Cell culture media containing cellulose derivatives and related methods

Abstract

A cell culture medium for growing cells in suspension includes a cellulose derivative, wherein the cellulose derivative has anhydroglucose units linked by 1-4 linkages, wherein hydroxyl groups of the anhydroglucose units are substituted with methyl or hydroxypropyl groups, such that s23 / s26 is 0.16 to 0.36. Related methods include providing such a cell culture medium, combining cells with the cell culture medium, and incubating the cells and medium to allow growth.

Description

Technical Field

[0001] This field involves cellulose derivatives used for cell growth. Background Technology

[0002] Upstream cell growth is a critical process in the manufacture of advanced therapeutic modalities, particularly cell, gene, and protein drug products. The cost of manufacturing these products is extremely high, resulting in very high drug prices. There is a need to increase cell growth output through process intensification to help reduce the manufacturing costs of these therapies. It is known in the art that protein drug titers are improved using cell engineering to obtain higher copy numbers, shorter culture times, and reduced proteolytic activity. Despite efforts in this area, protein drug prices remain high, partly due to manufacturing costs. Further increases in cell growth are needed to reduce costs and produce more protein, making drug products affordable for more patients.

[0003] Multiple cell types have been used in the biopharmaceutical industry. Chinese hamster ovary (CHO) cells are the most common cell type used for the production of therapeutic proteins. These proteins can include enzymes, growth factors, cytokines, hormones, insulin, and antibodies. Recently, variants of these proteins, including antibody-derived proteins and protein subunits, have also been developed. Immunoglobulin G (IgG) is a class of antibodies commonly used as a platform for the development of biotherapeutic agents. IgG1 is a subclass of IgG antibodies.

[0004] Other cell types used in the biopharmaceutical field include human endothelial kidney (HEK) cells and Vero cells, which are used to manufacture vaccines and gene therapies.

[0005] As the industry further develops, other animal-derived cell types are being considered for adaptation or direct application in suspension culture. Suspension culture is desirable for cell growth because it allows for more efficient space utilization compared to two-dimensional growth on a flat surface, and reduces contamination points, feeding and maintenance time, and variability due to operator differences.

[0006] In conventional suspension growth, cells are mass-produced in a bioreactor, where proteins, genes, or vaccines are generated by modifying the cells. In some cases, cells, cell aggregates, or cell parts such as extracellular vesicles are the intended products. In all cases, a high density of cells in the reactor is desirable.

[0007] Generally, all animal cells require highly specialized, customized, and complex culture media for optimal growth. Historically, serum has been a component of cell culture media to help improve cell growth. However, due to regulatory, quality, safety, and ethical concerns, there is a growing need to utilize culture media that lack serum and any animal-derived materials.

[0008] As regulators begin to require fully characterized cell culture media, it is also necessary to remove animal and human-derived components, including serum, from cell culture media. Serum traditionally helps stabilize cells from shear stress, including the effects of containers, other cells, and air bubbles. In addition to being animal-derived, serum is known in the field to tend to be variable and difficult to define precisely.

[0009] However, removing serum from the culture medium can pose a challenge to cell proliferation, potentially reducing cell growth and protein titers.

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

[0011] Poloxamer 188 is a polyether block copolymer surfactant. Polyether surfactants are formed by the 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 to be mild, water-soluble surfactants.

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

[0013] Poloxamer 188 is well-known and quite common in the field of suspension cell culture for Chinese hamster ovary cells. In suspension cell culture, poloxamer 188 has been used both in the presence and absence of serum. While effective in maintaining and improving cell growth rates, poloxamer 188 presents purity challenges, batch-to-batch variability, and potential problems requiring removal during downstream processing. A reference mentioning many issues concerning poloxamer 188 is "Development of Small Scale Cell Culture Models for Screening Poloxamer 188 Lot-to-Lot Variation" by Peng, H., Hall, KM, Clayton, B., Wiltberger, K., Hu, W., Hughes, E., Kane, J., Ney, R., & Ryll, T., Biotechnology Progress, 30, 1411-1418, 2014.

[0014] Methylcellulose is used in the food industry as a substitute for poloxamer, where poloxamer is not used due to regulatory restrictions. Understanding of methylcellulose for use in cell culture dates back to the 1960s (see Bryant, JC, “Methylcellulose Effect on Cell Proliferation and Glucose Utilization in Chemically Defined Medium in Large Stationary Cultures”, Biotechnology and Bioengineering, XI, 155-179, 1969.), and its use in suspension culture dates back to the early 1990s (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.). The use of methylcellulose alone as a substitute for poloxamer in suspension cell culture is disclosed in WO2021248141. Despite this understanding, the adoption of methylcellulose in the pharmaceutical industry is not believed to have occurred due to its inferior performance compared to poloxamer.

[0015] There remains a need for improved alternatives to serum and poloxamer, as well as for improved cell growth. Summary of the Invention

[0016] Cell culture media for suspension growth of cells contain a cellulose derivative having dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually replaced by methyl or hydroxypropyl groups, such that s23 / s26 is 0.16 to 0.36, wherein s23 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are replaced by methyl or hydroxypropyl groups, and wherein s26 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are replaced by methyl or hydroxypropyl groups.

[0017] A method for increasing cell growth includes: providing a cell culture medium containing a cellulose derivative having dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with methyl or hydroxypropyl groups, such that s23 / s26 is 0.16 to 0.36, wherein s23 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with methyl or hydroxypropyl groups, and wherein s26 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with methyl or hydroxypropyl groups; combining cells with the cell culture medium; and incubating the cells and culture medium to allow growth. Detailed Implementation

[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 construction or interpretation of the claims.

[0019] A method for suspending cells for growth is disclosed, which includes adding a cellulose derivative having a specific defined pattern of chemical substitution to a cell culture medium.

[0020] In one instance, the cell culture medium did not include serum.

[0021] The key concepts in the instruction manual are discussed below.

[0022] abbreviation. As stated in this specification, "MC" is an abbreviation for conventional methylcellulose, and "HPMC" is an abbreviation for conventional hydroxypropyl methylcellulose.

[0023] Defoamer. In another example, the cell culture medium optionally includes an antifoaming agent. The antifoaming agent is optionally a simethicone antifoaming agent.

[0024] Polyether surfactants. The use of polyether surfactants is optional. Exemplary polyether surfactants for use in cell culture media include poloxamer 124, poloxamer 188, poloxamer 338, and poloxamer 407.

[0025] Cellulose derivatives. Exemplary cellulose derivatives for use in cell culture media in this disclosure include methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethyl methylcellulose, and any combination thereof. Other cellulose derivatives include SG versions of methylcellulose, hydroxypropyl methylcellulose, and any combination thereof.

[0026] In one instance, the hydroxyl group of a cellulose derivative (including SG cellulose derivatives) may include an alkyl substituent or a hydroxyalkyl substituent or a combination thereof.

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

[0028] concentration. In one instance, the concentration of the cellulose derivative is less than 5%, with an optimal range of 0.01% to 4%. In another instance, the concentration of the cellulose derivative is less than 3%, with an optimal range of 0.02% to 1%.

[0029] Cells that can use the disclosed methods can be used. The disclosed methods can be used with any one or a combination 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 suitable for the disclosed methods can be used.

[0030] The following text describes methylcellulose and hydroxypropyl methylcellulose in more detail.

[0031] Methylcellulose and hydroxypropyl methylcellulose Methylcellulose is a cellulose ether formed through the methylation of cellulose. Cellulose is a naturally occurring polysaccharide produced by many plants, including trees and cotton. Polysaccharide polymers consist of dehydrated glucose units linked by β-1,4 bonds.

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

[0033] Cellulose derivatives are well-known across the pharmaceutical and food industries. Cellulose derivatives are defined in several ways. The United States Pharmacopeia (USP) defines them by their chemical derivatization and molecular weight. They can also optionally be further defined by a chemical derivatization pattern around the dehydrated glucose unit, such as by the s23 / s26 ratio.

[0034] Chemical derivatizing agents. The reaction of cellulose with derivatizing agents yields cellulose derivatives. Derivatizing agents include etherifying agents. For example, etherifying agents include methylating agents.

[0035] Cellulose ethers are examples of cellulose derivatives formed by the reaction of cellulose with an etherifying agent. For example, cellulose fibers are treated with an alkaline solution, followed by treatment with an etherifying agent such as chloromethane, to obtain cellulose ethers, methylcellulose, a cellulose derivative.

[0036] If a cellulose ether is substituted with hydroxypropyl and methyl groups, such a cellulose ether is called hydroxypropyl methylcellulose or hydroxypropyl methylcellulose (HPMC), an example of a cellulose derivative.

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

[0038] For example, methylcellulose is defined by the USP as having 26% and no more than 33% of its hydroxyl groups substituted with methylation.

[0039] Hydroxypropyl methylcellulose (HMCMC) is defined by the USP as having four different substitution types. The E chemistry is defined as follows: Type 2910 or E-type substitution has 28-30% methoxy group and 7-12% hydroxypropyl group substitution. The K chemistry is defined as follows: Type 2208 or K-type substitution has 19-24% methoxy group and 4-12% hydroxypropyl group substitution. The F chemistry is defined as follows: Type 2906 or F-type substitution has 27-30% methoxy group and 4-7.5% hydroxypropyl group substitution. The J chemistry is defined as follows: Type 1828 or J-type substitution has 16.5-20% methoxy group and 23-32% hydroxypropyl group substitution.

[0040] Molecular weight. The molecular weight of cellulose derivatives is typically described by the approximate viscosity of an aqueous solution of the polymer. These solutions are typically 2% by weight. For example, a 15 cP methylcellulose polymer is a methylcellulose in which a 2% aqueous solution of methylcellulose has a viscosity of approximately 15 cP when measured at 20 degrees Celsius. The methods used to determine the viscosity of this solution are well known to those skilled in the art and are defined by the United States Pharmacopeia, Chapter 912.

[0041] Chemical substitution patterns can be defined by the s23 / s26 ratio. Cellulose ether dehydrated glucose units may have more than one hydroxyl group substituted by a derivative. A chemical substitution pattern can be defined by the s23 / s26 ratio, where s23 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose unit are substituted, and where s26 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose unit are substituted. Further definitions of substitution patterns can be found in EP1171471, WO20000 / 59947, US6235893, and US6228416.

[0042] The ratio of hydroxyl positions on the 1,4-hydroglucose ring to their substitutions is not defined in the USP or other international pharmacopoeias.

[0043] Conventional methylcellulose and conventional hydroxypropyl methylcellulose. Most commercially available methylcellulose and hydroxypropyl methylcellulose have an s²⁃ / s²⁶ ratio of 0.37 to 0.42. These polymers are referred to as conventional methylcellulose or conventional hydroxypropyl methylcellulose.

[0044] The substitution modes of the present invention are defined as follows: In one example, the cellulose derivative is methylcellulose. Methylcellulose has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are replaced by methyl groups, such that s23 / s26 is 0.16 to 0.36, where s23 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are replaced by methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are replaced by methyl groups. The foregoing is an example of unconventional methylcellulose.

[0045] In another example, the cellulose derivative is methylcellulose. Methylcellulose has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are replaced by methyl groups, such that the s23 / s26 ratio is 0.26 to 0.32, where s23 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are replaced by methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are replaced by methyl groups. These polymers are called SG methylcellulose (“SG-MC”), an example of unconventional methylcellulose. Methylcellulose with an s23 / s26 ratio in the range of 0.23 to 0.32 is called SG methylcellulose.

[0046] In another example, the cellulose derivative is methylcellulose. Methylcellulose has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are replaced by methyl groups, such that the s23 / s26 ratio is 0.16 to 0.25, where s23 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are replaced by methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are replaced by methyl groups. These polymers are called LTG methylcellulose (“LTG-MC”), an example of unconventional methylcellulose. Methylcellulose with an s23 / s26 ratio in the range of 0.16 to 0.25 is called LTG methylcellulose.

[0047] In one instance, the favorable s23 / 26 is 0.23 to 0.32.

[0048] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is hydroxypropyl methylcellulose (HPMC). HPMC has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with hydroxypropyl or methyl groups, such that the s23 / s26 ratio is 0.16 to 0.36, where s23 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups. The foregoing is an example of unconventional HPMC. This unconventional HPMC includes both hydroxypropyl and methyl groups in its overall chemical structure.

[0049] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is hydroxypropyl methylcellulose. Hydroxypropyl methylcellulose has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with hydroxypropyl or methyl groups, such that the s23 / s26 ratio is 0.26 to 0.32, where s23 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups. These polymers are called SG hydroxypropyl methylcellulose (“SG-HPMC”), an example of unconventional HPMC. This unconventional SG-HPMC includes both hydroxypropyl and methyl groups in its overall chemical structure.

[0050] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is hydroxypropyl methylcellulose. Hydroxypropyl methylcellulose has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with hydroxypropyl or methyl groups, such that the s23 / s26 ratio is 0.16 to 0.25, where s23 is the molar fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups, and where s26 is the molar fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups. These polymers are called LTG hydroxypropyl methylcellulose (“LTG-HPMC”), an example of unconventional HPMC. This unconventional LTG-HPMC includes both hydroxypropyl and methyl groups in its overall chemical structure.

[0051] Unconventional cellulose derivatives. The aforementioned SG-MC, LTG-MC, SG-HPMC, and LTG-HPMC can also be referred to as "unconventional cellulose derivatives".

[0052] For example, exemplary SG methylcellulose and SG HPMC are manufactured as described in EP1171471, WO20000 / 59947, US6235893 and US6228416.

[0053] As used in the instructions, "conventional methylcellulose" has the same meaning as "conventional substituted methylcellulose".

[0054] Example 1 Compared to conventional methylcellulose and the additive-free control, SG methylcellulose resulted in a higher viable cell density. A proprietary CHO DG-44 cell line expressing IgG1 antibodies (referred to as "the Cells") was adapted and grown in a non-optimized medium (Hycell™, available from Cytiva). The adaptation and accompanying growth process is known to those skilled in the art. The Cells were engineered to grow optimally in PowerCHO2™ medium from Lonza, but because PowerCHO2™ medium contains poloxamer 188, the Cells were adapted to Hycell™ medium through sequential growth in a medium containing more Hycell™ and less PowerCHO2™ until the Cells grew in Hycell-only medium and therefore poloxamer 188 was absent. Therefore, PowerCHO2™ medium was not used for Example 1. The use of Hycell™ medium allows those skilled in the art to perform appropriate scientific controls to understand the growth effects of adding poloxamer or methylcellulose or both to the medium, although at the expense of optimal cell growth. Hycell™ medium has a viscosity of 2.23 cP when measured at 37°C.

[0055] Cells were grown in an ambr15 parallel 24-cell bioreactor system (Sartorius). Further details of the system are available at https: / / www.sartorius.com / en / products / fermentation-bioreactors / ambr-multi-parallel-bioreactors / ambr-15-cell-culture.

[0056] The ambr15 system continuously monitors the sample and adjusts parameters such as dissolved oxygen (DO) and feed conditions. The working volume is 14 mL, and cell culture is maintained for 14 days.

[0057] The samples were stirred by a stirring shaft in the reactor. In addition to the basal culture medium, custom-ordered Cytiva HyClone™ Cellboost™ 7a and 7b supplements, free of poloxamer 188, were used as supplemental feed to achieve a glucose concentration of 6 g / L.

[0058] Add Gibco Foam Away daily ® Irradiated AOF (animal-free) antifoaming agent to prevent foaming in the system. At the start of culture, add 0.3 x 10⁻⁶ ppm. 6 Cells were seeded at 100 cells / mL into each reactor and harvested after 14 days or after cell viability dropped to below 70% (whichever was earlier).

[0059] Live cell density was measured throughout the 14-day experiment. ® ViaCount™ reagents, along with flow cytometry and the Vi-Cell™ BLU cell viability analyzer (Beckman Coulter), are used to measure live cell density.

[0060] ViaCount™ works by differential nuclear staining of live and dead cells. The Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue rejection.

[0061] Peak viable cell density is the highest viable cell density measured during the fourteen-day culture period.

[0062] The experiment was run in duplicate for sample 1. The experiment was repeated four times for samples 2 and 3. The results are reported as the mean of the replicates for each sample.

[0063] Protein titer was measured by protein A. As is known in the art, protein A chromatography is used to measure IgG levels. Protein A-functionalized beads bind IgG, allowing IgG to be separated. After IgG is eluted from the beads, the protein content is measured to indicate IgG levels. Protein A chromatography using an Agilent Bio-Monolith Protein A affinity column and an HPLC system equipped with UV detection were used in this embodiment to measure protein titers.

[0064] The table below shows the differences in mean viable cell density and mean protein titer for the following: (1) the sample without methylcellulose (Sample 1), (2) the sample containing conventionally substituted methylcellulose (Sample 2), and (3) the sample containing SG methylcellulose (Sample 3). When measured as a 2% by weight aqueous solution at 20°C, the viscosity of the two methylcelluloses used was not significantly different.

[0065] Table 1 The change in live cell density (VCD) over the number of culture days was measured.

[0066] When compared with Sample 1 (control), Sample 2 (a culture medium containing 0.2% conventionally substituted methylcellulose 2cP) had an average peak viable cell density of approximately 5.9 times that of the control or approximately 592% of the control.

[0067] When compared with Sample 1 (control), Sample 3 (a culture containing 0.2% SG-substituted methylcellulose 2 cP, manufactured as described in EP1171471, WO20000 / 59947, US6235893 and US6228416 to obtain different polymer properties) showed a 9.4-fold increase in mean peak viable cell density, or approximately 942% of the control.

[0068] Peak viable cell density was higher when using SG MC than with conventional MC. When comparing the average peak viable cell density of Sample 2 and Sample 3, the peak viable cell density of Sample 3 increased by approximately 59% or 159% of the average peak viable cell density of Sample 2.

[0069] When compared with Sample 1 (control), the average IgG protein titer of Sample 2 (a culture medium containing 0.2% conventionally substituted methylcellulose) increased by approximately 12.6 times or approximately 1258% of the control.

[0070] When compared with Sample 1 (control), the average IgG protein titer of Sample 3 (a culture containing 0.2% SG-substituted methylcellulose, manufactured as described in EP1171471, WO20000 / 59947, US6235893, US6228416 to obtain different polymer properties) increased by 15.1 times or 15.14% of the control.

[0071] The average protein titer was higher when using SG MC than when using regular MC. When comparing the mean IgG protein titers of Sample 2 and Sample 3, the mean IgG protein titer of Sample 3 increased by approximately 20.3% or 120% of the mean peak viable cell density of Sample 2. Therefore, SG methylcellulose resulted in an increased protein titer compared to conventional methylcellulose.

[0072] The viscosity of the culture medium prepared with 0.2% conventional MC or SG MC remains unchanged at the incubation temperature. Therefore, the increase in cell density and protein titer observed with SG MC is not due to a change in the viscosity of the culture medium at the incubation temperature. Without being bound by theory, those skilled in the art would have expected that Sample 2 and Sample 3 would have comparable viable cell densities and protein IgG titers. This is because the two polymers have the same chemical derivatization and molecular weight, and the culture medium containing the polymers of Sample 2 and Sample 3 exhibits the same viscosity at the incubation temperature. Therefore, the aforementioned results are surprising and unexpected, and will be of interest to the industry.

[0073] Example 2 The use of SG methylcellulose increased the live cell density in high-shear shake flasks. Agarabi CRL3440 CHO (ATCC) cells were cultured in baffled shake flasks that were rotated on a shaker at 160 rpm. The presence of the baffles and the high rotational speed generated high shear forces, which could be damaging to the cells.

[0074] The culture medium was Hycell™, available from Cytiva, which was poloxamer-free. The incubator was maintained at 37°C and 8% CO2. The culture volume was 15 mL. At t=0, 3 x 10⁻⁶ cells / mL were added. 5 Cells were seeded at 10 cells / mL into flasks. Cells were cultured without additives, or with the addition of 0.2% methylcellulose (regular or SG grade), and / or with the addition of Gibco Foam Away® Irradiated AOF (animal-free) antifoamer (also known as “Foam Away™”) (10 uL / day). Cell counts were measured after 1 and 2 days of culture. The regular and SG methylcellulose were the same as described for samples 2 and 3.

[0075] The results are shown in the table below.

[0076] Table 2 Results of Day 1 When comparing cell / ml counts from Sample 5 and Sample 4 from Day 1, the cell / ml count from the conventional methylcellulose sample from Day 1 was 32 times that of the control (i.e., approximately 3235% of the control).

[0077] When comparing cell / ml counts from Sample 6 on Day 1 with Sample 4, the cell / ml counts of the conventional methylcellulose and Foam Away™ on Day 1 were approximately 31 times that of the control (i.e., approximately 3100% of the control).

[0078] When comparing the cell / ml counts from Sample 7 on Day 1 with Sample 4, the cell / ml count from SG MC (i.e., LV MC) on Day 1 was approximately 40.4 times that of the control (i.e., approximately 4043% of the control).

[0079] When comparing cell / ml counts from Sample 8 on Day 1 with Sample 4, the cell / ml counts of SG MC and Foam Away™ were approximately 37 times that of the control (i.e., approximately 3700% of the control).

[0080] For day 1, comparing samples 5 and 7, the use of SG MC resulted in a 25% improvement in mean viable cell density compared to the use of regular MC when Foam Away™ was not present.

[0081] For day 1, comparing samples 6 and 8, the use of SGMC resulted in a 21% improvement in mean viable cell density when Foam Away™ was present, compared to the use of conventional MC.

[0082] Results on Day 2 When comparing the cell / ml counts from Sample 5 on Day 2 with Sample 4, the cell / ml count from the routine methylcellulose on Day 2 was 197 times that of the control (i.e., approximately 19,745% of the control).

[0083] When comparing cell / ml counts from Sample 6 on Day 2 with Sample 4, the cell / ml counts of the conventional methylcellulose and Foam Away™ on Day 2 were approximately 155 times that of the control (i.e., approximately 15541% of the control).

[0084] When comparing the cell / ml counts from Sample 7 on Day 2 with that from Sample 4, the cell / ml count from SG MC on Day 2 was approximately 257 times that of the control (i.e., approximately 25,700% of the control).

[0085] When comparing cell / ml counts from Sample 8 on Day 2 with Sample 4, the cell / ml counts of SG MC and Foam Away™ were approximately 182 times that of the control (i.e., approximately 18,200% of the control).

[0086] For day 2, comparing samples 5 and 7, the use of SGMC resulted in a 30% improvement in mean viable cell density compared to the use of conventional MC when Foam Away™ was not present.

[0087] For day 2, comparing samples 6 and 8, the use of SG MC resulted in a 17% improvement in mean viable cell density when Foam Away™ was present, compared to the use of regular MC.

[0088] As can be seen from the above results, surprisingly and unexpectedly, the use of SG grade methylcellulose resulted in an increase in live cell density compared to the use of conventional methylcellulose.

[0089] Example 3 Higher molecular weight methylcellulose increases viable cell density and protein titer. This experiment was set up as in Example 1, but different polysaccharide polymers were added to the cell culture medium. Conventional methylcellulose polymers with different molecular weights were used. As with other methylcelluloses, the molecular weight was not measured directly, but rather expressed as the viscosity of a 2% solution at 20°C.

[0090] The polymers used and the results are described in Table 3.

[0091] In this embodiment, a protein A chromatography system using an Agilent Bio-Monolith Protein A affinity column and an HPLC system equipped with UV detection was used to measure protein titers.

[0092] The table below shows the samples tested in this embodiment and their corresponding peak viable cell density and protein titer.

[0093] Table 3 The results were compared with those of sample 2 in Example 1.

[0094] When comparing the viscosity of the culture medium in samples 2, 9, and 10, a slight increase in viscosity was observed as the molecular weight of methylcellulose increased.

[0095] When comparing the viscosity of the culture medium in samples 10 and 11, the viscosity is comparable and similar.

[0096] When comparing the peak viable cell density of Sample 2 (MC – 2 cP) and Sample 9 (MC – 338 cP) of Example 1, the peak viable cell density was 23.7% higher in Sample 9.

[0097] When comparing the peak viable cell density of Sample 2 (MC – 2 cP) of Example 1 with that of Sample 10 (MC – 2880 cP), the peak viable cell density in Sample 10 was 56.6% higher.

[0098] When comparing the protein IgG titers of sample 2 (conventional methylcellulose-2 cP, 0.2%) with those of sample 9 (MC-338 cP, 0.2%), the protein IgG titer in sample 9 was 9.2% higher.

[0099] When comparing the protein IgG titers of Sample 2 (conventional methylcellulose-2 cP, 0.2%) with those of Sample 10 (MC-2880 cP, 0.2%), the protein IgG titer in Sample 10 was 16.6% higher.

[0100] A comparison of the peak viable cell density of Sample 2 (conventional methylcellulose-2 cP, 0.2%) with that of Sample 11 showed an 8.2% increase in peak viable cell density for Sample 11, and a 2.4% MC compared to 0.2% MC in Sample 2.

[0101] A comparison of the protein IgG titers of Sample 2 and Sample 11 showed a 22.7% reduction in the protein IgG titer of Sample 11, with a 2.4% MC compared to 0.2% in Sample 2.

[0102] A comparison of peak viable cell density of Sample 10 (MC – 2880 cP, 0.2%) and Sample 11 (MC – 2 cP, 2.4%) showed that the peak viable cell density of Sample 10 was increased by 44.7% compared to Sample 11.

[0103] A comparison of protein IgG titers between Sample 10 (MC – 2880 cP, 0.2%) and Sample 11 (MC – 2 cP, 2.4%) showed that Sample 10 had a 49.1% increase in peak viable cell density compared to Sample 11.

[0104] The data in this embodiment show that higher molecular weight methylcellulose can improve live cell density and protein titer.

[0105] The data in this embodiment also show that the effectiveness of higher molecular weight methylcellulose in promoting greater peak viable cell density is not solely due to increased culture medium viscosity. For example, although both samples had similar viscosities, sample 11 (MC – 2 cP, 2.4%) was less effective than sample 10 (MC – 2880 cP, 0.2%) in promoting greater peak viable cell density.

[0106] The following are examples illustrating the effects of using poloxamer and HPMC or MC on cell growth.

[0107] Example 4 Compared to Px188 alone, the combination of Px188 with HPMC or MC yielded surprising and unexpected results in increasing mean viable cell density. The growth of the CHO DG-44 cell line using baffle-free cell culture shake flasks and PowerCHO2™ medium is described below.

[0108] Shake flasks are frequently used in the early stages of cell growth processes and in laboratory studies, making them industrially relevant and important. In addition to using standard methylcellulose and poloxamer 188, experiments were conducted involving the addition of a combination of standard hydroxypropyl methylcellulose (HPMC) and poloxamer 188.

[0109] The proprietary CHO DG-44 cell line expressing IgG1 antibody was grown in baffle-free 125 mL cell culture shake flasks with a working volume of 30 mL. Samples were agitated by rotating the flasks on a shaking plate. Oxygen and carbon dioxide levels were controlled by an incubator within which the shake flasks were placed, and there was no feedback loop. Baffles are generally not used in CHO cultures because their use imposes additional shear forces on the cells.

[0110] The cell culture medium was PowerCHO2™, available from Lonza, supplied with 0.1% poloxamer 188 as a component. This cell line had been optimized for growth in PowerCHO2™ medium. Working medium was prepared by combining 1000 mL of PowerCHO2™ medium, 20 mL of 200 mM glutamine, and 10 mL of anti-caking agent (Gibco™ product, catalog number 01-0057DG).

[0111] As indicated in Table 4 below, add another polymer, poloxamer 188 (Px188), methylcellulose (MC), or hydroxypropyl methylcellulose (HPMC) to the culture medium.

[0112] Methylcellulose is grade 15 cP.

[0113] Two conventional HPMC polymers were tested: HPMC with K chemistry and a viscosity of 3 cP as a 2% aqueous solution at 20°C, namely, "3cP HPMC-K", and HPMC with E chemistry and a viscosity of 5 cP as a 2% aqueous solution at 20°C, namely, "5cP HPMC-E". An exemplary 3cP HPMC-K is METHOCEL™ K3 Premium LV. An exemplary 5cP HPMC-E is METHOCEL™ E5 Premium LV.

[0114] Cytiva HyClone™ Cellboost™ 7a and 7b supplements containing Poloxamer 188 (0.1%) are used as supplements.

[0115] The cells were cultured for 14 days. Except for sample 7, which was tested once, all samples were tested in triplicate.

[0116] At the start of cultivation, add 0.3 x 10 6 10 cells / mL were seeded into each shake flask, and cells were harvested after 14 days or after cell viability dropped to below 70% (whichever was later). Starting from day 3 of culture, pH, glucose, ammonium, lactate, pCO2, and cell density were monitored daily.

[0117] Guava ® ViaCount™ reagents, along with flow cytometry and the Vi-Cell™ BLU cell viability analyzer (Beckman Coulter), are used to measure live cell density.

[0118] All other parameters were measured on the Beckman Coulter Vi-CELL MetaFLEX™ Bioanalyte Analyzer.

[0119] ViaCount™ works by differential nuclear staining of live and dead cells. The Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue rejection.

[0120] Peak viable cell density is the highest viable cell density measured during the fourteen-day culture period.

[0121] The following table compares the average viable cell density for individual Px188 (as included in the culture medium), additional Px188 for the initial Px188 value, and MC or different grades of HPMC values.

[0122] Table 4 The following section describes the analysis of the values ​​obtained from the table above.

[0123] Adding more poloxamer did not result in a significant increase in live cell density. When comparing cell culture media of sample 12 with 0.1% Px188 to cell culture media of sample 13 with a final concentration of 0.3% Px188, the addition of additional Px188 resulted in a slight 1.7% increase in peak viable cell density (VCD) for sample 13. Therefore, the addition of additional Px188 alone did not result in a significant increase in peak viable cell density (VCD).

[0124] Compared to Px188 alone, the combination of Px188 with HPMC grade or MC showed a surprising and significant improvement in live cell density. An unexpected result.However, comparisons were made between the viable cell density values ​​of 0.1% Px188 alone in Sample 12 and the results for: (1) the combination of 0.2% 3cP HPMC-K and 0.1% Px188 in Sample 14; (2) the combination of 0.2% 5cP HPMC-E and 0.1% Px188 in Sample 15; (3) the combination of 0.2% MC and 0.1% Px188 in Sample 16; and (4) the combination of 0.5% 3cP HPMC-K and 0.1% Px188 in Sample 17. These combinations showed a significant increase in peak viable cell density compared to Px188 alone. The increase in viable cell density ranged from 42.1% to 58.3% compared to 0.1% Px188 alone. The foregoing results are surprising and unexpected, as a significant increase in viable cell density was observed by combining the cellulose derivative with poloxamer 188, as shown in samples 14-17 respectively, compared to adding poloxamer 188 to sample 13. These results are unexpected because it is known in the art that Px188 is more effective than the cellulose derivative in increasing peak viable cell density.

[0125] Based on these preliminary results from Table 4, it is expected that using poloxamer 188 in combination with unconventional MC and HPMC polymers will result in higher cell growth than poloxamer alone or any of the aforementioned polymers.

[0126] Example 5 The protective effect of SG-MC alone or in combination with Px188 Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. As shown in Table 5, conventional MC or SG methylcellulose (SG-MC), poloxamer 188 (Px188), or a combination of such conventional MC or SG-MC and Px188 with a viscosity of 2 cP were added to the medium. Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) (10 μL / day) was added daily to prevent system foaming. At the start of culture, cells were introduced at a rate of 3 x 102 5Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. The seed chain cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 5 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using conventional trypan blue counting with a Countess™ 3 FL Automated CellCounter.

[0127] Table 5 below shows the VCD observed for the test samples.

[0128] Table 5 In Table 5, the percentages are based on the weight percentage of the total cell culture.

[0129] As shown in Table 5, 0.20% Px188 alone provides more protection than 0.20% conventional MC alone or 0.20% SG-MC alone. However, the protection provided by Px188 alone can be achieved by replacing a portion (here, 50%) of Px188 with conventional MC or SG-MC.

[0130] Example 6 The protective effect of the combination of LTG-MC or LTG-MC with Px188 This embodiment evaluates the protective effects of LTG-MC alone and LTG-MC in combination with poloxamer 188 (Px188).

[0131] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. As shown in Table 6, LTG methylcellulose (LTG-MC), Px188, or a combination of LTG-MC and Px188 with a viscosity of 2 cP were added to the medium. Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (ThermoFisher Scientific) (10 μL / day) was added daily to prevent system foaming. At the start of culture, cells were introduced at a rate of 3 x 102 5Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. The seeded cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 6 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0132] Table 6 below shows the VCD observed for the test samples.

[0133] Table 6 In Table 6, the percentages are based on the weight percentage of the total cell culture.

[0134] As shown in Table 6, LTG-MC provides more protection than Px188. Furthermore, the combination of LTG-MC and Px188 provides unexpected protection, exceeding the combined protection expected from LTG-MC and Px188 alone.

[0135] Example 7 The protective effect of SG-MC alone against cell damage caused by antifoaming agents. Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. As shown in Table 7, standard methylcellulose (2 cP viscosity), SG methylcellulose (SG-MC) (2 cP viscosity), and / or Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) were added. At the start of culture, cells were introduced at a rate of 3 x 10⁶ cells / mL. 5 Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. The seed chain cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 7 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0136] Table 7 below shows the VCD observed for the test samples.

[0137] Table 7 In Table 7, the percentages are based on the weight percentage of the total cell culture.

[0138] As shown in Table 7, both conventional MC and SG-MC provide protection against defoamer damage, with SG-MC providing slightly more protection than conventional MC.

[0139] Example 8 The protective effect of individual polymer additives against cell damage in defoamers In this embodiment, the protective effects of conventional methylcellulose (MC), SG-MC and LTG-MC alone were compared.

[0140] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. MC (15 cP viscosity), MC (2 cP viscosity), SG-MC (2 cP viscosity), or LTG-MC (2 cP viscosity) were added as shown in Table 8. Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) (10 μL / day) was added daily to prevent system foaming. At the start of culture, cells were introduced at a rate of 3 x 102 5 Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. The seed chain cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 8 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0141] Table 8 below shows the VCD observed for the test samples.

[0142] Table 8 In Table 8, the percentages are based on the weight percentage of the total cell culture.

[0143] As shown in Table 8, all three types of protective mechanisms—conventional MC, SG-MC, and LTG-MC—provide protection, with LTG-MC offering the greatest protection and SG providing even greater protection than conventional MC.

[0144] The numerical ranges listed in the specification include values ​​that are considered equivalent to the listed values ​​(e.g., having the same function or result) by those skilled in the art, such as + / - 5-10% of the listed values.

[0145] The claims are not limited to the preferred embodiments and examples, but are intended to cover many modifications and equivalents consistent with the written description as a whole.

Claims

1. A cell culture medium comprising a cellulose derivative for suspension growth of cells, wherein the cellulose derivative has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with methyl or hydroxypropyl groups, such that s23 / s26 is 0.16 to 0.36, wherein s23 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with methyl or hydroxypropyl groups, and wherein s26 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with methyl and / or hydroxypropyl groups.

2. The cell culture medium according to claim 1, wherein s23 / s26 is 0.26 to 0.

32.

3. The cell culture medium according to claim 1, wherein s23 / s26 is 0.16 to 0.

25.

4. The cell culture medium according to any one of claims 1-3, wherein the cell culture medium further comprises an antifoaming agent.

5. The cell culture medium according to any one of claims 1-4, wherein the defoamer comprises simethicone.

6. The cell culture medium according to any one of claims 1-5, wherein the cell culture medium further comprises a polyether surfactant.

7. The cell culture medium according to any one of claims 1-6, wherein the polyether surfactant is poloxamer.

8. The cell culture medium according to any one of claims 1-7, wherein the cellulose derivative has a 2% aqueous solution viscosity of less than 10,000 cP at 20 degrees Celsius.

9. The cell culture medium according to any one of claims 1-8, wherein the cellulose derivative has a 2% aqueous solution viscosity of less than 2,000 cP at 20 degrees Celsius.

10. The cell culture medium according to any one of claims 1-9, wherein the cellulose derivative has a 2% aqueous solution viscosity of less than 100 cP at 20 degrees Celsius.

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

12. A method for suspending cells for growth, comprising incubating the cells in a cell culture medium according to any one of claims 1-11.

13. A method for increasing cell growth, comprising: Provide a cell culture medium containing the following: A cellulose derivative having dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with methyl or hydroxypropyl groups, such that s23 / s26 is 0.16 to 0.36, wherein s23 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with methyl or hydroxypropyl groups, and wherein s26 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with methyl or hydroxypropyl groups; Combine the cells with the cell culture medium; and The cells and culture medium are incubated to allow growth.

14. The method of claim 13, wherein providing the cell culture medium comprises providing a cellulose derivative having an s23 / s26 ratio of 0.26 to 0.

32.

15. The method of claim 13, wherein providing the cell culture medium comprises providing a cellulose derivative having an s23 / s26 ratio of 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 defoamer 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 derived from the Chinese hamster ovary cell line.

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

22. The method of claim 13, wherein the cells are derived from the Vero cell line.

23. The method of claim 13, wherein the cell produces proteins.

24. The method of claim 13, wherein the cells produce antibodies.

25. The method of claim 13, wherein the cells produce antibody-derived proteins.

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

27. The method of claim 13, wherein the cells generate gene therapy.

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