Effects of differing viscosity-grade cellulose derivatives on cellular growth

Abstract

Cell culture media and methods relating to use of higher viscosity cellulose derivatives for improving cellular growth and protein titer levels are disclosed.

Description

Effects of Differing Viscosity-Grade Cellulose Derivatives on Cellular GrowthField of the Invention

[0001] The field relates to use of different grades of viscosity of cellulose derivatives on 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] 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 cellculture 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] It is noted in the art that lower viscosity grade hydroxypropyl methylcellulose reduces surface tension more than higher viscosity grades. Lower surface tension helps to protect cells from damage from bubble rupture. See, Chattopadhaya, D., et.al., “The protective Effect of Specific Medium Additives with Respect to Bubble Rupture,” Biotechnology and Bioengineering, 45, 473-480, 1995. A person skilled in the art would therefore presume that lower viscosity grade methylcellulose would be preferred for use in suspension cell culture.

[0016] There remains a need for alternatives in improving cellular growth in in-vitro cellular medias.Summary of the Invention

[0017] A cell culture media for growing cells in suspension comprises a cellulose derivative having a viscosity range from 20 cP to 8000 cP. An associated method for increasing cellular growth comprises: providing a cell culture media containing a cellulose derivative having a viscosity range from 20 cP to 8000 cP; 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 differing viscosity grade of cellulose derivative such as methylcellulose or hydroxypropyl methylcellulose to the cell culture media is disclosed.

[0020] The following describes certain key concepts in the specification.

[0021] In one example, the cell culture media does not include serum.

[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, and “Px188” is the abbreviation used for “poloxamer 188.”

[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 media in this disclosure include methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose, and any combinations thereof.

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

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

[0028] In one example, the cellulose derivative is a methylcellulose in the range of 20 cP to 8,000 cP. In one example, the cellulose derivative is a hydroxypropyl methylcellulose (HPMC) in the range of 20 cP to 8,000 cP.

[0029] 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%.

[0030] When poloxamer is used optionally, an exemplary concentration of poloxamer 188 is less than 5% and an optimal range is from 0.02% to 3%.

[0031] When poloxamer is used optionally, an exemplary concentration of the combination of polysaccharide and poloxamer is 0.05% to 1%. When poloxamer is used optionally, an exemplary concentration of the combination of a cellulose derivative and poloxamer is 0.05% to 1%.

[0032] 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.

[0033] The following describes more details about methylcellulose and hydroxypropyl methylcellulose.Methylcellulose and Hydroxypropyl Methylcellulose

[0034] 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.

[0035] Each anhydroglucose unit contains hydroxyl groups at the 2, 3, and 6 positions. Partial or complete substitution of these hydroxyl substituents creates cellulose derivatives.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Many cellulose derivatives are also defined by the United States Pharmacopeia (USP).

[0041] 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.

[0042] 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.

[0043] 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 at 20 degrees Celsius has a viscosity of approximately 15 cP. Methods for determining this solution viscosity are well known to those skilled in the art and are defined by the United States Pharmacopoeia, Chapter 912.

[0044] Chemical Derivatization 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 molarfraction 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.

[0045] 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.

[0046] 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.

[0047] Definition of the substitution pattern of the substituents on the cellulose by a s23 / s26 ratio to form the cellulose derivatives is optional.

[0048] The substitution pattern is optionally defined as follows:

[0049] 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.

[0050] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is a methylcellulose. The methylcellulose has anhydroglucose units joined by 1-4 linkages wherein hydroxy groups of anhydroglucose units are substituted with methyl groups such that s23 / s26 is from 0.26 to 0.32, wherein s23 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 3-positions of the anhydroglucose unit are substituted with methyl groups and wherein s26 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted with methyl groups. These polymers are referred to as SG methylcelluloses (“SG-MC”), 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.

[0051] In one example, a favored s23 / 26 is from 0.23 to 0.32.

[0052] 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.

[0053] 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.

[0054] Non-conventional cellulose derivatives. The foregoing SG MC and SG HPMC can be also referred to as “non-conventional cellulose derivatives.”

[0055] Exemplary SG methylcellulose and SG HPMC are manufactured as described in EP1171471 , WG20000 / 59947, US6235893 and US6228416, for example.Example 1Higher viscosities of methylcellulose leads to increased average peak viable cell density and average protein titer levels

[0056] 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.

[0057] Cells were grown in an ambr15 parallel 24 bioreactor system (Sartorius). Further details of the system are provided at

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue exclusion.

[0063] Peak viable cell density is the highest viable cell density that was measured over the fourteen-day culture.

[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 set of samples was run in quadruplicate, except for Sample 1 which was run in duplicate. Peak viable cell density and protein titer are reported as averages of the replicates for each sample.

[0066] The following table shows differences in average viable cell density and average protein titer for a sample containing no methylcellulose, a sample containing low viscosity methylcellulose, a sample containing medium viscosity methylcellulose and a high viscosity methylcellulose.

[0067] Molecular weights are not directly measured but are represented through their 2% solution viscosity in water at 20 degrees Celsius, as is typical for methylcelluloses.Table 1

[0068] A comparison of the average peak viable cell densities and the IgG protein titer in Samples 2-5 with Sample 1 demonstrates the need for addition of a polymer to the cell media to yield higher peak viable cell densities and higher protein titer, both of which are desired by those skilled in the art.

[0069] The increase in peak viable cell density with the addition of polymer in Samples 2-5 ranged from 892-927% as compared to Sample 1.

[0070] The increase in IgG protein titer with the addition of polymer to the cell media in Samples 2-5 ranged from 972-1450% as compared to Sample 1.

[0071] Comparing the viscosities of cell medias used in Samples 2, 3, and 4, the viscosity increased slightly as the molecular weight of the methylcellulose increased. For example, the viscosity of media of Sample 4 (MC - 2880 cP, 3.49 cP media as used) was greater than the viscosity of media of Sample 3 (MC - 338 cP, 2.94 cP media as used). The viscosity of media of Sample 3 (MC - 338 cP, 2.94 cP media as used) was greater than the viscosity of media of Sample 2 (MC - 2 cP, 2.18 cP media as used).

[0072] Comparing the viscosities of cell medias used in Samples 4 and 5, the viscosity was comparable for the two medias, though polymers of different molecular weight were used. This was accomplished by using a higher concentration of low viscosity MC - 2cP in Sample 5 (2.4%) to match the viscosity of the high viscosity MC - 2880 cP in Sample 4 (0.2%).

[0073] Average peak viable cell density increases with a change of viscosity from a low viscosity methylcellulose to a medium viscosity methylcellulose. The average peak viable cell density of a medium viscosity methylcellulose of Sample 3 (0.2% concentration) had a value thatwas 23.7% greater than the average peak viable cell density for Sample 2 having a low viscosity methylcellulose (0.2%).

[0074] Average peak viable cell density increases even more with a change of viscosity from a low viscosity methylcellulose to a high viscosity methylcellulose.The average peak viable cell density of a high viscosity methylcellulose of Sample 4 (0.2% concentration) had a value that was 56.6% greater than the average peak viable cell density for low viscosity methylcellulose of Sample 2 (0.2% concentration).

[0075] Average peak viable cell density increases with a change of viscosity from a medium viscosity methylcellulose to a high viscosity methylcellulose. Even when comparing with average peak viable cell density values for medium viscosity methylcellulose and a high viscosity methylcellulose, the average peak viable cell density of a high viscosity methylcellulose of Sample 4 had a value that was 26.5 % greater than the average peak viable cell density for Sample 3 having a medium viscosity methylcellulose.

[0076] Average peak viable cell density is higher for a higher viscosity methylcellulose compared to lower viscosity methylcellulose even when the medias have the same viscosity. The average peak viable cell density of a high viscosity methylcellulose of Sample 4 (0.2% concentration) had a value that was 44.7% greater than the average peak viable cell density for low viscosity methylcellulose of Sample 5 (2.4% concentration).

[0077] Analyses. The foregoing results demonstrate that a greater peak viable cell density is achieved with a higher viscosity methylcellulose and that the increase in peak viable cell density is not due to increases in media viscosity as used.

[0078] The following discusses the resulting protein titer levels achieved with different viscosity grades of methylcellulose.

[0079] Average IgG protein titer increases from changing from a low viscosity methylcellulose to a medium viscosity methylcellulose. Using a medium viscosity methylcellulose at 0.2% concentration instead of a low viscosity methylcellulose at 0.2% led to an increase of 9.2% in average IgG protein titer.

[0080] Average IgG protein titer increases even more when changing from a low viscosity methylcellulose to a high viscosity methylcellulose. Using a high viscosity methylcellulose instead of a medium viscosity methylcellulose in the foregoing example leads to even more impressive results. When one uses a high viscosity methylcellulose at 0.2% concentration instead of a low viscosity methylcellulose at 0.2%, an increase of 15.2% in average IgG protein titer was achieved.

[0081] A comparison of average IgG protein titer between using a high viscosity methylcellulose and a medium viscosity methylcellulose shows an increase in average IgG protein titer. A comparison of IgG protein titer in a high viscosity methylcellulose of Sample 4(0.2%) with a medium viscosity methylcellulose of Sample 3 showed an increase of 5.5% increase in protein titer.

[0082] Average IgG protein titer is higher for a higher viscosity methylcellulose compared to lower viscosity methylcellulose even when the medias have the same viscosity. The average IgG protein titer in a high viscosity methylcellulose media of Sample 4 (0.2% concentration) had a value that was 49.2% greater than the average IgG protein titer in a low viscosity methylcellulose media of Sample 5 (2.4% concentration).

[0083] While Samples 4 and 5 had very comparable media viscosities, they did not have comparable peak viable cell densities or IgG protein titer. This shows how differences in viable cell density or IgG protein titer are not caused by changes in media viscosity. Instead, as demonstrated by the testing of the samples above, the differences in the viscosity of the methylcellulose utilized accounted for the differences in values for peak viable cell densities and IgG protein titer obtained.

[0084] Surprising and unexpected results using higher grades of viscosity in methylcellulose. Changing from a low viscosity methylcellulose (Sample 2) to medium viscosity methylcellulose (Sample 3) or a high viscosity methylcellulose (Sample 4) led to a surprising and unexpected result of an improvement in average peak viable cell density and average IgG protein titer since it was expected in the art that use of higher viscosity methylcellulose would lead to reduced cell growth.Example 2Critical upper limit of higher molecular weight polymers for use in cell culture media as certain higher molecular weight is associated with buildup of viscosity

[0085] The results as shown in the following table demonstrate that certain higher molecular weight polymers are associated with buildup of viscosity and there is a critical upper limit for using higher molecular weight polymers in cell media. At a certain upper viscosity limit of the polymer utilized, higher viscosity will create more shear during stirring, thus leading to deleterious effects in cells. Higher viscosity may also lead to more persistent foam in a bioreactor, which is not desirable.

[0086] When incorporating polymers of higher molecular weight with certain polymer viscosity into a cell culture media, the cell culture media (“media”) utilized in this example will have resulting higher viscosity as well, as the following table will show.

[0087] The viscosity of the media for this example was measured when higher molecular weight methylcelluloses were incorporated into the media. Before addition of the polymer, the media is the same Hycell™ media as in Sample 1 and has the same viscosity as reported for the Hycell™ media of Sample 1. Thus, the media of this Example 2 had a viscosity of 2.23 cP when measured at 37°C before addition of the polymer.

[0088] After the addition of the respective polymer, the viscosity of the media increased to6.42 cP and 6.34 cP as shown in Table 2.Table 2

[0089] Comparison of the media viscosities of Samples 6 and 7 to the media viscosities of Samples 1-5, the media viscosities of Samples 6 and 7 are approximately in a range from 1.83 to 2.94 times as viscous as the media viscosities of Samples 1-5. This viscosity range differences of 1.83 to 2.94 are determined by respectively comparing the highest media viscosity of Sample 6 with (1) the lowest media viscosity of Sample 2 and (2) the highest media viscosity of Sample 4.

[0090] Analyses. Higher viscosity of the polymer at a certain upper limit will create more shear during stirring, thus leading to deleterious effects in cells. Higher viscosity may also lead to more persistent foam in the reactor, which is not desirable. Based on the teachings of the Table 2 above, therefore, a person skilled in the art would not want to add polymers which build significant viscosity to the cell media. Consequently, methylcelluloses with a viscosity of 10,000 cP and greater would not be desirable for use in cell media.Example 3SG MC is effective like MC in promoting a higher viable cell density and protein IgG titer over a control with no additives

[0091] This experiment was performed as in Example 1 but used an SG methylcellulose, 2 cP (SG-MC - 2cP). As in Example 1 , Samples 2-4, the foregoing SG methylcellulose was added at 0.2% to the HyCell™ media.

[0092] The control for this experiment is Sample 1.

[0093] The results are shown in the following table.Table 3

[0094] The following is an analysis of the above data.

[0095] When comparing Sample 8 with Sample 2, both medias have approximately the same viscosity.

[0096] When comparing Sample 8 with Sample 2, the use of SG-MC leads to a 59% increase in peak viable cell density as compared to using MC, a conventional methylcellulose.

[0097] When comparing Sample 8 with Sample 2, 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.

[0098] When comparing Sample 8 with Sample 1 and comparing Sample 2 with Sample 1 , both methylcelluloses are able to significantly improve viable cell density compared to a control media with no additive.

[0099] The data in this example show that both different substitution patterns of methylcellulose are able to improve viable cell density and protein titer over a control with no additives.

[0100] 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

[0101] The following describes growth of a CHO DG-44 cell-line grown using non-baffled cell culture shake flasks and the PowerCHO2™ media.

[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.

[0107] Two conventional HPMC polymers were tested: HPMC having K chemistry and a 3 cP viscosity of 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] ViaCount™ works via differential nuclear staining of live and dead cells. Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue exclusion.

[0114] Peak viable cell density is the highest viable cell density that was measured over the fourteen-day culture.

[0115] 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

[0116] The following describes analysis of the values obtained from the table above.

[0117] Adding more poloxamer does not lead to a significant increase in viable cell density. When comparing cell culture media of Sample 9 with 0.1% Px188 with the cell culture media of Sample 10 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 10. Thus, inclusion of an additional Px188 alone did not lead to an appreciable increase in peak viable cell density (VCD).

[0118] 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 9 with the results for (1) a combination of 0.2% 3cP HPMC-K and 0.1% Px188 in Sample 11 ; (2) a combination of 0.2% 5cP HPMC-E and 0.1% Px188 in Sample 12; (3) a combination of 0.2% MC and 0.1% Px188 in Sample 13 and (4) a combination of 0.5% 3cP HPMC-K and 0.1% Px188 in Sample 14, 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.

[0119] 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 11-14 compared to adding poloxamer 188 of Sample 10. 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.

[0120] Based on these preliminary results from Table 4, it is expected that using a combination of poloxamer 188 and higher molecular weight polymers of MC and HPMC as demonstrated by higher viscosity when measured as a 2% solution in water at 20 degrees Celsius will lead to higher cellular growth than either poloxamer or any of the foregoing polymers alone.

[0121] 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.

[0122] 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 having a solution viscosity at 2% in water of a range from 20 cP to 8000 cP at 20 degrees Celsius.

2. The cell culture media of claim 1, wherein the cellulose derivative is methylcellulose or hydroxypropyl methylcellulose or a combination thereof.

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

4. The cell culture media of any of claims 1-3 wherein the cellulose derivative has a solution viscosity at 2% in water of less than 3500 cP at 20 degrees Celsius.

5. The cell culture media of any of claims 1-4 wherein the cellulose derivative has a solution viscosity at 2% in water of less than 1000 cP at 20 degrees Celsius.

6. The cell culture media of any of claims 1-5 wherein the cellulose derivative is a methylcellulose.

7. The cell culture media of any of claims 1-6, wherein the methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.16 to 0.36.

8. The cell culture media of any of claims 1-7, wherein the methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.26 to 0.32.

9. The cell culture media of any of claims 1-5, wherein the cellulose derivative is a hydroxypropyl methylcellulose.

10. The cell culture media of any of claims 1-5 and 9 wherein the cellulose derivative is a hydroxypropyl methylcellulose having E chemistry.

11. The cell culture media of any of claims 1-5 and 9 wherein the cellulose derivative is hydroxypropyl methylcellulose having K chemistry.

12. The cell culture media of any of claims 1-5 and 9, wherein the hydroxypropyl methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.16 to 0.36.

13. The cell culture media of any of claims 1-5 and 12, wherein the hydroxypropyl methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.26 to 0.32.

14. The cell culture media of any of claims 1-13, wherein the cell culture media comprises an antifoaming agent.

15. The cell culture media of any of claims 1-14, wherein the antifoaming agent comprises simethicone.

16. The cell culture media of any of claims 1-15, wherein the cell media further comprises a polyether surfactant.

17. The cell culture media of any of claims 1-16, wherein the polyether surfactant is poloxamer.

18. A method for growing cells in suspension comprising incubating cells in a cell culture media of any of claims 1-17.

19. A method for increasing cellular growth comprising: providing a cell culture media containing a cellulose derivative having a solution viscosity at 2% in water of a range from 20 cP to 8000 cP at 20 degrees Celsius; combining cells with said cell culture media; and incubating the cells and media to enable growth.

20. The method of Claim 19, wherein the step of providing the cellulose derivative provides the cellulose derivative in a concentration range from 0.01% to 5%.

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

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

23. The method of Claim 22, wherein the methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.26 to 0.32.

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

25. The method of Claim 24, wherein the hydroxypropyl methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.16 to 0.36.

26. The method of Claim 25, wherein the hydroxypropyl methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is from 0.26 to 0.32.

27. The method of Claim 19, wherein the cells are from a Chinese hamster ovary cell line.

28. The method of Claim 19, wherein the cells are from a human endothelial kidney cell line.

29. The method of Claim 19, wherein the cells are from a Vero cell line.

30. The method of Claim 19, wherein the cells produce a protein.

31. The method of Claim 19, wherein the cells produce an antibody.

32. The method of Claim 19, wherein the cells produce an antibody-derived protein.

33. The method of Claim 19, wherein the cells produce a vaccine.

34. The method of Claim 19, wherein the cells produce a gene therapy.

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