Protective effect of polysaccharides on cell cultures containing antifoam

Abstract

A cell culture medium for suspending cells is disclosed, comprising a combination of polysaccharides and an antifoaming agent. Additionally, methods for increasing cell growth include providing a cell culture medium containing a combination of polysaccharides and an antifoaming agent. Exemplary polysaccharides include cellulose derivatives.

Description

Technical Field

[0001] This field relates to the effects of cellulose derivatives on promoting cell growth, whether used alone or in combination with defoamers. Background Technology

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

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

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

[0005] As the industry further develops, other cell types of animal origin are being considered for adaptation or direct application in suspension culture. Suspension culture is desirable for cell growth because it allows for more efficient space utilization compared to two-dimensional growth on a flat surface.

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

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

[0008] As regulatory agencies begin to require fully characterized cell culture media, it is also necessary to remove animal and human-derived components, including serum, from these media. 3D suspension cell cultures are prone to foaming due to significant agitation and the presence of interfacial active materials in bioreactors, which can lead to numerous process challenges. To mitigate this issue, defoamers, such as simethicone and simethicone formulations, are added as components to the bioreactor.

[0009] Simethicone defoamers can be toxic to cells; therefore, while their use is necessary to reduce the process challenges of foaming, solutions are needed to reduce toxicity and allow cell growth.

[0010] Methylcellulose is used in the food industry as a shear stabilizer for suspension cell culture. Understanding 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, XI, 155-179, 1969.), and its use in suspension culture dates back to the early 1990s (Goldblum, S, et al., “Protective Effect of Methylcellulose and Other Polymers on Insect Cells Subjected to Laminar Shear Stress,” Biotechnology Progress, 6, 373-390, 1990.). The use of methylcellulose alone in suspension cell culture is disclosed in WO2021248141. Despite this understanding, its adoption in the pharmaceutical industry is not thought to have occurred due to its inferior performance compared to poloxamer.

[0011] There is a need to use defoamers to improve the process of cell culture media. Summary of the Invention

[0012] Cell culture media for cell suspension growth contain a combination of polysaccharides and antifoaming agents. Additionally, methods for increasing cell growth include: providing a cell culture medium containing a combination of polysaccharides and antifoaming agents; combining cells with the cell culture medium; and incubating the cells and culture medium to allow growth. Disclosed methods include methods for increasing cell growth that include: providing a cell culture medium containing a combination of cellulose derivatives and antifoaming agents; combining cells with the cell culture medium; and incubating the cells and culture medium to allow growth. Detailed Implementation

[0013] The examples provided in the detailed description are merely examples and should not be used to limit the scope of the claims in any construction or interpretation of the claims.

[0014] A method for enabling cells to grow in suspension has been disclosed, which includes adding polysaccharides and antifoaming agents.

[0015] Methylcellulose is known in the art to be used in suspension cell culture, rather than to protect cells from the toxic effects of antifoaming agents.

[0016] In one instance, the cell culture medium did not include serum.

[0017] The following describes some of the key concepts in the instruction manual.

[0018] abbreviation. As stated in this specification, "MC" is an abbreviation for conventional methylcellulose, and "HPMC" is an abbreviation for conventional hydroxypropyl methylcellulose.

[0019] Defoamer. The cell culture medium includes an antifoaming agent. The antifoaming agent is optionally a simethicone antifoaming agent and includes emulsions such as Gibco Foam Away. ® .

[0020] As described on the website https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / FoamAwayAOF_man.pdf, Gibco FoamAway™ Irradiated AOF (also referred to as "FoamAway™" in the instructions) is manufactured using a 30% simethicone emulsion USP containing methylcellulose and contains no components derived from human or animal sources. When the defoamer is added to cell culture media, the resulting concentration of methylcellulose in the culture medium is less than 0.01%.

[0021] 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.

[0022] Polysaccharides. Exemplary polysaccharides used in cell culture media in this disclosure include cellulose derivatives, alginate, carrageenan, α-glucan, β-glucan, and dextran and their chemical derivatives 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.

[0023] Cellulose derivatives. Exemplary cellulose derivatives for use in cell culture media in this disclosure include methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethyl methylcellulose, and any combination thereof.

[0024] In one instance, the hydroxyl group of a cellulose derivative may include an alkyl substituent or a hydroxyalkyl substituent or a combination thereof.

[0025] In one instance, the cellulose derivative is methylcellulose. In another instance, the cellulose derivative is hydroxypropyl methylcellulose.

[0026] 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.

[0027] 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%.

[0028] Cells that can use the disclosed methods can be used. The disclosed methods can be used with any one or a combination of the following exemplary cells: animal cells, insect cells, plant cells, eukaryotic cells, prokaryotic cells, mammalian cells, cells adapted for suspension, immortalized cells, Chinese hamster ovary (CHO) cells, human endothelial kidney (HEK) 293 cells, and VERO cells. Other cells and cell lines known in the art and suitable for the disclosed methods can be used.

[0029] The following text describes methylcellulose and hydroxypropyl methylcellulose in more detail.

[0030] Methylcellulose and hydroxypropyl methylcellulose Methylcellulose is a cellulose ether formed through the methylation of cellulose. Cellulose is a naturally occurring polysaccharide produced by many plants, including trees and cotton. Polysaccharide polymers consist of dehydrated glucose units linked by β-1,4 bonds.

[0031] Each dehydrated glucose unit contains hydroxyl groups at positions 2, 3, and 6. Partial or complete substitution of these hydroxyl substituents produces cellulose derivatives.

[0032] Cellulose derivatives are well-known across the pharmaceutical and food industries. Cellulose derivatives are defined in several ways. The United States Pharmacopeia (USP) defines them by their chemical derivatization and molecular weight. They can also optionally be further defined by a chemical derivatization pattern around the dehydrated glucose unit, such as by the s23 / s26 ratio.

[0033] Chemical derivatizing agents. The reaction of cellulose with derivatizing agents yields cellulose derivatives. Derivatizing agents include etherifying agents. For example, etherifying agents include methylating agents.

[0034] Cellulose ethers are examples of cellulose derivatives formed by the reaction of cellulose with an etherifying agent. For example, cellulose fibers are treated with an alkaline solution, followed by treatment with an etherifying agent such as chloromethane, to obtain cellulose ethers, methylcellulose, a cellulose derivative.

[0035] If a cellulose ether is substituted with hydroxypropyl and methyl groups, such a cellulose ether is called hydroxypropyl methylcellulose or hydroxypropyl methylcellulose (HPMC), an example of a cellulose derivative.

[0036] Many cellulose derivatives are also defined by the United States Pharmacopeia (USP).

[0037] For example, methylcellulose is defined by the USP as having methylated substitutions of hydroxyl groups of not less than 26% and not more than 33%.

[0038] Hydroxypropyl methylcellulose (HMCMC) is defined by the USP as having four different substitution types. The E chemistry is defined as follows: Type 2910 or E-type substitution has 28-30% methoxy group and 7-12% hydroxypropyl group substitution. The K chemistry is defined as follows: Type 2208 or K-type substitution has 19-24% methoxy group and 4-12% hydroxypropyl group substitution. The F chemistry is defined as follows: Type 2906 or F-type substitution has 27-30% methoxy group and 4-7.5% hydroxypropyl group substitution. The J chemistry is defined as follows: Type 1828 or J-type substitution has 16.5-20% methoxy group and 23-32% hydroxypropyl group substitution.

[0039] Molecular weight. The molecular weight of cellulose derivatives is typically described by the approximate viscosity of an aqueous solution of the polymer. These solutions are typically 2% by weight. For example, a 15 cP methylcellulose polymer is a methylcellulose in which a 2% aqueous solution of methylcellulose has a viscosity of approximately 15 cP. The methods used to determine the viscosity of this solution are well known to those skilled in the art and are defined by the United States Pharmacopeia, Chapter 912.

[0040] Chemical substitution patterns can be defined by the s23 / s26 ratio. Cellulose ether dehydrated glucose units may have more than one hydroxyl group substituted by a derivative. A chemical substitution pattern can be defined by the s23 / s26 ratio, where s23 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose unit are substituted, and where s26 is the mole fraction of dehydrated glucose units in which only two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose unit are substituted. Further definitions of substitution patterns can be found in EP1171471, WO20000 / 59947, US6235893, and US6228416.

[0041] The ratio of hydroxyl positions on the 1,4-hydroglucose ring to their substitutions is not defined in the USP or other international pharmacopoeias.

[0042] 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.

[0043] Defining the substitution pattern of substituents on cellulose by the s23 / s26 ratio to form cellulose derivatives is optional.

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

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

[0046] In one instance, the favorable s23 / 26 is 0.23 to 0.32.

[0047] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is hydroxypropyl methylcellulose (HPMC). HPMC has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with hydroxypropyl or methyl groups, such that the s23 / s26 ratio is 0.16 to 0.36, where s23 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups. The foregoing is an example of unconventional HPMC. This unconventional HPMC includes both hydroxypropyl and methyl groups in its overall chemical structure.

[0048] In another example, the cellulose derivative in which the s23 / s26 ratio is defined is hydroxypropyl methylcellulose. Hydroxypropyl methylcellulose has dehydrated glucose units linked by 1-4 bonds, wherein the hydroxyl groups of the dehydrated glucose units are individually substituted with hydroxypropyl or methyl groups, such that the s23 / s26 ratio is 0.26 to 0.32, where s23 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 3-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups, and where s26 is the mole fraction of dehydrated glucose units in which only the two hydroxyl groups at the 2- and 6-positions of the dehydrated glucose units are substituted with hydroxypropyl or methyl groups. These polymers are called SG hydroxypropyl methylcellulose (“SG-HPMC”), an example of unconventional HPMC. This unconventional SG-HPMC includes both hydroxypropyl and methyl groups in its overall chemical structure.

[0049] Unconventional cellulose derivatives. Methylcellulose and hydroxypropyl methylcellulose with s23 / 26 in the range of 0.16 to 0.36 are considered unconventional cellulose derivatives.

[0050] For example, exemplary SG methylcellulose and SG HPMC are manufactured as described in EP1171471, WO20000 / 59947, US6235893 and US6228416.

[0051] The experimental conditions for validating the proposed cell culture medium and related methods are described below.

[0052] Example 1 Methylcellulose, for example, increases the number of viable cells and protects cell viability in cell cultures containing antifoaming agents. Agarabi CHO cells (ATCC, CRL3440) were expanded in baffle-free shake flasks using Hycell™ cell culture medium available from Cytiva. Baffles are generally not used in CHO cultures because their use imposes additional shear forces on the cells.

[0053] The culture medium was supplemented with 6 mM L-glutamine, and 1.5 mL of HyClone™ CellBoost™ 1 supplement was added every other day. Cells were maintained at 8% CO2 and 37°C.

[0054] The cell culture volume was 15 mL. Using the Countess™ 3 FL Automated Cell Counter, conventional trypan blue counting was performed, with 0.5 mL taken out at each time point for cell counting.

[0055] As is known in the art, the document provided at this link (https: / / www.sigmaaldrich.com / deepweb / assets / sigmaaldrich / marketing / global / documents / 331 / 916 / t8154use.pdf) teaches that "trypan blue is one of several dyes recommended for dye exclusion procedures in live cell counting." The aforementioned document also states that "this method is based on the principle that live (surviving) cells do not absorb certain dyes, while dead (non-surviving) cells absorb certain dyes. Staining facilitates the visualization of cell morphology." Therefore, it is known to those skilled in the art that trypan blue staining facilitates the visualization and differentiation of live and dead cells.

[0056] For samples marked "AF" in Table 1 below, add 10 µL of Gibco to the culture medium daily. ® FoamAway ™ Irradiated AOF (animal-free) defoamer (also known as "Foam Away™"). The abbreviation "AF" in this sample indicates cells treated with the defoamer.

[0057] For the samples marked "MC" in Table 1, the culture medium was supplemented with 0.2% of conventional methylcellulose 15cP.

[0058] Table 2 below reports the average total number of viable cells in the flasks. Each sample was performed in triplicate.

[0059] The following table shows the measurement of live cells / mL: Table 1 Defoamers inhibit cell growth. Compared to the initial cell levels, the addition of the defoamer (sample 2) resulted in a decrease in viable cell density per day and led to zero cell viability on day 7. For day 7, there was no reported mean total viable cell count in the sample containing AF (the defoamer). This surprisingly demonstrates that cells simply cannot grow in the presence of the defoamer.

[0060] Compared with the control, the inclusion of 0.2% methylcellulose resulted in an increase in the mean total viable cell count. When comparing the results on day 7 for the sample containing 0.2% methylcellulose (Sample 3), the mean total viable cell count was approximately 5% higher than that in the control (Sample 1). This demonstrates that methylcellulose has no adverse effect on cell growth in this culture system, but also no significant benefit.

[0061] Compared with the control, the combination of 0.2% methylcellulose and defoamer resulted in a comparable total live cell count.On day 7, the mixture of methylcellulose and AF (sample 4) had approximately 7% fewer mean viable cells than the control (sample 1). Surprisingly and unexpectedly, the combination of methylcellulose and antifoaming agent in sample 4 exhibited cell growth, unlike sample 2 which showed no visible cell growth with only antifoaming agent. This result indicates that methylcellulose has a cell-protective effect on mixtures containing methylcellulose and AF, as samples containing AF alone had no mean total viable cell count on day 7. This result is surprising and unexpected, as methylcellulose does not provide a significant benefit to cell growth in this culture system.

[0062] Example 2 The inclusion of methylcellulose in cell culture media containing defoamers leads to cell protection at high shear rates. This example demonstrates that methylcellulose protects cells from damage by antifoaming agents at various shear rates. The experiment was set up as in Example 1, except that a baffled flask was used and the rotation of the vibrator was varied to generate higher and more variable shear forces. The presence of the baffle generated higher shear forces, and higher rpm resulted in even greater shear forces. Cells / mL after 48 hours of culture are reported.

[0063] The sample with only defoamer added was not tested because the results of sample 2 in Example 1 determined that cells could not grow effectively in the presence of defoamer alone.

[0064] The results are reported in the table below.

[0065] Table 2 The control sample showed lower growth efficiency under higher shear forces. Comparing cell growth in sample 5, at 100 rpm, there was a 2% increase in cell number compared to 80 rpm. At 130 rpm, there was a 33.5% decrease in cell number compared to 80 rpm. At 160 rpm, there was a 97% decrease in cell number compared to 80 rpm. This indicates that the cells are sensitive to shearing and do not grow well at higher shear rates (expressed as higher rpm).

[0066] Under higher shear stress, the sample containing 0.2% methylcellulose showed increased cell growth compared to the control.At 80 rpm, comparing cell growth in the sample containing 0.2% methylcellulose (Sample 6) with the control without additives (Sample 5), there was a 31% increase in cell count with the presence of 0.2% methylcellulose. At 100 rpm, comparing cell growth in the sample containing 0.2% methylcellulose (Sample 6) with the control without additives (Sample 5), there was a 1% decrease in cell count with the presence of 0.2% methylcellulose. At 130 rpm, comparing cell growth in the sample containing 0.2% methylcellulose (Sample 6) with the control without additives (Sample 5), there was a 92% increase in cell count with the presence of 0.2% methylcellulose. At 160 rpm, comparing cell growth in the sample containing 0.2% methylcellulose (Sample 6) with the control without additives (Sample 5), there was a 35-fold increase in cell count with the presence of 0.2% methylcellulose, which is equivalent to a 3500% increase in cell count. This demonstrates that methylcellulose can alleviate the increased sensitivity of cells to shear stress.

[0067] Samples containing 0.2% methylcellulose showed comparable cell growth under different shear forces. Comparing cell growth in sample 6, at 100 rpm, there was a 23% reduction in cell number compared to 80 rpm. At 130 rpm, there was a 2.3% reduction in cell number compared to 80 rpm. At 160 rpm, there was an 18% reduction in cell number compared to 80 rpm. This demonstrates that methylcellulose can protect cells from shear sensitivity in this baffled flask culture system.

[0068] Samples containing 0.2% methylcellulose and 10 µL of defoamer showed comparable performance at 80, 100, and 130 rpm. Cell growth. Comparing cell growth in sample 7, at 100 rpm, there was an 11% decrease in cell number compared to 80 rpm. At 130 rpm, there was a 4.1% increase in cell number compared to 80 rpm. At 160 rpm, there was a 97% decrease in cell number compared to 80 rpm. This demonstrates that at 80, 100, and 130 rpm, methylcellulose can protect cells from damage caused by the antifoaming agent, while also protecting cells from some of their shear sensitivity in this baffled flask culture system.

[0069] Example 3 When defoamers are present in bioreactors, methylcellulose improves cell growth. A proprietary CHO DG-44 cell line expressing IgG1 antibodies (referred to as "cells") is adapted and grown in Hycell™ medium (available from Cytiva). The adaptation and accompanying growth process is known to those skilled in the art. Cells are adapted to Hycell™ medium through sequential growth in a medium containing more Hycell™ until they grow in Hycell-only medium. The use of Hycell™ medium allows those skilled in the art to perform appropriate scientific controls to understand the growth effects of adding various additives to the medium, as it does not contain shear protectants. Hycell™ medium has a viscosity of 2.23 cP when measured at 37°C.

[0070] 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.

[0071] 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.

[0072] The samples were stirred by a stirring shaft in the reactor. In addition to the basal culture medium, custom-ordered Cytiva HyClone™ Cellboost™ 7a and 7b supplements, free of poloxamer 188, were used as supplemental feed to achieve a glucose concentration of 6 g / L.

[0073] Add Gibco daily ® FoamAway™ Irradiated AOF (animal-free) defoamer (10 µL / day) is used to prevent foaming in the system. At the start of culture, add 0.3 x 10⁻⁶ ppm. 6 Cells were seeded at 100 cells / mL into each reactor and harvested after 14 days or after cell viability dropped to below 70% (whichever was earlier).

[0074] Live cell density was measured throughout the 14-day experiment. ® ViaCount™ reagents, along with flow cytometry and the Vi-Cell™ BLU cell viability analyzer (Beckman Coulter), are used to measure live cell density.

[0075] ViaCount™ works by differential nuclear staining of live and dead cells. The Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue rejection.

[0076] Peak viable cell density is the highest viable cell density measured during the fourteen-day culture period.

[0077] Harvest survival rate is the percentage of live cells at the end of 14 days of culture.

[0078] Protein titer was 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 is a measurement of turbidity following nanoprecipitation of IgG protein. Turbidity is proportional to IgG content, and IgG concentration is determined by comparing the turbidity with that of a solution of known IgG concentration.

[0079] In Sample 8, no additional additives were added to the culture medium. In Sample 9, 0.2% MC 15 cP was added to the culture medium.

[0080] Sample 8 was run twice and sample 9 was run four times. The average of these replicates is reported.

[0081] The table below measures the peak viable cell density, harvest survival rate, and IgG titer in Example 3.

[0082] Table 3 Compared to the use of defoamers alone, the combination of defoamer and methylcellulose resulted in approximately six times the peak active cell density. Cell density. With the addition of 0.2% methylcellulose (Sample 9), the mean peak viable cell density increased surprisingly and unexpectedly to nearly six times compared to the sample (Sample 8) grown in a medium containing only FoamAway™ and without methylcellulose, as shown in Table 3 above. In other words, the mean viable cell density in the combination of the antifoaming agent and 0.2% methylcellulose was approximately 600% of the control.

[0083] With the addition of 0.2% methylcellulose (Sample 9), the average cell viability at harvest increased by 61% compared to the sample (Sample 8) grown in a medium containing only FoamAway™. In other words, the average cell viability in the combination was approximately 161% of that in Sample 8.

[0084] In sample 8, no protein IgG was detected after incubation. In contrast, for sample 9, 588 µg / mL of protein IgG was detected after incubation.

[0085] The above text demonstrates that in bioreactor systems, the use of defoamers such as FoamAway™ is detrimental to cell growth, survival, and the ability to produce proteins. Surprisingly and unexpectedly, results have shown that methylcellulose can mitigate the damage caused by defoamers, leading to increased cell growth, higher survival rates, and protein production.

[0086] Example 4 Different polysaccharides can protect cells from the damaging effects of antifoaming agents.

[0087] This experiment was set up as in Example 3, but different polysaccharide polymers were added to the cell culture medium. Methylcellulose polymers with different molecular weights and different substitution patterns were used. As with other methylcelluloses, the molecular weight was not measured directly, but rather expressed as the viscosity of its 2% aqueous solution at 20°C.

[0088] The polymers used and the results are described in Table 4 below.

[0089] Protein titer was measured by protein A. As is known in the art, protein A chromatography is used to measure IgG levels. Protein A-functionalized beads bind IgG, allowing IgG to be separated. After IgG is eluted from the beads, the protein content is measured to indicate IgG levels. Protein A chromatography using an Agilent Bio-Monolith Protein A affinity column and an HPLC system equipped with UV detection were used in this embodiment to measure protein titers.

[0090] The table below shows the samples tested in this embodiment and their corresponding peak viable cell density and protein titer.

[0091] Table 4 The results were compared with those of Sample 8 from Example 3 (FoamAway™ only).

[0092] When comparing the viscosity of the culture medium in samples 10, 12 and 13, a slight increase in viscosity was observed as the molecular weight of methylcellulose increased.

[0093] When comparing the viscosity of the culture medium in samples 10 and 11, the viscosity is comparable and similar.

[0094] When comparing the viscosity of the culture medium in samples 13 and 14, the viscosity is comparable and similar.

[0095] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with that of Sample 10 (MC–2 cP), the peak viable cell density was 4.9 times higher in Sample 10.

[0096] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with that of Sample 11 (SG MC – 2 cP), the peak viable cell density was 8.4 times higher in Sample 11.

[0097] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with that of Sample 12 (MC – 338 cP), the peak viable cell density was 6.3 times higher in Sample 12.

[0098] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with that of Sample 13 (MC – 2880 cP), the peak viable cell density was 8.3 times higher in Sample 13.

[0099] When comparing the peak viable cell density of Sample 8 (FoamAway™ only) of Example 3 with that of Sample 14 (MC – 2 cP, 2.4%), the peak viable cell density was 5.4 times higher in Sample 14.

[0100] When comparing the peak viable cell density of sample 13 (MC – 2880 cP, 0.2%) with that of sample 14 (MC – 2 cP, 2.4%), the peak viable cell density was 45% higher in sample 13.

[0101] Culture medium viscosity is not a factor contributing to the peak increase in viable cell density. When comparing the average protein IgG titer of Sample 13 with that of Sample 14, the average protein IgG titer in Sample 13 was 49% higher. This indicates that the increase in peak viable cell density was not due to the higher viscosity of the culture medium in Sample 13 compared to other samples containing 0.2% methylcellulose.

[0102] When comparing the average protein IgG titers of samples 10-14 with those of sample 8, all samples 10-14 had measurable protein IgG titers, while sample 8 (FoamAway™ only) had no measurable protein IgG titers.

[0103] The results for sample 11 (SG MC - 2cP, 0.2%) confirmed that unconventional methylcellulose was surprisingly and unexpectedly able to mitigate the damage caused by the defoamer, leading to cell growth and protein production.

[0104] The results for samples 10, 12, and 13 confirmed that methylcellulose with different molecular weights (MC – 2 cP, 0.2%; MC – 338 cP, 0.2%; and MC – 2880 cP, 0.2%) was surprisingly and unexpectedly able to mitigate the damage caused by the defoamer, leading to cell growth and protein production.

[0105] Example 5 Unexpected benefits were observed from combining methylcellulose and poloxamer 188 across different methylcellulose / poloxamer 188 ratios. Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. As shown in Table 5, standard methylcellulose (MC), poloxamer 188 (Px188), or a combination of MC and Px188 with a viscosity of 15 cP were added to the medium. Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) (10 μL / day) was added daily to prevent system foaming. At the start of culture, cells were introduced at a rate of 3 x 102 5 Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. The seeded cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 5 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0106] Table 5 below shows the VCD observed for the test samples.

[0107] Table 5 In Table 5, 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 expected VCD if the effects of MC and Px188 on VCD are simply additive. Therefore, expected VCD = (VCD with MC only) x (MC fraction) + (VCD with Px188 only) x (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.

[0108] As shown in Table 5, 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 indicating an unexpected benefit from the combination across the entire ratio range.

[0109] Example 6 Effect of reduced additive concentration in high-shear systems Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask contained a 15 mL cell culture volume. Hycell™ cell culture medium (Cytiva) was used. As shown in Tables 6 and 7, standard methylcellulose (MC), poloxamer 188 (Px188), or a combination of MC and Px188 with a viscosity of 15 cP were added to the medium. Experiments in Table 6 were performed using a shaker speed of 160 RPM, while experiments in Table 7 were performed using a shaker speed of 130 RPM. Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) (10 μL / day) was added daily to prevent system foaming. At the start of culture, cells were introduced at a rate of 3 x 102 5 Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. The seed chain cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Tables 6 and 7 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0110] Table 6 below shows the VCD observed for test samples in a higher shear system in which the shaking speed was 160 RPM.

[0111] Table 6 Table 7 below shows the VCD observed for test samples in a lower shear system in which the shaking speed was 130 RPM.

[0112] Table 7 In Tables 6 and 7, the percentages are based on the weight percentage of the total cell culture.

[0113] As shown in Tables 6 and 7, total additive concentrations from 0.01% to 0.2% were tested using shake flasks at 160 or 130 RPM. In the higher shear system with a shake flask speed of 160 RPM, the protective effect of the additive tended to be more pronounced at total additive concentrations of at least 0.1%. In the higher shear system, MC alone provided protection even at concentrations as low as 0.1%. In the lower shear system with a shake flask speed of 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 at least 0.05%. At the tested concentrations, MC tended to be more protective than Px188 in the lower shear system. This data demonstrates that growth in different culture systems responds differently to the levels of additives included in the culture medium.

[0114] Example 7 The protective effect of SG-MC alone against cell damage caused by antifoaming agents. Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. Regular methylcellulose (2 cP viscosity), SG methylcellulose (SG-MC) (2 cP viscosity), and / or Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) were added as shown in Table 8. At the start of culture, cells were introduced at a rate of 3 x 10⁶ cells / mL. 5 Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. The seed chain cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 8 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0115] Table 8 below shows the VCD observed for the test samples.

[0116] Table 8 In Table 8, the percentages are based on the weight percentage of the total cell culture.

[0117] As shown in Table 8, both conventional MC and SG-MC provide protection against damage from defoamers, with SG-MC offering slightly more protection than conventional MC.

[0118] Example 8 The protective effect of individual polymer additives against cell damage in defoamers In this embodiment, the protective effects of conventional methylcellulose (MC), SG-MC, and LTG-MC were compared individually. LTG-MC is an unconventional methylcellulose with an s23 / s26 ratio in the range of 0.16 to 0.25.

[0119] Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. MC (15 cP viscosity), MC (2 cP viscosity), SG-MC (2 cP viscosity), or LTG-MC (2 cP viscosity) were added as shown in Table 9. Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) (10 μL / day) was added daily to prevent system foaming. At the start of culture, cells were introduced at a rate of 3 x 102 5 Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. Seeded cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 9 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0120] Table 9 below shows the VCD observed for the test samples.

[0121] Table 9 In Table 9, the percentages are based on the weight percentage of the total cell culture.

[0122] As shown in Table 9, all three types of protective mechanisms—conventional MC, SG-MC, and LTG-MC—provide protection, with LTG-MC offering the greatest protection and SG providing even greater protection than conventional MC.

[0123] Example 9 Comparison of the protective effects of different HPMC additives against cell damage from different defoamers In this embodiment, the protective effects of three different cellulose ethers against cell damage from two different defoamers were compared, as shown in Table 10 below.

[0124] The tested cellulose ethers were conventional MC (15 cP viscosity), K3 (K-chemical characteristic hydroxypropyl methylcellulose (HPMC) with 3 cP viscosity), and E5 (E-chemical characteristic HPMC with 5 cP viscosity). All cellulose ethers were sourced from International Flavors & Fragrances Inc. Conventional MC and HPMC had s23 / s26 ratios ranging from 0.37 to 0.42. E-chemical characteristic HPMC is defined as having 28-30% methoxy groups and 7-12% hydroxypropyl substitution, either type 2910 or type E. K-chemical characteristic HPMC is defined as having 19-24% methoxy groups and 4-12% hydroxypropyl substitution, either type 2208 or type K.

[0125] The defoamers tested were Gibco™ FoamAway™ Irradiated AOF (animal-free) defoamer (Thermo Fisher Scientific) and Liveo™ silicone defoamer (DuPont™).

[0126] In this experiment, Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 130 RPM. Hycell™ cell culture medium (Cytiva) was used. At the start of culture, cells were introduced at a rate of 3 x 10⁶ cells / mL. 5 The seeded cells were seeded into each reactor at a concentration of [number] cells / mL. The seeded cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 10 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated CellCounter with conventional trypan blue counting.

[0127] Table 10 below shows the VCD observed for the test samples.

[0128] Table 10 In Table 10, the percentages are based on the weight percentage of the total cell culture.

[0129] As shown in Table 10, all tested cellulose ethers individually provided protection against defoamer-induced cell damage.

[0130] Example 10 Protective effect of methylcellulose alone against cell damage caused by defoamers at different defoamer levels Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. All samples contained 0.20% by weight of standard MC (15 cP) added to the medium. Gibco™ FoamAway™ Irradiated AOF (animal-free) antifoaming agent (Thermo Fisher Scientific) was added in the amounts described in Table 11. At the start of culture, cells were introduced at a rate of 3 x 10⁶ cells / mL. 5 The seeded cells were seeded into each reactor at a concentration of [number] cells / mL. The seeded cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 11 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using a Countess™ 3 FL Automated Cell Counter with conventional trypan blue counting.

[0131] Table 11 below shows the VCD observed for the test samples.

[0132] Table 11 In Table 11, the percentages are based on the weight percentage of the total cell culture.

[0133] As shown in Table 11, the protective effect of MC alone against defoamer cell damage decreases as the amount of defoamer used increases.

[0134] Example 11 Comparison of the protective effects of conventional HPMC against cell damage from non-silicone defoamers In this experiment, Agarabi CHO cells (ATCC, CRL3440) were expanded in 125 mL baffled shake flasks. Each flask had a 15 mL cell culture volume and a rotation speed of 160 RPM. Hycell™ cell culture medium (Cytiva) was used. AF204 non-silicone antifoaming agent (Sigma-Aldrich) (7 ppm) was added daily to prevent foaming of the system. Regular MC (15 cP) was also included, as shown in Table 12.

[0135] At the start of the culture, the cells were introduced at a rate of 3 x 10⁻⁶. 5 Seeded cells were inoculated into each reactor at a concentration of [number] cells / mL. Seeded cells were centrifuged and resuspended in medium containing the desired additives, then added to shake flasks for that condition. The viable cell density (VCD) shown in Table 12 is the average of three replicates. After centrifugation and resuspending in HyCell™ medium without methylcellulose or poloxamer, cell counting was performed using conventional trypan blue counting with a Countess™ 3 FL Automated CellCounter.

[0136] Table 12 below shows the VCD observed for the test samples.

[0137] Table 12 In Table 12, percentages and concentrations are based on total cell culture.

[0138] As shown in Table 12, without the use of methylcellulose, the cultures did not survive to day 2 in the presence of the added defoamer.

[0139] Example 12 Unexpected benefits were observed from combining methylcellulose and poloxamer 188 across different methylcellulose / poloxamer 188 ratios (3L reactor). Under the conditions shown in Table 13, CHO DG-44 cells expressing IgG1 antibodies were amplified in an Applikon Biotechnology 3L glass dish bottom reactor (model Z611000310) equipped with an Applikon Biotechnology EZ-Control (model Z310110011) controller: Table 13 Hycell™ cell culture medium (Cytiva) was used. Custom-ordered Cytiva HyClone™ Cellboost™ 7a and 7b supplements without poloxamer 188 (Px188) were used as supplemental feed to achieve glucose concentrations as shown in the feed regimens in Table 14 (percentages are based on weight percentage of total cell culture, and the target glucose concentration is based on total cell culture).

[0140] Table 14 As shown in Table 15, add conventional methylcellulose (MC), Px188, or a combination of MC and Px188 with a viscosity of 15 cP to the culture medium. Add Gibco™ FoamAway™ Irradiated AOF (animal-free) defoamer (Thermo Fisher Scientific) as needed to prevent system foaming (total amounts are provided in Table 15).

[0141] Live cell density was measured throughout the 14-day experiment. ™ ViaCount™ reagents, along with flow cytometry and the Vi-Cell™ BLU cell viability analyzer (Beckman Coulter), are used to measure viable cell density. ViaCount™ works by differential nuclear staining of viable and dead cells. The Vi-Cell™ BLU cell viability analyzer works by measuring trypan blue rejection. Peak viable cell density is the highest viable cell density measured during a 14-day culture period.

[0142] As is known in the art, protein A chromatography is used to measure IgG levels. Protein A-functionalized beads bind IgG, allowing IgG to be separated. After IgG is eluted from the beads, the protein content is measured to indicate the IgG level.

[0143] At the end of the experiment, protein titers measured using the Cedex™ Bio HT analyzer were evaluated. IgG Bio HT is a test kit for determining IgG and is a measurement of turbidity following nanoprecipitation of IgG protein. Turbidity is proportional to IgG content, and IgG concentration is determined by comparing the turbidity of the solution with that of a known concentration of IgG.

[0144] Table 15 below shows the results.

[0145] Table 15 In Table 15, the percentages are based on the weight percentage of the total cell culture.

[0146] As shown in Table 15, methylcellulose, Px188, and combinations of both are all effective and allow for high VCD and protein production in conventional bioreactors. Surprisingly, the combination of MC and Px188 with 0.5% total additives is more effective than MC or Px188 alone in increasing peak VCD or protein titers, as can be seen by comparing sample 76 with samples 75 and 74. This data also confirms the effectiveness of methylcellulose in protecting cell cultures from damage by antifoaming agents, as shown by samples 75, 77, and 78.

[0147] The numerical ranges listed in the specification include values ​​that are considered equivalent to the listed values ​​(e.g., having the same function or result) by those skilled in the art, such as + / - 5-10% of the listed values.

[0148] The claims are not limited to the preferred embodiments and examples, but are intended to cover many modifications and equivalents consistent with the written description as a whole.

Claims

1. A cell culture medium for suspending cells for growth, comprising a combination of polysaccharides and an antifoaming agent.

2. The cell culture medium according to claim 1, wherein the defoamer comprises simethicone.

3. The cell culture medium according to claim 1, 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, further comprising a polyether surfactant.

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

7. The cell culture medium according to any one of claims 1-6, wherein the polysaccharide has a viscosity of less than 2,000 cP in a 2% aqueous solution at 20 degrees Celsius.

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

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

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

11. The cell culture medium according to any one of claims 1-10, wherein the polysaccharide is methylcellulose having a substitution pattern defined such that the s23 / s26 ratio is 0.16 to 0.

36.

12. The cell culture medium according to any one of claims 1-9, wherein the polysaccharide is hydroxypropyl methylcellulose with E chemical properties, having a 2% aqueous solution viscosity of 5 cP at 20 degrees Celsius.

13. The cell culture medium according to any one of claims 1-9, wherein the polysaccharide is hydroxypropyl methylcellulose with K chemical properties, having a 2% aqueous solution viscosity of 3 cP at 20 degrees Celsius.

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

36.

15. A method for suspending cells for growth, comprising incubating the cells in a cell culture medium according to any one of claims 1-14.

16. A method for increasing cell growth, comprising: Provide cell culture media containing a combination of cellulose derivatives and antifoaming agents; Combine the cells with the cell culture medium; and Incubate the cells and culture medium to allow them to grow.

17. The method of claim 16, wherein the step of providing the combination comprises providing a cellulose derivative in a concentration range of 0.01% to 5%.

18. The method of claim 16, wherein the defoamer comprises simethicone.

19. The method of claim 16, wherein the cellulose derivative is methylcellulose.

20. The method of claim 19, wherein the methylcellulose is methylcellulose 15 cP.

21. The method of claim 19, wherein the methylcellulose has a substitution pattern defined such that the s23 / s26 ratio is 0.16 to 0.

36.

22. The method of claim 16, wherein the cellulose derivative is hydroxypropyl methylcellulose.

23. The method according to claim 22, wherein the hydroxypropyl methylcellulose is a hydroxypropyl methylcellulose with K chemical properties, having a 2% aqueous solution viscosity of 3 cP at 20 degrees Celsius.

24. The method according to claim 22, wherein the hydroxypropyl methylcellulose is a hydroxypropyl methylcellulose with E chemical properties, having a 2% aqueous solution viscosity of 5 cP at 20 degrees Celsius.

25. The method of claim 22, 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 16, wherein the cells are derived from the Chinese hamster ovary cell line.

27. The method of claim 16, wherein the cells are derived from a human endothelial kidney cell line.

28. The method of claim 16, wherein the cells are derived from the Vero cell line.

29. The method of claim 16, wherein the cell produces proteins.

30. The method of claim 16, wherein the cells produce antibodies.

31. The method of claim 16, wherein the cells produce antibody-derived proteins.

32. The method of claim 16, wherein the cells produce the vaccine.

33. The method of claim 16, wherein the cells generate gene therapy.

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