Combinations of polysaccharides and polyether surfactants

Abstract

A method of increasing cell growth in a cell culture medium comprising contacting the cells with a combination of a polysaccharide and a polyether surfactant. Further, a cell culture medium comprising a polysaccharide and a polyether surfactant for increasing cell growth is disclosed. Exemplary embodiments include cellulose derivatives and poloxamers.

Description

Technical Field

[0001] The technical field involves the effects of polysaccharides on in vitro cell growth.

[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] Improved serum alternatives and reduced dependence on poloxamer are still needed. Summary of the Invention

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

[0017] A related method for growing cells in a suspension includes incubating cells in a cell culture medium. A method for increasing cell growth includes: providing a cell culture medium containing a combination of a cellulose derivative and a polyether surfactant; 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 cell culture medium formulation for improving the growth of suspended cells is disclosed, comprising polysaccharides and polyether surfactants.

[0020] A method for improving the growth of suspended cells is disclosed, which includes using a cell culture medium containing polysaccharides and polyether surfactants.

[0021] In specific examples of polysaccharides and polyether surfactants, combinations of methylcellulose or hydroxypropyl methylcellulose with poloxamer 188 are disclosed.

[0022] Although both poloxamer 188 and methylcellulose, as described in the background section, are known in the art, their combination in cell culture media for enhancing cell growth as disclosed herein has not been explored. In fact, methylcellulose is known in the art for use in food cultures but not in cell culture media, such as those containing CHO cells.

[0023] In one instance, the cell culture medium did not contain serum.

[0024] The following describes some of the key concepts in this specification.

[0025] 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".

[0026] Defoamer In another example, the cell culture medium optionally contains an antifoaming agent. The antifoaming agent is optionally a dimethicone antifoaming agent.

[0027] Polyether surfactants Exemplary polyether surfactants for use in cell culture media include poloxamer 124, poloxamer 188, poloxamer 338, and poloxamer 407.

[0028] polysaccharides Exemplary polysaccharides used in cell culture media of this disclosure include cellulose derivatives, alginates, carrageenan, α-glucan, β-glucan, and dextran, and their chemical derivatives, as mentioned below. In another example, the molecular weight of the polysaccharide is such that, when dissolved in water at 2% concentration and measured at 20°C, the solution viscosity is less than 10,000 centipoise (cP), less than 7,000 cP, less than 2,000 cP, less than 1,500 cP, less than 500 cP, less than 100 cP, or less than 30 cP.

[0029] Cellulose derivatives. Exemplary cellulose derivatives used in cell culture media of this disclosure include methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethyl methylcellulose, and any combination thereof.

[0030] In one instance, the hydroxyl groups of a cellulose derivative may include alkyl substituents or hydroxyalkyl substituents or combinations thereof.

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

[0032] In one example, the cellulose derivative is methylcellulose 15 cP. In another example, the cellulose derivative is HPMC E 5 cP. In yet another example, the cellulose derivative is HPMC K 3 cP.

[0033] 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%.

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

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

[0036] 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.

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

[0038] 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.

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

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

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

[0045] For example, methylcellulose is defined by the USP as having not less than 26% and not more than 33% of methylated hydroxyl groups substituted.

[0046] 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.

[0047] molecular weightThe 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, 15 cP methylcellulose polymer is methylcellulose in which a 2% solution of methylcellulose in water 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 in Chapter 912 of the United States Pharmacopeia.

[0048] Chemical substitution 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.

[0049] 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.

[0050] 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.

[0051] The substitution pattern of the substituents on the cellulose constituting the cellulose derivative is optionally defined by the s23 / s26 ratio.

[0052] 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.

[0053] 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 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. These polymers are referred to as SG methylcellulose (“SG-MC”).

[0054] In one instance, the favorable s23 / 26 is 0.23 to 0.32.

[0055] 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.

[0056] 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.

[0057] Unconventional cellulose derivatives. Methylcellulose and hydroxypropyl methylcellulose having s23 / 26 in the range of 0.16 to 0.36 are considered unconventional cellulose derivatives.

[0058] Exemplary SG methylcellulose and SG HPMC are manufactured, for example, as described in EP1171471, WO20000 / 59947, US6235893 and US6228416.

[0059] The experimental conditions for the control group, which did not use poloxamer 188 and methylcellulose, are described below.

[0060] Example 1 Comparison 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Add Gibco Foam Away daily ® Irradiate AOF (animal-free) defoamer (10 µL / day) 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).

[0065] 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.

[0066] 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.

[0067] Peak viable cell density is the highest viable cell density measured during fourteen days of culture.

[0068] 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.

[0069] Protein titers were measured using Bio HT. Protein titers measured using the Cedex Bio HT analyzer were evaluated at the end of the experiment. IgG Bio HT is a test kit for determining IgG and measures turbidity after IgG protein nanoprecipitation. Turbidity is proportional to IgG concentration and is determined by comparing the turbidity of a solution of IgG at a known concentration.

[0070] The experiment was run in duplicate.

[0071] Results: The mean peak viable cell density was 2.06 × 10⁻⁶. 6100 cells / mL, indicating that cells did not significantly expand in the absence of poloxamer 188 or methylcellulose. IgG1 protein was not detected by BioHT. 50 µg / mL IgG was detected by protein A chromatography using an Agilent Bio-Monolith Protein A column and an HPLC system equipped with UV detection. As will be shown, the amount of IgG recovered in the additive-free control medium was minimal compared to examples containing polysaccharides, poloxamer, or a combination of both.

[0072] Example 2 The combination of methylcellulose and poloxamer 188 surprisingly resulted in an increase in average viable cell density. Samples 1-6 used a similar Hycell as in Example 1. TM Culture medium was prepared, but conventional methylcellulose 15 cP (MC), poloxamer 188 (Px188), or a 1:1 ratio of MC and Px188 were added to the cell culture medium.

[0073] Each sample was tested in quadruplicate, and the average value was reported. Protein content was measured using Bio HT and protein A chromatography using an Agilent Bio-Monolith protein A column and an HPLC system equipped with UV detection, similar to that in Example 1.

[0074] HPLC was also used to perform dextran profiling to assess changes in protein quality.

[0075] The table below shows the peak viable cell density (VCD) for samples 1-6.

[0076] Table 1 The average peak viable cell density is the average of four replicates in the experiment (mean).

[0077] The following describes the analysis of the values ​​obtained from the table above.

[0078] Poloxamer 188 promotes a higher live cell density than methylcellulose. As will be shown below, poloxamer 188 (Px188) is more effective than methylcellulose in increasing cell density. This analysis is demonstrated by comparing the mean peak viable cell density between samples 1 and 2, and between samples 3 and 4, respectively.

[0079] Higher concentrations of poloxamer 188 result in higher viable cell density compared to lower concentrations of poloxamer 188. Notably, higher concentrations of Px188 resulted in greater efficiency in increasing viable cell density compared to lower concentrations. This is illustrated by comparing Sample 2 and Sample 4.

[0080] The combination of Px188 and MC at the same concentration (0.1%) was compared with the individual components at the same concentration. Surprisingly higher live cell density resultsSurprisingly and unexpectedly, when compared with the viable cell density of the same total concentration of Px188 (0.1%) in Sample 4, the combination of the same total concentration of Px188 and MC (0.1%) in Sample 6 resulted in a higher viable cell density.

[0081] These results are surprising and unexpected to those skilled in the art, as MC is less effective than Px188 in promoting cell density. Without being bound by theory, those skilled in the art would expect the viable cell density value of the combination of Px188 and MC (0.1%) to fall between the measured viable cell density values ​​of samples 3 and 4.

[0082] A combination of Px188 and MC at the same concentration (0.2%) and poloxamer 188 at the same concentration alone. (0.2%) yielded a surprisingly equal result in viable cell density. Sample 5 had the same total amount of additives as Samples 1 and 2. Surprisingly and unexpectedly, the combination of the same total concentration of Px188 and MC (0.2%) in Sample 5 resulted in the same viable cell density compared to the same total concentration of Px188 (0.2%) in Sample 2.

[0083] Without being bound by theory, those skilled in the art will expect the viable cell density value of Sample 5 (a combination of both Px188 and MC (0.2%)) to be between that of Sample 1 with 0.2% MC alone and Sample 2 with 0.2% Px188. This is because MC is less effective than Px188 in promoting cell density.

[0084] Given concerns about the quality control of Px188, there is an expectation to reduce the amount of Px188 in the culture medium. Therefore, the aforementioned results are both surprising and of interest to the industry.

[0085] The combination of lower total concentrations of MC and Px188 surprisingly exhibited peak activity similar to that of higher concentrations of Px188. Cell density The combination of Px188 and MC in Sample 6 (total concentration 0.1%, containing 0.05% Px188 and 0.5% MC) had less total additive (0.1%), and Px188 was one-quarter of the 0.2% Px188 alone in Sample 2.

[0086] Surprisingly, the combination mentioned has a similar peak viable cell density to 0.2% Px188 alone, thus achieving the industry goal of reducing Px188 levels in cell culture media.

[0087] Example 3 The combination of methylcellulose and poloxamer 188 surprisingly resulted in higher protein titer levels. Similar to that in Example 1, the protein A titer and BioHT titer levels in this example were measured.

[0088] The table below shows the average IgG protein levels of samples 1–6 as measured by protein A and BioHT at the end of cell culture.

[0089] Table 2 The following describes the analysis of the values ​​obtained from the table above.

[0090] The IgG protein titer in Px188 was higher than that in MC. As shown by comparing Sample 1 (0.2% MC) with Sample 2 (0.2% Px188) or Sample 3 (0.1% MC) with Sample 4 (0.1% Px188), at 0.2% and 0.1%, Px188 was more effective than MC at the same level in increasing IgG protein titers, resulting in higher yields of the target protein product (IgG protein).

[0091] Higher concentrations of both Px188 and MC resulted in higher IgG protein titers than at lower concentrations. When comparing Sample 1 and Sample 3, MC at the higher concentration (0.2% vs 0.1%) had a higher IgG protein titer than MC at the corresponding lower concentration. When comparing Sample 2 and Sample 4, Px188 at the higher concentration (0.2% vs 0.1%) had a higher IgG protein titer than Px188 at the corresponding lower concentration.

[0092] The combination of MC and Px188 at the same total concentration exhibits surprisingly higher levels compared to MC or Px188 alone. For example, IgG protein titers measured by Bio HT, or those with the same properties as Px188 alone, such as those measured by protein A chromatography. IgG protein titer Compared to the IgG protein titer (610 µg / mL) of MC alone (0.1% MC) in Sample 3, the IgG protein titer (919 µg / mL) of the combination of MC and Px188 (0.1%) in Sample 6, as measured by protein A chromatography, was higher. The IgG protein titer of the aforementioned combination was the same as that of Px188 alone (0.1% Px188) in Sample 4, with the Px188 concentration halved.

[0093] The combination of MC and Px188 (0.1%) in Sample 6 had the same total concentration of additive as MC alone in Example 3 and Px188 alone in Sample 4, but the IgG protein titer in Sample 6 as measured by Bio HT (i.e., 1138 µg / mL) was higher than that in Sample 3 (563 µg / mL) or Sample 4 (838 µg / mL).

[0094] The aforementioned results are surprising and unexpected, because MC is less effective than Px188 in promoting IgG protein titers. Therefore, those skilled in the art would expect the IgG protein titer level of the combination of MC and Px188 in Sample 6 to be between the measured IgG protein titer levels of MC alone in Sample 3 and Px188 alone in Sample 4.

[0095] Example 4 The combination of methylcellulose and poloxamer 188 surprisingly resulted in a reduced average M5 polysaccharide content. The table below shows the average M5 glycan content of samples 1-6. A higher M5 glycan content is not beneficial because it can lead to immunogenicity when IgG protein is administered as a therapeutic agent.

[0096] Table 3 The following describes the analysis of the values ​​obtained from the table above.

[0097] Px188 resulted in a lower M5 glycan content than MC. Comparing the MC concentration (0.2%) in sample 1 with the Px188 concentration (0.2%) in sample 2, and comparing the MC concentration (0.1%) in sample 3 with the Px188 concentration (0.1%) in sample 4, it was found that the presence of Px188 resulted in a lower M5 glycan content in the cell culture medium compared to the M5 glycan content indicated by the presence of MC in the cell culture medium.

[0098] The combination of MC and Px188 (0.1% total concentration) surprisingly resulted in lower levels of MC and Px188 at the same corresponding concentrations alone compared to individual products. M5 polysaccharide content of Px188 The combination of MC and Px188 in Sample 6 (0.1%) had the same total concentration as MC alone in Sample 3 and Px188 alone in Sample 4, but the M5 polysaccharide content in Sample 6 (3.1%) was lower than that in Sample 3 (4.7%) or Sample 4 (3.3%).

[0099] The aforementioned results for the combination of Sample 6 are surprising and unexpected, as the presence of MC contents results in a higher M5 content than Px188. Therefore, those skilled in the art would expect the M5 content of the M5 glycans in the aforementioned combination to be between the M5 glycan levels measured by MC alone in Sample 3 and by Px188 alone in Sample 4.

[0100] Example 5 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 same CHO DG-44 cell line as previously described is described below, but using baffle-free cell culture shake flasks and PowerCHO2. TM Culture medium growth. Example 2 used an ambr15 parallel 24-position bioreactor system (Sartorius).

[0101] 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.

[0102] The proprietary CHO DG-44 cell line expressing IgG1 antibodies 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 shake 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.

[0103] 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.

[0104] Add other polymers, namely poloxamer 188 (Px188), methylcellulose (MC), or hydroxypropyl methylcellulose (HPMC), to the culture medium as shown in Table 4 below.

[0105] The methylcellulose is grade 15 cP and is the same type of methylcellulose used in Example 2.

[0106] Two conventional HPMC polymers were tested: HPMC with the K chemistry and a viscosity of 3 cP as a 2% aqueous solution at 20°C (i.e., "3cP HPMC-K"), and HPMC with the E chemistry and a viscosity of 5 cP as a 2% aqueous solution at 20°C (i.e., "5cP HPMC-E"). An exemplary 3cP HPMC-K is METHOCEL. TM K3 PremiumLV. An exemplary 5cP HPMC-E is a METHOCEL. TM E5 Premium LV.

[0107] Using Cytiva HyClone containing poloxamer 188 (0.1%) TMCellboost TM Supplements 7a and 7b are supplements.

[0108] Cells were cultured for 14 days. Except for sample 7, which was tested once, all samples were tested in triplicate.

[0109] 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.

[0110] 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.

[0111] All other parameters are in Beckman Coulter Vi-CELL MetaFLEX TM Measurements were taken on a bioanalyte analyzer.

[0112] Peak viable cell density is the highest viable cell density measured during fourteen days of culture.

[0113] 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.

[0114] Table 4 The following describes the analysis of the values ​​obtained from the table above.

[0115] Adding more poloxamer did not result in a significant increase in live cell density. When the cell culture medium of Sample 7 with 0.1% Px188 was compared with that of Sample 8 with a final concentration of 0.3% Px188, the addition of Px188 resulted in a slight increase of 1.7% in the peak viable cell density (VCD) of Sample 8. Therefore, the addition of Px188 alone did not cause a significant increase in peak viable cell density (VCD).

[0116] 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 resultHowever, comparing the corresponding viable cell density values ​​of 0.1% Px188 alone in Sample 7 with the following results: (1) the combination of 0.2% 3cP HPMC-K and 0.1% Px188 in Sample 9; (2) the combination of 0.2% 5cP HPMC-E and 0.1% Px188 in Sample 10; (3) the combination of 0.2% MC and 0.1% Px188 in Sample 11; and (4) the combination of 0.5% 3cP HPMC-K and 0.1% Px188 in Sample 12, 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%.

[0117] The aforementioned results are surprising and unexpected, as, as shown in the corresponding samples 9-12, a significant increase in viable cell density was observed by combining the cellulose derivative with poloxamer 188 compared to sample 8 with added poloxamer 188. These results are unexpected because the previously mentioned comparisons of samples 1 and 2 with 0.2% MC and 0.2% Px188 indicate that Px188 is more effective than the cellulose derivative in increasing peak viable cell density.

[0118] Example 6 The combination of methylcellulose and poloxamer 188 resulted in an increase in mean peak viable cell density. Similar to Example 1, the proprietary CHO DG-44 cell line expressing IgG1 antibody was grown in the Ambr15 system, but PowerCHO2 was used. TM Cell culture medium, as described in samples 7-12 of Example 4.

[0119] Each condition is tested four times, and the average result of these four tests for each condition is reported.

[0120] The table below compares the average viable cell density of Px188 alone, Px188 plus an additional Px188 value, and MC plus an additional MC value. MC is 15 cP methylcellulose as used in the previous examples.

[0121] Table 5 The following describes the analysis of the values ​​obtained from the table above.

[0122] Adding more poloxamer does not lead to a significant increase in live cell density.Comparison of samples 13-14 showed that the peak viable cell density decreased slightly by 1.4% when the concentration of Px188 increased from 0.1% to a total concentration of 0.3%. These results indicate that including additional Px188 does not lead to an increase in peak viable cell density. These results are consistent with those found in Table 4.

[0123] The combination of the same total concentration of methylcellulose and poloxamer 188 resulted in an increase in viable cell density. Comparison of Sample 13, containing only Px188, with Sample 15, containing a combination of MC and Px188, showed that even when the total concentration of the combination was 0.3%, the peak viable cell density was increased by 7.4% compared to the concentration of Px188 alone in Sample 14 (0.3%) (where there was no increase in peak VCD). In contrast, the viable cell density achieved by including additional Px188 (Sample 14) to reach a total concentration of 0.3% was reduced, while the peak viable cell density achieved by including a combination of MC (0.2%) and 0.1% Px188 (Sample 15) was actually increased. These results are surprising and unexpected, as Samples 1 and 2 indicate that Px188 is more effective than cellulose derivatives in increasing peak viable cell density. This also suggests that the combination of Px188 and cellulose derivatives surprisingly and unexpectedly promotes cell growth.

[0124] Example 7 The combination of methylcellulose and poloxamer 188 surprisingly resulted in a high live cell density. This example uses Agarabi CHO cells (ATCC, CRL3440), which differ from the proprietary CHO DG-44 cell line expressing IgG1 antibody used in Example 4. Agarabi CHO cells (ATCC, CRL3440) were used in the CD FortiCHO... TM Culture medium (Gibco) TM The product line was amplified in 125 mL baffle-free shake flasks.

[0125] This commercial culture medium contained 0.074% poloxamer 188. The medium was supplemented with 8 mM L-glutamine. As detailed in the table below, the medium was further supplemented with polymer-free or 15 cP methylcellulose. Cells were maintained at 8% CO2 and 37°C. The initial (cell) culture volume was 10 mL, increased to 18 mL on day 3, and to 34 mL on day 7. At each time point, 0.5 mL was taken for cell counting using a trypan blue conventional count and a Countess 3FL counter. Each sample was tested in quadruplicate.

[0126] The table below reports the average total viable cells in the shake flasks and describes the effect of using methylcellulose on cell growth.

[0127] Table 6 The following discussion compares daily cell counts for the control (sample 16), 0.2% methylcellulose (sample 17), and 0.4% methylcellulose (sample 18).

[0128] On day 1, the cell count in the 0.2% methylcellulose group increased by 29% compared to the control group on day 1 (129% of the control group). On day 1, the cell count in the 0.4% methylcellulose group was the same as the control group on day 1.

[0129] On day 2, the cell count in the 0.2% methylcellulose group increased by 46% compared to the control group on day 2 (146% of the control group). On day 2, the cell count in the 0.4% methylcellulose group increased by 18% compared to the control group on day 2.

[0130] On day 3, the cell count in the 0.2% methylcellulose group increased by 96% compared to the control group on day 3 (196% of the control group). On day 3, the cell count in the 0.4% methylcellulose group increased by 79% compared to the control group on day 3 (179% of the control group).

[0131] On day 4, the cell count in the 0.2% methylcellulose group increased by 114% compared to the control group on day 4 (214% of the control group). On day 4, the cell count in the 0.4% methylcellulose group increased by 109% compared to the control group on day 4 (209% of the control group).

[0132] On day 7, the cell count in the 0.2% methylcellulose group increased by 59% compared to the control group on day 7 (159% of the control group). On day 7, the cell count in the 0.4% methylcellulose group increased by 51% compared to the control group on day 7 (151% of the control group).

[0133] On day 8, the cell count in the 0.2% methylcellulose group increased by 43% compared to the control group on day 8 (143% of the control group). On day 8, the cell count in the 0.4% methylcellulose group increased by approximately 43% compared to the control group on day 8 (143% of the control group).

[0134] On day 9, the cell count in the 0.2% methylcellulose group increased by approximately 34% compared to the control group on day 9 (134% of the control group). On day 9, the cell count in the 0.4% methylcellulose group increased by approximately 41% compared to the control group on day 9 (141% of the control group).

[0135] On day 10, the cell count in the 0.2% methylcellulose group increased by approximately 20% compared to the control group on day 10 (120% of the control group). On day 10, the cell count in the 0.4% methylcellulose group increased by approximately 27% compared to the control group on day 10 (127% of the control group).

[0136] On day 11, the cell count in the 0.2% methylcellulose group increased by approximately 37% compared to the control group on day 11 (137% of the control group). On day 11, the cell count in the 0.4% methylcellulose group increased by approximately 20% compared to the control group on day 11 (120% of the control group).

[0137] The following comparisons were made of the cell counts of the control group on day 1 and day 11, the cell counts of 0.2% methylcellulose on day 1 and day 11, and the cell counts of 0.4% methylcellulose on day 1 and day 11.

[0138] When compared to the control number on day 1, cells not treated with 0.2% or 0.4% methylcellulose showed an approximately 148-fold increase in cell growth by day 11. This also means an increase in cell growth of approximately 14,847% relative to the control cell number on day 1.

[0139] When compared to the cell count of cells treated with 0.2% methylcellulose on day 1, the cell growth of cells treated with 0.2% methylcellulose increased by approximately 158-fold on day 1, relative to the cell count of cells treated with 0.2% methylcellulose on day 1. This also means that cell growth increased by approximately 15,880% relative to the cell count on day 1.

[0140] Cell growth comparison between control and 0.2% MC Therefore, when compared with the increase in cell growth of the control from day 1 to day 11, the cell growth of cells treated with 0.2% methylcellulose increased by approximately 10% relative to the cell growth of the control (i.e., 158 times vs. 148 times the control).

[0141] When compared to the cell number on day 1 of cells treated with 0.4% methylcellulose, the cell growth of cells treated with 0.4% methylcellulose increased by approximately 179-fold on day 11. This also means that cell growth increased by approximately 17,900% relative to the cell number on day 1.

[0142] Cell growth comparison between control and 0.4% MC Therefore, when compared with the increase in cell growth of the control from day 1 to day 11, the cell growth of cells treated with 0.4% methylcellulose increased by approximately 30% relative to the cell growth of the control (i.e., 179 times vs. 148 times for the control).

[0143] The data shown in Examples 2-7 demonstrate the same trend, with the combination of Px188 with MC or Px188 with HPMC exhibiting a surprisingly positive effect on cell growth. This indicates that the surprising effects of combining methylcellulose or HPMC with poloxamer are applicable to various suspension cell culture systems, including different reactor types, culture medium choices, or cell lines. More broadly, this suggests that the surprising effects of combining polysaccharides with poloxamer are applicable to various suspension cell culture systems.

[0144] Example 8 SG MC was as effective as MC in promoting higher viable cell density and protein IgG titers compared to the additive-free control, but still had a lower effect than poloxamer 188. This experiment was conducted similarly to that in Example 2, but two different methylcelluloses were compared as culture medium additives. One methylcellulose used in this example was conventional methylcellulose 2 cP. The other methylcellulose used in this example was SG methylcellulose 2 cP, an example of an unconventional methylcellulose. Both were added to HyCell at 0.2%. TM In the culture medium.

[0145] The control in this experiment is Example 1.

[0146] The results are shown in the table below.

[0147] Table 7 The following is an analysis of the above data.

[0148] When comparing sample 19 and sample 20, the two culture media had approximately the same viscosity.

[0149] When comparing samples 19 and 20, the use of SG-MC resulted in a 59% increase in peak viable cell density compared to the use of conventional methylcellulose MC.

[0150] When comparing samples 19 and 20, the use of SG-MC also resulted in a 20.3% increase in protein IgG titer. Protein titer was determined using a protein A chromatography method similar to that described in Example 1.

[0151] When comparing sample 19 with Example 1 and sample 20 with Example 1, both types of methylcellulose significantly increased viable cell density compared to the control culture medium without additives.

[0152] As will be shown below, the use of unconventional methylcellulose still resulted in a lower mean peak viable cell density and a lower IgG protein titer than when using the same concentration of poloxamer 188.

[0153] When comparing the peak viable cell density of Sample 20 (0.2% SG methylcellulose) with that of Sample 2 (0.2% Px188) in Example 2, Px188 was more effective than SG-MC, with an average peak viable cell density of 3.09 × 10⁻⁶. 7 The peak viable cell density was 59% higher than that of the same concentration of SG-MC in sample 20.

[0154] When comparing the IgG protein titers of Sample 20 (757 µg / mL) with those of Sample 2 (1297 µg / mL) in Example 3, Px188 was 71% more effective than SG-MC in promoting the increase of the target IgG protein.

[0155] The data in this embodiment show that, compared to the control without additives, both methylcelluloses with different substitution modes can increase viable cell density and protein titer, but the effect is still not as good as Px188.

[0156] Example 9 Higher molecular weight methylcellulose was as effective as MC 15 cP in promoting higher viable cell density and IgG protein titers compared to the additive-free control. This experiment was conducted using the procedure of Example 2, but different molecular weights of conventional methylcellulose were compared as culture medium additives. Furthermore, the results were compared with those for sample 19 using conventional methylcellulose 2 cP (Example 8). Average protein titers were measured using protein A chromatography.

[0157] In this embodiment, conventional methylcelluloses of different molecular weights were used. Molecular weight is not measured directly, but rather expressed by their viscosity at 2% solution at 20 degrees Celsius; the same applies to other methylcelluloses.

[0158] One type of methylcellulose used in this embodiment is conventional methylcellulose 338 cP (sample 21). Another type of methylcellulose used in this embodiment is conventional methylcellulose 2880 cP (sample 22).

[0159] For the aforementioned samples, the corresponding methylcellulose was added to HyCell at 0.2%. TM In the culture medium.

[0160] The last additional sample in this embodiment was prepared using MC 2cP methylcellulose, which was added to HyCell at 2.4%. TM The viscosity of the culture medium was matched to that of the medium containing 0.2% MC 2880 cP (sample 22). The 2.4% MC 2cP methylcellulose was the same as that used in sample 19.

[0161] The control in this experiment is Example 1.

[0162] The results are shown in the table below.

[0163] Table 8 The following is an analysis of the above data.

[0164] When comparing the peak viable cell density of sample 19 (conventional methylcellulose-2 cP, 0.2%) with that of sample 21 (MC-338 cP, 0.2%), the peak viable cell density of sample 21 was 23.7% higher.

[0165] When comparing the peak viable cell density of sample 19 (conventional methylcellulose - 2 cP, 0.2%) with that of sample 22 (MC - 2880 cP, 0.2%), the peak viable cell density of sample 22 was 56.6% higher.

[0166] When comparing the protein IgG titers of sample 19 (conventional methylcellulose - 2 cP, 0.2%) with those of sample 21 (MC - 338 cP, 0.2%), the protein IgG titer of sample 21 was 9.2% higher.

[0167] When comparing the protein IgG titers of sample 19 (conventional methylcellulose - 2 cP, 0.2%) with those of sample 22 (MC - 2880 cP, 0.2%), the protein IgG titer of sample 22 was 16.6% higher.

[0168] A comparison of the peak viable cell density of Sample 19 (conventional methylcellulose-2 cP, 0.2%) with that of Sample 23 showed that the peak viable cell density of Sample 23, with a MC of 2.4%, was increased by 8.2% compared to that of Sample 19 (0.2% MC).

[0169] A comparison of the protein IgG titers of samples 19 and 23 showed that the protein IgG titer of sample 23, which had a MC of 2.4%, was reduced by 22.7% compared to 0.2% MC in sample 19.

[0170] A comparison of peak viable cell densities of Sample 22 (MC - 2880 cP, 0.2%) and Sample 23 (MC - 2 cP, 2.4%) showed that the peak viable cell density of Sample 22 increased by 44.7% compared to Sample 23.

[0171] A comparison of the culture medium viscosity of samples 19, 21 and 22 showed that the viscosity increased slightly with the increase of MC molecular weight.

[0172] A comparison of the viscosity of the culture medium in samples 22 and 23 showed that the two samples had similar viscosities.

[0173] Comparison of Sample 21 with Example 1 and Comparison of Sample 22 with Example 1 showed that both methylcelluloses significantly increased viable cell density compared to the control culture medium without additives.

[0174] Comparing the peak viable cell density of sample 21 with that of sample 2 in Example 2, and comparing the peak viable cell density of sample 22 with that of sample 2 in Example 2, shows that Px188 is still more effective than higher molecular weight MC alone in promoting higher peak viable cell density.

[0175] Comparing the IgG protein titers of Sample 21 (687 µg / ml) with Sample 2 (1297 µg / ml) of Example 3, and comparing the IgG protein titers of Sample 22 (725 µg / ml) with Sample 2 (1297 µg / ml) of Example 3, it is shown that Px188 is still more effective than high molecular weight MC in promoting higher IgG protein titers.

[0176] The data in this embodiment show that higher molecular weight methylcellulose can increase live cell density and protein titer, but the effect is still not as good as Px188.

[0177] The data in this embodiment also indicate that the effectiveness of higher molecular weight methylcellulose in promoting higher peak viable cell densities is not solely attributable to increased culture medium viscosity. For example, sample 23 (MC - 2 cP, 2.4%) was less effective than sample 22 (MC - 2880 cP, 0.2%) in promoting higher peak viable cell densities, despite both samples having similar viscosities.

[0178] Example 10 Unexpected benefits of combining methylcellulose and poloxamer 188 were observed at various methylcellulose / poloxamer 188 ratios. Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL shake flasks with baffles. Each shake flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell was used. TM Cell culture medium (Cytiva). As shown in Table 9, add conventional methylcellulose (MC), poloxamer 188 (Px188), or a combination of MC and Px188 with a viscosity of 15 cPs to the medium. Add Gibco daily. TM FoamAway TMIrradiate AOF (animal-free) defoamer (Thermo Fisher Scientific) (10 µL / day) to prevent foaming in the system. Start with 3 × 10⁻⁶ at the beginning of the culture. 5 Cells were seeded into each reactor at a concentration of 1 cell / mL. Before being added to shake flasks under these conditions, the seeded cells were centrifuged and resuspended in a medium containing the desired (or one) additives. The viable cell density (VCD) shown in Table 9 is the average of three replicates. Cell counting was performed after centrifugation and resuspending of cells in HyCell without methylcellulose or poloxamer. TM After adding to the culture medium, use Countess TM The 3FL automated cell counter was performed using conventional trypan blue counting.

[0179] Table 9 below shows the observation VCDs of the test samples.

[0180] Table 9 In Table 9, the percentages are based on the weight percentage of the total cell culture. MC fraction = (MC concentration) / (total concentration of MC + Px188). “Expected VCD” is the VCD expected if the effects of MC and Px188 on VCD are simply additive. Therefore, expected VCD = (VCD of MC only) × (MC fraction) + (VCD of Px188 only) × (Px188 fraction). In this equation, (MC fraction) + (Px188 fraction) = 1, and (Px188 fraction) = (Px188 concentration) / (total concentration of MC + Px188). All concentrations discussed here are weight concentrations.

[0181] As shown in Table 9, MC / Px188 ratios from 7 / 1 to 1 / 7 were tested. VCDs obtained using the combination of MC and Px188 exceeded the expected VCDs in every case, thus demonstrating the unexpected benefits of this combination across the entire ratio range.

[0182] Example 11 The effect of increasing additive concentration in high-shear systems Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL shake flasks with baffles. Each shake flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell was used. TM Cell culture medium (Cytiva). As shown in Table 10, add conventional methylcellulose (MC), poloxamer 188 (Px188), or a combination of MC and Px188 with a viscosity of 15 cPs to the medium. Add Gibco daily. TMFoamAway TM Irradiate AOF (animal-free) defoamer (Thermo Fisher Scientific) (10 µL / day) to prevent foaming in the system. Start with 3 × 10⁻⁶ at the beginning of the culture. 5 Cells were seeded into each reactor at a concentration of 1 cell / mL. Before being added to shake flasks under these conditions, the seeded cells were centrifuged and resuspended in a medium containing the desired (or one) additives. The viable cell density (VCD) shown in Table 10 is the average of three replicates. Cell counting was performed after centrifugation and cell resuspending in HyCell without methylcellulose or poloxamer. TM After adding to the culture medium, use Countess TM The 3FL automated cell counter was performed using conventional trypan blue counting.

[0183] Table 10 below shows the observation VCDs of the test samples.

[0184] Table 10 In Table 10, the percentages are based on the weight percentage of the total cell culture.

[0185] As shown in Table 10, total additive concentrations of 0.05% to 0.5% were tested in high-shear systems. By using a combination of methylcellulose and Px188 instead of either alone, an unexpected improvement in VCD performance was observed with total additive concentrations of 0.4% to 0.5%.

[0186] Example 12 The effect of reducing additive concentration in high-shear systems Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL shake flasks with baffles. Each shake flask contained 15 mL of cell culture. Hycell was used. TM Cell culture medium (Cytiva). As shown in Tables 11 and 12, add standard methylcellulose (MC), poloxamer 188 (Px188), or a combination of MC and Px188 with a viscosity of 15 cPs to the medium. Experiments in Table 11 were performed using a shaker speed of 160 RPM, while experiments in Table 12 were performed using a shaker speed of 130 RPM. Gibco was added daily. TM FoamAway TM Irradiate AOF (animal-free) defoamer (Thermo Fisher Scientific) (10 µL / day) to prevent foaming in the system. Start with 3 × 10⁻⁶ at the beginning of the culture. 5Cells were seeded into each reactor at a concentration of 1 cell / mL. Before being added to shake flasks under these conditions, the seeded chain cells were centrifuged and resuspended in a medium containing the desired (or one) additives. The viable cell density (VCD) shown in Tables 11 and 12 is the average of three replicates. Cell counting was performed after centrifugation and cell resuspending in HyCell without methylcellulose or poloxamer. TM After adding to the culture medium, use Countess TM The 3FL automated cell counter was performed using conventional trypan blue counting.

[0187] Table 11 below shows the observation VCDs of test samples conducted in a high-shear system (where the shaker speed is 160 RPM).

[0188] Table 11 Table 12 below shows the observation VCDs of test samples conducted in a low-shear system (where the shaker speed is 130 RPM).

[0189] Table 12 In Tables 11 and 12, the percentages are based on the weight percentage of the total cell culture.

[0190] As shown in Tables 11 and 12, total additive concentrations of 0.01% to 0.2% were tested at shake flask rotation speeds of 160 or 130 RPM. In higher shear systems (where shake flask rotation speed was 160 RPM), the protective effect of the additive tended to be more pronounced at total additive concentrations of at least 0.1%. In higher shear systems, MC alone provided protection even at concentrations as low as 0.1%. In lower shear systems (where shake flask rotation speed was 130 RPM), the protective effect of the additive tended to be more pronounced at total additive concentrations of at least 0.02%, and particularly at least 0.05%. At the concentrations tested, MC tended to be more protective than Px188 in lower shear systems. This data suggests that growth in different culture systems will respond differently to the levels of additives contained in the culture medium.

[0191] Example 13 The protective effect of SG-MC alone or in combination with Px188 Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL shake flasks with baffles. Each shake flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell was used. TMCell culture medium (Cytiva). As shown in Table 13, add to the medium conventional MC or SG methylcellulose (SG-MC), poloxamer 188 (Px188), or a combination of such conventional MC or SG-MC with Px188, having a viscosity of 2 cPs. Add Gibco daily. TM FoamAway TM Irradiate AOF (animal-free) defoamer (Thermo Fisher Scientific) (10 µL / day) to prevent foaming in the system. At the start of culture, use 3 × 10⁻⁶... 5 Cells were seeded into each reactor at a concentration of 1 cell / mL. Seeded cells were centrifuged and resuspended in a medium containing the desired (or one) additives before being added to the shake flasks under these conditions. The viable cell density (VCD) shown in Table 13 is the average of three replicates. Cell counting was performed after centrifugation and resuspension of cells in HyCell without methylcellulose or poloxamer. TM After adding to the culture medium, use Countess TM The 3FL automated cell counter was performed using conventional trypan blue counting.

[0192] Table 13 below shows the observation VCDs of the test samples.

[0193] Table 13 In Table 13, the percentages are based on the weight percentage of the total cell culture.

[0194] As shown in Table 13, 0.20% Px188 alone provides greater protection than 0.20% conventional MC alone or 0.20% SG-MC alone. However, the protective effect of Px188 alone can be achieved by replacing a portion (in this case, 50%) of Px188 with conventional MC or SG-MC. The improvements observed in S58 and S59 cannot be attributed to Px188 alone, as comparisons of samples 56 and 57 demonstrate that 0.10% Px188 is less effective than 0.20% Px188.

[0195] Example 14 The protective effect of combining LTG-MC or LTG-MC with Px188 This embodiment evaluated the protective effects of LTG-MC alone and in combination with poloxamer 188 (Px188). LTG-MC is an unconventional methylcellulose with an s23 / s26 ratio in the range of 0.16 to 0.25.

[0196] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL shake flasks with baffles. Each shake flask had a cell culture volume of 15 mL and a rotation speed of 160 RPM. Hycell was used. TM Cell culture medium (Cytiva). As shown in Table 14, add LTG methylcellulose (LTG-MC), Px188, or a combination of LTG-MC and Px188 with a viscosity of 2 cPs to the medium. Add Gibco daily. TM FoamAway TM Irradiate AOF (animal-free) defoamer (Thermo Fisher Scientific) (10 µL / day) to prevent foaming in the system. Start with 3 × 10⁻⁶ at the beginning of the culture. 5 Cells were seeded into each reactor at a concentration of 1 cell / mL. Before being added to shake flasks under these conditions, the seeded cells were centrifuged and resuspended in a medium containing the desired (or one) additives. The viable cell density (VCD) shown in Table 14 is the average of three replicates. Cell counting was performed after centrifugation and cell resuspending in HyCell without methylcellulose or poloxamer. TM After adding to the culture medium, use Countess TM The 3FL automated cell counter was performed using conventional trypan blue counting.

[0197] Table 14 below shows the observation VCDs of the test samples.

[0198] Table 14 In Table 14, the percentages are based on the weight percentage of the total cell culture.

[0199] As shown in Table 14, LTG-MC provides greater protection than Px188. Furthermore, the combination of LTG-MC and Px188 provides unexpected protection, exceeding the combined effect of LTG-MC and Px188 alone.

[0200] Example 15 Unexpected benefits of combining methylcellulose and poloxamer 188 were observed at various methylcellulose / poloxamer 188 ratios (3L reactor). CHO DG-44 cells expressing IgG1 antibody were amplified in an Applikon Biotechnology 3L glass dish-bottom reactor (model #Z611000310) using an Applikon Biotechnology EZ-Control controller (model #Z310110011) under the conditions shown in Table 15: Table 15 Using Hycell TM Cell culture medium (Cytiva). Custom-ordered Cytiva HyClone without poloxamer 188 (Px188) is used. TM Cellboost TM Supplements 7a and 7b are used as supplementary feeds to achieve the target glucose concentrations shown in the feed protocols in Table 16 (percentages refer to the weight percentage of total cell culture, and the target glucose concentration is based on total cell culture).

[0201] Table 16 As shown in Table 17, add conventional methylcellulose (MC), Px188, or a combination of MC and Px188 with a viscosity of 15 cPs to the culture medium. Add Gibco as needed. TM FoamAway TM Irradiated AOF (animal-free) defoamer (ThermoFisher Scientific) is used to prevent system foaming (total amounts are provided in Table 17).

[0202] Viable 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. 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. Peak viable cell density is the highest viable cell density measured during fourteen days of culture.

[0203] 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.

[0204] At the end of the experiment, the protein titers measured using the Cedex Bio HT analyzer were evaluated. IgG Bio HT is a test kit for determining IgG and measures the turbidity after IgG protein nanoprecipitation. Turbidity is proportional to IgG content, and IgG concentration is determined by comparing it to the turbidity of a solution of IgG at a known concentration.

[0205] Table 17 below shows the results.

[0206] Table 17 In Table 17, the percentages are based on the weight percentage of the total cell culture.

[0207] As shown in Table 17, methylcellulose, Px188, and combinations thereof are effective in conventional bioreactors and enable high VCD and protein production. Surprisingly, as can be seen by comparing sample 88 with samples 87 and 86, the combination of MC and Px188 at a total addition of 0.5% is more effective than MC or Px188 alone in increasing peak VCD or protein titers. This data also demonstrates the effectiveness of methylcellulose in protecting cell cultures from antifoaming agent damage, as shown in samples 87, 89, and 90.

[0208] 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.

[0209] 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, comprising polysaccharides and polyether surfactants.

2. The cell culture medium according to claim 1, wherein the polyether surfactant is poloxamer 188.

3. The cell culture medium according to claim 1 or 2, wherein the polysaccharide is a cellulose derivative.

4. The cell culture medium according to any one of claims 1-3, wherein the cellulose derivative is methylcellulose or hydroxypropyl methylcellulose or a combination thereof.

5. The cell culture medium according to any one of claims 1-4, wherein the polysaccharide has a solution viscosity of less than 10,000 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 polysaccharide has a solution viscosity of less than 2,000 cP in water at 2% concentration at 20 degrees Celsius.

7. The cell culture medium according to any one of claims 1-6, wherein the polysaccharide has a solution viscosity of less than 100 cP in water at 2% concentration at 20 degrees Celsius.

8. The cell culture medium according to any one of claims 1-7, wherein the polysaccharide has a solution viscosity of less than 20 cP in water at 2% concentration at 20 degrees Celsius.

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

10. The cell culture medium according to any one of claims 1-8, wherein the polysaccharide is hydroxypropyl methylcellulose of chemical type E with a viscosity of 5 cP in a 2% solution in water at 20 degrees Celsius.

11. The cell culture medium according to any one of claims 1-8, wherein the polysaccharide is hydroxypropyl methylcellulose with the K chemical form, having a viscosity of 3 cP in a 2% solution in water at 20 degrees Celsius.

12. The cell culture medium according to any one of claims 1-11, wherein the substitution pattern of the methylcellulose or hydroxypropyl methylcellulose is defined such that the s23 / s26 ratio is 0.16 to 0.

36.

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

14. A method for increasing cell growth, comprising: Provide cell culture media containing a combination of cellulose derivatives and polyether surfactants; Mix the cells with the cell culture medium; and The cells and culture medium are incubated to enable them to grow.

15. The method of claim 14, wherein the step of providing the combination of the cellulose derivative and the polyether surfactant provides the combination at a concentration ranging from 0.01% to 5%.

16. The method of claim 14, wherein the polyether surfactant is poloxamer 188.

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

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

19. The method of claim 17, wherein the substitution mode of the methylcellulose is defined such that the s23 / s26 ratio is 0.16 to 0.

36.

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

21. The method according to claim 20, wherein the hydroxypropyl methylcellulose is a hydroxypropyl methylcellulose having the K chemical type, with a solution viscosity of 3 cP in 2% water at 20 degrees Celsius.

22. The method according to claim 20, wherein the hydroxypropyl methylcellulose is an E-type hydroxypropyl methylcellulose with a viscosity of 5 cP in a 2% solution in water at 20 degrees Celsius.

23. The method of claim 20, wherein the substitution mode of the hydroxypropyl methylcellulose is defined as such that the s23 / s26 ratio is 0.16 to 0.

36.

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

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

26. The method of claim 14, wherein the cells are derived from the Vero cell line.

27. The method of claim 14, wherein the cell produces proteins.

28. The method of claim 14, wherein the cells produce antibodies.

29. The method of claim 14, wherein the cell produces antibody-derived proteins.

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

31. The method of claim 14, wherein the cells produce gene therapy.

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