Combinations of polysaccharides and polyether surfactants

Abstract

A method to increase cellular growth in a cell culture media includes contacting the cells with a combination of a polysaccharide and a polyether surfactant. In addition, a cell culture media including a polysaccharide and a polyether surfactant for increasing cellular growth is disclosed. Exemplary embodiments include a cellulose derivative and a poloxamer.

Description

Combinations of Polysaccharides and Polyether SurfactantsField of the Invention

[0001] The field relates to effects of polysaccharides on in vitro cellular growth.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, reduced culture time, and reduced 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, among others.

[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 and reduces points of contamination, time required for feeding and maintenance, and variability from operator differences.

[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. Serum traditionally helped to stabilize cells from shear stresses, including impact of vessels, other cells, and bubbles. Aside from being animal derived, it is known in the field that serum tends to be variable and hard to be well defined.

[0009] However, the elimination of serum from media can pose a challenge for cell proliferation, potentially reducing cell growth and protein titer.

[0010] One alternative known in the art is poloxamer 188.

[0011] Poloxamer 188 is a polyether block copolymer surfactant. Polyether surfactants are formed from sequential polymerization of ethylene oxide and propylene oxide. Different polyether surfactants have different block sizes and ratios, but they are generally known in the art for being mild, water-soluble surfactants.

[0012] Poloxamer 188 is also sold as Pluronic® F68 or Kolliphor® P188 BIO. It is an A-B-A triblock copolymer of ethylene oxide (A) and propylene oxide (B). Its molecular weight of the B block is approximately 1800 Daltons and the polymer is approximately 80 percent A block by weight. Other poloxamer examples include poloxamer 124, poloxamer 338 and poloxamer 407, which are sold under various trade names including Pluronic®, Kolliphor®, and Synperonic™.

[0013] Poloxamer 188 use is well known and fairly ubiquitous in the art for suspension cell cultures of Chinese hamster ovary cells, for example. Poloxamer 188 has been used both in the presence of serum and in the absence of serum in suspension cell culture. While effective in maintaining and improving cell growth rates, poloxamer 188 has purity challenges, batch-to-batch variability, and potentially causes problems during downstream processing and needs to be removed. A reference mentioning many of the problems with poloxamer 188 is “Development of Small Scale Cell Culture Models for Screening Poloxamer 188 Lot-to-Lot Variation”, Biotechnology Progress, 30, 1411-1418, 2014, by Peng, H., Hall, K.M., Clayton, B., Wiltberger, K., Hu, W., Hughes, E., Kane, J., Ney, R., & Ryll, T.

[0014] One alternative to poloxamer is methylcellulose. Methylcellulose is used as an alternative to poloxamer in the food industry, where poloxamer is not used because of regulatory limitations. 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 as a replacement for poloxamer. 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.

[0015] There remains a need for improved alternatives to serum and for reducing the reliance on poloxamer.Summary of the Invention

[0016] A cell culture media for growing cells in suspension comprises a polysaccharide and a polyether surfactant. In one example, the polyether surfactant is poloxamer 188. In one example, the polysaccharide is a cellulose derivative. In one example, the cellulose derivative is methylcellulose. In another example, the cellulose derivative is hydroxypropyl methylcellulose.

[0017] An associated method for growing cells in a suspension comprises incubating cells in the cell culture media as follows. A method for increasing cellular growth comprises: providing a cell culture media containing a combination of a cellulose derivative and a polyether surfactant; combining cells with said cell culture media; and incubating the cells and media to enable growth.Detailed Description of the Invention

[0018] 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. .

[0019] A cell culture media formulation for use in the improvement of growth of cells in suspension includes a polysaccharide and a polyether surfactant is disclosed.

[0020] A method to improve growth of cells in suspension includes using a cell culture media containing a polysaccharide and a polyether surfactant is disclosed.

[0021] In a specific example of a polysaccharide and a polyether surfactant respectively, a combination of methylcellulose or hydroxypropyl methylcellulose and poloxamer 188 is disclosed.

[0022] While poloxamer 188 as described in the background and methylcellulose are both known in the art, their combination in cell media has not been explored for improving cell growth as disclosed herein in this specification. Indeed, methylcellulose is known in the art for application to food cultures but not for use in cellular media such as culture media containing CHO cells, for example.

[0023] In one example, the cell culture media does not include serum.

[0024] The following describes certain key concepts in the specification.

[0025] Abbreviations. As stated in this specification, “MC” is the abbreviation that is used for conventional methylcellulose, “HPMC” is the abbreviation that is used for conventional hydroxypropyl methylcellulose, and “Px188” is the abbreviation used for “poloxamer 188.”

[0026] Antifoam. In another example, the cell culture media optionally includes antifoam. The antifoam is optionally a simethicone antifoam.

[0027] Polyether surfactants. Exemplary polyether surfactants used in the cell media include poloxamer 124, poloxamer 188, poloxamer 338 and poloxamer 407.

[0028] 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.

[0029] Cellulose derivatives. Exemplary cellulose derivatives used in the cell culture media in this disclosure include methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose, and any combinations thereof.

[0030] In one example, the hydroxyl groups of a cellulose derivative may include an alkyl substituent or a hydroxy alkyl substituents or combinations thereof.

[0031] In one example, the cellulose derivative is methylcellulose. In another example, the cellulose derivative is hydroxypropyl methylcellulose.

[0032] 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.

[0033] 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%.

[0034] The concentration of poloxamer 188 is less than 5% and an optimal range is from 0.02% to 3%.

[0035] In one example, the concentration of the combination of polysaccharide and poloxamer is 0.05% to 1%. In one example, the concentration of the combination of a cellulose derivative and poloxamer is 0.05% to 1%.

[0036] 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.

[0037] The following describes more details about methylcellulose and hydroxypropyl methylcellulose.Methylcellulose and Hydroxypropyl Methylcellulose

[0038] 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.

[0039] Each anhydroglucose unit contains hydroxyl groups at the 2, 3, and 6 positions. Partial or complete substitution of these hydroxyl substituents creates cellulose derivatives.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] If a cellulose ether is substituted with hydroxypropyl and methyl groups, such a cellulose ether is known as hydroxypropyl methylcellulose or hypromellose (HPMC), an example of a cellulose derivative.

[0044] Many cellulose derivatives are also defined by the United States Pharmacopeia (USP).

[0045] 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.

[0046] 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.

[0047] 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 when measured at 20 degrees Celsius. 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.

[0048] 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.

[0049] 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.

[0050] Conventional methylcellulose and conventional hydroxypropyl methylcelluloses. Most commercially available methylcelluloses and hydroxypropyl methylcelluloses have s23 / s26 ratios of 0.37 to 0.42. These polymers are referred to as conventional methylcelluloses or conventional hydroxypropyl methylcelluloses.

[0051] Definition of the substitution pattern of the substituents on the cellulose by a s23 / s26 ratio to form the cellulose derivatives is optional.

[0052] The substitution pattern is optionally defined as follows:

[0053] 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.

[0054] 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 twohydroxy 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”).

[0055] In one example, a favored s23 / 26 is from 0.23 to 0.32.

[0056] 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.

[0057] 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, 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. 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.

[0058] 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.

[0059] Exemplary SG methylcellulose and SG HPMC are manufactured as described in EP1171471, WG20000 / 59947, US6235893 and US6228416, for example.

[0060] The following describes experimental conditions regarding a control in which poloxamer 188 and methylcellulose was not utilized.Example 1 Control

[0061] A proprietary CHO DG-44 cell line expressing an lgG1 antibody (referred as “the cells”) was adapted to and grown in a non-optimized media (Hycell™, available from Cytiva). The adaption and accompanying growth process are known to persons of ordinary skill in the art. The cells are engineered to grow best in PowerCHO2™ media from Lonza, but because PowerCHO2™ media contains poloxamer 188, the cells were adapted to the Hycell™ media by sequential growth in media containing more Hycell™ and less PowerCHO2™ until the cells were growing in Hycell only and thus no poloxamer 188 was present. Thus, no PowerCHO2™ media was used for Example 1. Using the Hycell™ media allows a person of ordinary skill in the art the proper scientific control to understand growth impacts of addition of poloxamer or methylcellulose or both to the media, though at the expense of optimal cell growth. The Hycell™ media had a viscosity of 2.23 cP when measured at 37 °C.

[0062] Cells were grown in an ambr15 parallel 24 bioreactor system (Sartorius). Further details of the system are provided at

[0063] 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.

[0064] 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.

[0065] Gibco Foam Away® 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 reactor at the start of the culture and cells were harvested after the earlier of 14 days or after cell viability dropped below 70 percent.

[0066] 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.

[0067] ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue exclusion.

[0068] Peak viable cell density is the highest viable cell density that was measured over the fourteen-day culture.

[0069] 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.

[0070] 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.

[0071] The experiment was run in duplicate.

[0072] Results. Average peak viable cell density was 2.06 x 106cells / mL, which shows that the cells did not significantly expand in the absence of poloxamer 188 or methylcellulose. No lgG1 protein was detected by BioHT. 50pg / mL IgG was detected by Protein A chromatography using an Agilent Bio-Monolith Protein A column and HPLC system equipped with UV detection. As will be shown, the amount of IgG recovered in the control media with no additives was minimal as compared to examples which contained polysaccharide, poloxamer, or a combination of both.Example 2A combination of methylcellulose and poloxamer 188 surprisingly leads to increased average viable cell density

[0073] Samples 1-6 were prepared using the Hycell™ media as in Example 1 , but conventional methylcellulose 15 cP (MC), poloxamer 188 (Px188), or a 1 :1 ratio of MC and Px188 were added to the cell media.

[0074] Each example was tested in quadruplicate and average (mean) values are reported. Protein content was measured as in Example 1 by Bio HT and by Protein A chromatography using an Agilent Bio-Monolith Protein A column and HPLC system equipped with UV detection.

[0075] Glucan profiling was also performed to assess changes in protein quality using HPLC.

[0076] The following table shows peak viable cell density (VCD) for samples 1-6.Table 1

[0077] Average peak viable cell density is the average (mean) of the four replicates in an experiment.

[0078] The following describes analysis of the values obtained from the table above.

[0079] Poloxamer 188 promotes higher viable cell density than methylcellulose. As the following will show, Poloxamer 188 (Px188) is more efficient at increasing cell density than methylcellulose. This analysis is shown by comparing the average peak viable cell density between Samples 1 and 2 and Samples 3 and 4 respectively.

[0080] A higher concentration of poloxamer 188 lead to higher viable cell density than a lower concentration. Notably, a higher concentration of Px188 lead to higher efficiency in increasing viable cell density when compared with a lower concentration. This is shown by a comparison of Sample 2 and Sample 4.

[0081] Surprising and unexpected higher viable cell density results for a combination of Px188 and MC with same concentration (0.1%) than the individual components alone with the same concentration. Surprisingly and unexpectedly, the same total concentration of a combination of both Px188 and MC (0.1%) in Sample 6 leads to higher viable cell density when comparing the viable cell density for the same total concentration of Px188 (0.1 %) in Sample 4.

[0082] These results are surprising and unexpected in view of a person of ordinary skill in the art, as MC is less efficient in promoting cell density than Px188. Without being bound by theory, a person of ordinary skill in the art would have expected that the combination of Px188 and MC (0.1 %) would have a viable cell density value to be between the viable cell density values measured for Samples 3 and 4.

[0083] Surprising and unexpected equal viable cell density results for a combination of Px 188 and MC (0.2%) with same concentration than poloxamer 188 (0.2%) alone with the same concentration. Sample 5 has the same total amount of additive as Sample 1 and Sample 2. Surprisingly and unexpectedly, the same total concentration of a combination of both Px188 and MC (0.2%) in Sample 5 leads to the same viable cell density when compared to the same total concentration of Px188 (0.2%) in Sample 2.

[0084] Without being bound by theory, a person of ordinary skill in the art would have expected that Sample 5, a combination of both Px188 and MC (0.2%), would have a viable cell density value between the viable cell density for 0.2% MC alone in Sample 1 and 0.2% Px188 in Sample 2. This is because MC is less efficient in promoting cell density than Px188.

[0085] Reducing the amount of Px188 in the media is desired due to quality control concerns with Px188, so the foregoing results are both surprising and would be of interest to the industry.

[0086] A combination of lower total concentration of MC and Px188 surprisingly has similar peak viable cell density as a higher concentration of Px188. The combination of Px188 and MC (0.1% total concentration, with 0.05% Px188 and 0.5% MC) in Sample 6 has total less additive (0.1%) and has four times less Px188 than the 0.2% Px188 alone in Sample 2.

[0087] Surprisingly, the mentioned combination has similar peak viable cell density as the 0.2% Px188 alone and thereby accomplishing an industry goal of reducing Px188 levels in cell media.Example 3A combination of methylcellulose and poloxamer 188 surprisingly leads to higher protein titer levels

[0088] Protein A titer levels and Bio HT titer levels were measured for this example as performed for Example 1.

[0089] The following table shows the average IgG protein content levels measured by Protein A and Bio HT at the end of the cell culture for Samples 1-6.Table 2

[0090] The following describes analysis of the values obtained from the table above.

[0091] IgG protein titer is higher in Px188 than in MC. As shown by a comparison of Sample 1 (0.2% MC) with Sample 2 (0.2% Px188) or Sample 3 (0.1% MC) with Sample 4 (0.1% Px188), Px188 at both 0.2% and 0.1% is more efficient at increasing IgG protein titer than at the same levels for MC, leading to higher yield of the target protein product (an IgG protein).

[0092] Higher respective concentrations of Px188 and MC leads to higher IgG protein titer than lower concentrations. When comparing Sample 1 with Sample 3, MC at a higher concentration (0.2% vs 0.1%) has higher IgG protein titer than the respective lower concentration. When comparing Sample 2 with Sample 4, Px188 at a higher concentration (0.2% vs 0.1%) has higher IgG protein titer than the respective lower concentration.

[0093] A combination of MC and Px188 at same total concentration has surprisingly higher IgG protein titer than either MC or Px188 alone as measured by Bio HT or the same IgG proteintiter as Px188 alone as measured by Protein A chromatography. The IgG protein titer measured by Protein A chromatography (919 pg / ml) is higher for the combination of MC and Px188 (0.1 %) in Sample 6 as compared to the IgG protein titer of MC alone (0.1 % MC) of Sample 3 (610 pg / ml). The IgG protein titer for the mentioned combination is the same as Px188 alone (0.1% Px188) in Sample 4, while reducing the concentration of Px188 by half.

[0094] The combination of MC and Px188 (0.1%) in Sample 6 has the same amount of total concentration of additive as the MC alone in Example 3 and Px188 alone in Sample 4 yet the IgG protein titer measured by Bio HT ( / .e., 1138 pg / ml) is higher in Sample 6 than in Sample 3 (563 pg / ml) or in Sample 4 (838 pg / ml).

[0095] The foregoing results are surprising and unexpected, as MC is less efficient than Px188 in promoting IgG protein titer, so a person skilled in the art would expect the combination of MC and Px188 of Sample 6 to have IgG protein titer levels to be between the IgG protein titer levels measured for the MC alone as in Sample 3 and Px188 alone as in Sample 4.Example 4A combination of methylcellulose and poloxamer 188 surprisingly leads to lowered average M5 glycan content

[0096] The following table shows the average M5 glycan content for Samples 1-6. A higher MS glycan content is not beneficial as it can lead to immunogenicity when the IgG protein is administered as a therapeutic.Table 3

[0097] The following describes analysis of the values obtained from the table above.

[0098] Px188 leads to lower M5 glycan content than MC. A comparison of concentration ofMC in Sample 1 (0.2%) with a concentration of Px188 in Sample 2 (0.2%), and a comparison ofconcentration of MC in Sample 3 (0.1%) and a concentration of Px188 (0.1%) in Sample 4 shows that inclusion of Px188 leads to lower M5 glycan content in cell media than the M5 glycan content as shown by the inclusion of MC in cell media.

[0099] A combination ofMC and Px188 (0. 1% total concentration) surprisingly leads to lower M5 glycan content than the same respective concentration for MC and Px188 alone. The combination of MC and Px188 (0.1%) in Sample 6 has the same amount of total concentration as the MC alone in Sample 3 and Px188 alone in Sample 4 yet the M5 glycan content (3.1 %) is lower in Sample 6 than Sample 3 (4.7%) or Sample 4 (3.3%).

[0100] The foregoing results for the combination of Sample 6 are surprising and unexpected as inclusion of MC content leads to a higher M5 content than Px188, so a person skilled in the art would have expected that the M5 glycan levels for the mentioned combination to have a M5 content to be between the M5 glycan levels between the M5 glycan levels measured for the MC alone as in Sample 3 and Px188 alone as in Sample 4.Example 5A combination of Px188 and HPMC or MC has surprising and unexpected results for increasing average viable cell density as compared to Px188 alone

[0101] The following describes growth of the same CHO DG-44 cell line as previously described but grown using non-baffled cell culture shake flasks and the PowerCHO2™ media. Example 2 used an ambr15 parallel 24 bioreactor system (Sartorius).

[0102] Shake flasks are often used in early stages of cell growth processes and for laboratory studies, making them relevant and important for the industry. In addition to using conventional methylcellulose with poloxamer 188, experiments were conducted in which conventional hydroxypropyl methylcelluloses (HPMC) were added in combination with poloxamer 188.

[0103] A proprietary CHO DG-44 cell line expressing an lgG1 antibody was grown in nonbaffled 125 mL cell culture shake flasks with a 30mL working volume. Samples were agitated by rotation of the shake flasks on a shaker plate. Control of oxygen and carbon dioxide is maintained by the incubator in which the shake flasks are placed, and there is no feedback loop. Baffles are not generally used in CHO culture because such use of baffles would impart extra shear on the cells in the CHO culture.

[0104] The cellular media was PowerCHO2™, available from Lonza, which is supplied with 0.1 % poloxamer 188 as a component. This cell line has been optimized for growth in PowerCHO2™ media. 1000 mL PowerCHO2™ media, 20 mL 200 mM glutamine, and 10 mL of antidumping agent, Gibco™ product having catalog number of 01-0057DG, were combined to form the working media.

[0105] Additional polymer, poloxamer 188 (Px188) or methylcellulose (MC) or hydroxypropyl methylcellulose (HPMC), were added to the media as indicated in Table 4 below.

[0106] The methylcellulose was 15cP grade and is the same type of methylcellulose used in Example 2.

[0107] Two conventional HPMC polymers were tested: HPMC having K chemistry and a 3 cP viscosity as a 2% solution in water at 20 degrees Celsius, i.e., “3cP HPMC-K”) and HPMC having E chemistry and a 5 cP viscosity as a 2% solution in water at 20 degrees Celsius, i.e., “5cP HPMC-E.” An exemplary 3cP HPMC-K is METHOCEL™ K3 Premium LV. An exemplary 5cP HPMC-E is METHOCEL™ E5 Premium LV.

[0108] Cytiva HyClone™ Cellboost™ 7a and 7b supplements which contain Poloxamer 188 (0.1%), were used as supplements.

[0109] Cells were cultured for 14 days. All samples were tested in triplicate except for Sample 7, which was tested a single time.

[0110] 0.3 x 106cells / mL were inoculated into each shake flask at the start of the culture and cells were harvested after the sooner of 14 days or after cell viability dropped below 70 percent. pH, glucose, ammonium, lactate, pCO2, and cell density were monitored daily starting on day 3 of culture.

[0111] Guava® ViaCount™ reagent with flow cytometry and Vi-Cell™ BLU cell viability analyzer (Beckman Coulter) were used to measure viable cell density.

[0112] All other parameters were measured on a Beckman Coulter Vi-CELL MetaFLEX™ bioanalyte analyzer.

[0113] Peak viable cell density is the highest viable cell density that was measured over the fourteen-day culture.

[0114] The following is a table comparing average viable cell densities for Px188 alone (as included in the media), inclusion of additional Px188 to starting Px188 values and inclusion of MC or different grades of HPMC values.Table 4

[0115] The following describes analysis of the values obtained from the table above.

[0116] Adding more poloxamer does not lead to a significant increase in viable cell density. When comparing cell culture media of Sample 7 with 0.1% Px188 with the cell culture media of Sample 8 with a final concentration of 0.3% Px188, the inclusion of additional Px188 led to a slight, 1.7 percent increase in peak VCD for Sample 8. Thus, inclusion of an additional Px188 alone did not lead to an appreciable increase in peak viable cell density (VCD).

[0117] Surprising and unexpected results in viable cell density for a combination of Px188 and HPMC grades or MC as compared to Px188 alone. However, comparing the respective viable cell density values in 0.1 % Px188 alone of Sample 7 with the results for (1) a combination of 0.2% 3cP HPMC-K and 0.1% Px188 in Sample 9; (2) a combination of 0.2% 5cP HPMC-E and 0.1 % Px188 in Sample 10; (3) a combination of 0.2% MC and 0.1%Px188 in Sample 11 and (4) a combination of 0.5% 3cP HPMC-K and 0.1% Px188 in Sample 12, those mentioned combinations showed a significant increase in peak viable cell density compared to only Px188. The increase in viable cell density ranged from 42.1 to 58.3 percent increase in viable cell density as compared to 0.1% Px188 alone.

[0118] The foregoing results show surprising and unexpected results because there is a noticeable increase in viable cell density by combining the cellulose derivatives with poloxamer 188 as shown in respective Samples 9-12 compared to adding poloxamer 188 of Sample 8. These results are unexpected because the previously mentioned Samples 1 and 2 comparing 0.2% MC and 0.2% Px188 demonstrate that Px188 is more efficient than cellulose derivatives at increasing peak viable cell density.Example 6A combination of methylcellulose and poloxamer 188 leads to increased average peak viable cell density

[0119] Like Example 1 , a proprietary CHO DG-44 cell line expressing an lgG1 antibody was grown in the Ambr15 system but used PowerCHO2™ cellular media, as described for Samples 7-12 of Example 4.

[0120] Each condition was tested four times, and the average findings of those four tests for each condition are reported.

[0121] The following is a table comparing average viable cell densities for Px188 alone, inclusion of additional Px188 to starting Px188 values and inclusion of MC to starting Px188 values. The MC was 15 cP methylcellulose as used in previous examples.Table 5

[0122] The following describes analysis of the values obtained from the table above.

[0123] Adding more poloxamer does not lead to a significant increase in viable cell density. A comparison of Samples 13-14 shows that there’s a slight 1.4 percent decrease in peak viable cell density when increasing the concentration from 0.1 percent Px188 to a total concentration of 0.3%. These results demonstrate that inclusion of additional Px188 does not lead to an increase in peak viable cell density. These results are consistent with those found in Table 4.

[0124] Combination of methylcellulose and poloxamer 188 at same total concentration leads to increase in viable cell density. A comparison of Sample 13 having Px188 alone and Sample 15 having a combination of MC and PX188 shows that there is a 7.4 percent increase in peak viable cell density even when the total concentration of 0.3% for the combination is the same as the concentration of Px188 alone in Sample 14 (0.3%), which did not have any increase in peak VCD. By contrast with the lowered viable cell density achieved with the inclusion of additional Px188 (Sample 14) for a total concentration of 0.3%, peak viable cell density actually increasedwith inclusion of of a combination of the MC (0.2%) with 0.1% Px188 (Sample 15). These results are surprising and unexpected because Sample 1 and Sample 2 demonstrate that Px188 is more efficient than cellulose derivatives at increasing peak viable cell density. This also demonstrates that the combination of Px188 and the cellulose derivative surprisingly and unexpectedly facilitates cellular growth.Example 7A combination of methylcellulose and poloxamer 188 surprisingly leads to higher viable cell density

[0125] Agarabi CHO cells (ATCC, CRL3440), which are different from the proprietary CHO DG-44 cell line expressing an lgG1 antibody of Example 4, were used in this example. Agarabi CHO cells (ATCC, CRL3440 were expanded in 125 mL non-baffled shake flasks using CD FortiCHO™ media (Gibco™ product line).

[0126] This commercial media contains 0.074% Poloxamer 188. The media was supplemented with 8 mM L-glutamine. Media was further supplemented with no polymer or methylcellulose 15 cP as detailed in the table below. Cells were maintained at 8% CO2 and 37 degrees C. Initial (cell) culture volume was 10 mL, increasing to 18 mL on day 3 and to 34 mL on day 7. 0.5 mL were removed at each time point for cell counting, using traditional counting in trypan blue with Countess 3FL counter. Each example was tested in quadruplicate.

[0127] The following table reports average total live cells in the flask and describes the effects of using methylcellulose on cellular growth.Table 6

[0128] The following discusses a comparison of the cellular count for each day for the control (Sample 16), 0.2% methylcellulose (Sample 17) and 0.4% methylcellulose (Sample 18).

[0129] On day 1 , 0.2% methylcellulose had 29 percent increase in cellular count (129 percent of the control) over the control at day 1. On day 1, 0.4% methylcellulose had the same cell count as the control at day 1.

[0130] On day 2, 0.2% methylcellulose had 46 percent increase in cellular count over the control (146 percent of the control) at day 2. On day 2, 0.4% methylcellulose had 18 percent increase in cell count over the control at day 2.

[0131] On day 3, 0.2% methylcellulose had 96 percent increase in cellular count over the control (196 percent of the control) at day 3. On day 3, 0.4% methylcellulose had 79 percent increase in cell count (179 percent of the control) over the control at day 3.

[0132] On day 4, 0.2% methylcellulose had 114 percent increase in cellular count over the control (214 percent of the control) at day 4. On day 4, 0.4% methylcellulose had 109 percent increase in cell count (209 percent of the control) over the control at day 4.

[0133] On day 7, 0.2% methylcellulose had 59 percent increase in cellular count over the control (159 percent of the control) at day 7. On day 7, 0.4% methylcellulose had 51 percent increase in cell count over the control (151 percent of the control) at day 7.

[0134] On day 8, 0.2% methylcellulose had 43 percent increase in cellular count over the control (143 percent of the control) at day 8. On day 8, 0.4% methylcellulose had approximately 43 percent increase in cell count over the control (143 percent of the control) at day 8.

[0135] On day 9, 0.2% methylcellulose had approximately 34 percent increase in cellular count over the control (134 percent of the control) at day 9. On day 9, 0.4% methylcellulose had approximately 41 percent increase in cell count over the control (141 percent of the control) at day 9.

[0136] On day 10, 0.2% methylcellulose had approximately 20 percent increase in cellular count over the control (120 percent of the control) at day 10. On day 10, 0.4% methylcellulose had approximately 27 percent increase in cell count over the control (127 percent of the control) at day 10.

[0137] On day 11 , 0.2% methylcellulose had approximately 37 percent increase in cellular count over the control (137 percent of the control) at day 11. On day 11, 0.4% methylcellulose had approximately 20 percent increase in cell count over the control at day 11 (120 percent of the control).

[0138] The following compares the respective cell numbers at day 1 and at day 11 for the control, the respective cell numbers at day 1 and at day 11 for 0.2% methylcellulose and the respective cell numbers at day 1 and at day 11 for 0.4% methylcellulose.

[0139] When comparing with the control numbers at day 1, the cells that were not treated with either 0.2% methylcellulose or 0.4% methylcellulose had approximately 148-fold increase in cellular growth at day 11. This also means approximately 14,847 percent increase in cellular growth over the cellular numbers for the control at day 1.

[0140] When comparing with day 1 cellular numbers for the cells treated with 0.2% methylcellulose, the cells treated with 0.2% methylcellulose had approximately 158-fold increase in cellular growth at day 11 over the cellular numbers for the cells treated with 0.2% methylcellulose at day 1. This also means approximately 15,880 percent increase in cellular growth over the cellular numbers at day 1 .

[0141] Comparison of cellular growth for the control and 0.2% MC. Thus, when comparing with the increased cellular growth for the control starting from day 1 to day 11 , cells treated with 0.2% methylcellulose had approximately 10 percent increased cellular growth over that of the control ( / .e., 158-fold vs. 148-fold for the control.)

[0142] When comparing with day 1 cellular numbers for cells treated with 0.4% methylcellulose, the cells treated with 0.4% methylcellulose had approximately 179 -fold increase in cellular growth at day 11. This also means approximately 17,900 percent increase in cellular growth over the cellular numbers at day 1 .

[0143] Comparison of cellular growth for the control and 0.4% MC. Thus, when comparing with the increased cellular growth for the control starting from day 1 to day 11 , cells treated with 0.4% methylcellulose had approximately 30 percent increased cellular growth over that of the control ( / .e., 179-fold vs. 148-fold for the control.)

[0144] The data shown in Examples 2-7 demonstrate the same trend, with the combination of Px188 with MC or Px188 with HPMC having a surprising positive impact on cell growth. This demonstrates that the surprising effect of the combination of the methylcellulose or HPMC with poloxamer is relevant for different suspension cell culture systems, including different reactor types, media choices, or cell lines. More broadly, this demonstrates that the surprising effect of the combination of the polysaccharide with poloxamer is relevant for different suspension cell culture systems.Example 8SG MC is effective like MC in promoting a higher viable cell density and protein IgG titer over a control with no additives but has still lower effects than poloxamer 188

[0145] This experiment was performed as in Example 2 but compared two different methylcelluloses as the media additive. One methylcellulose used in this example was a conventional methylcellulose 2 cP. The other methylcellulose used in this example was an SG methylcellulose 2 cP, an example of a non-conventional methylcellulose. Both were added at 0.2% to the HyCell™ media.

[0146] The control for this experiment is Example 1.

[0147] The results are shown in the following table.Table 7

[0148] The following is an analysis of the above data.

[0149] When comparing Sample 19 with Sample 20, both medias have approximately the same viscosity.

[0150] When comparing Sample 19 with Sample 20, the use of SG-MC leads to a 59% increase in peak viable cell density as compared to using MC, a conventional methylcellulose.

[0151] When comparing Sample 19 to Sample 20, the use of SG-MC also leads to a 20.3% increase in protein IgG titer. Protein titer measurements were determined by using Protein A chromatography as in Example 1.

[0152] When comparing Sample 19 to Example 1 and comparing Sample 20 to Example 1 , both methylcelluloses are able to significantly improve viable cell density compared to a control media with no additive.

[0153] As the following will show, use of a non-conventional methylcellulose still led to a lower average peak viable cell density and lower IgG protein titer than use of poloxamer 188 at the same concentration.

[0154] When comparing the peak viable cell density of Sample 20 (0.2% SG methylcellulose) with Sample 2 (0.2% Px188) in Example 2, the Px188 is more effective than SG-MC, having an average peak viable cell density of 3.09 x 107, which is a 59% increase in peak viable cell density than the same concentration of SG-MC in Sample 20.

[0155] When comparing the IgG protein titer of Sample 20 (757 pg / ml) with Sample 2 (1297 pg / ml) in Example 3, Px188 is more effective than SG-MC by 71 percent in promoting an increase in target IgG protein.

[0156] The data in this example show that both different substitution patterns of methylcellulose alone are able to improve viable cell density and protein titer over a control with no additives, but yet still are not as effective as Px188.Example 9Higher molecular weight methylcelluloses are effective like MC 15 cP in promoting higher viable cell density and IgG protein titer over a control with no additives

[0157] This experiment was performed using the procedures in Example 2 but compared different molecular weight conventional methylcelluloses as the media additives. Additionally, results were compared with the results for Sample 19 directed to conventional methylcellulose 2 cP (Example 8). Protein A chromatography was used to measure average protein titer.

[0158] In this example, the following conventional methylcelluloses of different molecular weights are used. Molecular weights are not directly measured but are represented through their 2% solution viscosity at 20 degrees Celsius, as with other methylcelluloses.

[0159] One methylcellulose used for this example was a conventional methylcellulose 338 cP (Sample 21). Another methylcellulose used in this example was a conventional methylcellulose 2880 cP (Sample 22).

[0160] For of the foregoing samples, each respective methylcellulose was added at 0.2% to the HyCell™ media.

[0161] The final additional sample for this example was prepared using a MC 2cP methylcellulose, which was added at 2.4% to the HyCell™ media in order to match the viscosity of the media containing 0.2% of the MC 2880 cP (Sample 22). The MC 2cP methylcellulose at 2.4% was the same methylcellulose as was used in Sample 19.

[0162] The control for this experiment is Example 1.

[0163] The results are shown in the following table.Table 8

[0164] The following is an analysis of the above data.

[0165] When comparing peak viable cell density of Sample 19 (conventional methylcellulose- 2 cP, 0.2%) with Sample 21 (MC - 338 cP, 0.2%), the peak viable cell density was 23.7% greater in Sample 21.

[0166] When comparing peak viable cell density of Sample 19 (conventional methylcellulose- 2 cP, 0.2%) with Sample 22 (MC - 2880 cP, 0.2%), the peak viable cell density was 56.6% greater in Sample 22.

[0167] When comparing protein IgG titer of Sample 19 (conventional methylcellulose - 2 cP, 0.2%) with Sample 21 (MC - 338 cP, 0.2%), the protein IgG titer was 9.2 % greater in Sample21.

[0168] When comparing protein IgG titer of Sample 19 (conventional methylcellulose - 2 cP, 0.2%) with Sample 22 (MC - 2880 cP, 0.2%), the protein IgG titer was 16.6 % greater in Sample22.

[0169] A comparison of the peak viable cell density of Sample 19 (conventional methylcellulose - 2 cP, 0.2%) with Sample 23 shows an 8.2% increase in peak viable cell density for Sample 23, which has 2.4% MC as compared to 0.2% MC in Sample 19.

[0170] A comparison of the protein IgG titer of Sample 19 with Sample 23 shows a 22.7% decrease in protein IgG titer for Sample 23, which has 2.4% MC compared to 0.2% MC in Sample 19.

[0171] A comparison of the peak viable cell density of Sample 22 (MC - 2880 cP, 0.2%) with Sample 23 (MC - 2 cP, 2.4%) shows a 44.7% increase in peak viable cell density for Sample 22 as compared to Sample 23.

[0172] Comparison of the media viscosities for Sample 19, Sample 21 , and Sample 22 shows a slight increase in viscosity with higher molecular weight MC.

[0173] Comparison of the media viscosities for Sample 22 and Sample 23 shows similar viscosity for both samples.

[0174] Comparing Sample 21 to Example 1 and comparing Sample 22 to Example 1 shows that both methylcelluloses are able to significantly improve viable cell density as compared to a control media with no additive.

[0175] Comparing the peak viable cell density of Sample 21 to Sample 2 in Example 2 and comparing the peak viable cell density of Sample 22 to Sample 2 in Example 2 shows that Px188 is still more effective in promoting higher peak viable densities than higher molecular weight MCs’ alone.

[0176] Comparing the IgG protein titer of Sample 21 (687 pg / ml) to Sample 2 (1297 pg / ml) of Example 3 and comparing the IgG protein titer of Sample 22 (725 pg / ml) to Sample 2 (1297 pg / ml) in Example 3 shows that Px188 is still more effective than higher molecular weight MC’s in promoting greater IgG protein titer.

[0177] The data in this example show that higher molecular weights of methylcellulose are able to improve viable cell density and protein titer, but yet still are not as effective as Px188.

[0178] The data in this example also show that the effectiveness of higher molecular weights of methylcellulose in promoting greater peak viable cell density is not solely due to increase in media viscosity. For example, Sample 23 (MC - 2 cP, 2.4%) had lower effectiveness than Sample 22 (MC - 2880 cP, 0.2%) in promoting greater peak viable cell density even though both samples had similar viscosity.Example 10Unexpected benefit from combining methylcellulose and poloxamer 188 is observed across different methylcellulose / poloxamer 188 ratios

[0179] 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 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 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 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.

[0180] The following Table 9 shows the observed VCD for the tested samples.Table 9

[0181] In Table 9, 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.

[0182] As shown in Table 9, 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 11 Effect of increased additive concentration in a high shear system

[0183] 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 (MC) with 15 cPs viscosity, poloxamer 188 (Px188), or a combination of MC and Px188 was added to the media as shown in Table 10. 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 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.

[0184] The following Table 10 shows the observed VCD for the tested samples.Table 10

[0185] In Table 10, the percentages are weight percentages based on the total cell culture.

[0186] As shown in Table 10, total additive concentrations of from 0.05 to 0.5% were tested in the high shear system. Use of a total additive concentration of from 0.4 to 0.5% was observed to have an unexpected improvement of VCD by using the combination of methylcellulose and Px188 rather than either one individually.Example 12Effect of decreased additive concentration in a high shear system

[0187] 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 11 and 12. The experiments in Table 11 were carried out using a shake flask rotation speed of 160 RPM, while the experiments in Table 12 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 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 Tables 11 and 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.

[0188] The following Table 11 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 11

[0189] The following Table 12 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 12

[0190] In Tables 11 and 12, the percentages are weight percentages based on the total cell culture.

[0191] As shown in Table 11 and 12, 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 the lower 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 13Protective effect of SG-MC alone or SG-MC in combination with Px188

[0192] 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 MC or SG methylcellulose (SG-MC) with 2 cPs viscosity, poloxamer 188 (Px188), or a combination of such a conventional MC or SG-MC and Px188 was added to the media as shown in Table 13. 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 reactor at the start of the culture at a concentration of53x10 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 13 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.

[0193] The following Table 13 shows the observed VCD for the tested samples.Table 13

[0194] In Table 13, the percentages are weight percentages based on the total cell culture.

[0195] As shown in Table 13, 0.20% Px188 alone provided more protective effect than either 0.20% conventional MC alone or 0.20% SG-MC alone. But the protective effect of Px188 alone can be achieved by replacing a portion (here, 50%) of the Px188 with either conventional MC or SG-MC. The improvements observed in S58 and S59 cannot be attributed to the Px188 alone, because 0.10% Px188 is not as efficient as 0.20% Px188 as demonstrated by comparing Samples 56 and 57.Example 14Protective effect of LTG-MC or a combination of LTG-MC with Px188

[0196] This example assesses the protective effects of LTG-MC alone and in combination with poloxamer 188 (Px188). LTG-MC is a non-conventional methylcellulose with an s23 / s26 in the range of 0.16 to 0.25.

[0197] 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. LTG methylcellulose (LTG-MC) with 2 cPs viscosity, Px188, or a combination of LTG-MC and Px188 was added to the media as shown in Table 14. 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 reactor at the 5 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 14 is the average (mean) of three replicated experiments. Cell counting was carried out using traditional counting in Trypan Bluewith a Countess™ 3 FL Automated Cell Counter following centrifugation and resuspension of the cells in HyCell™ media with no methylcellulose or poloxamer.

[0198] The following Table 14 shows the observed VCD for the tested samples.Table 14

[0199] In Table 14, the percentages are weight percentages based on the total cell culture.

[0200] As shown in Table 14, LTG-MC provided more protective effect than Px188. And the combination of LTG-MC and Px188 provided an unexpected protective effect, i.e., a greater effect than would have been expected to be achieved by the sum of the effects of the LTG-MC alone and Px188 alone.Example 15Unexpected benefit from combining methylcellulose and poloxamer 188 is observed across different methylcellulose / poloxamer 188 ratios (3L reactor)

[0201] 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 15:Table 15

[0202] 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 16 (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 16

[0203] Conventional methylcellulose having 15 cPs viscosity (MC), Px188, or a combination of MC and Px188 was added to the media as shown in Table 17. 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 17).TM

[0204] 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. ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypanblue exclusion. Peak viable cell density is the highest viable cell density that was measured over the 14-day culture.

[0205] 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.

[0206] 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.

[0207] The following Table 17 shows the results.Table 17

[0208] In Table 17, the percentages are weight percentages based on the total cell culture.

[0209] As shown in Table 17, 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 88 to Samples 87 and 86. This data also demonstrates the effectiveness of methylcellulose to protect a cell culture from damage by antifoam, as shown by Samples 87, 89 and 90.

[0210] 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.

[0211] 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 polysaccharide and a polyether surfactant.

2. The cell culture media of claim 1 wherein the polyether surfactant is poloxamer 188.

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

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 2,000 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 100 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 20 cP at 20 degrees Celsius.

9. The cell culture media of any of claims 1-8 wherein the polysaccharide is methylcellulose with a solution viscosity at 2% in water of 15 cP at 20 degrees Celsius.

10. The cell culture media of any of claims 1-8 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.11 . The cell culture media of any of claims 1-8, wherein the polysaccharide is hydroxypropyl methylcellulose having K chemistry with a solution viscosity at 2% in water of 3 cP at 20 degrees Celsius.

12. The cell culture media of any of claims 1-11 wherein the methylcellulose or hydroxypropyl methylcellulose substitution pattern is defined such that the s23 / s26 ratio is from 0.16 to 0.36.

13. A method for growing cells in suspension comprising incubating cells in a cell culture media of any of claims 1-12.

14. A method for increasing cellular growth comprising: providing a cell culture media containing a combination of a cellulose derivative and a polyether surfactant; combining cells with said cell culture media; and incubating the cells and media to enable growth.

15. The method of Claim 14, wherein the step of providing the combination of the cellulose derivative and a polyether surfactant provides the combination in a concentration range from 0.01 % to 5%;16. The method of Claim 14, wherein the polyether surfactant is poloxamer 188.

17. The method of Claim 14, wherein the cellulose derivative is methylcellulose.

18. The method of Claim 17, wherein the methylcellulose is methylcellulose 15 cP.

19. The method of Claim 17, wherein the methylcellulose has a substitution pattern that is defined such that the s23 / s26 ratio is from 0.16 to 0.36.

20. The method of Claim 14, wherein the cellulose derivative is hydroxypropyl methylcellulose.

21. The method of Claim 20, 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.

22. The method of Claim 20, 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.

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

24. The method of Claim 14, wherein the cells are from a Chinese hamster ovary cell line.

25. The method of Claim 14, wherein the cells are from a human endothelial kidney cell line.

26. The method of Claim 14, wherein the cells are from a Vero cell line.

27. The method of Claim 14, wherein the cells produce a protein.

28. The method of Claim 14, wherein the cells produce an antibody.

29. The method of Claim 14, wherein the cells produce an antibody-derived protein.

30. The method of Claim 14, wherein the cells produce a vaccine.

31. the method of Claim 14, wherein the cells produce a gene therapy.

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