Cell culture medium

Abstract

The present invention relates to a serum-free cell culture medium, wherein the medium contains maltose as the sole carbohydrate source, or contains maltose and at least one other sugar as the carbohydrate source. In a preferred embodiment, the additional sugar is a monosaccharide, preferably glucose, and the medium comprises DMEM-F12. In a preferred embodiment, the cells to be cultured can be CHO cells or HEK293 cells. Also disclosed are methods of growing and / or culturing cells using a cell culture medium as described herein, methods of increasing protein yield using a cell culture medium as described herein, and kits thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Singapore Patent Application No. 10201601284Y, filed on February 22, 2016, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This invention relates to compositions for the propagation, preservation, or maintenance of cells. Specifically, this invention relates to culture media. The invention also relates to compositions for supporting protein production in cultured cells. Background Technology

[0004] From the very first day of its invention, cell culture media have been gradually improved and studied according to the intended purpose of cultivation. The initial cell culture solutions provided perfusion, water supply, inorganic anions required for cell metabolism, osmotic balance, buffering systems, and a source of carbohydrates for growth.

[0005] As research has progressed on how cell function can be improved, cell culture media have evolved to include a variety of components, which can be included depending on the purpose of the medium. However, what remains constant for cells (such as mammalian cells) is the provision of glucose as a carbohydrate source.

[0006] Therefore, there is a need to provide cell culture media that offer alternative carbohydrate sources. The potential use of alternative carbohydrate sources as energy sources could have practical significance in biopharmaceutical manufacturing. Specifically, the potential use of alternative carbohydrate sources could lead to improvements in carbohydrate loading in batch culture media and could result in reduced lactate accumulation, which can be toxic to cells.

[0007] Therefore, there is a need to provide cell culture media with alternative carbohydrate sources. Summary of the Invention

[0008] In the first aspect, a serum-free cell culture medium is provided. This cell culture medium contains maltose as the sole carbohydrate source.

[0009] In a second aspect, a serum-free cell culture medium is provided, the serum-free cell culture medium comprising maltose and at least one, at least two, at least three or more sugars as a carbohydrate source.

[0010] In a third aspect, methods for growing and / or culturing cells are provided, wherein the methods include growing and / or culturing cells in a serum-free cell culture medium as described in the first or second aspect.

[0011] In a fourth aspect, methods for growing and / or culturing cells are provided, wherein the methods include growing and / or culturing cells in a serum-free cell culture medium as described herein.

[0012] In a fifth aspect, methods for increasing protein yield are provided, wherein the methods include growing and / or culturing cells in a serum-free cell culture medium as described herein.

[0013] In a sixth aspect, a kit is provided that comprises components of a serum-free cell culture medium as described herein. Attached Figure Description

[0014] The invention will be better understood by referring to the detailed description when considered in conjunction with non-limiting embodiments and the accompanying drawings, in which:

[0015] Figure 1 Results of fitness tests of Chinese hamster ovary (CHO) cells in various disaccharide media are shown. In (A), CHO-K1 cells were cultured in serum-free, protein-free HyQ PF-CHO cell culture medium containing 3.6 g / L of different sugars (maltose, sucrose, lactose, trehalose, or glucose) as the carbohydrate source. Viable cell density (marked with lines) and culture viability (marked only) of these cultures were plotted at the beginning and end of each passage over a 74-day period. Cultures containing sucrose, lactose, or trehalose were terminated on day 31 due to decreased culture viability and viable cell density. A 1.0 × 10⁶ sucrose culture medium was used. 6 To obtain similar results, the experiment was repeated at a seeding cell density of 1.0 × 10⁶ cells / mL. In (B), at a seeding cell density of 1.0 × 10⁶ cells / mL... 6 CHO-DG44 cells were cultured at a seeding density of 10 cells / mL in serum-free, protein-free HyQ PF-CHO cell culture medium containing 10 g / L of different sugars (maltose, sucrose, lactose, trehalose, or glucose) as the carbohydrate source. Viable cell density (marked with lines) and cell viability (marked only) of these cultures were plotted at the beginning and end of each passage over a 22-day period. In (C), the seeding density was 1.0 × 10⁻⁶ cells / mL. 6 HEK293 cells were cultured at a seeding density of [number] cells / mL in a chemically defined, protein-free cell culture medium, PFCDM, containing 10 g / L of a different sugar (maltose, sucrose, lactose, trehalose, or glucose) as the carbohydrate source. Viable cell density (marked with lines) and cell viability (marked only) of these cultures were plotted at the beginning and end of each passage over a 22-day period. Cultures containing sucrose, lactose, or trehalose were terminated on day 14 due to decreased culture viability and viable cell density. Therefore, Figure 1 The results showed that CHO-K1 cells, CHO-DG44 cells, and HEK293 cells could proliferate using maltose as a glycogen source in serum-free culture medium, but could not proliferate using sucrose, lactose, or trehalose as a glycogen source.

[0016] Figure 2 Growth and biochemical curves of CHO-K1 cells adapted to maltose medium and cultured in maltose medium are shown compared to those of unadapted CHO-K1 cells cultured in glucose medium. CHO-K1 cells pre-adapted to serum-free, protein-free cell culture medium HyQPF-CHO with 3.6 g / L maltose as the carbohydrate source (square markers) and normal unadapted CHO-K1 cells cultured in the same medium but with glucose as the carbohydrate source (cross markers) were monitored over 14 days to obtain their (A) viable cell density (marked with lines) and culture viability (marked only), as well as (B) glucose curves, (C) lactate curves, (D) glutamine curves, and (E) ammonium curves. To illustrate the specific consumption and production rates of (F) glutamine and (G) ammonium, the concentrations of these components were also plotted against integral viable cell density (IVCD). The mean and standard deviation from three (3) parallel shake flask assays were plotted. Figure 2 This indicates that maltose can be used as a glucose substitute.

[0017] Figure 3Growth and biochemical profiles of the CHO-K1 cell line (SH87) producing recombinant monoclonal antibodies, cultured in a protein-free, chemically defined medium (PFCDM) with glucose and maltose as sugar sources, are shown. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDM containing 4 g / L glucose, 4 g / L glucose + 0.5 g / L maltose, 4 g / L glucose + 1 g / L maltose, 4 g / L glucose + 3 g / L maltose, or 6 g / L glucose. Cultures were monitored until viability fell below 50% to obtain their (A) viable cell density (labeled with lines) and culture viability (labeled only), as well as (B) glucose concentration, (C) lactate concentration, (D) maltose concentration, (E) IgG titer, (F) glutamine concentration, and (G) ammonium concentration in the medium. (H) SH87 cells were harvested from day 0 to day 5 in PFCDM culture containing 4 g / L glucose or 4 g / L glucose + 3 g / L maltose for LC-MS quantification of intracellular maltose concentration. Intracellular maltose was found to be absent (below the detection limit) in all samples obtained from the 4 g / L glucose culture and in day 0 samples from the 4 g / L glucose + 3 g / L maltose culture. For (D), the mean and standard deviation of two (2) parallel determinations from a set of shake flasks were plotted. For other curves, the mean and standard deviation of two (2) parallel determination shake flasks were plotted. Therefore, Figure 3 This shows that cells can switch to maltose metabolism to maintain cell growth and protein production when glucose is depleted.

[0018] Figure 4 Maltose and glucose profiles in cell-free conditioned medium (CM) are shown. In (A), SH87 cells routinely maintained in glucose-only PFCDM were passaged into PFCDM containing 4 g / L glucose or 4 g / L glucose + 3 g / L maltose. Cell-free conditioned medium (CM) was harvested from both cultures on days 2, 4, 6, and 8. Viable cell density (labeled with lines) and culture viability (labeled only) were also monitored. In (B), CM from maltose-supplemented cultures was incubated at 37°C for 3 days. Samples were harvested daily from these CMs starting from the day of harvest to determine glucose and maltose concentrations. In (C), maltose was added to CM harvested from glucose cultures to a final concentration of 3 g / L, followed by further incubation at 37°C. Samples were harvested daily from these CMs for three (3) days to determine glucose and maltose concentrations. Plot the mean and standard deviation of the results from two (2) parallel determinations of the shake flasks. Therefore, Figure 4The cultured cells utilized maltose, and maltose hydrolysis did not occur spontaneously in the conditioned medium, even when the culture viability was low.

[0019] Figure 5 Growth and biochemical profiles of SH87 cells cultured in a protein-free, chemically defined medium (PFCDM) with high concentrations of glucose and maltose are shown. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDMs containing 14 g / L glucose, 24 g / L glucose, 4 g / L glucose + 10 g / L maltose, or 4 g / L glucose + 20 g / L maltose. Cultures were monitored until viability was below 50% to obtain their (A) viable cell density (labeled with lines) and culture viability (labeled only), and (B) glucose, (C) lactate, (D) maltose, (E) glutamine, (F) telomeric osmolality, and (G) IgG titer profiles. For the maltose profile, the mean and standard deviation of two (2) technical parallel determinations from a set of shake flasks were plotted. For the other profiles, the mean and standard deviation of two (2) parallel determination shake flasks were plotted. therefore, Figure 5 Similar cell growth was shown in cultures with 10 g / L or 20 g / L maltose supplementation compared to 14 g / L glucose, thus demonstrating that higher maltose supplementation concentrations provide ample support for cell growth due to maltose metabolism. This allows for a lower initial glucose concentration in the culture compared to the culture with 14 g / L glucose, resulting in lactate consumption in the cell culture with maltose supplementation. Furthermore, the tbc molar osmolality was significantly lower than in the culture with 20 g / L glucose, removing the tbc molar osmolality limitation on carbohydrate loading in the cell culture medium. Additionally, Figure 5 It also showed an increase in the maximum recombinant protein yield (i.e., IgG titer) in cultures supplemented with maltose.

[0020] Figure 6 A Monod model of specific maltose consumption rate is shown. In (A), according to Equation 4, the specific maltose consumption rate of cultures with different initial maltose concentrations from different experiments is determined as the slope of a graph relating maltose concentration to cumulative integrated viable cell density (IVCD). In (B), the maximum specific maltose consumption rate (qs_max) and the affinity constant (Ks) are determined by nonlinear regression according to Equation 5. The Monod model of specific maltose consumption rate calculated using these parameters is then plotted with the experimental data used for the model. Therefore, Figure 6 New estimates of maltose metabolism kinetics in mammalian cells are presented.

[0021] Figure 7 Growth and biochemical profiles of SH87 fed-batch bioreactor cultures in basal media with a defined protein-free composition (PFCDM) containing only glucose or glucose + maltose are shown. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDM containing 4 g / L glucose or 4 g / L glucose + 20 g / L maltose in a 2-liter stirred tank bioreactor. Cultures in glucose-only basal media were supplemented daily with 100% of their calculated glucose requirement, while cultures in glucose and maltose basal media were supplemented daily with 50% of their calculated glucose requirement. Glutamine was used as a reference nutrient; other nutrients were similarly supplemented in separate feeds. Cultures were monitored until culture viability fell below 50% to obtain their (A) viable cell density (labeled with lines) and culture viability (labeled only), as well as (B) glucose curves, (C) lactate curves, (D) maltose curves, (E) glutamine curves, (F) molar osmolality curves, and (G) IgG titer curves. Two-stage bioreactor cultures were performed for each fed-batch condition, and data from all four cultures were plotted. Therefore, Figure 7 The presence of maltose shows that less glucose supplementation is needed, which can result in higher maximum recombinant protein yield (i.e., IgG titer).

[0022] Figure 8 Glycosylation profiles of anti-Her2 antibodies produced from SH87 fed batch bioreactor cultures in basal media with a protein-free composition defined by glucose only or glucose + maltose (PFCDM) are shown. (SH87 fed batch cultures from PFCDM containing 4 g / L glucose or 4 g / L glucose + 20 g / L maltose are also shown.) Figure 7 Anti-Her2 monoclonal antibodies were purified from samples from day 10 and day 15 and subjected to glycosylation analysis. (A) is a representative fluorescence chromatogram of four day 10 samples with symbolic representations of the selected structures. (B) shows the relative abundance of glycan structures. The mean and standard deviation of two (2) biological parallel assays are shown. The mean difference between glucose + maltose samples and glucose-only samples, as well as the mean difference between day 15 samples and day 10 samples, were also calculated. The effects of maltose supplementation and late day 15 harvest on the relative abundance of glycan structures are bolded and underlined. Paired one-tailed Stuttgart's t-test was used to calculate p-values ​​for data from individual samples. Therefore, Figure 8Maltose supplementation has been shown to be a means of fine-tuning protein co-translation or post-translational modification processes (e.g., in monoclonal antibody glycosylation profiles, particularly in slightly reducing sialylation levels, which is known to enhance antibody-dependent cytotoxicity (ADCC) of therapeutic antibodies).

[0023] Figure 9 Growth and biochemical profiles of SH87 fed batch bioreactor cultures in a basal medium of glucose + maltose with a defined protein-free composition (PFCDM) with glucose-only or glucose + maltose feed are shown. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDM containing 4 g / L glucose + 20 g / L maltose in a 2 L stirred tank bioreactor. A glucose-only feed or a 1:1 ratio of glucose and maltose was used, supplementing the culture daily with 50% of its calculated glucose requirement. Glutamine was used as a reference nutrient; other nutrients were similarly supplemented in separate feeds. Cultures were monitored until culture viability fell below 50% to obtain their (A) viable cell density (labeled with lines) and culture viability (labeled only), as well as (B) glucose curves, (C) lactate curves, (D) maltose curves, (E) glutamine curves, (F) osmolality curves, and (G) IgG titer curves. Two-stage bioreactor cultures were performed for each fed-batch condition, and the mean and standard deviation from the parallel assays were plotted. Therefore, Figure 9 The results showed that supplementing the fed medium with maltose slightly reduced lactate concentration compared to cultures fed only with glucose. This characteristic would be advantageous in cultures where lactate buildup can lead to culture collapse. Figure 9 It was also shown that adding maltose to the fed culture medium could maintain a higher maltose concentration in the culture without adversely affecting cell growth and recombinant protein production.

[0024] Figure 10 The growth and biochemical profiles of CHO-K1 cells in a protein-free, chemically defined medium (PFCDM) are shown as the maltose concentration increases. CHO-K1 cells adapted to PFCDM with 10 g / L maltose were cultured in batch shake flasks in PFCDM with 10 g / L, 20 g / L, 30 g / L, and 40 g / L maltose. Cultures were monitored until viability fell below 50% to obtain their (A) viable cell density (labeled with lines) and culture viability (labeled only), and (B) osmolality (wt%) curves, (C) glucose curves, (D) lactate curves, (E) glutamine curves, and (F) ammonium curves. Therefore, Figure 10The results showed that CHO-K1 cells could grow faster in PFCDM culture medium with 20 g / L or more maltose as the sole carbohydrate. Even at higher maltose concentrations, lactate concentrations remained low until viability began to decline on day 6. This supports... Figure 2 The findings described therein confirm that maltose can be used as a glucose substitute in the absence of serum and hydrolysates during routine culture of cells (such as mammalian cells), with the added advantage of low lactate accumulation.

[0025] Figure 11 The specific IgG productivity of SH87 cells cultured in a protein-free, chemically defined medium (PFCDM) with high concentrations of glucose and maltose is shown. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDM containing 14 g / L glucose, 24 g / L glucose, 4 g / L glucose + 10 g / L maltose, or 4 g / L glucose + 20 g / L maltose. An IgG titer versus IVCD graph was plotted according to Equation 3. The slope of the graph indicates the specific IgG productivity of the culture over the stated time period. Therefore, Figure 11 Supported Figure 5 The data shows that, in some instances, maltose can increase protein production (i.e., IgG production) after glutamine depletion.

[0026] Figure 12 The specific maltose consumption rate of SH87 cells cultured in a protein-free, chemically defined culture medium (PFCDM) is shown. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDMs containing 4 g / L glucose and supplemented with 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 10 g / L, and 20 g / L maltose. A graph showing maltose concentration versus IVCD was plotted according to Equation 4. The slope of the graph indicates the specific maltose consumption rate of the cultures during the stated time period. Cultures with 0.5 g / L, 1 g / L, and 3 g / L maltose are described in... Figure 3 The 10 g / L maltose (1) culture and the 20 g / L maltose (1) culture are described in [the text]. Figure 5 Detailed culture data for 2 g / L maltose, 10 g / L maltose (2), and 20 g / L maltose (2) are not described in this report. Therefore, Figure 12 Supported Figure 6 The data in the study confirms that the Mono equation can be used to model maltose consumption rates.

[0027] Figure 13Growth and biochemical profiles of SH87 fed-batch bioreactor cultures in a glucose-only, protein-free chemistomeric medium (PFCDM) basal medium with glucose supplementation at 100% or 50% of their calculated glucose requirements are shown. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDM containing 4 g / L glucose in a 2-liter stirred tank bioreactor. The cultures were supplemented daily with either 100% or 50% of their calculated glucose requirements. Glutamine was used as a reference nutrient, and other nutrients were similarly supplemented in separate feeds. Cultures were monitored until culture viability fell below 50% to obtain their (A) viable cell density (labeled with lines) and culture viability (labeled only), and (B) glucose, (C) lactate, (D) IgG titer, (E) glutamine, and (F) ammonium profiles. For each feed batch condition, bioreactor cultures were performed in duplicate, and the mean and standard deviation of the cultures from parallel determinations were plotted. The weight molar osmolality was maintained below 381 mOsm / kg (data not shown). Therefore, Figure 13 The results showed that using a lower glucose supplement alone, without maltose supplementation, resulted in a lower cell growth curve and lower recombinant protein yield (i.e., IgG titer). This supports... Figure 7 The results confirmed that the presence of maltose enabled the use of lower glucose feeds, which resulted in the observed higher cell production yield (i.e., higher maximum IgG titers). Detailed Implementation

[0028] Most cultured cells (such as mammalian cells) are chemoheterotrophic and typically require a carbohydrate source to grow in the culture. Because carbohydrates have low permeability through the phospholipid bilayer that makes up most of the cell membrane (Bresseleers et al., 1984; Wood et al., 1968), the transport of sugars into the cell is facilitated by transport proteins (Jones and Nickson, 1982; Thorens, 1996; Wright et al., 1994). Therefore, glucose is the only commonly used carbohydrate for cell culture (such as mammalian cells) because it can be efficiently transported into the cell via two major families of monosaccharide transporters: sodium-glucose cotransporters (SGLTs) (Wright et al., 1994) and glucose transporters (GLUTs).

[0029] Besides glucose, other carbohydrate sources have been tested to support the growth of animal cell cultures (Altamirano et al., 2000; Morgan and Faik, 1981). In these reports, the monosaccharides galactose, fructose, and mannose were demonstrated to be utilized by most cell types in both serum- and serum-free media, consistent with the availability of transport proteins that internalize these sugars (Mueckler and Thorens, 2013; Wright, 2013). Polysaccharides have also been shown to support cell growth in serum-supplemented cell cultures because serum contains glycoenzymes necessary for the breakdown of complex carbohydrates in the medium (Morgan and Faik, 1981). In another study, heat-inactivated serum lacking amylase and / or maltase activity and culture dishes coated with serum-containing medium were used to isolate Chinese hamster ovary (CHO) cell variants capable of utilizing maltose or starch (Scannell and Morgan, 1982). The authors confirmed that coating culture dishes with serum-containing medium did not promote glycoenzyme activity, and therefore they hypothesized that only endogenous carbohydrate hydrolases, additionally expressed in the small intestine, were induced in these isolates to allow them to grow in media containing maltose and starch (Scannell and Morgan, 1982). However, in this study, serum containing glycoenzymes was used to coat the culture dishes, and how this promoted cellular utilization of maltose and starch was not evaluated.

[0030] In this study, the use of disaccharides to support the growth of the mammalian Chinese hamster ovary (CHO) cell line CHO-K1 in serum-free and protein-free cultures was evaluated. CHO-K1 cells were found to utilize maltose for growth in the absence of glucose. Using a CHO-K1 cell line producing recombinant monoclonal antibodies, it was confirmed that these cells could utilize maltose in a biphasic manner after glucose depletion when using a medium containing both glucose and maltose. Furthermore, it was confirmed that maltose was internalized in the conditioned medium and did not spontaneously hydrolyze. Maltose supplementation also resulted in a 15% increase in cellular protein yield (i.e., the recombinant monoclonal antibody titer from batch cultures). Specific maltose consumption was then determined and fitted to a Monon model to obtain a maximum specific maltose consumption rate (qs_max) of 0.257 ng / cell / day and an affinity constant (Ks) of 7.03 g / L. It was then demonstrated that, compared to fed-batch cultures containing only glucose, the application of maltose supplementation in fed-batch bioreactor cultures resulted in a 23% increase in maximum monoclonal antibody titer and a 55% increase in specific monoclonal antibody productivity. Therefore, maltose supplementation can be applied as a simple bioreactor process improvement to increase the yield of cellular protein production (such as monoclonal antibodies) in current manufacturing processes.

[0031] The inventors of this disclosure have surprisingly discovered that, although serum-free and protein-free cultures of mammalian cells have been reported since the 1970s and 1980s, respectively (Hayashi and Sato, 1976; Okabe et al., 1984), there have been no reports to date of using polysaccharides to support cell growth in serum-free cultures of cells such as mammalian cells. This is not surprising, as only one known animal disaccharide sucrose transporter has been recently reported (Meyer et al., 2011). Whether polysaccharides can support mammalian cell growth in serum-free cultures is a concern for both basic and applied sciences. To gain a fundamental understanding of the mammalian cell metabolism of polysaccharides, the use of serum-free cultures completely eliminates the role of serum-derived glycoenzymes in facilitating the survival of cells utilizing only polysaccharides, a role that could not be ruled out in previous reports (Scannell and Morgan, 1982). If serum-free mammalian cell cultures utilizing polysaccharides are obtained, then the cultures can serve as models for explaining previously unknown mechanisms of polysaccharide transport and metabolism in mammalian cells, such as the recent discovery of the first known animal sucrose transporter in Drosophila melanogaster (Meyer et al., 2011).

[0032] For practical applications, the use of polysaccharides in mammalian cell culture can offer advantages for serum-free suspension cell culture of commonly used transformed cell lines or for the biomanufacturing of recombinant protein therapeutics. Such cell cultures typically encounter two glucose-related problems: First, glucose is often a limiting substrate in serum-free suspension batch cultures due to its high consumption rate and the high cell density achieved in suspension cell cultures. This is also true of high initial glucose concentrations in the medium, which are typically the most abundant nutrient in most medium formulations used for mammalian cell batch cultures (Sinacore et al., 2000), at concentrations 2 to 10 times higher than the second most abundant nutrient utilized by cells during cell growth. Further addition of glucose to the cell culture medium is limited by the total weight molar osmolality of the medium, as hypertonic media have been shown to be detrimental to cell growth (Kurano et al., 1990; Ozturk and Palsson, 1991). Secondly, glucose is known to lead to high lactate levels in cultures because these transformed cell lines exhibit high rates of glycolysis and lactate production, a phenotype described as the Warburg effect (Warburg, 1956). This becomes a productivity limitation for both batch and fed-batch cultures, as lactate is toxic to cells and increased lactate concentrations in the bioreactor lead to decreased cell growth rates (Hassell et al., 1991; Lao and Toth, 1997).

[0033] The inventors of this disclosure therefore hypothesize that the use of polysaccharides in serum-free suspension cell cultures could potentially solve both problems: since polysaccharides help reduce the osmotic pressure concentration per unit mass concentration, these sugars could increase the availability of sugars to cells in batch media, as higher mass concentrations can be used. Depending on the conversion rate of polysaccharides to monosaccharides, it could also mitigate lactate accumulation in bioreactors by providing a sugar source that is not readily available to cells, thereby limiting glycolysis and lactate production. In theory, this would be somewhat similar to maintaining low glucose concentrations in bioreactors, as achieved through dynamic online feeding strategies (Wong et al., 2005), although this is practically simpler in terms of setup and implementation.

[0034] In view of the foregoing, this disclosure describes the use of disaccharides (the simplest polysaccharides) to support the growth of mammalian cell lines in serum-free, protein-free cultures. Therefore, in a first aspect, the present invention relates to a serum-free cell culture medium comprising maltose as the sole carbohydrate source. In another aspect, the present invention relates to a serum-free cell culture medium comprising maltose and at least one, at least two, at least three, or more additional sugars as carbohydrate sources.

[0035] As shown in the experimental section below, the inventors of this disclosure have surprisingly demonstrated the successful use of maltose as a carbohydrate source in mammalian cell culture. This surprisingly demonstrates that animal cells are capable of utilizing disaccharides. The results are surprising because it was previously unknown that mammalian cells express disaccharide transporters, and only selected mammalian cells (such as intestinal cells) express maltase on their cell membranes to allow the digestion of maltose.

[0036] As used herein, the term "serum-free" refers to a cell culture medium that does not contain fetal calf serum (FCS), fetal bovine serum (FBS), newborn calf serum (NBS), or serum from any other human or animal source. As illustrated or demonstrated in the Experimental Section below, the inventors of this invention have been able to use maltose as a carbohydrate source in serum-free cultures. The use of maltose as a carbohydrate source in this disclosure contrasts with known uses in the art, where maltose is generally used as a carbohydrate source for cell (mammalian cell) cultures in serum-containing media. The reason for using maltose in serum-containing media for cell (mammalian cell) cultures is that serum has been shown to contain maltase, which breaks down maltose into glucose in vitro, which is then metabolized by the cells. The inventors of this disclosure have developed the remarkable use of disaccharides as a carbohydrate source for serum-free cell cultures (such as animal cell cultures). Furthermore, it has been remarkably demonstrated that cells (such as mammalian cells) can be routinely cultured in maltose-containing media in the absence of glucose, just as they can be cultured in glucose-containing media.

[0037] The advantage of using maltose in serum-free cell cultures (such as mammalian cell cultures) is that it can be added to cell media with a smaller osmotic loading due to its disaccharide nature. Therefore, more carbohydrates can be loaded into the medium compared to monosaccharides such as glucose. Furthermore, as demonstrated herein, the use of maltose in batch media can induce a reduction in lactate accumulation, which is known to be detrimental to cell culture. It has also been demonstrated in this disclosure that the use of maltose in both batch and fed-batch cultures can increase protein yields (such as recombinant proteins or recombinant monoclonal antibodies).

[0038] In some instances, cell cultures as described herein can provide a carbohydrate source that may consist solely of maltose. In other instances, cell cultures as described herein can provide a carbohydrate source that may contain at least one additional sugar (i.e., maltose plus at least one other), or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or more sugars. As described in the experimental section below, sugars are loaded into batch media so that carbohydrates are no longer limiting. Therefore, in some instances, sugars may be present, independently or in combination with each other, at concentrations of: 0.5 g / L to 40 g / L, 10 g / L to 15 g / L, 15 g / L to 20 g / L, 20 g / L to 25 g / L, 25 g / L to 30 g / L, 30 g / L to 35 g / L, 35 g / L to 40 g / L, at least about 1 g / L, at least about 2 g / L, at least about 3 g / L, at least about 4 g / L, at least about 5 g / L. Liters, at least about 8 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, about 1.5 g / L, about 3.5 g / L, about 3.6 g / L, about 5.5 g / L, about 8 g / L, about 11 g / L, about 14 g / L, about 18 g / L, about 23 g / L, about 28 g / L, about 33 g / L, or about 38 g / L.

[0039] In some instances, the sugars described herein can be polysaccharides. Polysaccharides described herein can be dextran or disaccharides.

[0040] As used herein, the term "glucan" refers to a polysaccharide, which is a polymer composed of D-glucose monomers linked together by glycosidic bonds. In some instances, glucans as described herein may include, but are not limited to, cellobiose, kosperidin, aspergillus niger, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminarin, gentiobiose, dextran, glycogen, amylopectin, starch, cellulose, fumarose, gel polysaccharide, kelp polysaccharide, lentinan, lichen polysaccharide, oat β-glucan, oyster mushroom β-glucan (pleuran), yeast polysaccharide, and combinations thereof.

[0041] In some instances, the "disaccharide" as described herein may include, but is not limited to, cellobiose, chitobiose, kojibiose, aspergillus niger, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminarin, gentiobiose, pinobiose, maltulose, paragine, gentiobiulose, mannobiose, melibiose, plantain, rutin, xylobiose, and combinations thereof.

[0042] In some instances, sugars as described herein can be components of unspecified cell culture supplements, including but not limited to soybean hydrolysates, yeast self-solutions, whey protein hydrolysates, casein hydrolysates, gelatin hydrolysates, gluten hydrolysates, liver hydrolysates, vegetable hydrolysates, wheat hydrolysates, peptones from animal tissues, trypsin, protein peptones, and combinations thereof.

[0043] In some instances, sugars as described herein can be monosaccharides. As used herein, the term "monosaccharide" refers to any of a class of carbohydrates that cannot be broken down by hydrolysis into simpler sugars and constitute building blocks of oligosaccharides and polysaccharides. Monosaccharides may have at least three carbon atoms, one of which is attached to an oxygen atom to form an aldehyde (CHO) or ketone, and the other carbon atoms are each attached to a hydroxyl group (OH). A monosaccharide containing three carbons per molecule is called a triose. A monosaccharide containing four carbons per molecule is called a tetrosaccharide. A monosaccharide containing five carbons per molecule is called a pentose. A monosaccharide containing six carbons per molecule is called a hexose. Monosaccharides can exist as chains or rings. Monosaccharides can include, but are not limited to, tagatose, glucose, galactose, ribose, fructose, and xylose. As shown in the experimental section, an increase in protein titers was observed when maltose was used. Therefore, in some instances, monosaccharides can be glucose. As shown in the experimental section of this disclosure, in some instances, culture media as described herein may contain glucose and additional maltose supplements. Therefore, in some instances, such as those described herein, cell culture media may contain maltose and glucose as the sole carbohydrate source. As demonstrated in the experimental section below, the use of maltose in cell cultures (e.g., in fed-batch processes) allows for lower glucose levels to be controlled, thereby increasing recombinant protein yield. Not to be bound by theory, the combination of maltose and glucose as the sole carbohydrate source reduces the risk of complete carbohydrate depletion, which could lead to culture burnout (collapse).

[0044] As described in the experimental section below, batch media are loaded with maltose so that carbohydrates are no longer limiting. Therefore, in some instances, maltose may be present in media as described herein at the following concentrations: 0.5 g / L to 40 g / L, 10 g / L to 15 g / L, 15 g / L to 20 g / L, 20 g / L to 25 g / L, 25 g / L to 30 g / L, 30 g / L to 35 g / L, 35 g / L to 40 g / L, at least about 1 g / L, at least about 2 g / L, at least about 3 g / L, at least about 4 g / L, at least about 5 g / L. g / L, at least about 8 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, about 1.5 g / L, about 3.5 g / L, about 3.6 g / L, about 5.5 g / L, about 8 g / L, about 11 g / L, about 14 g / L, about 18 g / L, about 23 g / L, about 28 g / L, about 33 g / L, or about 38 g / L.

[0045] In some instances, serum-free cell cultures, as described herein, can be protein-free. As used herein, the term "protein-free" refers to a cell culture medium that does not contain insulin or transferrin or growth factors, or any protein purified from any organism, or any recombinant protein. For clarity, hydrolysates or hydrolyzed proteins may be present in protein-free cell cultures. Because serum contains protein, all protein-free media are also serum-free.

[0046] In some instances, serum-free cell cultures, as described herein, can be chemically defined media. As used herein, the term "chemically defined" refers to a growth medium suitable for in vitro cell culture in which all chemical components are identified and their precise concentrations are known. A chemically defined medium must also be completely free of any unidentified components, such as fetal bovine serum or serum from any other human or animal source, or soybean hydrolysate, yeast hydrolysate, or any hydrolysate in which the exact identity and concentration of the components are unknown. This means that chemically defined media can contain recombinant forms of proteins, such as, but not limited to, albumin and growth factors, typically derived from rice or *E. coli*. Because serum contains unidentified components, all chemically defined media are also serum-free.

[0047] In some instances, serum-free cell culture media, as described herein, may be protein-free chemically defined media (PFCDM). As used herein, the term "protein-free chemically defined" refers to a growth medium suitable for in vitro cell culture in which all chemical components are known and free from insulin or transferrin or growth factors or any protein purified from any organism or any recombinant protein.

[0048] As shown throughout the following experimental sections, the cell culture media described herein may further include, but are not limited to, basal cell culture media selected from the group consisting of: basal medium Eagle (BME), Eagle's minimum essential medium (MEM or Eagle's MEM), Earle's balanced salt solution (EBSS), Dulbecco's modified Eagle's medium (DMEM), HAM's F-10 medium, HAM's F-12 medium, DMEM-F12 medium, Roswell Park Memorial Institute 1640 (RPMI 1640 or RPMI), Leiboitz's medium (L-15 medium), combinations thereof, or modified forms thereof. In some instances, cell culture media as described herein may include Iscove's Modified Dulbecco's Medium (IMDM); IMDM containing HEPES and L-glutamine; IMDM containing HEPES and without L-glutamine; RPMI 1640 containing L-glutamine; RPMI 1640 containing HEPES, L-glutamine, and / or penicillin-streptomycin; Minimum Essential Medium-α (MEM-α); DMEM:F12 1:1 containing L-glutamine; DME / F12; Eagle's Basal Medium containing Earl's BSS; GMEM (Glasgow's MEM). MEM); GMEM containing L-glutamine; F-10; F-12; Ham's F-10 containing L-glutamine; Ham's F-12 containing L-glutamine; L-15 (Lebovitz) (2×) without L-glutamine or phenol red; L-15 (Lebovitz) without L-glutamine; McCoy's 5A Modified Medium; Medium 199; Eagle's MEM (2×) without L-glutamine or phenol red; Eagle's MEM-Earl's BSS containing L-glutamine; Eagle's MEM-Earl's BSS without L-glutamine; Eagle's MEM-Hanks BSS without L-glutamine; NCTC-109 containing L-glutamine; Richter's CM medium containing L-glutamine. CMMedium); and culture media containing hydrolysates.

[0049] Furthermore, depending on the cells to be cultured, it is considered advantageous if the cell culture medium, as described herein, can further contain at least one additional component, including but not limited to at least one amino acid, at least one vitamin, at least one inorganic salt, at least one trace element, adenine sulfate, ATP, deoxyribose, ethanolamine, ethanolamine·HCl, glutathione, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), hypoxanthine, linoleic acid, lipoic acid, phenol red, ethanolamine phosphate, putrescine, sodium pyruvate, cholesterol, dextran sulfate, β-mercaptoethanol, methotrexate (MTX), methionine sulfoxide imine (MSX), thymidine, uracil, xanthine, combinations thereof, etc.

[0050] In some instances, the amino acid component may include, but is not limited to, one or more amino acids, such as, but not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof.

[0051] The vitamin components described herein may include, but are not limited to, one or more vitamins, such as ascorbic acid, biotin, choline chloride, D-Ca++-pantothenate, folic acid, i-inositol, menadione, nicotinamide, nicotinic acid, para-aminobenzoic acid (PABA), pyridoxal, pyridoxine, riboflavin, thiamine, vitamin A acetate, vitamin B12, vitamin D2, etc., or combinations thereof.

[0052] The inorganic salt components described herein may include, but are not limited to, one or more inorganic salts, such as CaCl2, KCl, MgCl2, MgSO4, NaCl, NaHCO3, Na2HPO4, NaH2PO4, KH2PO4, Ba(C2H3O2)2, KBr, CoCl2, KI, MnCl2, Cr(SO4)3, CuSO4, NiSO4, H2SeO3, NaVO3, TiCl4, GeO2, (NH4)6Mo7O 24 Na2SiO3, FeSO4, NaF, AgNO3, RbCl, SnCl2, ZrOCl2, CdSO2, ZnSO4, Fe(NO3)3, AlCl3, iron citrate chelate, or combinations thereof.

[0053] The trace element components described herein may include, but are not limited to, ions of one or more trace elements, such as barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or combinations thereof.

[0054] It is also envisioned that the cell culture medium as described herein can be further combined with at least one, or at least two, or at least all of the components, such as hydrolysates, or enzyme digests, or yeast cell extracts.

[0055] In some instances, cell culture media, as described herein, may be further combined with any one or more of zwitterionic, ionic, or nonionic surfactants. Suitable surfactants can be ionic surfactants, which can be anionic or cationic. Anionic surfactants have a negatively charged group, based on permanent anions such as sulfate, sulfonate, or phosphate, or based on pH-dependent anions such as carboxylates. Examples of anionic surfactants include, but are not limited to, alkyl sulfates, alkyl sulfonates, alkyl ether sulfates, alkyl phosphates, alkyl phosphonates, docusate, sulfonate fluorinated surfactants, alkylbenzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, alkyl polyoxyethylene sulfates, carboxylate fluorinated surfactants, ammonium dodecyl sulfate, sodium dodecyl sulfate (SDS), sodium dioctyl sulfosuccinate, sodium deoxycholate, sodium alginate, sodium n-dodecylbenzene sulfonate, sodium lauryl sulfate, sodium lauryl ether sulfate (SLES), and nutmeg. Sodium polyether sulfate, sodium dioctyl sulfosuccinate, phosphatidylglycerol, potassium laurate, phosphatidylinosine, phosphatidylinositol, perfluorooctane sulfonate (PFOS), perfluorobutane sulfonate, sodium stearate, triethanolamine stearate, diphosphatidylglycerol, phosphatidylserine, phosphatidic acid and their salts, sodium carboxymethyl cellulose, cholic acid and other bile acids (e.g., cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid) and their salts (e.g., sodium deoxycholate), sodium lauroyl sarcosinate, perfluorononanoate, and perfluorooctanoate (PFOA or PFO), etc.

[0056] In some instances, nonionic surfactants may include, but are not limited to, glycerides, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters (polysorbates), polyoxyethylene fatty acid esters, sorbitan esters, glyceryl monostearate, polyethylene glycol, polypropylene glycol, cetyl alcohol, cetearyl alcohol, stearyl alcohol, arylalkyl polyether alcohols, polyoxyethylene-polyoxypropylene copolymers (poloxam), poloxamer, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, amorphous cellulose, polysaccharides, including starch and starch derivatives such as hydroxyethyl starch (HES); polyvinyl alcohol, polyvinylpyrrolidone, etc.

[0057] Cationic surfactants contain positively charged groups, and pH-dependent cationic surfactants are based on primary, secondary, or tertiary amines, while permanently charged cationic surfactants are based on quaternary ammonium cations. Examples of cationic surfactants include, but are not limited to, natural phospholipids, synthetic phospholipids, quaternary ammonium compounds, benzalkonium chloride, cetyltrimethylammonium bromide, chitosan, lauryldimethylbenzylammonium chloride, acylcarnitine hydrochloride, dimethyl dioctadecylammonium bromide (DDAB), dioleoyltrimethylammonium propane (DOTAP), dimyristoyltrimethylammonium propane (DMTAP), dimethylaminoethane carbamoylcholine (DC-Chol), 1,2-diacylglycerol-3-(O-alkyl)phosphocholine, O-alkylphosphatidylcholine, and alkylpyridine. Halides, or long-chain alkylamines, such as n-octylamine, oleylamine, etc.

[0058] In some instances, the surfactant may be an amphoteric surfactant, which is an electrically neutral surfactant having localized positive and negative charges within the same molecule. Examples of suitable amphoteric surfactants include, but are not limited to, amphoteric phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, diacylglycerol-phosphate ethanolamine (e.g., dimyristoylglycerol-phosphate ethanolamine (DMPE), dipalmitoylglycerol-phosphate ethanolamine (DPPE), distearate-glycerol-phosphate ethanolamine (DSPE), dioleoylglycerol-phosphate ethanolamine (DOPE)), etc. In some instances, the culture medium as described herein may include mixtures of phospholipids, including anionic and amphoteric phospholipids. Such mixtures include, but are not limited to, lysophospholipids, lecithin, or soybean lecithin, or any combination thereof. Phospholipids, whether anionic, amphoteric, or mixtures thereof, may be salted or desalted, hydrogenated or partially hydrogenated, or natural, semi-synthetic, or synthetic.

[0059] In some examples, the surfactant may include, but is not limited to, fatty alcohols; polyoxyethylene glycol octylphenol ether; and polyoxyethylene glycol alkyl sorbitol. In other examples, the surfactant may be a nonionic surfactant. In some examples, the surfactant may include, but is not limited to, polysorbate 80 (PS80), polysorbate 20 (PS20), and poloxamer 188 (P188).

[0060] To prevent unwanted growth, it may be advantageous for cell culture media as described herein to contain agents that prevent the unwanted growth of contaminants. Therefore, in some instances, cell culture media as described herein may further contain antibiotics. The antibiotics may include, but are not limited to, hygromycin B, puromycin, blasticidin, bleomycin sulfate, geneticin (G418), zeocin, amphotericin B, ampicillin, penicillin, chloramphenicol, gentamycin, kanamycin, neomycin, streptomycin, tetracycline, polymyxin B, actinomycin D, amikacin, bacitracin, carbenicillin, ceftazidime, and coumermycin A1. A1), D-cycloserine, cyclohexamide, dihydrostreptomycin sesquisulfate, kasugamycin, mycophenolic acid, nalidixic acid, nourseothricin sulfate, oxytetracycline, paromomycin sulfate, phleomycin, mitomycin C, ribostamycin, rifampicin, rifamycin, spectinomycin, tazobactam, thiostrepton, ticarcillin, and combinations thereof.

[0061] Those skilled in the art will understand that each component described herein will be present in an amount suitable for the desired cell culture in vitro. Therefore, in one example, each component of the culture medium as described herein can support the presence of a quantity of cells for in vitro culture. As will be understood, it is contemplated that increasing the concentration of other nutrients in the batch culture medium could further improve protein (e.g., recombinant protein) productivity.

[0062] To facilitate the transport and / or storage of cell culture media as described herein, the cell culture media described herein may be provided as cell culture media concentrates. Therefore, in some instances, the cell culture media described herein may be 1× to 100× culture media formulations, or 1×, or 2×, or 5×, or 10×, or 50×, or 100× culture media formulations. The culture media described herein may be provided in powder or liquid form.

[0063] In some instances, the cell culture medium may include, but is not limited to, Duchenne Minimum Essential Medium (DMEM), F-12 Basal Medium (DMEM-F12), L-glutamine, nonionic surfactants, genimycin, and maltose, or modified forms thereof.

[0064] As shown in the experimental section below, the cell culture medium may further include glucose. Therefore, in some instances, the cell culture medium may include, but is not limited to, Duchenne Minimum Essential Medium (DMEM), F-12 Basal Medium (DMEM-F12), L-glutamine, nonionic surfactants, genistein, maltose, and glucose, or modified forms thereof.

[0065] Depending on the purpose of cell culture, the cells to be cultured in the culture medium described herein may include, but are not limited to, vertebrate cells, arthropod cells, annelid cells, mollusc cells, sponge cells, jellyfish cells, insect cells, avian cells, mammalian cells, and fish cells.

[0066] When the cells to be cultured are insect cells, the insect cells may be derived from species of the genus *Spodoptera* spp. or *Trichoplusa* spp.

[0067] In some instances, the cells to be cultured in the culture medium described herein may be mammalian cells. These mammalian cells may include, but are not limited to, human cells, mouse cells, rat cells, hamster cells, rabbit cells, dog cells, monkey cells, hybridoma cells, CHO cells, CHO-K1 cells, CHO-DG44 cells, CHO-S cells, CHO-DXB11 cells, CHO-GS cells, SH87 cells, BHK cells, COS cells, VERO cells, HeLa cells, 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, CAP cells, AGE1.HN cells, or derivatives thereof. In some instances, the mammalian cells may be CHO-K1 cells, or CHO-DG44 cells, or derivatives of their proteins (such as recombinant proteins), such as SH87.

[0068] Advantageously, as demonstrated in the Experimental Section below, the culture media described herein can be used to culture any cells without any modification to enable the metabolism of alternative carbohydrate sources (such as maltose). Therefore, in some instances, cells that can be used in cell culture media as described herein may be able to metabolize only one or more carbohydrate sources without prior adaptation to said one or more sources. The term "adaptation" as used herein refers to the regulation or alteration of an organism's physiology to better suit its environment. This regulation or alteration can occur, for example, through genetic modification of the organism, but can also occur spontaneously through natural selection and environmental selection pressures.

[0069] In another aspect, a method for growing and / or culturing cells is provided, wherein the method includes growing and / or culturing cells in a serum-free cell culture medium as described herein. In some instances, the method may include growing and / or culturing cells in the serum-free cell culture medium, which may contain maltose as the sole carbohydrate source or may contain maltose and at least one, at least two, at least three, at least four, or more sugars as carbohydrate sources. In some instances, the method may include growing and / or culturing cells as described herein, wherein the culture medium may contain maltose and glucose as the sole carbohydrate sources or the culture medium may contain Duchenne Minimum Essential Medium (DMEM), F-12 Basal Medium (DMEM-F12), L-glutamine, a nonionic surfactant, genistein, and maltose, or a modified form thereof. As shown in the experimental section below, the method for growing and / or culturing cells as described herein increases protein yield. Therefore, in some instances, the method described herein increases protein yield.

[0070] The methods for growing and / or culturing cells as described herein can utilize cell culture methods known to others. In some instances, cells as described herein can be grown and / or cultured in cultures including, but not limited to, adherent cultures, suspension cultures, T-flask cultures, spin flask cultures, shake flask cultures, spiral tube cultures, microbioreactor cultures, bioreactor cultures, batch cultures, fed-batch cultures, continuous cultures, perfusion cultures, etc. In some instances, the growth and / or culture of the cells can be performed under conditions suitable for supporting the growth and / or culture of the cells.

[0071] As shown in the experimental section, the methods disclosed herein can improve protein yield. Therefore, in another aspect, a method for improving protein yield is provided. The method includes growing and / or culturing cells in a serum-free cell culture medium as described herein. In some instances, the method may include growing and / or culturing cells in the serum-free cell culture medium, which may contain maltose as the sole carbohydrate source or may contain maltose and at least one, at least two, at least three, at least four, or more sugars as carbohydrate sources. In some instances, the method may include growing and / or culturing cells as described herein, wherein the culture medium may contain maltose and glucose as the sole carbohydrate sources or the culture medium may contain Duchenne Minimum Essential Medium (DMEM), F-12 Basal Medium (DMEM-F12), L-glutamine, a nonionic surfactant, genistein, and maltose, or a modified form thereof.

[0072] In some instances, the increased protein yield may be an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some instances, the increase in protein yield may be an increase in antibody production, wherein the antibody titer may increase by about 2% to about 300%, or about 2%, or about 4%, or about 6%, or about 8%, or about 10%, or about 12%, or about 14%, or about 16%, or about 18%, or about 20%, or about 22%, or about 24%, or about 26%, or about 28%, or about 30%, or about 32%, or about 34%. %, or approximately 36%, or approximately 38%, or approximately 40%, or approximately 43%, or approximately 45%, or approximately 48%, or approximately 50%, or approximately 55%, or approximately 60%, or approximately 65%, or approximately 70%, or approximately 75%, or approximately 80%, or approximately 90%, or approximately 100%, or approximately 125%, or approximately 150%, or approximately 175%, or approximately 200%, or approximately 230%, or approximately 250%, or approximately 300%.

[0073] In some instances, the methods described herein can increase cell specific productivity by about 2% to about 300%, or about 2%, or about 4%, or about 6%, or about 8%, or about 10%, or about 12%, or about 14%, or about 16%, or about 18%, or about 20%, or about 22%, or about 24%, or about 26%, or about 28%, or about 30%, or about 32%, or about 34%, or about 30%. 6%, or about 38%, or about 40%, or about 43%, or about 45%, or about 48%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 90%, or about 100%, or about 125%, or about 150%, or about 175%, or about 200%, or about 230%, or about 250%, or about 300%.

[0074] In some instances, cell culture media and / or methods as described herein can increase the maximum viable cell density / number by about 2% to about 300%, or about 2%, or about 4%, or about 6%, or about 8%, or about 10%, or about 12%, or about 14%, or about 16%, or about 18%, or about 20%, or about 22%, or about 24%, or about 26%, or about 28%, or about 30%, or about 32%, or about 34%, or about 36%. %, or about 38%, or about 40%, or about 43%, or about 45%, or about 48%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 90%, or about 100%, or about 125%, or about 150%, or about 175%, or about 200%, or about 230%, or about 250%, or about 300%, as measured over, for example, a 14-day period.

[0075] like Figure 8 As described in Table 3, the cell culture media as described herein and / or the methods described herein can be used to fine-tune the glycosylation profile of protein products (e.g., recombinant glycoprotein products). In one aspect, a method for modulating the glycosylation profile of a protein is provided, wherein the method comprises culturing cells expressing the protein in a cell culture medium as described herein, thereby producing cells expressing a protein having a modulated glycosylation profile. In some instances, modulating the glycosylation profile of a protein can be modulating glycosylation, such as, but not limited to, antennae, fucosylation, mannosylation, sialylation, or combinations thereof.

[0076] As used herein, the term "regulation" refers to an increase or decrease in a specific type of glycosylation of a protein compared to a control. A "control" culture medium, as used herein, can be a cell culture medium identical to that described herein, except that the control does not contain maltose. That is, the control can be an equivalent cell culture medium that does not contain maltose, as described herein. The only difference between the control cell culture medium and the cell culture medium described herein is the presence of a disaccharide (such as maltose) in the cell culture medium described herein.

[0077] In some instances, the regulated glycosylation profile includes, but is not limited to, decreased levels of fucosylated glycans, decreased levels of dibranched glycans, increased levels of mannosylated glycans, increased levels of monobranched glycans, decreased levels of sialylated glycans, or combinations thereof. Therefore, in one instance, a method is provided for generating a protein having at least one, at least two, at least three, or all of the regulated glycosylations, said regulated glycosylations including, but not limited to, decreased levels of fucosylated glycans, decreased levels of dibranched glycans, increased levels of mannosylated glycans, increased levels of monobranched glycans, and decreased levels of sialylated glycans. In some instances, as shown in Table 3, the protein may be an antibody, an immunoglobulin, or a fragment thereof.

[0078] In some instances, regulating fucosylation levels can involve reducing the level of fucosylation in the protein. In some instances, the reduction in the level of fucosylated glycans can be a reduction of approximately 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%.

[0079] In some instances, regulating the level of mannosylated glycans can involve increasing the mannosylation level of a protein. In some instances, the mannosylation level can be increased by approximately 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0080] In some instances, regulating sialylation levels can involve reducing the sialylation level in the protein. In some instances, the reduction in sialylated glycan levels can be approximately 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%.

[0081] In some instances, regulating the level of monobranched glycans can be achieved by increasing the level of monobranched glycans in the protein. In some instances, the level of monobranched glycans can be increased by approximately 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0082] In some instances, regulating the level of dibranched glycans can be achieved by reducing the level of dibranched glycans in the protein. In some instances, the reduction in the level of dibranched glycans can be a reduction of approximately 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%.

[0083] As used in this paper, the term "antennae" refers to the addition of a GlcNAc sequence to the core of a heterozygous and complex glycan. Thus, a "single-branched glycan" refers to a GlcNAc branch connected to the core glycan, a "two-branched glycan" refers to two GlcNAc branches connected to the core glycan, and so on.

[0084] In one example, a method is provided for producing a composition comprising an antibody or an antigen-binding fragment thereof having a regulated glycosylation profile. The method may include culturing host cells expressing the antibody or an antigen-binding fragment thereof in a cell culture medium as described herein, thereby producing a composition comprising the antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment having at least one (at least two or all) selected from the group consisting of: a 0.1%-50% reduction in the level of fucosylated glycans, a 0.1%-50% reduction in the level of sialylated glycans, and an 0.1%-50% increase in the level of mannosylated glycans compared to a control. In some examples, the control is a composition comprising an antibody or an antigen-binding fragment thereof, produced by culturing host cells expressing the antibody or an antigen-binding fragment thereof in a cell culture medium not in a culture medium as described herein. In one example, the antibody is an anti-Her2 antibody or an antigen-binding fragment thereof. In one example, the culture medium contains glucose and / or maltose.

[0085] like Figure 8 As shown in A, maltose supplementation may also reduce the sialylation level in the expressed antibody. It is known that reducing the sialylation level of an antibody can increase antibody-dependent cytotoxicity (ADCC) of a therapeutic antibody. Therefore, in some instances, this disclosure also provides a method for producing a composition comprising an antibody or an antigen-binding fragment thereof having a regulated glycosylation profile, the method being achieved by culturing host cells expressing the antibody or an antigen-binding fragment thereof in a cell culture medium as described herein, thereby producing a composition comprising the antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof exhibits a 0.1%-30% increase in antibody-dependent cytotoxicity (ADCC) response compared to a control, wherein the control is a composition comprising the antibody or an antigen-binding fragment thereof, the composition being produced by culturing host cells expressing the antibody or an antigen-binding fragment thereof in a cell culture medium other than that described herein. In one instance, the antibody is an anti-Her2 antibody or an antigen-binding fragment thereof. In one instance, the culture medium contains glucose and / or maltose.

[0086] The cell culture media described herein and the methods described herein can be used to culture cells and / or promote cell growth and / or increase the protein yield of cells, such as vertebrate cells, arthropod cells, annelid cells, mollusc cells, sponge cells, jellyfish cells, insect cells, avian cells, mammalian cells, fish cells, etc. In some instances, the cell culture media described herein and the methods described herein can be used to culture eukaryotic cells or tissues and / or promote the growth of eukaryotic cells or tissues and / or increase the protein yield of eukaryotic cells or tissues, including animal cells, human cells, insect cells, plant cells, avian cells, fish cells, mammalian cells, etc.

[0087] In some instances, the mammalian cells may include, but are not limited to, human cells, mouse cells, rat cells, hamster cells, rabbit cells, dog cells, monkey cells, hybridoma cells, CHO cells, CHO-K1 cells, CHO-DG44 cells, CHO-S cells, CHO-DXB11 cells, CHO-GS cells, SH87 cells, BHK cells, COS cells, VERO cells, HeLa cells, 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, CAP cells, AGE1.HN cells, or derivatives thereof.

[0088] The culture media described herein enhance the production of immunoglobulins in the cells. Therefore, in some instances, the cells to be cultured in the cell cultures and methods described herein may be antibody-producing cells. In some instances, the mammalian cells may be CHO-K1 cells, or CHO-DG44 cells, or recombinant protein-producing derivatives thereof, such as SH87.

[0089] For convenience, the culture medium can be provided as a kit. Therefore, in one aspect, a kit is provided comprising components of a cell culture medium as described herein and / or components for performing the methods described herein. In some instances, the kit may further comprise cells. For example, the cells may be cells to be cultured. The cells may include, but are not limited to, vertebrate cells, arthropod cells, annelid cells, mollusc cells, sponge cells, jellyfish cells, insect cells, avian cells, mammalian cells, fish cells, etc. In some instances, the mammalian cells may include, but are not limited to, human cells, mouse cells, rat cells, hamster cells, rabbit cells, dog cells, monkey cells, hybridoma cells, CHO cells, CHO-K1 cells, CHO-DG44 cells, CHO-S cells, CHO-DXB11 cells, CHO-GS cells, SH87 cells, BHK cells, COS cells, VERO cells, HeLa cells, 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, CAP cells, AGE1.HN cells, etc., or derivatives thereof.

[0090] As illustrated in the experimental section, the mammalian cells may be CHO-K1 cells, CHO-DG44 cells, or derivatives of their proteins (such as recombinant proteins), such as SH87.

[0091] In some instances, the cells to be cultured in the culture medium as described herein and / or in the methods described herein may be protein-producing cells, such as, but not limited to, recombinant proteins, antibodies, etc. As shown in the experimental section below, the protein may be a monoclonal antibody.

[0092] The disclosure described herein can be suitably implemented in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing,” for example, should be interpreted broadly and without limitation. Furthermore, the terminology used herein has been used as descriptive rather than restrictive, and is not intended to exclude any equivalents of the shown and described features or portions thereof, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been explicitly disclosed through preferred embodiments and optional features, modifications and variations of the invention embodied herein can be made by those skilled in the art, and such modifications and variations are considered to fall within the scope of the invention.

[0093] This invention has been described broadly and generally herein. Each of the more narrower contents and subclasses falling within the general disclosure also forms part of this invention. This includes describing the invention generally by removing any incidental or negative limiting conditions from said class, regardless of whether the excluded contents are specifically described herein.

[0094] Other embodiments fall within the scope of the following claims and non-limiting embodiments. Furthermore, where features or aspects of the invention are described in the Markush group, those skilled in the art will recognize that the invention is therefore also described in the context of any single member or subgroup of the Markush group.

[0095] Experimental Section

[0096] Materials and methods

[0097] Cell lines and cell culture

[0098] The CHO-K1 cell line was pre-acclimated to an environment supplemented with 2 g / L sodium bicarbonate (Sigma-Aldrich, St. Louis, MO), 3.6 g / L D-(+)-glucose (Sigma-Aldrich), 6 mM L-glutamine (Sigma-Aldrich), and 0.1% sodium bicarbonate. F-68 (Life Technologies, Carlsbad, CA) was cultured in serum-free, protein-free HyQ PF-CHOMPS (Hyclone, Logan, UT) medium. SH87 was a suspension CHO-K1 cell line producing anti-Her2 monoclonal antibodies (Ho et al., 2012), pre-acclimated to a chemically defined DMEM / F12-based protein-free medium (PFCDM) supplemented with 6 g / L D-(+)-glucose (Sigma-Aldrich), 8 mM L-glutamine (Sigma-Aldrich), 0.1% Pluronic F-68 (Life Technologies), and 600 μg / ml G418 pyrosulfate (Sigma-Aldrich). CHO-K1 and SH87 cells were routinely passaged every 3 to 4 days.

[0099] Unless otherwise stated, the cell cultures of this disclosure were performed in disposable conical flasks (Corning, Acton, MA) and the flasks were incubated in a humidified incubator (Climo-Shaker ISF-1-W, Kuhner, Switzerland) at 37°C, 8% CO2, and 110 rpm.

[0100] Analysis of cell culture samples

[0101] Viable cell density and culture viability were determined using a Vi-Cell XR cell viability analyzer (Beckman Coulter, Brea, CA) via trypan blue exclusion method, following the manufacturer's instructions. For biochemical and other cell culture parameter analyses, 1 ml of culture sample was centrifuged at 8000 g for 10 minutes to obtain a clear supernatant.

[0102] The concentrations of ammonium, glutamine, glucose, and lactate were analyzed using a BioProfile 100Plus (NovaBiomedical, Waltham, MA). Molar osmolality was measured using a vapor pressure osmometer (Vapro 5520, Wescor, Logan, UT) according to the manufacturer's instructions. Maltose concentration was quantified using a maltose colorimetric / fluorescence assay kit (Biovision, Milpitas, CA) according to the manufacturer's instructions. Monoclonal IgG antibody titers were determined by turbidity assay using an IMMAGE 800 (Beckman Coulter) according to the manufacturer's instructions.

[0103] Adapting CHO cell lines to culture media with different disaccharides

[0104] HyQ PF-CHO disaccharide medium was prepared by replacing the 3.6 g / L glucose normally added to the medium with the same mass concentration of maltose, sucrose, lactose, or trehalose during medium preparation. CHO-K1 cells were then inoculated at 0.3 × 10⁻⁶ cells / mL. 6 1.0 × 10⁶ cells / mL and 1.0 × 10⁶ cells / mL 6 Cells were seeded at a density of 10 cells / mL into these disaccharide media and passaged every 3 to 4 days. At each passage during the acclimation process, viable cell density and culture viability were determined before passage and after seeding into fresh medium.

[0105] Shake flask batch culture sampling and characterization

[0106] To compare the growth curves of CHO-K1 cells in maltose and glucose media, CHO-K1 cells adapted to maltose medium and those adapted to maltose medium were seeded into HyQ PFCHO disaccharide medium containing 3.6 g / L maltose and normal HyQ PF-CHO medium containing 3.6 g / L glucose, respectively. To evaluate maltose utilization using the adapted SH87, cells were seeded into PFCDM medium containing 4 g / L, 6 g / L, 14 g / L, or 24 g / L glucose, or 4 g / L glucose supplemented with 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 10 g / L, or 20 g / L maltose. Cells were seeded in duplicate at a rate of 0.3 × 10⁻⁶ cells / mL. 6Cells were seeded at a density of [number] cells / mL in disposable Erlenmeyer flasks (Corning). Cell culture samples were collected and analyzed daily throughout the growth curve experiment until culture viability dropped below 50%. If necessary, culture supernatant samples were stored at -20°C for further analysis.

[0107] Quantitative intracellular maltose

[0108] First, quench with ice-cold 150mM NaCl (Merck, ACS reagent grade) solution for 10 minutes. 7 Samples for intracellular maltose quantification were prepared using 10 μL of 0.4 mM 13C-maltose (UL- ). After centrifugation to precipitate the cells, the supernatant was removed by aspiration, and 10 μL of 0.4 mM 13C-maltose (UL- ) was added. 13 C 12Maltose monohydrate (Omicron Biochemicals, South Bend, IN) was used as a reference standard. Intracellular maltose was extracted using a two-phase liquid extraction protocol involving a solution of methanol (Fisher Scientific, premium grade), chloroform (Merck), and tris(hydroxymethyl)methylglycine (Merck) (40:35:25 v / v) (Selvarasu et al., 2012). The extract was stored at -80°C, dried under vacuum at 4°C (CentriVap, Labconco, US), and reconstituted in a water-methanol mixture (95:5 v / v), followed by analysis by liquid chromatography-mass spectrometry (LC-MS) (Acquity UPLC-Xevo TQ-S MS, Waters, Milford, MA). Separation was performed using a C18 reversed-phase column (Waters Corporation, HSS T3 column, 2.1 mm × 50 mm, 1.8 μm particle size) with the following solvents: A: water containing 0.1% formic acid (Sigma-Aldrich, 98%); B: methanol at a flow rate of 0.4 mL / min. Intracellular maltose was quantified via multiple reaction monitoring experiments, where the integrated peak areas of maltose and 13C-maltose were obtained for each sample. The actual concentration of maltose in each sample was quantified by directly comparing the relative integrated peak area of ​​maltose with that of a 13C-maltose reference standard. The limit of detection for maltose was observed to be 7.5 ng per injection. Intracellular maltose concentration was calculated based on the amount of maltose within the detection limit, the number of cells used, and the average cell diameter obtained from a Vi-Cell XR cell viability analyzer. Samples were analyzed in triplicate on LC-MS, and a relative standard deviation of less than 15% was observed for the integrated peak area obtained for each sample.

[0109] Feed-in culture sampling and characterization in bioreactors

[0110] For each culture condition to be tested, SH87 was scaled up in PFCDM and at 3 × 10⁻⁶. 5Cells were seeded at a viable cell density of 10 cells / mL into two (2) liter glass bioreactors (Sartorius, Germany) in duplicate. Culture temperature, pH, dissolved oxygen, and stirring rate were maintained at 37°C, 7.1%, 50%, and 120 rpm, respectively. Glucose and glutamine culture setpoints were 0.5 g / L and 0.5 mM, respectively, and these concentration levels were maintained using a predictive feed by adding concentrated DMEM-based protein-free feed and 150 g / L glucose solution. Cell culture samples were collected and analyzed daily throughout the growth curve experiment until culture viability dropped below 50%. If necessary, culture supernatant samples were stored at -20°C for further analysis.

[0111] Antibody glycosylation analysis

[0112] With modifications, antibody glycosylation analysis was performed according to a previously published protocol (Chan et al., 2015). Briefly, the IgG sample purified from protein A was first desalted using a PD 10 column (GE Healthcare, Pittsburgh, PA) following the manufacturer's protocol. The glycan was then released and labeled using RapiFluor MS (RFMS) (Lauber et al., 2015) according to the manufacturer's protocol (Waters Corporation, Milford, MA). After labeling, excess RFMS was removed by passing the labeled mixture through a MiniTrap G-10 desalting column (GE Healthcare), and the purified RFMS-labeled glycan was then dried under vacuum. The sample was restored in 200 μl of restoration buffer containing 42.8 μl water, 50 μl dimethylformamide, and 107.2 μl acetonitrile, and analyzed using the UNIFI biopharmaceutical platform (Waters Corporation, Milford, MA). The raw retention times of each obtained chromatographic peak were converted into glucose units (GU) by fitting them to a calibration curve established using a dextran standard ladder labeled with RFMS (Waters Corporation). The observed GU values ​​and associated masses of each peak were then used to search an experimental database of N-glycans embedded in the UNIFI biopharmaceutical platform, which contains information on expected GU values ​​and masses for over 300 N-glycan species. Structures were then assigned to each peak based on two orthogonal criteria: 1) the observed GU value matched the expected GU value within a 0.2 GU deviation; and 2) the observed mass matched the expected mass of the glycan within a 5 ppm mass error. Furthermore, knowledge of CHO glycosylation characteristics was applied as a biofilter to remove irrelevant candidate structures, such as glycans with bipartite GlcNAc and α2,6-linked sialic acid.

[0113] calculate

[0114] According to Equation 1, the specific growth rate (μ) is determined by plotting ln(VCD) versus t, where VCD is the viable cell density, VCD0 is the initial viable cell density, and t is the culture time.

[0115] VCD = VCD0e μt

[0116] ln(VCD / VCD0)=μt---Equation 1

[0117] Based on Equation 2, the cumulative integral live cell density (IVCD) is calculated using the trapezoidal rule.

[0118] IVCD t =IVCD t-1 +0.5×(VCD t +VCD t-1 Equation 2

[0119] According to Equation 3, the specific IgG productivity (qp) between culture times t1 and t2 is determined by plotting the relationship between IgG titer (P) and IVCD.

[0120] q p =(P t2 -P t1 ) / IVCD

[0121] P t2 =q p ×IVCD+P t1 ---Equation 3

[0122] According to Equation 4, the specific substrate consumption rate (qs) between culture times t1 and t2 is determined by plotting the relationship between substrate concentration (S) and IVCD.

[0123] -q s =(S t2 -S t1 ) / IVCD

[0124] S t2 =-q s ×IVCD+S t1 ---Equation 4

[0125] The specific substrate consumption rate (qs) is fitted into the Mono model according to Equation 5, where qs_max is the maximum specific substrate consumption rate, S is the substrate concentration, and Ks is the affinity constant.

[0126] q s =(q s_max×S) / (K s +S)---Equation 5

[0127] Results and Discussion

[0128] Evaluation of disaccharide support for CHO-K1 cell growth

[0129] To evaluate the use of disaccharides to support mammalian cell growth, 0.3 × 10⁻⁶ cells were used. 6 The seeding cell density of Chinese hamster ovary (CHO) cell line CHO-K1 was determined by culturing it in serum-free and protein-free HyQ PF-CHO cell medium at a seeding density of 3.6 g / L of maltose, sucrose, lactose, trehalose, or glucose. The osmotic pressure concentration of these media was determined to be between 308 mOsm / kg and 324 mOsm / kg, well within the optimal range for mammalian cell culture. The viable cell density and culture viability of these cultures at the beginning and end of each passage over a 74-day period are shown in [data missing]. Figure 1 While cells grew to high culture viability and viable cell density at each passage in glucose-containing media, those parameters decreased and remained stagnant in disaccharide media, respectively. However, culture viability and viable cell density in maltose cultures began to increase on day 14. Cell proliferation in maltose cultures then persisted for the studied period, while in the other disaccharides it ceased on day 31 due to lack of cell growth and decreased culture viability. Using 1.0 × 10⁻⁶... 6 Repeated experiments at higher seeding densities of 10 cells / mL yielded similar results (data not shown). Therefore, this disclosure confirms that CHO-K1 cells can proliferate using maltose as a glycogen source in serum-free, protein-free media, but not using sucrose, lactose, or trehalose as a glycogen source.

[0130] To verify the observation results, then 0.3 × 10⁻⁶ was used. 6 With similar initial cell density of 1 cell / mL and glucose concentration of 3.6 g / L, the growth curves of the adapted CHO-K1 cell line in maltose-free protein medium were compared with those of CHO-K1 cells cultured in glucose-free protein medium. Figure 2 Cells cultured using glucose as an energy source (reaching 5.9 × 10⁻⁶ on day 6) were compared with those cultured using glucose as an energy source. 6 Compared to cells / mL, cells cultured using maltose as an energy source grew to 1.4 × 10⁻⁶ cells / mL on day 7. 6 The maximum viable cell density is much lower than that of cells / mL. Figure 2A). Since the initial sugar concentrations are the same, the inventors of this disclosure hypothesize that the difference may be due to the depletion of other nutrients such as glutamine, since glutamine is consumed at a similar rate in both glucose and maltose cultures until it is fully utilized on days 4 and 5, respectively. Figure 2 D). Using live cell density data up to day 4, the doubling time of maltose cultures was 53.7 hours, compared to 22.3 hours for glucose cultures, suggesting that the rate of energy metabolism may limit cell growth rate in maltose cultures. Interestingly, maltose cultures maintained higher culture viability (greater than 80%) for a longer period compared to cells cultured in glucose. Figure 2 A). This may be partly because maltose cultures achieve lower maximum cell densities, which in turn may result in higher levels of unmetabolized nutrients and lower accumulation of toxic metabolites at subsequent time points in culture. Another reason may be that cells in maltose cultures have lower metabolism due to the limiting rate of energy uptake, leading to less cellular stress and slower cell death.

[0131] When examining key biochemical substances in a culture medium, glucose is undetectable in maltose cultures. Figure 2 B), which confirms the absence of glucose in the culture medium from the start of the culture. Since the glucose concentration remained undetectable throughout the culture, this suggests that maltose was not hydrolyzed into glucose by secreted maltases in the medium, but rather consumed by the cells. This consumption may occur via one of three (3) possible mechanisms: (1) maltose may be transported into the cells and subsequently hydrolyzed by intracellular maltases, such as acid α-glucosidase (GAA); or (2) maltose may be hydrolyzed by plasma membrane-bound maltases, such as intestinal maltase-glucosidase (MGAM) and sucrase-isomaltase (SI), and immediately taken up by glucose transporters also present on the plasma membrane. These additional steps of energy metabolism may be rate-limiting, resulting in the slow growth rate of maltose cultures, as described above. The lack of lactate production in maltose cultures ( Figure 2 C) supports this hypothesis because it confirms that cells in maltose cultures utilize a more energy-efficient Krebs cycle, rather than ending the glycolytic pathway by producing lactate, as demonstrated by lactate production in glucose cultures. Figure 2 C).

[0132] The study investigated glutamine consumption, although this was slower in maltose cultures than in glucose cultures. Figure 2(D) However, this may be because the maltose culture grows more slowly. This was verified by comparing the specific glutamine consumption rates of the glucose and maltose cultures: by plotting the relationship between the glutamine characteristics of the cultures and IVCD, it was observed that the specific glutamine consumption rates, given by the slope of the curves, were similar between the two cultures. Figure 2 F). This confirms that the glutamine consumption rate of each cell type is unaffected by the use of maltose. When compared to glucose culture, the similar specific glutamine consumption rate, given the slower growth rate and lower maximum cell density of maltose culture, suggests that more glutamine in maltose culture is being diverted to energy metabolism rather than cell replication. This hypothesis is supported by the ammonium production curves: although the specific ammonium production rates of the two cultures are initially similar, the specific ammonium production rate of glucose culture slows significantly from day 2 onwards. Figure 2 G) to achieve a final ammonium concentration of 0.14 g / L. Figure 2 E), while the specific ammonium production rate of maltose cultures remained at a similar rate until day 5 to reach a higher final ammonium concentration of 0.20 g / L. The slower specific ammonium production rate of glucose cultures from day 2 onwards can be attributed to the higher cell growth rate (E). Figure 2 A) and similar ammonium production rates, indicating that more amino acids were used for cell replication compared to maltose cultures. The difference in final ammonium concentration further supports this, as it confirms that more amino acids in maltose cultures underwent deamidation to replenish cellular energy expenditure compared to glucose cultures, since both cultures had the same initial concentrations of glutamine and other amino acids. It was also noted that the time to reach steady-state ammonium production (day 4 for glucose cultures and day 6 for maltose cultures) corresponded to the glutamine depletion time of the corresponding cultures, suggesting that ammonia accumulation may be partly due to spontaneous glutamine deamidation. When these maltose-adapted CHO-K1 cells were further acclimatized to a protein-free, chemically defined medium (PFCDM) based on DMEM / F12 and tested with increasing concentrations of maltose from 10 g / L to 40 g / L, cells proliferated more rapidly, suggesting that maltose can be used as a glucose substitute in the absence of hydrolysates in routine mammalian cell cultures. Figure 10 ).

[0133] In summary, these data confirm that cells do indeed proliferate in maltose medium in the absence of serum or protein supplements, although at a slower rate, likely due to rate-limiting energy metabolism. The specific glutamine consumption rate remained similar in maltose culture compared to glucose culture, suggesting that essential nutrients may be depleted to limit the maximum viable cell density achievable in maltose culture. The similar specific glutamine consumption rate and increased ammonium production in maltose culture compared to glucose culture further confirm that more amino acids may be deamidinated in maltose culture to supplement energy metabolism.

[0134] The survival and proliferation of CHO-K1 cells in a protein-free medium containing maltose is remarkable, given the absence of known mammalian maltose transporters and the fact that mammalian cells are generally known to be unable to metabolize disaccharides unless, for example, secretory or transmembrane maltases are expressed in intestinal cells. Although CHO cells have been shown to utilize polysaccharides as an energy source for survival, these experiments have been conducted in serum-containing media (Morgan and Faik, 1981), and it has been demonstrated that enzymes in serum break down these polysaccharides for cellular metabolism (Scannell and Morgan, 1982). Therefore, this disclosure remarkably demonstrates that serum-free, protein-free mammalian cell cultures, as disclosed herein, can provide a sufficient energy source for cell growth when containing disaccharides.

[0135] Maltose is used to maintain culture viability when glucose is depleted.

[0136] Because CHO-K1 cells can utilize maltose as an energy source and maintain high cell culture viability for a longer period of time in the absence of glucose in a protein-free medium containing maltose. Figure 2 Therefore, the inventors of this disclosure further evaluated whether maltose could be utilized to maintain the culture viability of maladaptive CHO-K1 production cell lines in the event of glucose depletion. Cells were cultured in a medium containing both glucose and maltose to investigate whether the cells could utilize glucose for normal cell growth in the medium, subsequently switching to maltose metabolism to maintain culture viability and protein production. This potentially extends the application of maltose supplementation to maladaptive cell lines and is also a simple method for extending cell culture viability in batch cultures to achieve higher recombinant protein productivity.

[0137] For this evaluation, the CHO-K1 cell line (SH87) producing anti-Her2 monoclonal antibodies was used (Ho et al., 2012). These cells were monitored in batch shake-flask cultures in DMEM / F12-based, protein-free, chemically defined media (PFCDM) containing 4 g / L glucose, 6 g / L glucose, or 4 g / L glucose supplemented with different concentrations of maltose until culture viability decreased to below 50%. Figure 3 A basal glucose concentration of 4 g / L was chosen because this would allow for early but controlled cell growth restriction, primarily due to glucose depletion in the batch cultures of the PFCDM. Figure 3 This controlled growth restriction was observed in culture A, where the 4 g / L glucose culture reached 8.5 × 10⁸ on day 5. 6 The maximum viable cell density (VCD) was achieved one day earlier than the 6 g / L glucose culture, which reached 11.9 × 10⁶ cells / mL on day 6. 6 The maximum VCD is [number] cells / mL. This corresponds to glucose depletion in both cultures, which occurs one day before reaching the maximum VCD. Figure 3 B). Therefore, by supplementing the 4 g / L glucose medium with maltose, the effect of additional maltose on this unadapted CHO-K1 production cell line can be determined without the additional complications of other nutrient depletion.

[0138] from Figure 3 In A, all cultures supplemented with maltose were observed to have higher maximum VCD and longer culture viability compared to cultures containing only 4 g / L glucose, even though glucose in these media was simultaneously depleted. Figure 3 B).

[0139] Compared to cultures containing only 4 g / L glucose, cultures supplemented with maltose also maintained recombinant protein production, resulting in higher IgG titers. Figure 3 E). When compared with cultures containing only 6 g / L glucose, cultures supplemented with maltose had lower maximum VCD and IgG titers. Figure 3 A and Figure 3 G), except for the IgG titer of the culture supplemented with 3 g / L maltose, this IgG titer matched the IgG titer of the culture supplemented with 6 g / L glucose. This confirms that maltose can support cell growth even when glucose is depleted before the 6 g / L glucose culture. Figure 3 B), but metabolism is slower in maltose-supplemented cultures, resulting in a slower growth rate. This is further supported by the observation that the growth enhancement caused by maltose is concentration-dependent. Figure 3A), indicating that the rate of maltose metabolism may be limiting at these concentrations. This also corresponds to the ammonium production curve ( Figure 3 G), which showed that when compared with cultures containing 6 g / L glucose or 3 g / L maltose supplement, cultures containing 0 g / L, 0.5 g / L, and 1 g / L maltose supplement had higher ammonium production, although the glutamine consumption curves were similar. Figure 3 F). This indicates that in cultures containing no maltose or with low concentrations of maltose, more amino acids undergo deamidation to replenish energy expenditure. On the other hand, cultures supplemented with 3 g / L maltose exhibited lower ammonia production, similar to cultures supplemented with 6 g / L glucose. Figure 3 (G). This indicates that during the later stages of batch culture, at this maltose concentration, the maltose metabolic rate was sufficient to meet the reduced energy requirements of the cells.

[0140] When studying lactate curves, it was noted that lactate consumption occurred in all cultures under conditions of glucose depletion. Figure 3 C). In contrast to glucose-only cultures (where lactate consumption was partial), all lactate was consumed in maltose-supplemented cultures by day 6. It is presumed that complete lactate consumption may be facilitated by maltose metabolism, which sustains culture survival and allows for lactate utilization over a longer period in a low-glucose environment. This characteristic of maltose-supplemented cultures could potentially be useful for biopharmaceutical production, as lactate accumulation in fed-batch bioreactor production often leads to cytotoxicity (Hassell et al., 1991; Lao and Toth, 1997). Interestingly, lactate production was again observed from day 6 in cultures supplemented with 3 g / L maltose, supporting the hypothesis that at this maltose concentration, the rate of maltose metabolism is sufficient for cell survival via glycolysis.

[0141] When analyzing the maltose concentration in the culture supernatant, it was observed that maltose was actually consumed by the cells, and most of the consumption occurred when glucose was depleted. Figure 3 D). This indicates that these cells preferentially utilize glucose for growth, and when glucose is depleted, the culture switches to maltose metabolism, which helps maintain culture viability. Figure 3 A). Interestingly, maltose was depleted on approximately the same day, despite variations in initial maltose concentrations of up to 6-fold. This may be partly attributed to differences in VCD among various maltose-supplemented cultures, but it also suggests that maltose metabolism at these maltose concentrations may be concentration-dependent, leading to higher maltose utilization at higher concentrations.

[0142] To determine whether maltose could be internalized by cells, SH87 cell samples were obtained from both cultures containing only 4 g / L glucose and cultures supplemented with 3 g / L maltose for LC-MS analysis. Figure 3 H). Although intracellular maltose was detected 1 day after inoculation into maltose-supplemented medium, it was absent (below the detection limit) in cells cultured in glucose-only medium or immediately after inoculation into maltose-supplemented medium. This confirms the presence of an intracellular maltose pool in cultures supplemented with 3 g / L maltose and demonstrates that maltose does indeed enter these cells despite the lack of a known transport mechanism, adding further evidence that maltose is indeed utilized by cells in maltose-supplemented cultures. Furthermore, the intracellular maltose concentration provided further insight into maltose transport; over the 5 days monitored, the intracellular maltose concentration was maintained at approximately 1 / 100th of the level in the medium. This suggests that despite the concentration gradient, maltose transport may be actively regulated to prevent its intracellular accumulation, as cells only switch to maltose metabolism when glucose is depleted. Figure 3 D).

[0143] To further verify that maltose is indeed utilized by cells rather than hydrolyzed in the culture medium, cell-free conditioned medium (CM) was obtained from cultures supplemented with 3 g / L maltose and 4 g / L glucose cultures on days 2, 4, 6, and 8. 3 g / L maltose was incorporated into the CM from the glucose culture, and changes in glucose and maltose concentrations in both CM groups were monitored at 37°C for 3 days. Figure 4 The growth curve of the culture is similar to... Figure 3 Of those in A, the maltose-supplemented cultures, compared to glucose cultures, showed higher viable cell density and prolonged culture viability on days 6 and 8. Figure 4 A). The glucose and maltose curves of the maltose-supplemented cultures were also similar, with glucose depleted on day 6 and maltose depleted between day 4 and day 8. Figure 4 B). On the other hand, despite incubation at the same temperature for 3 days, CMs harvested from maltose-supplemented cultures maintained the same glucose and maltose concentrations as when they were harvested. Figure 4 B). Similarly, despite low culture viability on days 6 and 8, CM derived from glucose cultures incorporating 3 g / L maltose maintained consistent glucose and maltose concentrations. Figure 4C). This confirms that the presence of cells is essential for the utilization of maltose, and that maltose hydrolysis does not occur spontaneously in conditioned media, even when culture viability is low. Since maltose can be hydrolyzed by pancreatic and salivary α-amylases of secretory proteins, by intestinal maltase-glucosidase and sucrose-isomaltase of transmembrane proteins, and by lysosomal α-glucosidase and neutral α-glucosidase C of intracellular proteins, this data confirms that secreted maltases are not involved in maltose metabolism in these CHO cells.

[0144] Because the culture medium described herein is free of serum, hydrolysates, and proteins, this disclosure excludes the role of ambiguous culture medium components in these observations and ultimately demonstrates that the mammalian cell line CHO-K1 can utilize maltose for growth and recombinant protein production. Furthermore, this data shows that the cells do not require prior adaptation to utilize maltose in a biphasic manner and maintain culture viability.

[0145] Comparison of batch cultures with high glucose and maltose concentrations

[0146] Since maltose metabolism at concentrations of 3 g / L or lower is observed to be potentially limiting, the culture curves of SH87 cells in media supplemented with 4 g / L glucose and 10 g / L or 20 g / L maltose will be compared with those of the same cells in media containing 14 g / L and 24 g / L glucose. Figure 5 Compared to the other three cultures, the 24 g / L glucose culture grew more slowly, reaching a lower 10.6 × 10⁻⁶. 6 Maximum VCD of cells / mL, and reaches low viability below 50% more quickly. Figure 5 A). The slower growth of the 24 g / L glucose culture can be attributed to the higher osmotic pressure concentration of the medium (360 mOsm / kg). Figure 5 F), while the other three cultures had similar weight molar osmolality concentrations of 318 mOsm / kg to 331 mOsm / kg, which are within the optimal range for mammalian cell culture. This confirms the limitation of high glucose loading in batch media, as glucose significantly increases weight molar osmolality and can affect cell growth at high concentrations. Conversely, the same mass concentration of maltose resulted in a smaller increase in weight molar osmolality and therefore had no negative impact on cell growth.

[0147] Compare the growth curves of cultures with 10 g / L maltose, 20 g / L maltose, and 14 g / L glucose. Figure 5A) They are similar, even after the glucose concentration in maltose-supplemented cultures reaches its minimum on day 5. Figure 5 B). This indicates that the rate of maltose metabolism at these concentrations did not limit cell growth, compared to the slower growth observed after glucose depletion at maltose concentrations below 3 g / L. Figure 3 A and Figure 3 B) Conversely. Glucose accumulation was observed in cultures supplemented with 10 g / L and 20 g / L maltose. Figure 5 B), this is a phenomenon that has not been observed before. Figure 3 B) supports the hypothesis that maltose metabolism is higher at these concentrations. This indicates that maltose hydrolysis occurs faster than the cells' metabolic needs during the corresponding culture periods: after day 5, glucose accumulation was observed in the culture supplemented with 20 g / L maltose while cells were still growing at a similar rate to the 14 g / L glucose culture, confirming that maltose metabolism did not limit cell growth at this concentration. At the lower maltose concentration of 10 g / L, glucose accumulation was only observed after day 8, when cells were entering the death phase. Cells grew similarly to the 14 g / L glucose culture, suggesting that at 10 g / L maltose, the rate of maltose metabolism was just sufficient to support cell growth without excessive glucose accumulation in the culture medium.

[0148] It is also noteworthy that lactate concentrations accumulated in glucose-only cultures throughout the entire culture duration, whereas maltose-supplemented cultures consumed most of their lactate from day 5 to day 8. Figure 5 (C) Although a second lactate production phase was observed in cultures supplemented with 10 g / L and 20 g / L maltose after day 9 and day 10, respectively, lactate concentrations reached significantly lower levels in these cultures compared to glucose cultures. This confirms another limitation of glucose loading in batch media: the lack of glucose consumption under high glucose loading may limit cell culture growth because lactate accumulation can lead to cytotoxicity (Hassell et al., 1991; Lao and Toth, 1997). In this case, this lactate toxicity may have led to the faster death phase observed in the 24 g / L glucose culture.

[0149] The study investigated the maltose curves of cultures supplemented with maltose, showing that most of the maltose was consumed by day 5 after glucose depletion. Figure 5 D), which is similar to the previous data ( Figure 3 D), and for both maltose-supplemented cultures, maltose was not depleted until the end of the run on day 14. Similarly, for both glucose cultures, glucose was not depleted ( Figure 5B). Although sugar was present in excess, other nutrients may have limited the growth of these cells; for example, glutamine was depleted at a similar rate on day 6 for all four cultures. Figure 5 E). These other limitations may similarly affect growth so that the growth curves of cultures supplemented with maltose and cultures with 14 g / L glucose are similar.

[0150] Interestingly, given similar growth curves, it was noted that the culture supplemented with 20 g / L maltose produced a maximum IgG titer of 298 mg / L, which was 15% higher than the maximum IgG titers obtained from the 14 g / L glucose culture and the culture supplemented with 10 g / L maltose (providing maximum titers of 259 mg / L and 263 mg / L, respectively). Figure 5 To investigate this, the specific IgG production rate of the culture was determined according to Equation 3 (Table 1 and ). Figure 11 It was noted that the specific IgG production of all four cultures decreased after day 7 when glutamine concentrations reached their minimum, suggesting that glutamine may limit IgG production from day 7 onwards. Regardless of glutamine limitation, specific IgG production was observed to be 21% to 29% higher in cultures containing more glucose or more maltose, indicating that higher sugar concentrations can improve specific IgG production. When the specific IgG production of maltose-supplemented cultures was compared to their corresponding glucose cultures with the same total sugar concentration, the maltose-supplemented cultures had 13% to 17% lower specific IgG production up to day 6, although these became 25% to 26% higher than their corresponding glucose cultures from day 7 onwards. Therefore, the presence of maltose negatively impacts specific IgG production before glutamine depletion but contributes to improved specific IgG production after glutamine depletion. Therefore, when compared with cultures supplemented with 14 g / L glucose and 10 g / L maltose, the higher sugar content of cultures supplemented with 20 g / L maltose resulted in 7% to 53% higher IgG production, respectively, and also resulted in higher maximum IgG titers in cultures supplemented with 20 g / L maltose, despite having similar growth curves.

[0151] Table 1: Specific IgG productivity of SH87 cells cultured in a protein-free, chemically defined medium (PFCDM) with high concentrations of glucose and maltose. SH87 cells, routinely maintained in glucose-only PFCDM, were passaged into PFCDMs containing 14 g / L glucose, 24 g / L glucose, 4 g / L glucose + 10 g / L maltose, or 4 g / L glucose + 20 g / L maltose. IgG titers were then plotted against IVCD according to Equation 3 to obtain the specific IgG productivity as the slope of the graph.

[0152]

[0153]

[0154] Characterization of maltose metabolic kinetics

[0155] To characterize maltose metabolism in maltose-supplemented cultures, the specific maltose consumption rate was obtained from a graph of maltose concentration versus cumulative integrated viable cell density (IVCD) according to Equation 4. These graphs showed linear trend lines with R² values ​​ranging from 0.853 to 0.999 when 3 to 5 data points were used after glucose depletion. Figure 12 This indicates that the specific maltose consumption rate is fairly constant during maltose metabolism. The slope of these trend lines gives the average specific maltose consumption rate of these cultures and... Figure 6 The data is presented in a table in section A. The specific maltose consumption rate increases with increasing initial maltose concentration, confirming that maltose metabolism at these concentrations is indeed concentration-dependent. Furthermore, it was observed that the increase in specific consumption rate decreases at higher initial maltose concentrations, suggesting the possible existence of a maximum in this relationship. Therefore, according to Equation 5, the data is fitted to a Monod model via nonlinear regression. The data is fitted to the model (…). Figure 6 B) To obtain the maximum specific maltose consumption rate (qs_max) of 0.257 ng / cell / day and the affinity constant (Ks) of 7.03 g / L. While it may be meaningful to model the effect of maltose concentration on the relative growth rate, it is not feasible to use this data because cell growth is mostly minimized after glucose depletion. The maximum specific maltose consumption rate determined here is similar to that of fructose (0.21 ng / cell / day) and galactose (0.21 ng / cell / day) as reported using 3.6 g / L monosaccharide (Altamirano et al., 2000), but lower than the measured specific consumption rates of glucose (0.76 ng / cell / day) and mannose (0.88 ng / cell / day). This suggests that maltose can be metabolized at rates similar to fructose and galactose, while maltose metabolism will be slower than glucose and mannose. While the inventors of this application note that these parameters can be determined more accurately in continuous culture, this can be practically challenging at low maltose concentrations because the growth rate will be low. Therefore, the current data provide compelling evidence that mammalian cells may possess maltose metabolic kinetics.

[0156] Application of maltose in fed-batch culture

[0157] Since fed-batch culture is a common mode for manufacturing monoclonal antibodies, the use of maltose in fed-batch culture was evaluated. Unlike batch culture, where the initial glucose concentration is limited by the adverse effects on cell growth due to an increase in osmotic pressure exceeding a certain limit, fed-batch culture does not have this limitation because glucose can be continuously added to the culture. Therefore, the use of maltose to supplement glucose in fed-batch cultures to drive cells toward a slower but more efficient metabolism was evaluated: using a common glucose concentration setpoint of 0.5 g / L, SH87 cells in a medium supplemented with 4 g / L glucose and 20 g / L maltose were supplemented daily with 50% of their calculated glucose requirement, while the same cells in a medium containing 4 g / L glucose were supplemented daily with 100% of their calculated glucose requirement. Two bioreactor cultures were performed for each condition, and growth, biochemical, and IgG titer profiles of these cultures were plotted. Figure 7 middle.

[0158] Compared with lactate consumption observed in batch cultures ( Figure 3 and Figure 5 Conversely, there is no lactate consumption in fed-batch cultures, even in cultures supplemented with maltose and containing 50% glucose. Figure 7 C). It is speculated that this may be due to the consistent nutrient supplementation in fed batch cultures, where glucose, glutamine, or other nutrients may be depleted, leading to the observed lactate consumption. Nevertheless, the IgG titer in fed batch cultures ( Figure 7 G) is more than 5 times the IgG titer obtained from batch cultures. Figure 5 This indicates that the fed-batch strategy was successful in increasing the expected IgG yield in fed-batch cultures.

[0159] Comparing fed-batch cultures, the maltose-supplemented cultures showed similar viable cell density, culture viability, and lactate profiles to glucose-only culture #2, while glucose-only culture #1 exhibited higher maximum viable cell density, faster culture viability decline, and faster lactate accumulation. Figure 7 A and Figure 7 C). The osmotic concentration of culture 1, which contains only glucose, increases more rapidly by weight molar (C). Figure 7F) This could be due to pH correction caused by faster lactate accumulation. These illustrate possible variations in these parameters in SH87 fed batch cultures. On the other hand, the glucose and glutamine profiles were similar between maltose-supplemented and glucose-only cultures. Furthermore, IgG titers were fairly consistent between parallel cultures, although variations in viable cell density, lactate, and osmolality were observed in glucose-only batch cultures.

[0160] Comparing the glucose curves, it is interesting to note that despite being fed only 50% of its calculated glucose requirement, the maltose-supplemented culture maintained a similar culture glucose concentration to the glucose-only fed batch cultures. Nevertheless, due to this reduced glucose feeding, the specific glucose consumption rate of the maltose-supplemented culture was 0.115 ± 0.007 ng / cell / day, which is 45% of the specific glucose consumption rate of the glucose-only culture (0.254 ± 0.013 ng / cell / day) (Table 2). These suggest that the maltose-supplemented culture most likely utilized a secondary energy source in addition to glucose to achieve growth and culture viability curves comparable to those of the glucose-only culture (Culture 2). This secondary energy source is likely maltose, because the rate of maltose consumption is similar between maltose-supplemented cultures and glucose-only cultures (Table 2), and maltose consumption was observed in maltose-supplemented cultures from day 7 (one day after glucose feeding began in maltose-supplemented fed batch cultures). Figure 7 D).

[0161] Table 2: Maximum IgG titers, specific productivity, and specific consumption of SH87 cells cultured in fed-batch bioreactor cultures in chemically defined, protein-free media (PFCDM) with and without maltose supplementation. SH87 cells routinely maintained in glucose-only PFCDM were passaged in duplicate fed-batch bioreactor cultures (denoted as No. 1 and No. 2). Figure 7 As described in [the document]. The specific growth rate was determined according to Equation 1. A graph was plotted showing the cumulative amount of biochemical substances produced by the cells versus the cumulative integral number of living cells (IVCs) to obtain the specific productivity as the slope of the graph.

[0162]

[0163]

[0164] With maltose-supplemented metabolism, improvements of 23% and 55% were observed in the maximum IgG titer (1.483 ± 0.067 g / L) and specific IgG productivity (28.8 ± 3.5 pcd) compared to the glucose-only fed batch culture (18.6 ± 4.1 pcd), respectively (Table 2). When compared to the glucose-only culture 2 (with a more comparable growth curve of 1.53 g / L and 21.5 pcd), the improvements in maximum IgG titer and specific IgG productivity were 20% and 34%, respectively.

[0165] One possible mechanism for the observed increase in IgG production is due to the additional 20 g / L maltose, resulting in a higher initial osmolar concentration in the maltose-supplemented culture. This could lead to a 22% to 40% lower specific growth rate in the maltose-supplemented culture compared to glucose-only cultures (Table 2), potentially allowing for slower and higher yields in the maltose-supplemented culture. Nevertheless, when comparing maltose-supplemented batch cultures with glucose-only batch cultures having similar initial osmolar concentrations... Figure 5 G), a strikingly similar increase in IgG titers was observed. This suggests that the increase in maximum IgG titers observed in maltose-supplemented fed batch cultures is not solely due to the telomeric osmotic concentration effect: not wanting to be bound by theory, it is hypothesized that the lower glucose availability achieved through the presence of maltose may induce more efficient cellular metabolism in maltose-supplemented cultures, thus also contributing to the observed higher maximum IgG titers.

[0166] To verify whether this lower glucose availability would have the same effect in the absence of maltose, separate experiments were conducted comparing SH87 fed batch cultures containing only glucose with 100% or 50% of their calculated glucose requirements. Figure 13 Although similar maximum viable cell densities were reached before glucose feeding was initiated under both conditions, the viability of glucose-only cultures with 50% calculated glucose feeding decreased to below 50% four days earlier than that of cultures with 100% calculated glucose feeding. The maximum IgG titer of the 50% fed cultures was also only 46.4% of that of the 100% fed cultures. These findings confirm that the presence of maltose allows for the use of lower glucose feeding levels to induce... Figure 7 The higher maximum IgG titer observed in G.

[0167] Since glycosylation is a key property of therapeutic IgG products, glycosylation profiling was performed on purified IgG from glucose-only cultures and maltose-supplemented cultures from days 10 and 15. Figure 8 Representative fluorescence chromatogram of the sample on day 10 (). Figure 8 A) The glycosylation profiles of IgG from both glucose-only and maltose-supplemented cultures were similar, indicating that maltose supplementation generally does not affect the glycan profiles of the resulting monoclonal antibodies. When the relative abundance of glycan structures in IgG from maltose-supplemented cultures was compared to that from glucose-only cultures ( Figure 8 B) Note that fucosylation, sialylation, G1F, G2F, and bibranched glycans were slightly less, while G0F and monobranched glycans were more abundant. Most of these differences were also observed when glycans from day 15 samples were compared to those from day 10 samples, suggesting that maltose supplementation affects the glycan profile in a manner very similar to later harvests. An exception to this similarity is the relative abundance of sialylation and high-mannose glycans: while the later day 15 harvest provided higher levels of high-mannose glycans and similar levels of sialylated glycans compared to day 10 harvests, maltose supplementation provided lower levels of sialylated glycans and similar levels of high-mannose glycans compared to glucose-only cultures. This data suggests that maltose supplementation can also be used as a means of fine-tuning the glycosylation profile of monoclonal antibodies, particularly by slightly reducing sialylation levels, which are known to improve antibody-dependent cytotoxicity (ADCC) of therapeutic antibodies. Furthermore, this could also be important in the field of biosimilar manufacturing, where matching the glycan profile of biosimilars with that of innovative drugs is a crucial criterion.

[0168] The effect of maltose supplementation in both basal and fed media in fed-batch cultures.

[0169] Furthermore, this disclosure investigates whether supplementing the fed medium with maltose can have a further impact on fed batch cultures with a maltose-supplemented basal medium. Using a common glucose concentration setpoint of 0.5 g / L, SH87 was supplemented daily with a sugar feed consisting solely of glucose or a 1:1 ratio of glucose and maltose to add 50% of its calculated glucose requirement to a medium supplemented with 4 g / L glucose and 20 g / L maltose.

[0170] For each condition, two copies of the bioreactor were cultured, and the growth curves, biochemical curves, and IgG titer curves of these cultures were plotted. Figure 9In this study, additional maltose supplementation in the fed medium was observed to have no additional effect on cell growth curves, viability curves, glutamine curves, osmolar concentration curves, and IgG titer curves. This is likely because the initial maltose supplementation was not exhausted in cultures with glucose-only supplementation. It would be expected that adding maltose to the fed medium could be effective if the initial maltose is exhausted during longer fed batch runs (which could be achieved if temperature and / or pH variations are implemented). It was noted that additional maltose supplementation in the fed medium increased glucose concentration and slightly decreased lactate concentration compared to cultures with glucose-only supplementation. This could be advantageous in cultures where lactate buildup can lead to culture collapse.

[0171] Similarly, glycosylation analysis was performed on samples from these cultures. The differences in glycosylation profiles are shown in Table 3.

[0172] Table 3: Effect of maltose supplementation in fed culture media on glycosylation profiles. Differences in glycosylation profiles of anti-Her2 antibodies produced from SH87 fed batch bioreactor cultures using glucose-only (glucose + maltose) or glucose + maltose-only (glucose + maltose_BF) basal media with protein-free chemical composition defined by glucose-only or glucose + maltose (PFCDM) feed. Anti-Her2 monoclonal antibodies were purified from samples from day 10 and day 15 of SH87 fed batch cultures and glycosylation profiles were analyzed. Differences in the relative abundance of glycan structures were calculated using data from two parallel biological assays. The difference in the effect of maltose supplementation on the relative abundance of glycan structures compared to maltose cultures with glucose-only feed is shown in bold.

[0173]

[0174]

[0175] Although maltose supplementation in the fed medium had no effect on the titers of the produced antibodies, it provided slightly fewer fucoidan and dibranched glycans and more high-mannose and monobranched glycans compared to maltose cultures with glucose-only supplementation. Comparing columns 3 and 4 in Table 3, it is noted that the presence of maltose in the feed resulted in a glycosylation profile of the maltose-supplemented culture that was more similar to that of later-harvested cultures. These findings confirm that maltose supplementation in the fed medium can be used to supplement maltose-supplemented basal media to fine-tune the glycosylation profiles of recombinant glycoprotein products.

[0176] Evaluation of disaccharide support for the growth of CHO-DG44 cells

[0177] To determine whether the same method could be applied to other CHO cell lines, the use of disaccharide-supported growth of CHO-DG44 cells was evaluated. These cells were cultured in serum-free, protein-free HyQPF-CHO cell culture medium using 10 g / L maltose, sucrose, lactose, trehalose, or glucose as an energy source. The viable cell density and culture viability of these cultures at the beginning and end of each passage over a 22-day period are shown in [data missing]. Figure 1 In section B, while cells grew to high culture viability and live cell density at each passage in media containing glucose and maltose, those parameters decreased and remained stagnant in other disaccharide media, respectively. Therefore, it is confirmed that CHO-DG44 cells can also proliferate using maltose as a glycogen source in serum-free and protein-free media, but cannot proliferate using sucrose, lactose, or trehalose as a glycogen source. It is expected that, with maltose supplementation, the growth of CHO-DG44 producing cells and product titers will exhibit similar improvements, as demonstrated in this disclosure using CHO-K1 cells.

[0178] This disclosure surprisingly demonstrates that CHO-K1 cells can grow using maltose in the absence of serum or protein supplementation, although the cells grow at a slower rate. Furthermore, when using a medium containing both glucose and maltose, prior cell adaptation is not necessary for maltose utilization, which occurs biphasically after glucose depletion. Maltose utilization depends on the presence of cells in the culture because maltose is internalized by cells, and maltose hydrolysis does not occur spontaneously in conditioned media. Practical application of supplementing with maltose to increase the carbohydrate content of the cell culture medium is also shown, as the increase in glucose concentration is limited by a corresponding increase in t-molar osmotic pressure. Maltose utilization in batch cell cultures has the added advantage of promoting lactate consumption, which would otherwise accumulate and become toxic to cells. These factors resulted in a 15% increase in recombinant monoclonal antibody titers from batch cultures. The specific maltose consumption rate obtained from batch cultures was fitted into a Mono model to obtain a maximum specific maltose consumption rate (qs_max) of 0.257 ng / cell / day and an affinity constant (Ks) of 7.03 g / L.

[0179] Further confirmation indicates that maltose supplementation can also be applied to fed-batch bioreactor cultures, resulting in a 23% increase in maximum monoclonal antibody titer and a 55% increase in specific monoclonal antibody productivity compared to fed-batch cultures containing only glucose. This demonstrates that maltose supplementation can be applied as a simple bioreactor process improvement to increase monoclonal antibody yields in current fed-batch monoclonal antibody manufacturing processes. Glycosylation profiling analysis of antibodies produced from fed-batch cultures showed that maltose supplementation slightly affected the glycan profile, similar to later harvests, with a slight decrease in sialylation levels without the additional effect of increased high-mannose glycans. This suggests that maltose supplementation can also be applied to slightly affect the glycosylation profile of monoclonal antibodies, which is important for influencing ADCC of antibody therapeutics and the physical matching of biosimilar glycan profiles with innovative drugs.

[0180] Beyond the practical significance of maltose supplementation for biopharmaceutical production, the ability of CHO-K1 cells to utilize maltose in protein-free media is itself remarkable, given the absence of known mammalian maltose transporters and the generally accepted inability of mammalian cells to metabolize disaccharides. Therefore, this disclosure provides surprising evidence of serum-free, protein-free mammalian cell cultures utilizing disaccharides as an energy source. Estimates of maltose metabolic kinetics in mammalian cells are also disclosed.

[0181] Furthermore, this disclosure has demonstrated that maltose supplementation in fed culture media may have a negligible effect on the glycosylation profile of glycoprotein products, and that CHODDG44 cells can also utilize maltose as a carbohydrate source.

[0182] References

[0183] Altamirano, C., Paredes, C., Cairo, JJ, Godia, F., 2000. Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine, Biotechnol Prog. 16, 69-75.

[0184] Bresseleers, GJ, Goderis, HL, Tobback, PP, 1984. Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellarvesicles. Effect of membrane composition and temperature, Biochim Biophys Acta. 772, 374-82.

[0185] Chan, KF, Shahreel, W., Wan, C., Teo, G., Hayati, N., Tay, SJ, Tong, WH, Yang, Y., Rudd, PM, Zhang, P., Song, Z., 2015. Inactivation of GDP-fucosetransporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies, Biotechnol J.

[0186] Hassell, T., Gleave, S., Butler, M., 1991. Growth inhibition in animal cellculture. The effect of lactate and ammonia. Appl Biochem Biotechnol. 30, 29-41.

[0187] Hayashi, I., Sato, GH, 1976. Replacement of serum by hormones permits growth of cells in a defined medium, Nature. 259, 132-4.

[0188] Ho, SC, Bardor, M., Feng, H., Mariati, Tong, YW, Song, Z., Yap, MG, Yang, Y., 2012. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines, J Biotechnol. 157, 130-9.

[0189] Jones, MN, Nickson, JK, 1982. The human erythrocyte monosaccharide transporter in bilayer lipid membranes, Biochem Soc Trans. 10, 5-6.

[0190] Kurano, N., Leist, C., Messi, F., Kurano, S., Fiechter, A., 1990. Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor: 1. Effects of physical and chemical environments, J Biotechnol. 15, 101-111.

[0191] Lao, MS, Toth, D., 1997. Effects of ammonium and lactate on growth and metabolism of a recombinant Chinese hamster ovary cell culture, Biotechnol Prog. 13, 688-91.

[0192] Lauber, MA, Yu, YQ, Brousmiche, DW, Hua, Z., Koza, SM, Magnelli, P., Guthrie, E., Taron, CH, Fountain, KJ, 2015. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection, Anal Chem. 87, 5401-9.

[0193] Lodish H, BA, Zipursky SL, et al., 2000. Molecular Cell Biology, 4th ed. New York: WH Freeman.

[0194] Meyer, H., Vitavska, O., Wieczorek, H., 2011. Identification of an animals ucrose transporter, J Cell Sci. 124, 1984-91.

[0195] Morgan, MJ, Faik, P., 1981. Carbohydrate metabolism in cultured animal cells, Biosci Rep. 1, 669-86.

[0196] Mueckler, M., Thorens, B., 2013. The SLC2(GLUT) family of membrane transporters, Mol Aspects Med. 34, 121-38.

[0197] Okabe, T., Fujisawa, M., Takaku, F., 1984. Long-term cultivation and differentiation of human erythroleukemia cells in a protein-free, chemically defined medium, Proc Natl Acad Sci US A. 81, 453-5.

[0198] Ozturk, SS, Palsson, BO, 1991. Effect of medium osmolarity on hybridoma growth, metabolism, and antibody production. Biotechnol Bioeng. 37, 989-93.

[0199] Scannell, J., Morgan, MJ, 1982. The regulation of carbohydrate metabolism in animal cells: isolation of starch-and maltose-utilizing variants, Biosci Rep. 2, 99-106.

[0200] Selvarasu, S., Ho, YS, Chong, WP, Wong, NS, Yusufi, FN, Lee, YY, Yap, MG, Lee, DY, 2012. Combined in silico modeling and metabolomics analysis to characterize fed-batch CHO cell culture, Biotechnol Bioeng. 109, 1415-29.

[0201] Sinacore, MS, Drapeau, D., Adamson, SR, 2000. Adaptation of mammalian cells to growth in serum-free media, Mol Biotechnol. 15, 249-57.

[0202] Thorens, B., 1996. Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes, Am J Physiol. 270, G541-53.

[0203] Warburg, O., 1956. On the origin of cancer cells, Science. 123, 309-14.

[0204] Wong, D., Wong, K., Goh, L.-T., Heng, C., Yap, M., 2005. Impact of dynamic online fedbatch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures, Biotechnol Bioeng. 89, 164-77.

[0205] Wood, RE, Wirth, FP, Jr., Morgan, HE, 1968. Glucose permeability of lipid bilayer membranes, Biochim Biophys Acta. 163, 171-8.

[0206] Wright, EM, 2013. Glucose transport families SLC5 and SLC50, Mol Aspects Med. 34, 183-96.

[0207] Wright, EM, Loo, DD, Panayotova-Heiermann, M., Lostao, MP, Hirayama, BH, Mackenzie, B., Boorer, K., Zampighi, G., 1994. 'Active' sugar transport ineukaryotes, J Exp Biol. 196, 197-212.

Claims

1. A method for prolonging cell culture viability and increasing protein yield from mammalian cells, wherein the method comprises culturing mammalian cells in a serum-free cell culture medium containing maltose and glucose as carbohydrate sources, wherein maltose is present at a concentration of 3 g / L to 20 g / L and glucose is present at a concentration of 2 g / L to 8 g / L, wherein the serum-free cell culture medium is a chemically defined protein-free medium (PFCDM), and wherein the mammalian cells are CHO cells or HEK293 cells.

2. The method of claim 1, wherein maltose is present at a concentration of 10 g / L to 20 g / L.

3. The method of claim 1, wherein maltose is present at a concentration of 10 g / L or 20 g / L.

4. The method of claim 1, wherein the serum-free cell culture medium contains maltose and glucose as the sole carbohydrate source.

5. The method of claim 1, wherein the cell culture medium is further combined with an ionic surfactant or a nonionic surfactant.

6. The method of claim 1, wherein the cell culture medium further comprises an antibiotic agent.

7. The method of claim 1, wherein the serum-free cell culture medium comprises Duchenne Minimum Essential Medium (DMEM), F-12 Basal Medium (DMEM-F12), L-glutamine, nonionic surfactant, or genimycin.

8. The method of claim 1, wherein the serum-free cell culture medium is in powder or liquid form.

9. The method of claim 1, wherein the cells are capable of metabolizing the carbohydrate source without prior adaptation to the source.

10. The method of claim 1, wherein the CHO cells are selected from the group consisting of: CHO-K1 cells, CHO-DG44 cells, CHO-S cells, CHO-DXB11 cells, CHO-GS cells, or SH87 cells.

11. The method of claim 1, wherein the CHO cells are CHO-K1 cells.

12. The method of claim 1, wherein the CHO cells are SH87 cells.

13. A serum-free cell culture medium for use in the method of any one of claims 1 to 12, wherein the serum-free cell culture medium is PFCDM.

14. A method for modulating the glycosylation profile of a protein, wherein the method comprises culturing cells expressing the protein in a serum-free cell culture medium as defined in claim 1 or any one of claims 5 to 12, thereby producing cells expressing the protein having a modulated glycosylation profile, wherein the modulated glycosylation profile includes a reduced level of sialylated glycans and wherein the culture medium comprises glucose and maltose as the sole carbohydrate source, and wherein maltose is present at a concentration of 20 g / L and glucose is present at a concentration of 4 g / L.

15. The method of any one of claims 1-12 or claim 14, wherein the cell is an antibody-producing cell.

16. A kit for use in the method of any one of claims 1-12 or 14-15, the kit comprising the components of any one of claims 1-12 or 14-15, wherein the components comprise at least the serum-free cell culture medium and the cells.

Images