quality;Docket No. P39212-WO-1 optionally wherein the improved product quality comprises a higher percentage of the main peak (by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium that not comprising suramin; further optionally wherein the higher percentage in the main peak is about 10-20% higher than with a similar eukaryotic cell culture medium that does not comprise suramin.

52. The method of any of claims 31-50, wherein the lx eukaryotic cell culture medium comprises suramin, thereby providing the recombinant protein with an improved product quality; optionally wherein the improved product quality comprises a higher percentage of the monomer (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture medium not comprising suramin; further optionally wherein the higher percentage in the monomer is about 2-20% higher than with a similar eukaryotic cell culture medium that does not comprise suramin.

53. Use of a eukaryotic cell culture medium of any of claims 1-30 for culturing a eukaryotic cell; optionally wherein the culturing provides enhanced growth and / or viability and / or productivity compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.

54. Use of a eukaryotic cell culture medium of any of claims 1-30 for the production of a recombinant protein from a cultured cell line comprising a polynucleotide encoding the recombinant protein; optionally wherein the titer of the recombinant protein is enhanced and / or the product quality of the recombinant protein is enhanced compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin; and / orDocket No. P39212-WO-1 optionally wherein the culture viability is enhanced compared to a said use of a similar eukaryotic cell culture medium without ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin, further optionally wherein the increase in culture viability is about 5-30% higher than with a similar eukaryotic cell culture medium that does not comprise ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.

55. The use of claim 54, wherein the enhanced product quality comprises that an increased proportion of the recombinant protein is present as the main peak (by charge distribution).

56. The use of claim 54, wherein the enhanced product quality comprises that an increased proportion of the recombinant protein is present as the monomer (by size distribution).

57. The use of any of claims 54-56, wherein the cell line is a CHO cell.

58. The use of any of claims 54-56, wherein the cell is a cell therapy product.

59. Use of hypotaurine for enhancing growth and / or viability and / or productivity of a eukaryotic cell culture, wherein the hypotaurine is present in the eukaryotic cell culture medium, wherein the eukaryotic cell culture medium comprises iron at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally wherein the iron is adapted to be present at a level of from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to about 350 pM; further optionally wherein the iron is adapted to be present at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, or from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation.

60. Use of hypotaurine for enhancing the titer and / or product quality of a recombinant protein product, wherein the recombinant protein product is produced by a eukaryoticDocket No. P39212-WO-1 cell culture, and wherein the hypotaurine is present in the eukaryotic cell culture medium, wherein the eukaryotic cell culture medium comprises iron at a level of from about 50 pM to about 5000 pM when the medium is provided as a IX medium formulation; optionally wherein the iron is adapted to be present at a level of from about 50 pM to about 1000 pM, or from about 50 pM to about 500 pM, or from about 50 pM to about 350 pM; further optionally wherein the iron is adapted to be present at a level of from about 100 pM to about 5000 pM, or from about 100 pM to about 1000 pM, or from about 100 pM to about 500 pM, or from about 100 pM to about 350 pM, or from about 150 pM to about 5000 pM, or from about 150 pM to about 1000 pM, or from about 150 pM to about 500 pM, or from about 150 pM to about 350 pM when the medium is provided as a IX medium formulation.

61. The use of hypotaurine according to claim 59 or claim 60, wherein the hypotaurine is present at a level of from about 0.1 mM to about 20 mM when the medium is provided as a IX medium formulation; optionally wherein the hypotaurine is present at a level of from about 1 mM to about 20 mM when the medium is provided as a IX medium formulation; further optionally wherein the hypotaurine is present at a level of from about 1 mM to about 10 mM when the medium is provided as a IX medium formulation.

62. Use of ISRIB for enhancing growth and / or viability and / or productivity of a eukaryotic cell culture, wherein the ISRIB is present in the eukaryotic cell culture medium.

63. Use of ISRIB for enhancing the titer and / or product quality of a recombinant protein product, wherein the recombinant protein product is produced by a eukaryotic cell culture, and wherein the ISRIB is present in the eukaryotic cell culture medium.

64. Use of suramin for enhancing growth and / or viability and / or productivity of a eukaryotic cell culture, wherein the suramin is present in the eukaryotic cell culture medium.

65. Use of suramin for enhancing the titer and / or product quality of a recombinant protein product, wherein the recombinant protein product is produced by a eukaryotic cell culture, and wherein the suramin is present in the eukaryotic cell culture medium.Docket No. P39212-WO-166. The use according to any one of claims 59-65, wherein enhancing the growth and / or viability and / or productivity of the eukaryotic cell culture comprises increasing growth and / or viability and / or productivity of the eukaryotic cell culture compared to what would be obtained performing a comparative method with a similar cell culture medium not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin.

67. The use of claim 66, wherein growth and / or viability is measured as viable cell density and / or final viability and / or viable cell count, optionally integrated viable cell count, optionally wherein the final viability and / or integrated viable cell count is at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% higher than that what would be obtained performing the comparative method.

68. The use of claim 59-67, wherein productivity is measured as the titer of a recombinant polypeptide expressed by the mammalian cells.

69. The use of any of claims 59-68, wherein the enhancing the product quality comprises: a higher percentage of the main peak (by charge distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture that not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin, optionally wherein the higher percentage of the main peak (by charge distribution) is about 10-20% higher than with the similar eukaryotic cell culture medium; and / or a lower percentage of the high molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin, optionally wherein the lower percentage in the high molecular weight species / forms is about 1- 10% lower than with the similar eukaryotic cell culture medium; and / or a lower percentage of the low molecular weight species / forms (by size distribution) of the recombinant protein than what would be obtained performing a comparative method with a similar eukaryotic cell culture not comprising ISRIB and / orDocket No. P39212-WO-1 hypotaurine (and optionally increased level of iron) and / or suramin, optionally wherein the lower percentage in the low molecular weight species / forms is about 1- 10% lower than with the similar eukaryotic cell culture medium.

70. The use of any of claims 59-69, wherein enhancing the product titer comprises enhancing the titer of the recombinant polypeptide expressed by the mammalian cells compared to that what would be obtained performing a comparative method with a similar cell culture medium not comprising ISRIB and / or hypotaurine (and optionally increased level of iron) and / or suramin optionally wherein enhancing the product titer comprises enhancing the titer of the recombinant polypeptide expressed by the mammalian cells by at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to that what would be obtained performing the comparative method.

71. The use of any of claims 59-70, wherein the eukaryotic cell culture is a CHO cell culture.

72. The use of any of claims 59-70, wherein the eukaryotic cell culture is a culture of a cell therapy product.

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