Protective effect of polysaccharides on cell cultures containing Anti-foam agents

Abstract

A cell culture media for growing cells in suspension including a combination of a polysaccharide and an antifoaming agent is disclosed. In addition, a method for increasing cellular growth includes providing a cell culture media containing a combination of a polysaccharide and an antifoaming agent. An exemplary polysaccharide includes a cellulose derivative.

Description

Protective effect of polysaccharides on cell cultures containing anti-foam agentsField of the Invention

[0001] The field relates to effects of cellulose derivatives on promoting cellular growth when used alone or in combination with an antifoaming agent.Background

[0002] Upstream cell growth is a key process in the manufacture of advanced therapeutic modalities, particularly cell, gene, and protein drug products. The cost to manufacture these products is very high and is passed along in very high drug prices. There is a need to increase cell growth output through process intensification to help reduce the cost to produce these therapies. It is known in the art that protein drug titer was improved using cell engineering to obtain more copies, reduce culture time, and reduce proteolysis. Despite efforts in the field, protein drug prices remain high, in part due to cost to manufacture. There continues to be a need to increase cell growth to reduce costs and produce more protein to make the drug product affordable for more patients.

[0003] Many cell types have been adopted for use in the biopharmaceutical industry. Chinese hamster ovary (CHO) cells are the most common cell type for production of therapeutic proteins. These proteins can include enzymes, growth factors, cytokines, hormones, insulins, and antibodies. More recently, variations on these proteins including antibody-derived proteins and protein subunits are also being produced. Immunoglobulin G (IgG) is one type of antibody class that is commonly used as a platform for the development of biotherapeutics. lgG1 is a subclass of IgG antibodies.

[0004] Other cell types that are used in the biopharmaceutical space include human endothelial kidney (HEK) cells and Vero cells, used for manufacturing vaccines and gene therapies.

[0005] As the industry develops further, additional cell types of animal origin are being considered for adaptation to or direct use in suspension culture. Suspension culture is desirable for cell growth as it enables more efficient use of space compared to two- dimensional growth on flat surfaces.

[0006] In a conventional suspension growth process, cells are grown to a large number in a bioreactor, where they produce the protein, gene, or vaccine for which the cells were engineered to produce. In some cases, the cells, cellular aggregates, or parts of the cells such as extracellular vesicles are the intended product. In all cases, a high density of cells in the reactor is desired.

[0007] Generally, all animal cells require highly-specialized, tailored, complex media in order to grow the most efficiently. Historically, serum has been an ingredient included in cell culture media to help improve cell growth. However, due to regulatory, quality, safety, and ethicalconcerns, there is increasing demand to utilize media devoid of serum and any animal derived materials.

[0008] As regulatory agencies start to demand well characterized cell culture media, there is also a need to eliminate animal and human derived components from cell culture media, including serum. Due to the significant agitation in bioreactors and presence of interfacially active materials, 3D suspension cell cultures have a tendency to foam, which can cause many process challenges. To alleviate this problem, anti-foam agents, such as simethicone and simethicone formulations, are added to bioreactors as an ingredient.Simethicone antifoaming agents can be toxic to cells, so while their use is required to reduce the process challenges of foaming, solutions are required to reduce the toxicity and enable cell growth.

[0009] Methylcellulose is used as a shear stabilizer for suspension cell culture in the food industry. Knowledge of methylcellulose for use in cell culture dates back to the 1960’s (see Bryant, J.C., “Methylcellulose Effect on Cell Proliferation and Glucose Utilization in Chemically Defined Medium in Large Stationary Cultures”, Biotechnology and Bioengineering, XI, 155-179, 1969.) and use in suspension culture to the early 1990’s (Goldblum, S, et.al. , “Protective Effect of Methylcellulose and Other Polymers on Insect Cells Subjected to Laminar Shear Stress”, Biotechnology Progress, 6, 373-390, 1990.) Use of methylcellulose use alone in suspension cell culture is disclosed in WO2021248141. Despite this knowledge, it is believed that adoption of methylcellulose in the pharmaceutical industry has not taken place due to its inferior performance to poloxamer 188.

[0010] There needs to be improved processes for cell media using antifoam agents.Summary of the Invention

[0011] A cell culture media for growing cells in suspension comprising a combination of a polysaccharide and an antifoaming agent. In addition, a method for increasing cellular growth comprises: providing a cell culture media comprising a combination of a polysaccharide and an antifoaming agent; combining cells with said cell culture media; and incubating the cells and media to enable growth. A disclosed method includes a method for increasing cellular growth comprising: providing a cell culture media comprising a combination of a cellulose derivative and an antifoaming agent; combining cells with said cell culture media; and incubating the cells and media to enable growth.Detailed Description of the Invention

[0012] The examples provided in the detailed description are merely examples and should not be used to limit the scope of the claims in any claim construction or interpretation.

[0013] A process to grow cells in suspension that includes addition of a polysaccharide and an antifoam agent is disclosed.

[0014] Methylcellulose is known in the art for application to suspension cell cultures but not for use in protecting cells from the toxic effects of antifoaming agents.

[0015] In one example, the cell culture media does not include serum.

[0016] The following describes certain key concepts in the specification.

[0017] Abbreviations. As stated in this specification, “MC” is the abbreviation that is used for conventional methylcellulose and “HPMC is the abbreviation that is used for conventional hydroxypropyl methylcellulose.

[0018] Antifoam. The cell culture media includes antifoam. The antifoam is optionally a simethicone antifoam and includes emulsions such as Gibco Foam Away®.

[0019] As described in the website,Assets / LSG / manuals / FoamAwayAOF man. pdf, Gibco FoamAway™ Irradiated AOF (also referred to in the specification as “FoamAway™” is manufactured with 30% Simethicone Emulsion USP containing methylcellulose and does not contain any components derived from human or animal sources. When the antifoam is added to cell culture media, the concentration of the resulting methylcellulose in the media is less than 0.01%.

[0020] Polyether surfactants. The use of polyether surfactants is optional. Exemplary polyether surfactants used in the cell media include poloxamer 124, poloxamer 188, poloxamer 338 and poloxamer 407.

[0021] Polysaccharide. Exemplary polysaccharides used in the cell culture media in this disclosure include cellulose derivatives mentioned below, alginates, carrageenans, alphaglucans, beta-glucans, and dextrans, and chemical derivatives thereof. In another example, the molecular weight of the polysaccharide is such that when dissolved at 2% in water and measured at 20 degrees Celsius, the solution viscosity is less than 10,000 centipoise (cP), less than 7,000 cP, less than 2,000 cP, less than 1 ,500 cP less than 500 cP, less than 100 cP, or less than 30 cP.

[0022] Cellulose derivatives. Exemplary cellulose derivatives used in the cell media in this disclosure include methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose, and any combinations thereof.

[0023] In one example, the hydroxyl groups of a cellulose derivative may include an alkyl substituent or a hydroxy alkyl substituents or combinations thereof.

[0024] In one example, the cellulose derivative is methylcellulose. In another example, the cellulose derivative is hydroxypropyl methylcellulose.

[0025] In one example, the cellulose derivative is methylcellulose 15 cP. In another example, the cellulose derivative is HPMC E 5 cP. In another example, the cellulose derivative is HPMC K 3 cP.

[0026] Concentrations. In one example, the concentration of polysaccharide is less than 5% and an optimal range is from 0.01 to 4%. In one example, the concentration of a cellulose derivative is less than 3% and an optimal range is from 0.02 to 1%.

[0027] Cells in which the disclosed process can be used. The disclosed process can be used for any one or combinations of the following exemplary cells: animal cells, insect cells, plant cells, eukaryotic cells, prokaryotic cells, mammalian cells, cells adapted for suspension, immortalized cells, Chinese hamster ovary (CHO) cells, human endothelial kidney (HEK) 293 cells and VERO cells. Other cells and cell lines known in the art and suited for the disclosed process may be used.

[0028] The following describes more details about methylcellulose and hydroxypropyl methylcellulose.Methylcellulose and Hydroxypropyl Methylcellulose

[0029] Methylcellulose is a cellulose ether formed by the methylation of cellulose. Cellulose is a naturally occurring polysaccharide produced by many plants including trees and cotton. The polysaccharide polymer is composed of anhydroglucose units joined by beta 1-4 linkages.

[0030] Each anhydroglucose unit contains hydroxyl groups at the 2, 3, and 6 positions. Partial or complete substitution of these hydroxyl substituents creates cellulose derivatives.

[0031] Cellulose derivatives are well known across the pharmaceutical and food industries. Cellulose derivatives are defined in many ways. Cellulose derivatives are defined by the United States Pharmacopeia (USP) by their chemical derivatization and molecular weight. They may optionally also be additionally defined by the pattern of chemical derivatization around the anhydroglucose units as defined by s23 / s26 ratio.

[0032] Chemical derivatization agents. A reaction of a cellulose with a derivatization agent yields a cellulose derivative. Derivatization agents include etherification agents. Etherification agents include methylating agents, for example.

[0033] A cellulose ether is an example of a cellulose derivative that is formed by reaction of cellulose with etherification agents. For example, treatment of cellulosic fibers with an alkaline solution, followed by an etherification agent such as methyl chloride, yields a cellulose ether, a methylcellulose, a cellulose derivative.

[0034] If a cellulose ether is substituted with hydroxypropyl and methyl groups, such a cellulose ether is known is hydroxypropyl methylcellulose or hypromellose (HPMC), an example of a cellulose derivative.

[0035] Many cellulose derivatives are also defined by the United States Pharmacopeia (USP).

[0036] For example, a methylcellulose is defined by the USP to have not less than 26% and not more than 33% substitution of the hydroxy groups being methylated.

[0037] Hydroxypropyl methylcellulose (Hypromellose) is defined by the USP into four different substitution types. E chemistry is defined as follows. Type 2910 or Type E substitution has 28-30% methoxy and 7-12% hydroxypropyl substitution. K chemistry is defined as follows. Type 2208 or Type K substitution has 19-24% methoxy and 4-12% hydroxypropyl substitution. F chemistry is defined as follows. Type 2906 or Type F substitution has 27-30% methoxy and 4- 7.5% hydroxypropyl substitution. J chemistry is defined as follows. Type 1828 or Type J has 16.5- 20% methoxy and 23-32% hydroxypropyl substitution.

[0038] Molecular weight. The molecular weight of a cellulose derivative is typically described by the approximate viscosity of a solution of that polymer in water. These solutions are typically 2 percent by weight. For example, a methylcellulose 15 cP polymer is a methylcellulose in which a 2% solution of methylcellulose in water has a viscosity of approximately 15 cP. Methods for determining this solution viscosity are well known to those skilled in the art and are defined by the United States Pharmacopoeia, Chapter 912.

[0039] Chemical Substitution Patterns as defined by s23 / s26 ratio. The cellulose ether anhydroglucose units can have more than one of its hydroxyls substituted with derivatives. The chemical substitution pattern can be defined by the s23 / s26 ratio, wherein s23 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 3-positions of the anhydroglucose unit are substituted and wherein s26 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted. Further definition of the substitution pattern can be found in EP1171471 , W020000 / 59947, US6235893, and US6228416.

[0040] The ratio of which hydroxyl positions on the 1 ,4 anhydroglucose ring are substituted relative to each other is not defined in the USP or other international pharmacopoeias.

[0041] Conventional methylcellulose and conventional hydroxypropyl methylcelluloses. Most commercially available methylcelluloses and hydroxypropyl methylcelluloses have s23 / s26 ratiosof 0.37 to 0.42. These polymers are referred to as conventional methylcelluloses or conventional hydroxypropyl methylcelluloses.

[0042] Definition of the substitution pattern of the substituents on the cellulose by a s23 / s26 ratio to form the cellulose derivatives is optional.

[0043] The substitution pattern is optionally defined as follows:

[0044] In one example, the cellulose derivative in which the s23 / s26 ratio is defined is a methylcellulose. The methylcellulose has anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are substituted with methyl groups such that s23 / s26 is from 0.16 to 0.36, wherein s23 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 3-positions of the anhydroglucose unit are substituted with methyl groups and wherein s26 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted with methyl groups. The foregoing is an example of a non-conventional methylcellulose.

[0045] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is a methylcellulose. The methylcellulose has anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are substituted with methyl groups such that s23 / s26 is from 0.26 to 0.32, wherein s23 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 3-positions of the anhydroglucose unit are substituted with methyl groups and wherein s26 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted with methyl groups. These polymers are referred to as SG methylcelluloses (“SG-MC”).

[0046] In one example, a favored s23 / 26 is from 0.23 to 0.32.

[0047] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is a hydroxypropyl methylcellulose. The hydroxypropyl methylcellulose has anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are individually substituted with hydroxypropyl or methyl groups such that s23 / s26 is from 0.16 to 0.36, wherein s23 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 3-positions of the anhydroglucose unit are substituted with hydroxypropyl or methyl groups and wherein s26 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted with hydroxypropyl or methyl groups. The foregoing is an example of non-conventional hydroxypropyl methylcellulose. This non-conventional HPMC includes both hydroxypropyl and methyl groups in an overall chemical structure.

[0048] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is a hydroxypropyl methylcellulose. The hydroxypropyl methylcellulose has anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are individually substituted with hydroxypropyl or methyl groups such that s23 / s26 is from 0.26 to 0.32, whereins23 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 3-positions of the anhydroglucose unit are substituted with hydroxypropyl or methyl groups and wherein s26 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted with hydroxypropyl or methyl groups. These polymers are referred to as SG hydroxypropyl methylcelluloses (“SG- HPMC”), an example of a non-conventional HPMC. This non-conventional SG-HPMC includes both hydroxypropyl and methyl groups in an overall chemical structure.

[0049] Non-conventional cellulose derivatives. Methylcelluloses and hydroxypropyl methylcelluloses with s23 / 26 in the range of 0.16 to 0.36 are considered non-conventional cellulose derivatives.

[0050] Exemplary SG methylcellulose and SG HPMC are manufactured as described in EP1171471, WG20000 / 59947, US6235893 and US6228416, for example.

[0051] The following describes experimental conditions which demonstrate an embodiment of the claimed cell culture media and associated method.Example 1Inclusion of methylcellulose leads to increased viable cell count and exerts a protective effect for cell viability for cell cultures containing antifoam

[0052] Agarabi CHO cells (ATCC, CRL3440) were expanded in non-baffled shake flasks using Hycell™ cell media, available from Cytiva. Baffles are not generally used in CHO culture because such use of baffles would impart extra shear on the cells in the CHO culture.

[0053] The media was supplemented with 6 mM L-glutamine, and 1.5 mL of HyClone™ Cell Boost™ 1 Supplement-was added every other day. Cells were maintained at 8% CO2 and 37°C.

[0054] Cell culture volume was 15 mL. 0.5 mL were removed at each time point for cell counting, using traditional counting in Trypan Blue using Countess™ 3 FL Automated Cell Counter.

[0055] As known in the art, htps: / / www.sigmaaldrich.eom / deepweb / assets / sigmaaldrich / marketinq / qlobal / documents / 331 / 9S4 se. df, the document provided at this link, teaches that “Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting.” The foregoing document also states that “[t]his method is based on the principle that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do. Staining facilitates the visualization of cell morphology.” Thus, a person of ordinary skill in the art knows that Trypan Blue’s staining facilitates the visualization and distinguishing of cells that are alive and cells that are dead.

[0056] For Samples labeled “AF” in the following Table 1, 10 pL of Gibco® FoamAway™ Irradiated AOF (animal origin-free) antifoam (also referred to as “Foam Away™”)was added to media each day. The abbreviation “AF” in this sample indicates cells treated with an antifoaming agent.

[0057] For samples labeled “MC” in Table 1 , the media was supplemented with a concentration of 0.2% conventional methylcellulose 15 cP.

[0058] The following Table 2 reports the average alive total cells in the flask. Each sample was performed in triplicate.

[0059] The following table measures live cells / mL:Table 1

[0060] Antifoaming agent blocks cells from growing. Addition of antifoaming agent (Sample 2) leads to reduced viable cell density every day compared to the initial cell level and leads to zero cell viability at day 7. For day 7, there was no reported average total live cell count in a sample containing AF (antifoaming agent). This surprisingly shows that cells are not able to grow at all in the presence of antifoaming agent.

[0061] Inclusion of 0.2% methylcellulose leads to increased average live total cell count compared to control. When comparing the results for day 7 for samples containing 0.2% methylcellulose (Sample 3), the average live total cell count was approximately five percent more than the average live total cell count in the control (Sample 1). This shows that in this culture system, methylcellulose does not have a detrimental effect on cell growth but also does not have a significant benefit for cell growth.

[0062] Inclusion of a combination of 0.2% methylcellulose and an antifoaming agent leads to comparable live total cell count compared to control. For day 7, a mixture of methylcellulose and AF (Sample 4) had approximately seven percent lower average live cells than the control (Sample 1). Compared to only antifoam in Sample 2 which saw no cell growth, the combination of methylcellulose and antifoam in Sample 4 surprisingly and unexpectedly had cell growth. This result indicates that methylcellulose had a protective cell effect on a mixture containing methylcellulose and AF because the sample containing AF alone had no average total live cell count at day 7. This result is surprising and unexpected as methylcellulose did not have a significant benefit on cell growth in this culture system.Example 2Inclusion of methylcellulose in a cell culture media containing antifoaming agent leads to protection of cells at high shear rates

[0063] This example shows that methylcellulose protects cells from antifoam damage at a variety of shear rates. The experiment was set up as in Example 1, except that baffled flasks were used, and the rotation of the shaker was varied to create higher and variable shear. The presence of baffles creates higher shear, and higher rpm leads to more shear. Reported are cells / mL after 48 hours of culture.

[0064] A sample with only antifoam added was not tested as it was established by the results of Sample 2 of Example 1 that cells cannot effectively grow in the presence of antifoam only.

[0065] The following table reports the results.Table 2

[0066] Control samples grow less effectively at higher shear. Comparing cellular growth for Sample 5, at 100 rpm, there was a 2 percent increase in the number of cells compared to at 80 rpm. At 130 rpm, there was a 33.5 percent decrease in the number of cells compared to at 80 rpm. At 160 rpm, there was a 97 percent decrease in the number of cells compared to at 80 rpm. This shows that the cells are sensitive to shear and do not grow well at higher shear rates, represented by higher rpm.

[0067] Samples containing 0.2% methylcellulose had increased cellular growth compared to control at higher shear. Comparing cellular growth for samples containing 0.2% methylcellulose (Sample 6) to control containing no additive (Sample 5) at 80 rpm, there was a 31% increase in cells with the 0.2% methylcellulose present. Comparing cellular growth for samples containing 0.2% methylcellulose (Sample 6) to control containing no additive (Sample 5) at 100 rpm, there was a 1% decrease in cells with the 0.2% methylcellulose present. Comparing cellular growth for samples containing 0.2% methylcellulose (Sample 6) to control containing no additive (Sample 5) at 130 rpm, there was a 92% increase in cells with the 0.2% methylcellulose present. Comparing cellular growth for samples containing 0.2% methylcellulose (Sample 6) to control containing no additive (Sample 5) at 160 rpm, there was a 35-fold increase in cells with the 0.2% methylcellulose present. That is a 3500% increase in cells. This shows that methylcellulose is able to alleviate the cells’ increased sensitivity to shear.

[0068] Samples containing 0.2% methylcellulose had comparable cellular growth at different shear. Comparing cellular growth for Sample 6, at 100 rpm, there was a 23 percent decrease in the number of cells compared to at 80 rpm. At 130 rpm, there was a 2.3 percent decrease in the number of cells compared to at 80 rpm. At 160 rpm, there was an 18 percent decrease in the number of cells compared to at 80 rpm. This shows that methylcellulose is able to protect the cells from their shear sensitivity in this baffled flask culture system.

[0069] Samples containing 0.2% methylcellulose and 10 pL antifoaming agent had comparable cellular growth at 80, 100, and 130 rpm. Comparing cellular growth for Sample 7, at 100 rpm, there was a 11 percent decrease in the number of cells compared to at 80 rpm. At 130 rpm, there was a 4.1 percent increase in the number of cells compared to at 80 rpm. At 160 rpm, there was a 97 percent decrease in the number of cells compared to at 80 rpm. This shows that at 80, 100, and 130 rpm, the methylcellulose is able protect the cells from damage caused by antifoaming agent while also able to protect the cells from some of their shear sensitivity in this baffled flask culture system.Example 3Methylcellulose improves cell growth when antifoaming agent is present in a bioreactor

[0070] A proprietary CHO DG-44 cell line expressing an lgG1 antibody (referred as “the cells”) was adapted to and grown in Hycell™ media (available from Cytiva). The adaption and accompanying growth process are known to persons of ordinary skill in the art. The cells were adapted to the Hycell™ media by sequential growth in media containing more Hycell™ until the cells were growing in Hycell only. Using the Hycell™ media allows a person of ordinary skill the art the proper scientific control to understand growth impacts of addition various additives to the media, as it contains no shear protectant. The Hycell™ media had a viscosity of 2.23 cP when measured at 37 °C.

[0071] Cells were grown in an ambr15 parallel 24 bioreactor system (Sartorius). Further details of the system are provided atbioreactors / ambr-muSti-parallel-bioreactors / ambr-15-celi-culture

[0072] The ambr15 system continuously monitors samples and adjusts parameters such as dissolved oxygen (DO) and feed conditions. The working volume is 14 mL, and cell cultures were maintained for 14 days.

[0073] Samples are agitated by a stir shaft in the reactor. In addition to the base media, custom-ordered Cytiva HyClone™ Cellboost™ 7a and 7b supplements without poloxamer 188 were used as a supplemental feed to target 6 g / L glucose concentration.

[0074] Gibco® FoamAway™ Irradiated AOF (animal origin-free) antifoam was added daily (10 pL / day) to prevent foaming of the system. 0.3 x 106cells / mL were inoculated into each reactorat the start of the culture and cells were harvested after the earlier of 14 days or after cell viability dropped below 70 percent.

[0075] Viable cell density was measured throughout the 14-day experiment. Guava® ViaCount™ reagent with flow cytometry and Vi-Cell™ BLU cell viability analyzer (Beckman Coulter) were used to measure viable cell density.

[0076] ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue exclusion.

[0077] Peak viable cell density is the highest viable cell density that was measured over the fourteen-day culture.

[0078] Harvest viability is the percentage of cells that are alive at the conclusion of the fourteen-day culture.

[0079] Protein titer measurement by Bio HT. Protein titer measured using a Cedex Bio HT analyzer was assessed at the end of the experiment. IgG Bio HT is a test kit for determination of IgG and is a turbidity measurement following a nanoprecipitation of IgG protein. Turbidity is proportional to IgG content and the IgG concentration is determined via comparison to turbidity of IgG solutions of known concentration.

[0080] In Sample 8, no additional additives were added to the media. In Sample 9, 0.2% MC 15 cP was added to the media.

[0081] Sample 8 was run two times and Sample 9 was run four times. The averages of those replicates are reported.

[0082] The following table measures peak viable cell density, harvest viability, and IgG titer for Example 3.Table 3

[0083] Combination of anti foaming agent and methylcellulose leads to approximately six-fold peak viable cell density as compared to use of antifoaming agent alone. With the addition of 0.2% methylcellulose (Sample 9), the average peak viable cell density surprisingly and unexpectedly increased close to six-fold compared to samples grown in media containing only FoamAway™ and not methylcellulose (Sample 8) as shown in Table 3 above. In other words, the average viable cell density in the combination of the antifoaming agent and 0.2% methylcellulose was approximately 600 percent of the control.

[0084] With the addition of 0.2% methylcellulose (Sample 9), the average cell viability at harvest increased 61 percent as compared to samples grown in media containing only FoamAway™ (Sample 8). In other words, the average cell viability in the combination was approximately 161 percent of Sample 8.

[0085] In Sample 8, no protein IgG was detected following the culture. In comparison, for Sample 9, 588 pg / mL of protein IgG was detected following the culture.

[0086] The above shows that in a bioreactor system, use of an antifoaming agent, such as FoamAway™ is detrimental to cell growth, viability, and ability to produce protein. Surprisingly and unexpectedly, the results have shown that methylcellulose can alleviate the damage caused by antifoaming agent, leading to cell growth, higher viability, and protein production.Example 4Different polysaccharides are able to protect cells from the damaging effects of antifoaming agents.

[0087] This experiment was set up as in Example 3, but different polysaccharide polymers were added to the cell culture media. Methylcellulose polymers of different molecular weights and different substitution patterns were utilized. Molecular weights are not directly measured but are represented through their 2% solution viscosity in water at 20 degrees Celsius, as with other methylcelluloses.

[0088] The polymers used are described with the results in the following Table 4.

[0089] Protein titer measurement by Protein A. Protein A chromatography, as known in the art, is used to measure IgG levels. Protein A-functionalized beads bind IgG, enabling isolation of the IgG. Measuring protein content following elution of the IgG from the beads is indicative of IgG levels. Protein A chromatography using an Agilent Bio-Monolith Protein A affinity chromatography column and HPLC system equipped with UV detection was used in this Example to measure protein titer.

[0090] The following table shows the samples tested in this Example and their corresponding peak viable cell densities and protein titers.Table 4

[0091] Results are compared to Sample 8 (FoamAway™ only) of Example 3.

[0092] When comparing the viscosities of the medias in Samples 10, 12, and 13, there is a slight increase in viscosity as the methylcellulose molecular weight is larger.

[0093] When comparing the viscosities of the medias in Samples 10 and 11, the viscosities are comparable and similar.

[0094] When comparing the viscosities of the medias in Samples 13 and 14, the viscosities are comparable and similar.

[0095] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with Sample 10 (MC -2 cP), the peak viable cell density was 4.9 fold higher in Sample 10.

[0096] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with Sample 11 (SG MC - 2 cP), the peak viable cell density was 8.4 fold higher in Sample 11.

[0097] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with Sample 12 (MC - 338 cP), the peak viable cell density was 6.3 fold higher in Sample 12

[0098] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with Sample 13 (MC - 2880 cP), the peak viable cell density was 8.3 fold higher in Sample 13.

[0099] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with Sample 14 (MC - 2 cP, 2.4%), the peak viable cell density was 5.4 fold higher in Sample 14.

[0100] When comparing the peak viable cell density of Sample 13 (MC - 2880 cP, 0.2%), with Sample 14 (MC - 2 cP, 2.4%), the peak viable cell density was 45% higher in Sample 13.

[0101] Media viscosity is not a factor for creating increased peak viable cell density

[0102] When comparing the average protein IgG titer of Sample 13 with Sample 14, the average protein IgG titer was 49% higher in Sample 13. This shows that the increase in peak viable cell density is not due to the higher media viscosity of Sample 13 as compared to other samples containing 0.2% methylcellulose.

[0103] When comparing the average protein IgG titer of Samples 10-14 to Sample 8, Samples 10-14 all had measurable protein IgG titers, while Sample 8 (Foam Away™ only) had no measurable protein IgG titer.

[0104] The results of Sample 11 (SG MC - 2cP, 0.2%) demonstrate that a non-conventional methylcellulose is surprisingly and unexpectedly able to alleviate the damage caused by an antifoaming agent, leading to cell growth, and protein production.

[0105] The results of Samples 10, 12, and 13 respectively demonstrate that a methylcelluloses of different molecular weights (MC - 2 cP, 0.2%; MC - 338 cP, 0.2% and MC - 2880 cP, 0.2%) are surprisingly and unexpectedly able to alleviate the damage caused by an antifoaming agent, leading to cell growth, and protein production.Example 5Unexpected benefit from combining methylcellulose and poloxamer 188 is observed across different methylcellulose / poloxamer 188 ratios

[0106] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell™ cell media (Cytiva) was used. Conventional methylcellulose with 15 cPs viscosity (MC), poloxamer 188 (Px188), or a combination of MC and Px188 was added to the media as shown in Table 5. Gibco™ FoamAway™ Irradiated AOF (animal origin-free) antifoam (Thermo Fisher Scientific) was added daily (10 pL / day) to prevent foaming of the system. Cells were inoculated into each 5 reactor at the start of the culture at a concentration of 3x10 cells / mL. Seed train cells were centrifuged and resuspended in media containing the desired additive(s) before addition to the shake flasks for that condition. Each viable cell density (VCD) shown in Table 5 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Blue with a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell™ media with no methylcellulose or poloxamer.

[0107] The following Table 5 shows the observed VCD for the tested samples.Table 5

[0108] In Table 5, the percentages are weight percentages based on the total cell culture. The fraction MC = (concentration MC) I (total concentration of MC + Px188). The “Expected VCD” is the VCD that would be expected if the effects of the MC and Px188 on VCD were simply additive. Accordingly, the Expected VCD = (VCD with MC only) x (fraction MC) + (VCD with Px188 only) x (fraction Px188). In this equation, (fraction MC) + (fraction Px188) = 1 , and (fraction Px188) = (concentration Px188) I (total concentration of MC + Px188). All concentrations discussed here are weight concentrations.

[0109] As shown in Table 5, MC / Px188 ratios of from 7 / 1 to 1 / 7 were tested. The VCDs obtained using combinations of MC and Px188 exceeded the Expected VCDs in every instance, thus indicating an unexpected benefit from the combination across the entire range of ratios.Example 6 Effect of decreased additive concentration in a high shear system

[0110] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a cell culture volume of 15 mL. Hycell™ cell media (Cytiva) was used. Conventional methylcellulose (MC) with 15 cPs viscosity, poloxamer 188 (Px188), or a combination of MC and Px188 was added to the media as shown in Tables 6 and 7. The experiments in Table 6 were carried out using a shake flask rotation speed of 160 RPM, while the experiments in Table 7 were carried out using a shake flask rotation speed of 130 RPM. Gibco™ FoamAway™ Irradiated AOF (animal origin-free) antifoam (Thermo Fisher Scientific) was added daily (10 pL / day) to prevent foaming of the system. Cells were inoculated into each 5 reactor at the start of the culture at a concentration of 3x10 cells / mL. Seed train cells werecentrifuged and resuspended in media containing the desired additive(s) before addition to the shake flasks for that condition. Each viable cell density (VCD) shown in Tables 6 and 7 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Blue with a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell™ media with no methylcellulose or poloxamer.

[0111] The following Table 6 shows the observed VCD for the tested samples carried out in the higher shear system where the shaker flask rotation speed was 160 RPM.Table 6

[0112] The following Table 7 shows the observed VCD for the tested samples carried out in the lower shear system where the shaker flask rotation speed was 130 RPM.Table 7

[0113] In Tables 6 and 7, the percentages are weight percentages based on the total cell culture.

[0114] As shown in Table 6 and 7, total additive concentrations of from 0.01% to 0.2% were each tested with a shaker flask rotation speed of 160 or 130 RPM. The protective effects of the additive tended to be more pronounced at total additive concentrations of at least 0.1% in the higher shear system where the shaker flask rotation speed was 160 RPM. MC alone provided protective effects even as low as 0.1% in the higher shear system. In the lower shear system where the shaker flask rotation speed was 130 RPM, the protective effects of the additive tended to be more pronounced at total additive concentrations of at least 0.02%, and particularly at total additive concentrations of at least 0.05%. MC tended to be more protective than Px188 in thelower shear system at the tested concentrations. This data shows that growth in different culture systems will have different responses to the levels of additives included in the media.Example 7Protective effect of SG-MC alone against antifoam cell damage

[0115] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell™ cell media (Cytiva) was used. Conventional methylcellulose (2 cPs viscosity), SG methylcellulose (SG-MC) (2 cPs viscosity) and / or Gibco™ FoamAway™ Irradiated AOF (animal origin-free) antifoam (Thermo Fisher Scientific) was added as shown in Table 8. Cells were inoculated into5 each reactor at the start of the culture at a concentration of 3x10 cells / mL. Seed train cells were centrifuged and resuspended in media containing the desired additive(s) before addition to the shake flasks for that condition. Each viable cell density (VCD) shown in Table 8 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Blue with a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell™ media with no methylcellulose or poloxamer.

[0116] The following Table 8 shows the observed VCD for the tested samples.Table 8

[0117] In Table 8, the percentages are weight percentages based on the total cell culture.

[0118] As shown in Table 8, both conventional MC and SG-MC provided protection from antifoam damage, with SG-MC providing slightly more protective effect than the conventional MC.Example 8Protective effect of different polymer additives alone against antifoam cell damage

[0119] In this example, the protective effects of conventional methylcellulose (MC), SG-MC and LTG-MC alone were compared. LTG-MC is a non-conventional methylcellulose with an s23 / s26 in the range of 0.16 to 0.25.

[0120] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell ™ cell media (Cytiva) was used. MC (15 cPs viscosity), MC (2 cPs viscosity), SG-MC (2 cPs viscosity), or LTG-MC (2 CPs viscosity) was added as shown in Table 9. Gibco™ FoamAway™ Irradiated AOF (animal origin-free) antifoam (Thermo Fisher Scientific) was added daily (10 L / day) to prevent foaming of the system. Cells were inoculated into each reactor at the start of the culture5 at a concentration of 3x10 cells / mL. Seed train cells were centrifuged and resuspended in media containing the desired additive(s) before addition to the shake flasks for that condition. Each viable cell density (VCD) shown in Table 9 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Blue with a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell ™ media with no methylcellulose or poloxamer.

[0121] The following Table 9 shows the observed VCD for the tested samples.Table 9

[0122] In Table 9, the percentages are weight percentages based on the total cell culture.

[0123] As shown in Table 9, the conventional MC’s, SG-MC and LTG-MC all provided a protective effect, with LTG-MC providing the greatest protective effect and SG providing a greater effect than conventional MC.Example 9Comparison of protective effect of different HPMC additives against cell damage from different antifoams

[0124] In this example, the protective effects of three different cellulose ethers against cell damage from two different antifoams as shown in the below Table 10 were compared.

[0125] The tested cellulose ethers were a conventional MC with 15 cPs viscosity, K3 (a K chemistry hydroxypropyl methylcellulose (HPMC) with 3 cPs viscosity) and E5 (an E chemistry HPMC with 5 cPs viscosity). All the cellulose ethers were from International Flavors & Fragrances Inc. Conventional MC’s and HPMCs have s23 / s26 ratios of from 0.37 to 0.42. E chemistry HPMCs are defined as follows: Type 2910 or Type E substitution having 28-30% methoxy and 7-12% hydroxypropyl substitution. And K chemistry is defined as follows: Type 2208 or Type K substitution has 19-24% methoxy and 4-12% hydroxypropyl substitution.

[0126] The tested antifoams were Gibco™ FoamAway™ Irradiated AOF (animal origin- free) antifoam (Thermo Fisher Scientific) and Liveo™ silicone antifoam (DuPont™).

[0127] In this experiment, Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a cell culture volume of 15 mL and a rotation speed of 130 RPM. Hycell™ cell media (Cytiva) was used. Cells were inoculated into each reactor at the start5 of the culture at a concentration of 3x10 cells / mL. Seed train cells were centrifuged and resuspended in media containing the desired additive(s) before addition to the shake flasks for that condition. Each viable cell density (VCD) shown in Table 10 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Blue with a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell ™ media with no methylcellulose or poloxamer.

[0128] The following Table 10 shows the observed VCD for the tested samples.Table 10

[0129] In Table 10, the percentages are weight percentages based on the total cell culture.

[0130] As shown in Table 10, all the tested cellulose ethers alone provided a protective effect against antifoam cell damage.Example 10Protective effect of methylcellulose alone against antifoam cell damage at different antifoam levels

[0131] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell™ cell media (Cytiva) was used. All samples contained 0.20 weight percent conventional MC (15 cPs) added to the media. Gibco™ FoamAway™ Irradiated AOF (animal origin-free) antifoam (Thermo Fisher Scientific) was added in amounts described in Table 11. Cells were inoculated5 into each reactor at the start of the culture at a concentration of 3x10 cells / mL. Seed train cells were centrifuged and resuspended in media containing the desired additive(s) before addition to the shake flasks for that condition. Each viable cell density (VCD) shown in Table 11 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Blue with a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell™ media with no methylcellulose or poloxamer.

[0132] The following Table 11 shows the observed VCD for the tested samples.Table 11

[0133] In Table 11, the percentages are weight percentages based on the total cell culture.

[0134] As shown in Table 11 , the protective effect of MC alone against antifoam cell damage decreases as the amount of antifoam used increases.Example 11Comparison of protective effect of conventional HPMC against cell damage from a nonsilicone antifoam

[0135] In this experiment, Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell™ cell media (Cytiva) was used. AF204 non-silicone antifoam (Sigma-Aldrich) was added daily (7 ppm) to prevent foaming of the system. Conventional MC (15 cPs) also was included as shown in Table 12.

[0136] Cells were inoculated into each reactor at the start of the culture at a concentration of 53x10 cells / mL. Seed train cells were centrifuged and resuspended in media containing the desired additive(s) before addition to the shake flasks for that condition. Each viable cell density (VCD) shown in Table 12 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Blue with a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell™ media with no methylcellulose or poloxamer.

[0137] The following Table 12 shows the observed VCD for the tested samples.Table 12

[0138] In Table 12, the percentages and concentrations are based on total cell culture.

[0139] As shown in Table 12, the culture did not survive to Day 2 in the presence of the added antifoam without the use of the methylcellulose.Example 12Unexpected benefit from combining methylcellulose and poloxamer 188 is observed across different methylcellulose / poloxamer 188 ratios (3L reactor)

[0140] CHO DG-44 cells expressing an lgG1 antibody were expanded in an Applikon Biotechnology 3L glass dish-bottom reactor (model #Z611000310) with an Applikon Biotechnology EZ-Control (model #Z310110011) controller under the conditions shown in Table 13:Table 13

[0141] Hycell™ cell media (Cytiva) was used. Custom-ordered Cytiva HyClone™ Cellboost™ 7a and 7b supplements without poloxamer 188 (Px188) were used as supplemental feeds to target the glucose concentration as shown in the feeding scheme in Table 14 (the percentages refer to the weight percents based on total cell culture, and the glucose target concentrations are based on the total cell culture).Table 14

[0142] Conventional methylcellulose having 15 cPs viscosity (MC), Px188, or a combination of MC and Px188 was added to the media as shown in Table 15. Gibco™ FoamAway™ Irradiated AOF (animal origin-free) antifoam (Thermo Fisher Scientific) was added as needed to prevent foaming of the system (total amounts provided in Table 15).TM

[0143] Viable cell density was measured throughout the 14-day experiment. GuavaViaCount™ reagent with flow cytometry and Vi-Cell™ BLU cell viability analyzer (Beckman Coulter) were used to measure viable cell density. ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue exclusion. Peak viable cell density is the highest viable cell density that was measured over the 14-day culture.

[0144] Protein A chromatography, as known in the art, is used to measure IgG levels. Protein A-functionalized beads bind IgG, enabling isolation of the IgG. Measuring protein content following elution of the IgG from the beads is indicative of IgG levels.

[0145] Protein titer measured using a Cedex™ Bio HT analyzer was assessed at the end of the experiment. IgG Bio HT is a test kit for determination of IgG and is a turbidity measurement following a nanoprecipitation of IgG protein. Turbidity is proportional to IgG content and the IgG concentration is determined via comparison to turbidity of IgG solutions of known concentration.

[0146] The following Table 15 shows the results.Table 15

[0147] In Table 15, the percentages are weight percentages based on the total cell culture.

[0148] As shown in Table 15, methylcellulose, Px188, and a combination of the two are all effective and enabling high VCD and protein production in a conventional bioreactor. Surprisingly, the combination of MC and Px188 with 0.5% total additive is more effective than MC or Px188 alone at increasing peak VCD or protein titer, as seen by comparing Sample 76 to Samples 75 and 74. This data also demonstrates the effectiveness of methylcellulose to protect a cell culture from damage by antifoam, as shown by Samples 75, 77, and 78.

[0149] The range of recited numerical values disclosed in the specification includes values , e.g., + / — 5-10% of the recited value, that a person of ordinary skill in the art would consider equivalent to the recited value, e.g., having the same function or result.

[0150] The claims are not limited by the preferred embodiments and examples but will cover many modifications and equivalents consistent with the written description as a whole.

Claims

What is claimed is:

1. A cell culture media for growing cells in suspension comprising a combination of a polysaccharide and an antifoaming agent.

2. The cell culture media of claim 1 , wherein the antifoaming agent comprises simethicone.

3. The cell culture media of claim 1 , wherein the polysaccharide is a cellulose derivative.

4. The cell culture media of any of claims 1-3, wherein the cellulose derivative is methylcellulose or hydroxypropyl methylcellulose or a combination thereof.

5. The cell culture media of any of claims 1-4, further comprising a polyether surfactant.

6. The cell culture media of any of claims 1-5, wherein the polysaccharide has a solution viscosity at 2% in water of less than 10,000 cP at 20 degrees Celsius.

7. The cell culture media of any of claims 1-6, wherein the polysaccharide has a solution viscosity at 2% in water of less than 2,000 cP at 20 degrees Celsius.

8. The cell culture media of any of claims 1-7, wherein the polysaccharide has a solution viscosity at 2% in water of less than 100 cP at 20 degrees Celsius.

9. The cell culture media of any of claims 1-8, wherein the polysaccharide has a solution viscosity at 2% in water of less than 20 cP at 20 degrees Celsius.

10. The cell culture media of any of claims 1-9, wherein the polysaccharide is methylcellulose with a solution viscosity at 2% in water of 15 cP at 20 degrees Celsius.

11. The cell culture media of any of claims 1-10, wherein the polysaccharide is methylcellulose having a substitution pattern defined such that the s23 / s26 ratio is from 0.16 to 0.36.

12. The cell culture media of any of claims 1-9, wherein the polysaccharide is a hydroxypropyl methylcellulose having E chemistry with a solution viscosity at 2% in water of 5 cP at 20 degrees Celsius.

13. The cell culture media of any of claims 1-9, wherein the polysaccharide is hydroxypropyl methylcellulose having K chemistry with a solution viscosity at 2% in water of 3 cP at 20 degrees Celsius.

14. The cell culture media of any of claims 1-9, wherein the polysaccharide is hydroxypropyl methylcellulose having a substitution pattern defined such that the s23 / s26 ratio is from 0.16 to 0.36.

15. A method for growing cells in suspension comprising incubating cells in a cell culture media of any of claims 1-14.

16. A method for increasing cellular growth comprising: providing a cell culture media comprising a combination of a cellulose derivative and an antifoaming agent; combining cells with said cell culture media; andincubating the cells and media to enable growth.

17. The method of Claim 16, wherein the step of providing the combination includes providing the cellulose derivative in a concentration range from 0.01% to 5%.

18. The method of Claim 16, wherein the antifoaming agent comprises simethicone.

19. The method of Claim 16, wherein the cellulose derivative is methylcellulose.

20. The method of Claim 19, wherein the methylcellulose is methylcellulose 15 cP.21 . The method of Claim 19, wherein the methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.16 to 0.36.

22. The method of Claim 16, wherein the cellulose derivative is hydroxypropyl methylcellulose.

23. The method of Claim 22, wherein the hydroxypropyl methylcellulose is a hydroxypropyl methylcellulose having K chemistry with a solution viscosity at 2% in water of 3 cP at 20 degrees Celsius.

24. The method of Claim 22, wherein the hydroxypropyl methylcellulose is a hydroxypropyl methylcellulose having E chemistry with a solution viscosity at 2% in water of 5 cP viscosity at 20 degrees Celsius.

25. The method of Claim 22, wherein the hydroxypropyl methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.16 to 0.36.

26. The method of Claim 16, wherein the cells are from a Chinese hamster ovary cell line.

27. The method of Claim 16, wherein the cells are from a human endothelial kidney cell line.

28. The method of Claim 16, wherein the cells are from a Vero cell line.

29. The method of Claim 16, wherein the cells produce a protein.

30. The method of Claim 16, wherein the cells produce an antibody.

31. The method of Claim 16, wherein the cells produce an antibody-derived protein.

32. The method of Claim 16, wherein the cells produce a vaccine.

33. the method of Claim 16, wherein the cells produce a gene therapy.

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