Effect of cellulose derivatives of different viscosity grades on cell growth

Abstract

Cell culture media and methods involving the use of higher viscosity cellulose derivatives to improve cell growth and protein titer levels are disclosed.

Description

Technical Field

[0001] The technical field involves the use of cellulose derivatives of different viscosity grades 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 very high, and this is passed on to very high drug prices. Process intensification is needed to increase cell growth output to help reduce the cost of producing these therapies. Cell engineering is known in the art to improve protein drug titers by obtaining more copies, reducing culture time, and minimizing proteolysis. Despite efforts in this area, protein drug prices remain high, partly due to manufacturing costs. Increased cell growth is still needed to reduce costs and produce more protein, making drug products 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 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, are also being produced. Immunoglobulin G (IgG) is a class of antibodies commonly used as a platform for the development of biotherapeutic drugs. 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 particularly used in the manufacture of vaccines and gene therapies.

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

[0006] In conventional suspension growth processes, cells are grown in large quantities in a bioreactor to produce proteins, genes, or vaccines that these cells are engineered to manufacture. In some cases, cells, cell aggregates, or portions of cells (e.g., extracellular vesicles) are the intended product. In all cases, a high density of cells in the reactor is required.

[0007] Typically, all animal cells require highly specialized, customized, and complex culture media for optimal growth. Historically, serum has been a component included in cell culture media to aid in improving cell growth. However, due to regulatory, quality, safety, and ethical considerations, the need for culture media that do not contain serum or any animal-derived materials is increasing.

[0008] As regulatory agencies begin to require fully characterized cell culture media, there is also a need to eliminate 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 art to tend to be volatile and difficult to define adequately.

[0009] However, removing serum from the culture medium can pose a challenge to cell proliferation and may reduce 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 continuous polymerization of ethylene oxide and propylene oxide. Different polyether surfactants have different block sizes and ratios, but they are generally considered in the art to be mild, water-soluble surfactants.

[0012] Pluronic 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). Its B block has a molecular weight of approximately 1800 Daltons, and the polymer contains approximately 80% by weight of the A block. Other poloxamer examples include poloxamer 124, poloxamer 338, and poloxamer 407, which are sold under various trade names, including Pluronic. ® Kolliphor ® and Synperonic TM .

[0013] For example, the use of poloxamer 188 in suspension cell culture of Chinese hamster ovary cells is well-known and quite common in the art. In suspension cell culture, poloxamer 188 is used in both 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 the potential to cause problems in downstream processing, necessitating its removal. A reference mentioning many issues with poloxamer 188 is “Development of Small Scale CellCulture 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, Vol. 30, pp. 1411-1418, 2014.

[0014] One alternative to poloxamer is methylcellulose. Methylcellulose is used as a substitute for poloxamer in the food industry where it is not used due to regulatory restrictions. Knowledge of the use of methylcellulose 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, Vol. XI, pp. 155-179, 1969), while knowledge of its use in suspension culture dates back to the early 1990s (Goldblum, S et al., “Protective Effect of Methylcellulose and Other Polymerson Insect Cells Subjected to Laminar Shear Stress”, Biotechnology Progress, Vol. 6, pp. 373-390, 1990). WO2021248141 discloses the use of methylcellulose alone as a substitute for poloxamer in suspension cell culture. Despite this knowledge, methylcellulose is believed to have not yet been adopted in the pharmaceutical industry because its performance is inferior to poloxamer 188.

[0015] It is noted in the art that lower viscosity grades of hydroxypropyl methylcellulose reduce surface tension more significantly compared to higher viscosity grades. Lower surface tension helps protect cells from damage caused by bubble rupture. See Chattopadhaya, D. et al., “The protective effect of Specific Medium Additives with Respect to Bubble Rupture,” Biotechnology and Bioengineering, Vol. 45, pp. 473-480, 1995. Therefore, those skilled in the art will consider that lower viscosity grades of methylcellulose would be preferred for use in suspension cell culture.

[0016] There is still a need to improve alternatives for cell growth in in vitro cell culture media. Summary of the Invention

[0017] A cell culture medium for suspension growth of cells comprising a cellulose derivative having a viscosity range of 20 cP to 8000 cP. A related method for increasing cell growth includes: providing a cell culture medium comprising a cellulose derivative having a viscosity range of 20 cP to 8000 cP; mixing cells with the cell culture medium; and incubating the cells and the culture medium to enable them to grow. 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 interpretation or description of the claims.

[0019] A process for suspension growth of cells is disclosed, which includes adding cellulose derivatives of different viscosity grades, such as methylcellulose or hydroxypropyl methylcellulose, to a cell culture medium.

[0020] The following describes some of the key concepts in this specification.

[0021] In one instance, the cell culture medium did not contain serum.

[0022] abbreviation As stated in this specification, "MC" is an abbreviation for conventional methylcellulose, "HPMC" is an abbreviation for conventional hydroxypropyl methylcellulose, and "Px188" is an abbreviation for "poloxam 188".

[0023] Defoamer In another example, the cell culture medium optionally contains an antifoaming agent. The antifoaming agent is optionally a dimethicone 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 used in the cell culture media of this disclosure include methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethyl methylcellulose, and any combination thereof.

[0026] In one instance, the hydroxyl groups of a cellulose derivative may include alkyl substituents or hydroxyalkyl substituents or combinations thereof.

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

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

[0029] concentration In one instance, the concentration of the polysaccharide 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%.

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

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

[0032] Cells using the disclosed process can be used. The disclosed process can be used in any 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 process can be used.

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

[0034] 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. This polysaccharide polymer contains dehydrated glucose units linked by β1-4 linkages.

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

[0036] Cellulose derivatives are well-known throughout the pharmaceutical and food industries. Cellulose derivatives are defined in several ways. They are defined by the United States Pharmacopeia (USP) based on their chemical derivatization and molecular weight. They can also optionally be further defined by the chemical derivatization pattern surrounding the dehydrated glucose unit, defined by the s23 / s26 ratio.

[0037] Chemical derivatizing agents. The reaction of cellulose with derivatizing agents produces cellulose derivatives. Derivatizing agents include etherifying agents. For example, etherifying agents include methylating agents.

[0038] 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 and then with an etherifying agent (such as chloromethane) to produce cellulose ethers, a type of methylcellulose, which is a cellulose derivative.

[0039] If a cellulose ether is substituted with hydroxypropyl and methyl groups, such a cellulose ether is called hydroxypropyl methylcellulose or hydroxypropyl methylcellulose (HPMC), which is an example of a cellulose derivative.

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

[0041] For example, methylcellulose is defined by the USP as having not less than 26% and not more than 33% of methylated hydroxyl groups substituted.

[0042] Hydroxypropyl methylcellulose (HMCMC) is defined by the USP as having four different substitution types. The E chemotype is defined as follows: Type 2910 or E-type substitution has 28-30% methoxy substitution and 7-12% hydroxypropyl substitution. The K chemotype is defined as follows: Type 2208 or K-type substitution has 19-24% methoxy substitution and 4-12% hydroxypropyl substitution. The F chemotype is defined as follows: Type 2906 or F-type substitution has 27-30% methoxy substitution and 4-7.5% hydroxypropyl substitution. The J chemotype is defined as follows: Type 1828 or J-type substitution has 16.5-20% methoxy substitution and 23-32% hydroxypropyl substitution.

[0043] molecular weight The molecular weight of cellulose derivatives is typically described by the approximate viscosity of the polymer in aqueous solution. These solutions are typically 2% by weight. For example, a 15 cP methylcellulose polymer is methylcellulose in which a 2% solution of methylcellulose in water has a viscosity of about 15 cP 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 in Chapter 912 of the United States Pharmacopeia.

[0044] Chemical derivatization patterns 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 the two hydroxyl groups at positions 2 and 3 of the dehydrated glucose unit are substituted, and where s26 is the mole fraction of dehydrated glucose units in which the two hydroxyl groups at positions 2 and 6 of the dehydrated glucose unit are substituted. Further definitions of substitution patterns can be found in EP1171471, WO20000 / 59947, US6235893, and US6228416.

[0045] The ratio of which hydroxyl positions on the 1,4-hydroglucose ring are substituted relative to each other is not defined in the USP or other international pharmacopoeias.

[0046] Most commercially available methylcellulose and hydroxypropyl methylcellulose have an s23 / s26 ratio of 0.37 to 0.42. These polymers are referred to as conventional methylcellulose or conventional hydroxypropyl methylcellulose.

[0047] The substitution pattern of the substituents on the cellulose constituting the cellulose derivative is optionally defined by the s23 / s26 ratio.

[0048] The replacement pattern can be optionally defined as follows: In one example, the cellulose derivative in which the s23 / s26 ratio is defined is methylcellulose. This methylcellulose has dehydrated glucose units linked by 1-4 linkages, wherein the hydroxyl groups of the dehydrated glucose units are substituted with 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 the two hydroxyl groups at positions 2 and 3 of the dehydrated glucose units are substituted with methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which the two hydroxyl groups at positions 2 and 6 of the dehydrated glucose units are substituted with methyl groups. This is an example of unconventional methylcellulose.

[0049] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is methylcellulose. This methylcellulose has dehydrated glucose units linked by 1-4 linkages, wherein the hydroxyl groups of the dehydrated glucose units are substituted with 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 positions 2 and 3 of the dehydrated glucose units are substituted with methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at positions 2 and 6 of the dehydrated glucose units are substituted with methyl groups. These polymers are called SG methylcellulose (“SG-MC”), which is an example of unconventional methylcellulose. Methylcellulose having an s23 / s26 ratio in the range of 0.23 to 0.32 is called SG methylcellulose.

[0050] In one instance, the favorable s23 / 26 is 0.23 to 0.32.

[0051] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is hydroxypropyl methylcellulose (HPMC). This HPMC has dehydrated glucose units linked by 1-4 linkages, wherein the hydroxyl groups of the dehydrated glucose units are 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 the two hydroxyl groups at positions 2 and 3 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 the two hydroxyl groups at positions 2 and 6 of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups. This is an example of unconventional HPMC. This unconventional HPMC contains both hydroxypropyl and methyl groups in its overall chemical structure.

[0052] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is hydroxypropyl methylcellulose. This hydroxypropyl methylcellulose has dehydrated glucose units linked by 1-4 linkages, wherein the hydroxyl groups of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups, such that the s23 / s26 ratio is 0.26 to 0.32, where s23 is the molar fraction of dehydrated glucose units in which only the two hydroxyl groups at positions 2 and 3 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 positions 2 and 6 of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups. These polymers are called SG hydroxypropyl methylcellulose (“SG-HPMC”), which is an example of unconventional HPMC. This unconventional SG-HPMC contains both hydroxypropyl and methyl groups in its overall chemical structure.

[0053] Unconventional cellulose derivatives. The above SG MC and SG HPMC can also be referred to as "unconventional cellulose derivatives".

[0054] Exemplary SG methylcellulose and SG HPMC are manufactured, for example, as described in EP1171471, WO20000 / 59947, US6235893 and US6228416.

[0055] Example 1 The higher viscosity of methylcellulose leads to an increase in mean peak viable cell density and mean protein titer levels. The proprietary CHO DG-44 cell line expressing IgG1 antibodies (referred to as "the cell") was adapted to an unoptimized culture medium (Hycell). TM The cells, obtained from Cytiva, are grown therein. The adaptation and accompanying growth process are known to those skilled in the art. The cells were engineered to grow in PowerCHO2 cells from Lonza. TM It grows best in the culture medium, but due to PowerCHO2 TM The culture medium contains poloxamer 188, and the cells are adapted to Hycell culture in the following ways. TM Culture medium: containing more Hycells TM And less PowerCHO2 TM Continuous growth in a culture medium until the cells are in Hycell-only culture medium. TM It grows in [the environment], and therefore poloxamer 188 is absent. Therefore, Example 1 did not use PowerCHO2. TM Culture medium. Although it comes at the cost of optimal cell growth, using Hycell... TM The culture medium provides a suitable scientific control for those skilled in the art to understand the effect of adding poloxamer or methylcellulose, or both, to the medium on growth. When measured at 37°C, Hycell... TM The culture medium has a viscosity of 2.23 cP.

[0056] 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.

[0057] 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.

[0058] The samples were agitated using a stirring shaft within the reactor. In addition to the basal culture medium, custom-ordered poloxamer 188-free Cytiva HyClone was used. TM Cellboost TM Supplements 7a and 7b were used as supplemental feed to achieve the target glucose concentration of 6 g / L.

[0059] Add Gibco Foam Away daily ® Irradiate AOF (animal-free) defoamer to prevent system foaming. At the start of culture, add 0.3 × 10 6 10 cells / mL were seeded into each reactor and the cells were harvested after 14 days or when the cell viability dropped to below 70% (whichever came first).

[0060] Live cell density was measured throughout the 14-day experiment. Guava was used. ® ViaCount TM Reagents, flow cytometer, and Vi-Cell TM The BLU cell viability analyzer (Beckman Coulter) is used to measure live cell density.

[0061] ViaCount TM It works by differentially staining live and dead cells. Vi-Cell TM The BLU Cell Viability Analyzer works by measuring trypan blue rejection.

[0062] Peak viable cell density is the highest viable cell density measured during fourteen days of culture.

[0063] Protein titers are measured using protein A. As is known in the art, protein A chromatography is used to measure IgG levels. Protein A functionalized beads bind IgG, thereby enabling the separation of IgG. The protein content, measured after eluting IgG from the beads, indicates the IgG level. In this embodiment, protein A chromatography using an Agilent Bio-Monolith protein A affinity column and an HPLC system equipped with UV detection is employed to measure protein titers.

[0064] Except for Sample 1, which was run in duplicate, the sample groups were run in quadruplicate. For each sample, peak viable cell density and protein titer were reported as the average of the replicates.

[0065] The table below shows the differences in mean viable cell density and mean protein titer between samples without methylcellulose, samples containing low-viscosity methylcellulose, samples containing medium-viscosity methylcellulose, and samples containing high-viscosity methylcellulose.

[0066] The molecular weight is not measured directly, but is expressed by the viscosity of a 2% solution in water at 20 degrees Celsius, which is typical for methylcellulose.

[0067] Table 1 A comparison of the average peak viable cell density and IgG protein titer between samples 2-5 and sample 1 indicates the need to add polymers to the cell culture medium to produce higher peak viable cell density and higher protein titer, both of which are desirable for those skilled in the art.

[0068] Compared to sample 1, the peak viable cell density of samples 2-5 with added polymer increased by 892-927%.

[0069] Compared to sample 1, the IgG protein titers of samples 2-5, to which polymers were added to the cell culture medium, increased by 972-1450%.

[0070] Comparison of the viscosity of the cell culture media used in samples 2, 3, and 4 revealed that viscosity increased slightly with increasing molecular weight of methylcellulose. For example, the viscosity of the culture medium in sample 4 (MC – 2880 cP, used medium 3.49 cP) was greater than that in sample 3 (MC – 338 cP, used medium 2.94 cP). The viscosity of the culture medium in sample 3 (MC – 338 cP, used medium 2.94 cP) was greater than that in sample 2 (MC – 2 cP, used medium 2.18 cP).

[0071] Comparing the viscosity of the cell culture media used in samples 4 and 5 revealed that, despite the use of polymers with different molecular weights, the viscosities of the two media were comparable. This was achieved by using a higher concentration (2.4%) of the low-viscosity MC-2cP in sample 5 to match the viscosity of the high-viscosity MC-2880 cP in sample 4 (0.2%).

[0072] The average peak viable cell density changes as the viscosity of methylcellulose changes from low viscosity to medium viscosity. Increase The mean peak viable cell density of sample 3, which has medium viscosity methylcellulose (0.2% concentration), is 23.7% higher than that of sample 2 (0.2%), which has low viscosity methylcellulose.

[0073] The average peak viable cell density increases as the viscosity of methylcellulose changes from low viscosity to high viscosity. Add even more .

[0074] The average peak viable cell density of high-viscosity methylcellulose (0.2% concentration) in Sample 4 was 56.6% higher than that of low-viscosity methylcellulose (0.2% concentration) in Sample 2.

[0075] The average peak viable cell density increased as the viscosity of the methylcellulose changed from medium viscosity to high viscosity. Even when comparing the average peak viable cell density values ​​of medium viscosity and high viscosity methylcellulose, the average peak viable cell density of sample 4, which had high viscosity methylcellulose, was 26.5% higher than that of sample 3, which had medium viscosity methylcellulose.

[0076] Even when the culture medium has the same viscosity, the average peak viable cell density is higher for higher viscosity methylcellulose compared to lower viscosity methylcellulose. The average peak viable cell density of high viscosity methylcellulose (0.2% concentration) in Sample 4 is 44.7% higher than that of low viscosity methylcellulose (2.4% concentration) in Sample 5.

[0077] Analysis. The above results indicate that a higher peak viable cell density was achieved using methylcellulose with higher viscosity, and that the increase in peak viable cell density was not due to an increase in the viscosity of the culture medium used.

[0078] The following discussion covers the protein titer levels achieved using methylcellulose of different viscosity grades.

[0079] The average IgG protein titer increased as the viscosity of methylcellulose changed from low-viscosity to medium-viscosity methylcellulose. Replacing 0.2% low-viscosity methylcellulose with 0.2% medium-viscosity methylcellulose resulted in a 9.2% increase in the average IgG protein titer.

[0080] When switching from low-viscosity methylcellulose to high-viscosity methylcellulose, the average IgG protein titer increases even more. More In the above embodiments, the use of high-viscosity methylcellulose instead of medium-viscosity methylcellulose resulted in even more impressive results. When 0.2% of low-viscosity methylcellulose was replaced with 0.2% of high-viscosity methylcellulose, the average IgG protein titer increased by 15.2%.

[0081] The ratio between the average IgG protein titers using high-viscosity methylcellulose and medium-viscosity methylcellulose The comparison showed an increase in average IgG protein titer. A comparison of the IgG protein titers of high-viscosity methylcellulose (0.2%) in Sample 4 with that in medium-viscosity methylcellulose in Sample 3 showed an increase of 5.5%.

[0082] Even when the culture medium has the same viscosity, the higher viscosity methylcellulose is superior to the lower viscosity methylcellulose. Vitamin B1 also had a higher average IgG protein titer.The average IgG protein titer of the high-viscosity methylcellulose medium (0.2% concentration) in Sample 4 was 49.2% higher than that of the low-viscosity methylcellulose medium (2.4% concentration) in Sample 5.

[0083] Although samples 4 and 5 had very similar culture medium viability, they did not have similar peak viable cell density or IgG protein titer. This indicates that the difference in viable cell density or IgG protein titer was not caused by the change in culture medium viscosity. Instead, as the tests on the samples above have shown, the difference in the viscosity of the methylcellulose used explains the difference in the obtained peak viable cell density and IgG protein titer values.

[0084] Using a higher viscosity grade of methylcellulose yielded surprising and unexpected results. The surprising and unexpected result of switching from low-viscosity methylcellulose (Sample 2) to medium-viscosity methylcellulose (Sample 3) or high-viscosity methylcellulose (Sample 4) was the improvement in average peak viable cell density and average IgG protein titer, as it is expected in the art that the use of higher viscosity methylcellulose would lead to decreased cell growth.

[0085] Example 2 There is a critical upper limit for higher molecular weight polymers used in cell culture media because some higher molecular weights are associated with viscosity buildup. The results shown in the table below indicate that certain higher molecular weight polymers are associated with viscosity buildup, and there is a critical upper limit to the use of higher molecular weight polymers in cell culture media. At a specific upper limit of viscosity for the polymer used, higher viscosity will generate greater shear forces during stirring, leading to detrimental effects on cells. Higher viscosity can also result in more persistent foaming in bioreactors, which is undesirable.

[0086] When a higher molecular weight polymer with a certain polymer viscosity is incorporated into the cell culture medium, the cell culture medium (“culture medium”) used in this embodiment will also produce a higher viscosity, as shown in the table below.

[0087] The viscosity of the culture medium in this embodiment was measured when higher molecular weight methylcellulose was incorporated into it. Prior to the addition of the polymer, the culture medium was the same Hycell medium as in Sample 1. TM Culture medium, and with Hycell for sample 1 TM The culture medium reported the same viscosity. Therefore, prior to the addition of the polymer, the culture medium of Example 2 had a viscosity of 2.23 cP when measured at 37°C.

[0088] After the addition of their respective polymers, the viscosity of the culture medium increased to 6.42 cP and 6.34 cP, as shown in Table 2.

[0089] Table 2 The viscosity of the culture medium in samples 6 and 7 was compared with that in samples 1-5. The viscosity of the culture medium in samples 6 and 7 was approximately 1.83 to 2.94 times that of the culture medium in samples 1-5. This difference in viscosity range of 1.83 to 2.94 was determined by comparing the highest viscosity of the culture medium in sample 6 with the lowest viscosity of the culture medium in (1) sample 2 and the highest viscosity of the culture medium in (2) sample 4, respectively.

[0090] Analysis. Higher viscosity of polymers at certain upper limits generates more shear forces during stirring, which can have a detrimental effect on cells. Higher viscosity can also lead to more persistent foaming in the reactor, which is undesirable. Therefore, based on the teachings of Table 2 above, those skilled in the art would not want to add polymers that establish significant viscosity to cell culture media. Thus, using methylcellulose with a viscosity of 10,000 cP and greater in cell culture media is undesirable.

[0091] Example 3 Similar to MC, SG MC is effective in promoting higher viable cell density and protein IgG titers compared to the additive-free control. This experiment was conducted similarly to that in Example 1, but SG methylcellulose, 2 cP (SG-MC – 2 cP) was used. Similar to samples 2-4 in Example 1, the aforementioned SG methylcellulose was added to HyCell at 0.2%. TM In the culture medium.

[0092] The control for this experiment was sample 1.

[0093] The results are shown in the table below.

[0094] Table 3 The following is an analysis of the above data.

[0095] When sample 8 was compared with sample 2, the two culture media had approximately the same viscosity.

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

[0097] When comparing Sample 8 with Sample 2, the use of SG-MC also resulted in a 20.3% increase in the protein IgG titer. Similar to Example 1, the protein titer was determined using protein A chromatography.

[0098] When comparing Sample 8 with Sample 1 and Sample 2 with Sample 1, both methylcelluloses significantly improved viable cell density compared to the control medium without additives.

[0099] The data in this example show that, compared with the additive-free control, both different substitution modes of methylcellulose can improve live cell density and protein titer.

[0100] The following are examples demonstrating the effects of using poloxamer and HPMC or MC on cell growth.

[0101] 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 following describes the use of baffleless cell culture shake flasks and PowerCHO2. TM Growth of CHO DG-44 cell line grown in culture medium.

[0102] Shake flasks are commonly used in the early stages of cell growth processes and laboratory studies, making them relevant and important to the industry. In addition to using regular methylcellulose with poloxamer 188, experiments were conducted involving the combined addition of regular hydroxypropyl methylcellulose (HPMC) and poloxamer 188.

[0103] 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. The sample was agitated by rotating the flasks on a shaker plate. Oxygen and carbon dioxide control was maintained by an incubator in which the shake flasks were placed, and there was no feedback loop. Baffles are typically not used in CHO cultures because their use would impose additional shear forces on the cells.

[0104] The cell culture medium is PowerCHO2, which is available from Lonza. TM It is supplied with 0.1% poloxamer 188 as a component. This cell line has been targeted for use in PowerCHO2. TM Growth in the culture medium was optimized. 1000 mL PowerCHO2 TM Culture medium, 20 mL 200 mM glutamine, and 10 mL anti-caking agent (Gibco with catalog number 01-0057DG). TM The products are mixed to form a working culture medium.

[0105] Add other polymers, namely poloxamer 188 (Px188), methylcellulose (MC), or hydroxypropyl methylcellulose (HPMC), to the culture medium as shown in Table 4 below.

[0106] Methylcellulose is graded at 15 cP.

[0107] Two conventional HPMC polymers were tested: HPMC with the K chemistry and a viscosity of 3 cP in a 2% aqueous solution at 20°C (i.e., "3cP HPMC-K"), and HPMC with the E chemistry and a viscosity of 5 cP in a 2% aqueous solution at 20°C (i.e., "5cP HPMC-E"). An exemplary 3cP HPMC-K is METHOCEL. TM K3 Premium LV. An exemplary 5cP HPMC-E is a METHOCEL. TM E5 Premium LV.

[0108] Using Cytiva HyClone containing poloxamer 188 (0.1%) TM Cellboost TM Supplements 7a and 7b are supplements.

[0109] Cells were cultured for 14 days. Except for sample 7, which was tested once, all samples were tested in triplicate.

[0110] At the start of cultivation, add 0.3 × 10 6 10 cells / mL were seeded into each shake flask, and cells were harvested after 14 days or when cell viability dropped below 70% (whichever was earlier). pH, glucose, ammonium, lactate, pCO2, and cell density were monitored daily starting from day 3 of culture.

[0111] Using Guava ® ViaCount TM Reagents, flow cytometer, and Vi-Cell TM The BLU cell viability analyzer (Beckman Coulter) is used to measure live cell density.

[0112] All other parameters are in Beckman Coulter Vi-CELL MetaFLEX TM Measurements were taken on a bioanalyte analyzer.

[0113] ViaCount TM It works by differentially staining live and dead cells. Vi-Cell TM The BLU Cell Viability Analyzer works by measuring trypan blue rejection.

[0114] Peak viable cell density is the highest viable cell density measured during fourteen days of culture.

[0115] The following table compares the average viable cell density of Px188 alone (included in the culture medium), with additional Px188 on top of the initial Px188 value, and with MC or different grades of HPMC values.

[0116] Table 4 The following describes the analysis of the values ​​obtained from the table above.

[0117] Adding more poloxamer did not result in a significant increase in live cell density. When the cell culture medium of sample 9 with 0.1% Px188 was compared with that of sample 10 with a final concentration of 0.3% Px188, the inclusion of additional Px188 resulted in a slight increase of 1.7% in the peak viable cell density (VCD) of sample 10. Therefore, the inclusion of additional Px188 alone did not cause a significant increase in peak viable cell density (VCD).

[0118] Compared to Px188 alone, the combination of Px188 with HPMC grade or MC showed a surprisingly good improvement in viable cell density. A surprising and unexpected result However, comparing the corresponding viable cell density values ​​of 0.1% Px188 alone in Sample 9 with the following results: (1) the combination of 0.2% 3cP HPMC-K and 0.1% Px188 in Sample 11; (2) the combination of 0.2% 5cP HPMC-E and 0.1% Px188 in Sample 12; (3) the combination of 0.2% MC and 0.1% Px188 in Sample 13; and (4) the combination of 0.5% 3cP HPMC-K and 0.1% Px188 in Sample 14, it was found that the aforementioned combinations showed a significant increase in peak viable cell density relative to Px188 alone. The increase in viable cell density relative to 0.1% Px188 alone ranged from 42.1% to 58.3%.

[0119] The aforementioned results are surprising and unexpected, as, as shown in corresponding samples 11-14, a significant increase in viable cell density was observed by combining the cellulose derivative with poloxamer 188 compared to sample 10 with poloxamer 188. 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.

[0120] Based on these preliminary results in Table 4, it is expected that the combination of poloxamer 188 with higher molecular weight MC and HPMC polymers (as indicated by the higher viscosity when measured as a 2% aqueous solution at 20 degrees Celsius) will result in higher cell growth than poloxamer alone or any of the aforementioned polymers.

[0121] The range of numerical values ​​disclosed in the specification includes values ​​that a person skilled in the art would consider equivalent to the recorded value (e.g., + / - 5-10% of the recorded value), such as values ​​that have the same function or result.

[0122] The claims are not limited to the preferred embodiments and examples, but are intended to cover numerous modifications and equivalents consistent with the written description as a whole.

Claims

1. A cell culture medium for suspension growth of cells, the cell culture medium comprising a cellulose derivative having a solution viscosity in water at 2% concentration ranging from 20 cP to 8000 cP at 20 degrees Celsius.

2. The cell culture medium according to claim 1, wherein the cellulose derivative is methylcellulose or hydroxypropyl methylcellulose or a combination thereof.

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

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

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

6. The cell culture medium according to any one of claims 1-5, wherein the cellulose derivative is methylcellulose.

7. The cell culture medium according to any one of claims 1-6, wherein the methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is 0.16 to 0.

36.

8. The cell culture medium according to any one of claims 1-7, wherein the methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is 0.26 to 0.

32.

9. The cell culture medium according to any one of claims 1-5, wherein the cellulose derivative is hydroxypropyl methylcellulose.

10. The cell culture medium according to any one of claims 1-5 and 9, wherein the cellulose derivative is hydroxypropyl methylcellulose having the E chemical type.

11. The cell culture medium according to any one of claims 1-5 and 9, wherein the cellulose derivative is hydroxypropyl methylcellulose having the K chemical form.

12. The cell culture medium according to any one of claims 1-5 and 9, wherein the hydroxypropyl methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is 0.16 to 0.

36.

13. The cell culture medium according to any one of claims 1-5 and 12, wherein the hydroxypropyl methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is 0.26 to 0.

32.

14. The cell culture medium according to any one of claims 1-13, wherein the cell culture medium comprises an antifoaming agent.

15. The cell culture medium according to any one of claims 1-14, wherein the defoamer comprises dimethicone.

16. The cell culture medium according to any one of claims 1-15, wherein the cell culture medium further comprises a polyether surfactant.

17. The cell culture medium according to any one of claims 1-16, wherein the polyether surfactant is poloxamer.

18. A method for suspending cells for growth, the method comprising incubating the cells in a cell culture medium according to any one of claims 1-17.

19. A method for increasing cell growth, the method comprising: A cell culture medium containing a cellulose derivative having a solution viscosity of 2% in water at 20 degrees Celsius, ranging from 20 cP to 8000 cP. Mix the cells with the cell culture medium; and The cells and culture medium are incubated to enable them to grow.

20. The method of claim 19, wherein the step of providing the cellulose derivative provides the cellulose derivative at a concentration ranging 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 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 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 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 0.26 to 0.

32.

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

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

29. The method of claim 19, wherein the cells are derived from the Vero cell line.

30. The method of claim 19, wherein the cell produces proteins.

31. The method of claim 19, wherein the cells produce antibodies.

32. The method of claim 19, wherein the cells produce antibody-derived proteins.

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

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

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