Cell culture media containing cellulose derivatives and associated method

Abstract

A cell culture media for growing cells in suspension includes a cellulose derivative where the cellulose derivative has anhydroglucose units joined by 1-4 linkages where the hydroxy groups of anhydroglucose units are substituted with methyl or hydroxypropyl groups such that s23 / s26 is from 0.16 to 0.36. An associated method includes providing such a cell culture media, combining cells with said cell culture media and incubating the cells and media to enable growth.

Description

Cell Culture Media ContainingCellulose Derivatives and Associated MethodField of the Invention

[0001] The field relates to cellulose derivatives for 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, 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, 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 culturemedia to help improve cell growth. However, due to regulatory, quality, safety, and ethical concerns, 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] 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 areplacement 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 poloxamer and for improving cellular growth.Summary of the Invention

[0016] A cell culture media for growing cells in suspension comprises a cellulose derivative wherein the cellulose derivative has anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are individually substituted with methyl or hydroxypropyl 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 or hydroxypropyl 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 or hydroxypropyl groups.

[0017] A method for increasing cellular growth comprises: providing a cell culture media containing a cellulose derivative having anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are individually substituted with methyl or hydroxypropyl 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 or hydroxypropyl 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 or hydroxypropyl groups; 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 process to grow cells in suspension that includes addition of a cellulose derivative having certain defined chemical substitution patterns to the cell culture media is disclosed.

[0020] In one example, the cell culture media does not include serum.

[0021] The following discusses key concepts in the specification.

[0022] 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.

[0023] Antifoam. In another example, the cell culture media optionally includes antifoam. The antifoam is optionally a simethicone antifoam.

[0024] 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.

[0025] 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. Additional cellulose derivatives include SG versions of methylcellulose, hydroxypropyl methylcellulose, and any combinations thereof.

[0026] In one example, the hydroxyl groups of a cellulose derivative including SG cellulose derivatives may include an alkyl substituent or a hydroxy alkyl substituents or combinations thereof.

[0027] In one example, the cellulose derivative is SG methylcellulose. In another example, the cellulose derivative is SG hydroxypropyl methylcellulose.

[0028] Concentrations. In one example, the concentration of cellulose derivative 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%.

[0029] 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.

[0030] The following describes more details about methylcellulose and hydroxypropyl methylcellulose.Methylcellulose and Hydroxypropyl Methylcellulose

[0031] 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.

[0032] Each anhydroglucose unit contains hydroxyl groups at the 2, 3, and 6 positions.Partial or complete substitution of these hydroxyl substituents creates cellulose derivatives.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Many cellulose derivatives are also defined by the United States Pharmacopeia (USP).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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 anhydroglucoseunits 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.

[0042] 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.

[0043] 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.

[0044] The substitution pattern for the invention is defined as follows:

[0045] In one example, the cellulose derivative 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.

[0046] In another example, the cellulose derivative 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”), an example of a non-conventional methylcellulose. Methylcelluloses with an s23 / s26 in the range of 0.23 to 0.32 are known as SG methylcellulose.

[0047] In another example, the cellulose derivative 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 0.25, 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 LTG methylcelluloses (“LTG-MC”), an example of a non-conventionalmethylcellulose. Methylcelluloses with an s23 / s26 in the range of 0.16 to 0.25 are known as LTG methylcellulose.

[0048] In one example, a favored s23 / 26 is from 0.23 to 0.32.

[0049] 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.

[0050] 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.

[0051] 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 0.25, 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 LTG hydroxypropyl methylcelluloses (“LTG- HPMC”), an example of a non-conventional HPMC. This non-conventional LTG-HPMC includes both hydroxypropyl and methyl groups in an overall chemical structure.

[0052] Non-conventional cellulose derivatives. The foregoing SG-MC, LTG-MC, SG-HPMC, and LTG-HPMC can be also referred to as “non-conventional cellulose derivatives.”

[0053] Exemplary SG methylcellulose and SG HPMC are manufactured as described in EP1171471 , WG20000 / 59947, US6235893 and US6228416, for example.

[0054] As used in the specification, “conventional methylcellulose” has the same meaning as “conventionally substituted methylcellulose.”Example 1SG methylcellulose leads to higher viable cell density than a conventional methylcellulose and a control with no additives

[0055] 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 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.

[0056] Cells were grown in an ambr15 parallel 24 bioreactor system (Sartorius). Further details of the system are provided atbioreactors / ambr-multi-parallel-bioreactors / ambr-15-cell-culture

[0057] 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.

[0058] 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.

[0059] Gibco Foam Away® Irradiated AOF (animal origin-free) antifoam was added daily 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.

[0060] 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.

[0061] ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue exclusion.

[0062] Peak viable cell density is the highest viable cell density that was measured over the fourteen-day culture.

[0063] The experiment was run in duplicate for Sample 1. The experiment was repeated 4 times for Sample 2 and Sample 3. Results are reported as averages (mean) of the replicates for each sample.

[0064] 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.

[0065] The following table shows differences in average viable cell density and average protein titer for (1) a sample containing no methylcellulose (Sample 1), (2) a sample containing conventionally substituted methylcellulose (Sample 2), and (3) a sample containing SG methylcellulose (Sample 3). The viscosity of both methylcelluloses used was not appreciably different when measured as a 2 wt% solution in water at 20 degrees Celsius.Table 1

[0066] Viable cell density (VCD) was measured as a function of day.

[0067] When compared with Sample 1 , (the control), average peak viable cell density for Sample 2, cultures containing 0.2% conventionally-substituted methylcellulose 2cP in the media was approximately 5.9 fold or was approximately 592 percent of the control.

[0068] When compared with Sample 1 , (the control), average peak viable cell density for Sample 3, cultures containing 0.2%, a SG substituted methylcellulose 2 cP, manufactured asdescribed in EP1171471 , W020000 / 59947, US6235893 and US6228416 to yield different polymer properties, increased 9.4-fold or was approximately 942 percent of the control.

[0069] Peak viable cell density higher when using SG MC than conventional MC. When comparing the average peak viable cell density of Sample 2 and Sample 3, Sample 3 peak viable cell density increased approximately 59 percent over the average peak viable cell density of Sample 2 or was 159 percent of Sample 2.

[0070] When compared with Sample 1 , (the control), average IgG protein titer for Sample 2, cultures containing 0.2% conventionally-substituted methylcellulose in the media, increased approximately 12.6 fold or was approximately 1258 percent of the control.

[0071] When compared with Sample 1 , (the control), average IgG protein titer for Sample 3, cultures containing 0.2%, a SG substituted methylcellulose, manufactured as described in EP1171471, WG20000 / 59947, US6235893, US6228416 to yield different polymer properties, increased 15.1 -fold or was 1514 percent of the control.

[0072] Average protein titer is higher when using SG MC than conventional MC. When comparing the average IgG protein titer of Sample 2 and Sample 3, Sample 3 average IgG protein titer increased approximately 20.3 percent over the average peak viable cell density of Sample 2 or was 120 percent of Sample 2. Thus, a SG methylcellulose leads to increased protein titer over a conventional methylcellulose.

[0073] The viscosity of media prepared with 0.2% of either conventional MC or SG MC is unchanged at incubation temperature. The increase in cell density and protein titer seen with the SG MC is therefore not due to changes in media viscosity at the incubation temperature. Without being bound by theory, a person of ordinary skill in the art would have expected Sample 2 and Sample 3 to have comparable viable cell densities and protein IgG titers. This is because both polymers have the same chemical derivatization and molecular weight, and medias including the polymers of Sample 2 and Sample 3 exhibit the same viscosity at incubation temperatures. Therefore, the foregoing results are surprising and unexpected and would be of interest to the industry.Example 2Use of SG methylcellulose increases viable cell density in high shear shake flasks

[0074] Agarabi CRL3440 CHO (ATCC) cells were cultured in baffled shake flasks, rotating at 160 rpm on a shaker. The presence of baffles and high rate of rotation creates high shear which can be damaging to cells.

[0075] Media was Hycell™, available from Cytiva, which contains no poloxamer. The incubator was maintained at 37 degrees C° and 8% CO2. Culture volume was 15 mL. 3 x105cells / mL were inoculated into the flasks at t=0. Cells were cultured with no additive or addition of 0.2% methylcellulose (Conventional or SG grade) and / or addition of Gibco Foam Away®Irradiated AOF (animal origin-free) antifoam (also referred to as “Foam Away™”) (10 uL / day). Cell counts were measured after 1 and 2 days of culture. The conventional and SG methylcelluloses were the same as those described for Samples 2 and 3.

[0076] The following table provides the results.Table 2Day 1 Results

[0077] When comparing the cells / ml count for Sample 5 with Sample 4 from day 1 , the cells / ml count of the conventional methylcellulose from day 1 is 32-fold over that of the control ( / .e.; approximately 3235% of the control.)

[0078] When comparing the cells / ml count for Sample 6 compared to Sample 4 from day 1, the cells / ml count of the conventional methylcellulose and Foam Away™ of day 1 is approximately 31-fold over that of the control (i.e.; approximately 3100% of the control.)

[0079] When comparing the cells / ml count for Sample 7 compared to Sample 4 from day 1, the cells / ml count of the SG MC (i.e., LV MC) from day 1 is approximately 40.4-fold over that of the control (i.e., approximately 4043% of the control.)

[0080] When comparing the cells / ml count for Sample 8 compared to Sample 4 from day 1 , the cells / ml count of the SG MC and Foam Away™ is approximately 37-fold over that of the control (i.e.; approximately 3700% of the control.)

[0081] For day 1 , comparing Samples 5 and Sample 7, the use of SG MC when compared with the use of conventional MC led to a 25% improvement in average viable cell density when no Foam Away™ was present.

[0082] For day 1 , comparing Samples 6 and Sample 8, the use of SG MC when compared with the use of conventional MC led to a 21% improvement in average viable cell density when Foam Away™ was present.Day 2 Results

[0083] When comparing the cells / ml count for Sample 5 compared to Sample 4 from day 2, the cells / ml count of the conventional methylcellulose from day 2 is 197-fold over that of the control ( / .e.; approximately 19745% of the control.)

[0084] When comparing the cells / ml count for Sample 6 compared to Sample 4 from day 2, the cells / ml count of the conventional methylcellulose and Foam Away™ of day 2 is approximately 155-fold over that of the control (i.e.; approximately 15541% of the control.)

[0085] When comparing the cells / ml count for Sample 7 compared to Sample 4 from day 2, the cells / ml count of the SG MC from day 2 is approximately 257-fold over that of the control (i.e., approximately 25700% of the control.)

[0086] When comparing the cells / ml count for Sample 8 compared Sample 4 from day 2, the cells / ml count of the SG MC and Foam Away™ is approximately 182-fold over that of the control (i.e.; approximately 18200% of the control.)

[0087] For day 2, comparing Samples 5 and 7, the use of SG MC when compared with the use of conventional MC led to a 30% improvement in average viable cell density when no Foam Away™ was present.

[0088] For day 2, comparing Samples 6 and 8, the use of SG MC when compared with the use of conventional MC led to a 17% improvement in average viable cell density when Foam Away™ was present.

[0089] As seen from the above results, it was surprising and unexpected that the use of SG grade of methylcellulose leads to an increased viable cell density compared to use of a conventional methylcellulose.Example 3Higher molecular weight methylcelluloses increase viable cell density and protein titer

[0090] This experiment was set up as in Example 1 , but different polysaccharide polymers were added to the cell culture media. Conventional methylcellulose polymers of differentmolecular weights were utilized. Molecular weights are not directly measured but are represented through their 2% solution viscosity at 20 degrees Celsius, as with other methylcelluloses.

[0091] The polymers used are described with the results in Table 3.

[0092] 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.

[0093] The following table shows the samples tested in this Example and their corresponding peak viable cell densities and protein titers.Table 3

[0094] Results are compared to Sample 2 of Example 1.

[0095] When comparing the viscosities of the medias in Samples 2, 9, and 10, there is a slight increase in viscosity as the methylcellulose molecular weight is larger.

[0096] When comparing the viscosities of the medias in Samples 10 and 11 , the viscosities are comparable and similar.

[0097] When comparing the peak viable cell density of Sample 2 of Example 1 (MC - 2 cP) with Sample 9 (MC - 338 cP), the peak viable cell density was 23.7 percent higher in Sample 9.

[0098] When comparing the peak viable cell density of Sample 2 of Example 1 (MC - 2 cP) with Sample 10 (MC - 2880 cP), the peak viable cell density was 56.6 percent higher in Sample 10.

[0099] When comparing protein IgG titer of Sample 2 (conventional methylcellulose - 2 cP, 0.2%) with Sample 9 (MC - 338 cP, 0.2%), the protein IgG titer was 9.2 % greater in Sample 9.

[0100] When comparing protein IgG titer of Sample 2 (conventional methylcellulose - 2 cP, 0.2%) with Sample 10 (MC - 2880 cP, 0.2%), the protein IgG titer was 16.6 % greater in Sample 10.

[0101] A comparison of the peak viable cell density of Sample 2 (conventional methylcellulose - 2 cP, 0.2%) with Sample 11 shows an 8.2% increase in peak viable cell density for Sample 11 , which has 2.4% MC as compared to 0.2% MC in Sample 2.

[0102] A comparison of the protein IgG titer of Sample 2 with Sample 11 shows an 22.7% decrease in protein IgG titer for Sample 11 , which has 2.4% MC compared to 0.2% MC in Sample 2.

[0103] A comparison of the peak viable cell density of Sample 10 (MC - 2880 cP, 0.2%) with Sample 11 (MC - 2 cP, 2.4%) shows a 44.7% increase in peak viable cell density for Sample 10 as compared to Sample 11 .

[0104] A comparison of the protein IgG titer of Sample 10 (MC - 2880 cP, 0.2%) with Sample 11 (MC - 2 cP, 2.4%) shows a 49.1% increase in peak viable cell density for Sample 10 as compared to Sample 11 .

[0105] The data in this example show that higher molecular weights of methylcellulose are able to improve viable cell density and protein titer.

[0106] 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 11 (MC - 2 cP, 2.4%) had lower effectiveness than Sample 10 (MC - 2880 cP, 0.2%) in promoting greater peak viable cell density even though both samples had similar viscosity.

[0107] The following is an example showing the effects on cellular growth using poloxamer and HPMC or MC.Example 4A combination of Px188 and HPMC or MC has surprising and unexpected results for increasing average viable cell density as compared to Px188 alone

[0108] The following describes growth of a CHO DG-44 cell-line grown using non-baffled cell culture shake flasks and the PowerCHO2™ media.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Additional polymer, poloxamer 188 (Px188) or methylcellulose (MC) or hydroxypropyl methylcellulose (HPMC), were added to the media as indicated in Table 4 below.

[0113] The methylcellulose was 15cP grade.

[0114] 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.

[0115] Cytiva HyClone™ Cellboost™ 7a and 7b supplements which contain Poloxamer 188 (0.1%), were used as supplements.

[0116] Cells were cultured for 14 days. All samples were tested in triplicate except for Sample 7, which was tested a single time.

[0117] 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.

[0118] Guava® ViaCount™ reagent with flow cytometry and Vi-Cell™ BLU cell viability analyzer (Beckman Coulter) were used to measure viable cell density.

[0119] All other parameters were measured on a Beckman Coulter Vi-CELL MetaFLEX™ bioanalyte analyzer.

[0120] ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue exclusion.

[0121] Peak viable cell density is the highest viable cell density that was measured over the fourteen-day culture.

[0122] 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

[0123] The following describes analysis of the values obtained from the table above.

[0124] Adding more poloxamer does not lead to a significant increase in viable cell density. When comparing cell culture media of Sample 12 with 0.1% Px188 with the cell culture media of Sample 13 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 13. Thus, inclusion of an additional Px188 alone did not lead to an appreciable increase in peak viable cell density (VCD).

[0125] 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 12 with the results for (1 ) a combination of 0.2% 3cP HPMC-K and 0.1% Px188 in Sample 14; (2) a combination of 0.2% 5cP HPMC-E and 0.1 % Px188 in Sample 15; (3) a combination of 0.2% MC and 0.1%Px188 in Sample 16 and (4) a combination of 0.5% 3cP HPMC-K and 0.1% Px188 in Sample 17, 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. 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 14-17 compared to adding poloxamer 188 of Sample 13. These results are unexpected because it is known in the art that Px188 is more efficient than cellulose derivatives at increasing peak viable cell density.

[0126] Based on these preliminary results from Table 4, it is expected that using a combination of poloxamer 188 and polymers of nonconventional MC and HPMC will lead to higher cellular growth than either poloxamer or any of the foregoing polymers alone.Example 5Protective effect of SG-MC alone or SG-MC in combination with Px188

[0127] 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 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 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 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.

[0128] The following Table 5 shows the observed VCD for the tested samples.Table 5

[0129] In Table 5, the percentages are weight percentages based on the total cell culture.

[0130] As shown in Table 5, 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.Example 6Protective effect of LTG-MC or a combination of LTG-MC with Px188

[0131] This example assesses the protective effects of LTG-MC alone and in combination with poloxamer 188 (Px188).

[0132] 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 6. 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 the5 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 6 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.

[0133] The following Table 6 shows the observed VCD for the tested samples.Table 6

[0134] In Table 6, the percentages are weight percentages based on the total cell culture.

[0135] As shown in Table 6, 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 7Protective effect of SG-MC alone against antifoam cell damage

[0136] 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 7. 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 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.

[0137] The following Table 7 shows the observed VCD for the tested samples.Table 7

[0138] In Table 7, the percentages are weight percentages based on the total cell culture.

[0139] As shown in Table 7, 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

[0140] In this example, the protective effects of conventional methylcellulose (MC), SG-MC and LTG-MC alone were compared.

[0141] 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 8. Gibco™ FoamAway™ IrradiatedAOF (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 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 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.

[0142] The following Table 8 shows the observed VCD for the tested samples.Table 8

[0143] In Table 8, the percentages are weight percentages based on the total cell culture.

[0144] As shown in Table 8, 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.

[0145] 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.

[0146] 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 cellulose derivative wherein the cellulose derivative has anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are individually substituted with methyl or hydroxypropyl 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 or hydroxypropyl 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 and / or hydroxypropyl groups.

2. The cell culture media of Claim 1 , wherein s23 / s26 is from 0.26 to 0.32.

3. The cell culture media of Claim 1 , wherein the s23 / s26 is from 0.16 to 0.25.

4. The cell culture media of any of claims 1-3, wherein the cell culture media further comprises an antifoaming agent.

5. The cell culture media of any of claims 1-4, wherein the antifoaming agent comprises simethicone.

6. The cell culture media of any of claims 1-5, wherein the cell culture media further comprises a polyether surfactant.

7. The cell culture media of any of claims 1-6, wherein the polyether surfactant is poloxamer.

8. The cell culture media of any of claims 1-7, wherein the cellulose derivative has a solution viscosity at 2% in water of less than 10,000 cP at 20 degrees Celsius.

9. The cell culture media of any of claims 1-8, wherein the cellulose derivative has a solution viscosity at 2% in water of less than 2,000 cP at 20 degrees Celsius.

10. The cell culture media of any of claims 1-9 where the cellulose derivative has a solution viscosity at 2% in water of less than 100 cP at 20 degrees Celsius.

11. The cell culture media of any of claims 1-10, wherein the cellulose derivative is SG methylcellulose or SG hydroxypropyl methylcellulose or a combination thereof.

12. A method for growing cells in suspension comprising incubating cells in a cell culture media of any of claims 1-11.

13. A method for increasing cellular growth comprising: providing a cell culture media containing a cellulose derivative having anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are individually substituted with methyl or hydroxypropyl 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 or hydroxypropylgroups 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 or hydroxypropyl groups; combining cells with said cell culture media; and incubating the cells and media to enable growth.

14. The method of Claim 13, wherein providing the cell culture media comprises providing the cellulose derivative having a s23 / s26 from 0.26 to 0.32.

15. The method of Claim 13, wherein providing the cell culture media comprises providing the cellulose derivative having a s23 / s26 from 0.16 to 0.25.

16. The method of Claim 13, wherein the method further comprises adding an antifoaming agent.

17. The method of Claim 14, wherein the antifoaming agent comprises simethicone.

18. The method of Claim 13, wherein the cellulose derivative is SG methylcellulose.

19. The method of Claim 13, wherein the cellulose derivative is hydroxypropyl methylcellulose.

20. The method of Claim 13, wherein the cells are from a Chinese hamster ovary cell line.

21. The method of Claim 13, wherein the cells are from a human endothelial kidney cell line.

22. The method of Claim 13, wherein the cells are from a Vero cell line.

23. The method of Claim 13, wherein the cells produce a protein.

24. The method of Claim 13, wherein the cells produce an antibody.

25. The method of Claim 13, wherein the cells produce an antibody-derived protein.

26. The method of Claim 13, wherein the cells produce a vaccine.

27. The method of Claim 13, wherein the cells produce a gene therapy.

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