A cell culture method for modulating antibody charge heterogeneity and glycoform

Abstract

The application discloses a cell culture method for producing trastuzumab, which can effectively reduce the acid peak of the antibody, improve the main peak of CE-SDS of the antibody, and reduce G0F and other glycosylation modifications without changing other key quality attributes of trastuzumab by adjusting the components of the feed and the culture temperature. The culture method is simple in operation and suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a cell culture process for regulating the charge heterogeneity and glycoform of trastuzumab biosimilars. Background Technology

[0002] Antibodies have complex molecular structures and large molecular weights. During production and storage, they can undergo a large number of modifications, resulting in a variety of isomers. The combination of these isomers leads to charge heterogeneity, glycosylation modification heterogeneity, and other properties of antibodies.

[0003] Glycosylation modification can be influenced by various process parameters, thus easily leading to heterogeneity. Furthermore, antibody glycosylation is closely related to antibody half-life, immunogenicity, ADCC, and CDC. Studies have shown that the main glycoforms of trastuzumab are G0F, G1F, and G2F, with G0F being the most abundant. High G0F modification can promote complement pathway activity and accelerate clearance rate. Therefore, in the production of generic trastuzumab, it is necessary to control the glycoform modification of trastuzumab, especially the proportion of G0F modification.

[0004] Charge heterogeneity arises from post-translational modifications and degradation in antibody molecules, altering their surface charge properties such as isoelectric point or space charge distribution, leading to the formation of charge variants. Heterogeneity often causes changes in antibody structure and function, including biological activity, pharmacokinetics, thermal stability, and immunogenicity, and is a key quality attribute of antibody drugs. While many in vitro chemical mechanisms inducing charge variants are relatively well understood, the fundamental causes of charge variant formation during cell culture remain unclear due to the involvement of multiple factors, particularly various culture medium components and their interactions. Therefore, for each generic drug, further investigation into the ultimate impact of various components on the antibody is necessary.

[0005] Changing cell lines and screening or developing culture medium components pose significant challenges in terms of time and cost. Therefore, optimizing cell culture processes is the preferred strategy for controlling charge heterogeneity and glycoside modification. Fed-batch culture is the most widely used animal cell culture process, and process parameters such as temperature, dissolved oxygen (DO), pH, accumulation of metabolic waste during culture, and culture time can all affect charge heterogeneity. Currently, the mechanisms by which these parameters affect charge heterogeneity are not fully understood, and their regulatory effects on different products are not entirely consistent, making it difficult to develop a widely applicable control strategy.

[0006] Therefore, for specific antibodies or cell lines, it is still necessary to study cell culture processes that match them. Summary of the Invention

[0007] This disclosure provides a cell culture process suitable for producing trastuzumab. By optimizing the composition and concentration of the feed and the culture temperature, the charge heterogeneity of trastuzumab (effectively reducing the acidic peak of the antibody) and sugar modification, especially G0F modification, are adjusted, while simultaneously increasing the CE-SDS main peak content of the antibody. Furthermore, it does not alter other key quality properties of trastuzumab. The culture method of this invention is simple to operate and suitable for large-scale production.

[0008] This disclosure provides a cell culture method for producing trastuzumab, the method comprising a seed culture stage and a fed culture stage, the fed culture stage comprising the following steps:

[0009] a. Inoculate cells in basal culture medium and incubate at a constant temperature of 35°C;

[0010] b. Replenishment:

[0011] i) Supplementing with feed medium containing Maxfeed 401 at a concentration of 9.0 g / L to 50 g / L; ii) Supplementing with amino acids, namely tyrosine, cysteine, and asparagine, wherein the amino acids are added simultaneously with the feed medium;

[0012] c. Harvest antibodies.

[0013] In some embodiments, the concentration of Maxfeed 401 is 9.0 g / L to 50 g / L, including but not limited to 9.0 g / L, 10.0 g / L, 15 g / L, 20 g / L, 25.0 g / L, 30 g / L, 35.0 g / L, 40.0 g / L, 45.0 g / L, 50.0 g / L, and any range between the above values. In some embodiments, the concentration of Maxfeed 401 is 9.0 g / L to 48 g / L. In some embodiments, the concentration of Maxfeed 401 is 9.0 g / L to 25 g / L. In some embodiments, the concentration of Maxfeed 401 is 9.5 g / L to 20 g / L. In some embodiments, the concentration of Maxfeed 401 is 18 g / L to 20 g / L. In some embodiments, the concentration of Maxfeed 401 is 9.51 g / L. In some formulations, the concentration of Maxfeed 401 is 19.02 g / L.

[0014] In some embodiments, the concentration of tyrosine is 0.5 g / L to 2.0 g / L, the concentration of cysteine ​​is 0.5 g / L to 2.0 g / L, and / or the concentration of asparagine is 0.8 g / L to 2.5 g / L.

[0015] In some embodiments, the concentration of tyrosine is 0.85 g / L to 1.7 g / L, the concentration of cysteine ​​is 0.75 g / L to 1.5 g / L, and / or the concentration of asparagine is 1.12 g / L to 2.24 g / L.

[0016] In some embodiments, the concentration of tyrosine is 0.5 g / L to 1.0 g / L, the concentration of cysteine ​​is 0.5 g / L to 1.0 g / L, and / or the concentration of asparagine is 0.8 g / L to 1.5 g / L.

[0017] In some embodiments, the concentration of tyrosine is 0.85 g / L, the concentration of cysteine ​​is 0.75 g / L, and / or the concentration of asparagine is 1.12 g / L.

[0018] In some implementations, galactose is also added during the fed culture phase, and the galactose is added simultaneously with the fed culture medium.

[0019] In some embodiments, the concentration of galactose is 21 g / L to 55 g / L. In some embodiments, the concentration of galactose is 30 g / L to 55 g / L. In some embodiments, the concentration of galactose is 45 g / L to 50 g / L. In some embodiments, the concentration of galactose is 48 g / L.

[0020] In this disclosure, the concentrations of Maxfeed 401, tyrosine, cysteine, asparagine, and galactose are calculated based on feed volume.

[0021] In some embodiments, the cell culture time during the fed-batch phase is 10–14 days. In some embodiments, the culture time during the fed-batch phase is 12 days.

[0022] In some implementations, the feeding is performed on days 3, 6, and 9 after the start of the fed-feeding culture phase.

[0023] In some embodiments, the feed volume is 8% to 12% (v / v) of the initial culture volume. In some embodiments, the feed volume is 10% (v / v) of the initial culture volume.

[0024] In some embodiments, the cell seeding density in step a is 1.0 × 10⁻⁶. 6 cells / mL ~1.8×10 6 cells / mL. In some embodiments, the cell seeding density in step a is 1.09 × 10⁻⁶ cells / mL. 6 cells / mL ~1.5×10 6cells / mL. In some embodiments, the cell seeding density in step a is 1.09 × 10⁻⁶ cells / mL. 6 cells / mL.

[0025] In some embodiments of the present invention, the cell culture medium may contain serum-free and / or animal-free products or components.

[0026] In some specific embodiments, the cell culture medium may have a defined chemical composition, wherein all chemical components are known. As those skilled in the art will understand, a culture medium can be determined by those skilled in the art without excessive experimentation, provided it is suitable for the specific cells being cultured.

[0027] Commercially available culture media can be used, including but not limited to: Hyclone's Maxpro 301, Hyclone's Maxpro 303, Millipore's EX-CELL Advanced CHO basal medium, Gibco's ExpiCHO basal medium, Zhongshan Kangcheng CHO CD04, CD OptiCHO, Hycell, Shanghai Duoning DN Feed1, Zhongshan Kangcheng CHO Feed 02, GrowthA, Dynamis, Iscove's Modified Dulbecco's Medium, GMEM containing glutamine, or any other culture media known to those skilled in the art for specific cell types. Supplementary components or ingredients, including optional components, may be added to the above exemplary culture media in appropriate concentrations or amounts, as needed or as required and as known and practiced by those skilled in the art using conventional skills.

[0028] In some embodiments, the basal culture medium is Maxpro 301, Maxpro 303, AdvancedCHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed1, CHO Feed 02, GrowthA, or Dynamis.

[0029] In some implementations, the basal culture medium is Maxpro 301.

[0030] In some embodiments, during the fed-batch culture phase, the glucose concentration in the culture medium is 3.5 g / L–4.5 g / L, calculated based on the initial culture volume. In some embodiments, during the fed-batch culture phase, the glucose concentration in the culture medium is 4.0 g / L.

[0031] In some embodiments, the pH during the fed-batch culture phase is 6.8–7.0. In some embodiments, the dissolved oxygen level during the fed-batch culture phase is 20%–60%. In some embodiments, the dissolved oxygen level is 40%.

[0032] In some implementations, the cell culture method includes a seed culture stage.

[0033] In some implementations, the culture medium for the seed culture stage is Maxpro 201, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed1, CHO Feed 02, GrowthA, or Dynamis.

[0034] In some implementations, the culture medium for the seed culture stage is Maxgrow 201.

[0035] In some implementations, the cell inoculation density during the seed culture stage is 0.35 × 10⁻⁶. 6 ~0.8×10 6 Cells / mL. In some embodiments, the cell seeding density during the seed culture phase is 0.35 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0036] In some embodiments, the seed culture stage is carried out at a constant temperature of 37°C for 2–3 days. In some embodiments, the seed culture stage is carried out when the cell density reaches 1.5 × 10⁻⁶ cells / year. 6 ~3.0×10 6 Passaging is performed when the cell density reaches 2.0 × 10⁶ cells / mL; in some embodiments, continuous passaging is performed at a 1:4 ratio. In some embodiments, during the seed culture stage, passage is performed when the cell density reaches 2.0 × 10⁶ cells / mL. 6 ~3.6×10 6 When the cell / mL ratio is reached, it is mixed with the production culture medium at a 1:1 ratio to start fed culture and enter the fed culture stage; the day is called day 0 of the fed culture stage.

[0037] In some embodiments, the cells are CHO cells. In some embodiments, the CHO cells are selected from CHO-K1, CHO DG44, and CHO-S cells. In some embodiments, the cells are CHO-K1 cells.

[0038] In some implementations, the culture conditions during the seed culture phase include one or more of the following conditions:

[0039] i) The culture medium is Maxgrow 201, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed1, CHO Feed 02, GrowthA or Dynamis;

[0040] ii) The cell seeding density is 0.35 × 10⁻⁶. 6 ~0.8×10 6 cells / mL;

[0041] iii) pH is 6.8–7.0; and

[0042] iv) Incubate at a constant temperature of 36.5℃~37.5℃ for 2~3 days.

[0043] In some embodiments, this disclosure provides a cell culture method for producing trastuzumab, the method comprising a seed culture stage and a fed culture stage, the fed culture stage comprising the following steps:

[0044] a. Inoculate cells in basal culture medium (Maxpro 301) and incubate at a constant temperature of 35°C. The inoculation density is 1.0 × 10⁻⁶ cells / year. 6 cells / mL ~1.8×10 6 cells / mL;

[0045] b. Replenishment:

[0046] i) Supplement with feed medium, wherein the feed medium is Maxfeed 401, and the concentration of Maxfeed 401 is 9.5 g / L to 20 g / L; ii) Supplement with amino acids, wherein the amino acids are 0.5 g / L to 1.0 g / L tyrosine, 0.5 g / L to 1.0 g / L cysteine, and 0.8 g / L to 1.5 g / L asparagine, wherein the amino acids are added simultaneously with the feed medium, and the concentration of the amino acids is calculated based on the feed volume; iii) Supplement with 45 g / L to 50 g / L galactose, wherein the galactose is added simultaneously with the feed medium, and the concentration of the galactose is calculated based on the feed volume;

[0047] c. The fed culture period is 10-14 days, and antibodies are harvested.

[0048] This invention uses a cell culture method with CHO-K1 expression system, optimizes the composition and culture temperature of the feed, and cultured cell lines for antibody expression. This method can optimize antibody G0F glycoform modification and improve the CE-SDS main peak while reducing the proportion of acidic peaks, so that the acidic peak and glycosylation level of the antibody are consistent with the original drug standard, thus ensuring the efficacy of the antibody. Detailed Implementation

[0049] The mammalian cell expression system disclosed herein is a cell line capable of stably expressing IgG1 monoclonal antibodies. It is derived from CHO-K1 cells by transfecting a single expression vector containing the trastuzumab encoding gene. Specifically, the single expression vector contains gene sequences encoding the light and heavy chains of trastuzumab, as well as different selection marker genes. These vectors are then transfected into CHO-K1 cells via electroporation at a specific ratio, followed by passage in a culture medium containing selection pressure. Monoclonal antibodies are then obtained through limiting dilution sorting combined with single-cell imaging technology. The CHO-K1 cell line used is preserved in the applicant's laboratory.

[0050] The trastuzumab in this invention, marketed as Herceptin, has the CDR region defined in U.S. Patent 5,821,337. Its full-length sequence can be obtained by referring to Figures 4A (light chain) and 4B (heavy chain) in CN106163558A.

[0051] definition

[0052] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields.

[0053] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0054] The term "cell culture medium" refers to an aqueous solution of nutrients that can be used to grow cells for extended periods. Typically, a cell culture medium contains the following components: an energy source, usually carbohydrates, preferably glucose; amino acids, preferably the basic group of amino acids, including all essential and non-essential amino acids; vitamins and / or other organic compounds required in low concentrations; free fatty acids; and inorganic compounds, including trace elements, inorganic salts, buffer compounds, and nucleosides and bases.

[0055] The term "basal medium" refers to a culture medium used to culture cells. This medium is used directly for culturing cells and is not used as an additive to other culture media, but various components can be added to a basal medium. For example, if CHO cells are cultured in DMEM (a well-known, commercially available mammalian cell culture medium) and periodically fed with glucose or other nutrients, DMEM would be considered a basal medium.

[0056] "Feed culture medium" is a medium used as a feed in cell culture, which can be a fed batch cell culture. Similar to basal medium, fed culture medium is designed based on the needs of the specific cells being cultured, and it can contain a high concentration of most, but not all, of the components of basal medium. For example, some components, such as nutrients including amino acids or carbohydrates, can be approximately 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 400, 600, 800, or even approximately 1000 times their normal concentration in basal medium. Some components, such as salts, can be maintained at approximately the same concentration as basal medium to keep the feed isotonic. Some components are added to maintain the physiological properties of the feed, while others are added because they replenish nutrients to the culture.

[0057] The term "seeding" refers to seeding cells into a culture medium provided for cell culture. Specifically, the culture medium can be provided before cell transfer or simultaneously with cell transfer to a cell bioreactor. In large-scale animal cell culture, the conventional practice is to pre-provide the culture medium, maintain the temperature and oxygen saturation within predetermined ranges, and then transfer the cells into the bioreactor.

[0058] The term "cell density" refers to the number of cells present in a given volume of culture medium.

[0059] The term "bioreactor" refers to any container used for the growth of mammalian cell cultures. A bioreactor can be of any size, as long as it is used to culture mammalian cells. Typically, such bioreactors will be at least 1 liter and can be 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000 liters or more, or any volume in between. Internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen, and temperature, are typically controlled during culture. The bioreactor can be composed of any material (including glass, plastic, or metal) suitable for containing mammalian cell cultures suspended in a culture medium under the culture conditions of the present invention.

[0060] The terms "feed-batch culture" or "feed culture" refer to a method of culturing cells in which additional components are provided to the culture one or more times after the culture process has begun. The components thus provided typically include the nutrient components of the cells that are consumed during the culture process. In this disclosure, the fed-batch culture phase begins after the cells are seeded into basal medium following the completion of the seed culture phase, with the day of cell seeding designated as day 0 of the fed-batch culture.

[0061] The term "initial culture volume" refers to the culture volume after cells have been inoculated in basal medium.

[0062] The present invention will be further illustrated by specific embodiments below, but the scope of the invention is not limited thereto. Unless otherwise specified, the reagents and instruments used in the present invention are conventional reagents and instruments that can be obtained commercially; the methods used are conventional techniques, and those skilled in the art can undoubtedly complete the experiments and obtain the corresponding results based on the contents described in the specification.

[0063] reagents

[0064] The reagents used in this invention and their sources are as follows:

[0065] Table 1. Main Reagents

[0066] Culture medium / reagent factory use Maxgrow 201 Hyclone Seed culture medium Maxpro 301 Hyclone basal culture medium Maxfeed 401 Hyclone Feeding culture medium L-glutamine Sigma replenishment L-cysteine ​​hydrochloride monohydrate SIAL replenishment L-Tyrosine disodium salt dihydrate Sigma replenishment L-Asparagine (monohydrate) Applichem replenishment D-galactose Sigma replenishment Herceptin Luo Reference drug

[0067] Measurement and detection methods

[0068] (1) Determination of cell viability and density: Cell viability was determined by trypan blue staining using a cell viability analyzer.

[0069] (2) Purification of trastuzumab:

[0070] The antibody was purified using a one-step affinity chromatography method. The steps were as follows: centrifugation (12000g for 10 min) was performed to collect the cell culture supernatant stably transfected with the recombinant plasmid. The supernatant was then purified using a ChromaFlow 600 affinity chromatography column (MabSelect SuRe packing material) on a protein purification chromatography system (Cytiva, AKTA process). The target protein was eluted with acetic acid solution at pH 2.8.

[0071] (3) Determination of trastuzumab concentration:

[0072] The determination was performed by HPLC. The HPLC system was equilibrated with phosphate buffer (0.05M disodium hydrogen phosphate + 0.15M sodium chloride, pH=7.0) at a flow rate of 3 mL / min for 15 min until the baseline was stable. The standard curve method program was set in the system program. 30 μL of sample was injected and eluted with 0.15M sodium chloride buffer (pH=1.9) at a flow rate of 3 mL / min. Relevant data were recorded and processed.

[0073] (4) Determination of charge heterogeneity of trastuzumab:

[0074] The determination was performed by HPLC: column: Propac WCX-10, 4.0*250nm, injection volume: 50μL, flow rate: 0.8mL / min, detection wavelength: 214nm, sample tray temperature: 4℃, column temperature: 25℃, run time: 48min.

[0075] (5) Determination of trastuzumab purity by SEC-HPLC:

[0076] The determination was performed by HPLC: First, the column was equilibrated by using 100% mobile phase at a flow rate of 0.5 mL / min until the baseline was stable. Then, the sample was injected and analyzed. Chromatographic conditions: Column: Propac WCX-10, 4.0*250 nm, injection volume: 25 μL, flow rate: 0.5 mL / min, detection wavelength: 280 nm, column temperature: 25 ℃, run time: 30 min.

[0077] (6) Determination of CE-SDS purity of trastuzumab:

[0078] Detection was performed using capillary electrophoresis. The capillary electrophoresis system was washed with 1M NaOH for 3 min, 1M HCl for 2 min, and water for 1 min. Before each injection, the system was filled with running buffer at 4.0 bar for 10 min before injection. The capillary was 33 cm long with an effective length of 8.5 cm. The detection wavelength was 214 nm, the column temperature was 20 °C, and the sample tray was 15 °C.

[0079] (7) HPLC-MS detection of trastuzumab glycosylation:

[0080] Chromatographic conditions (column: XBridge Glycan BEH Amide 130A, 2.1x150mm, 2.5μm): injection volume 20μL, excitation wavelength: 285nm, emission wavelength: 345nm, PMT value: 10, flow rate 0.3ml / min, column temperature 50℃, sample tray temperature 10℃;

[0081] Mass spectrometry conditions: Instrument mode Auto MS / MS, ion mode Dual AJS ESI source, cation, dry gas N2, dry gas flow rate (temperature) 8 L / min, 325℃; collision voltage 150 V; capillary voltage (VCap) 3500 V; jacket gas temperature (flow rate): 350℃, 11 L / min; nozzle voltage 400 V; separator voltage 45 V; OCT 1RF Vpp voltage 750 V; mass range 310-3200 m / z.

[0082] Example 1: Effect of feed composition on trastuzumab

[0083] 1.1 Cell Culture

[0084] Recombinant CHO-K1 cells were revived in seed culture medium until the cell density reached 1.5 × 10⁻⁶ cells / year. 6 ~3.0×10 6 Passage was performed when the cell density reached 2.0 × 10⁶ cells / mL, with a 1:4 continuous passaging ratio.6 ~3.6×10 6 When the inoculum density is 1.0–1.8 × 10⁶ cells / mL, mix it with the basal medium at a 1:1 ratio (i.e., the inoculum density is 1.0–1.8 × 10⁶ cells / mL). 6 (cells / mL), with an initial culture volume of 20 mL in Cultured in Bioreactor 50 (i.e. fed culture), with glucose concentration controlled at 4 g / L based on the results of biochemical analyzer testing, and culture conditions of 37℃, 125 rpm and 5% CO2.

[0085] On days 3, 6, and 9 of culture, 10% (v / v) of the initial culture volume of feed was added to investigate the effects of the amount of Maxfeed401 powder and the concentrations of L-tyrosine, L-cysteine, and L-asparagine in the feed on the expression level and purity of trastuzumab. The culture was harvested after 12 days. The specific composition of the feed medium is shown in Table 2.

[0086] After the culture was completed, the supernatant was harvested for expression level analysis. The supernatant was purified by affinity chromatography and its purity was determined (SEC-HPLC, CE-SDS(NR)).

[0087] Table 2. Feed Composition Table

[0088]

[0089] Note: The concentrations of the above components are the concentrations in the feed, i.e., calculated based on the feed volume.

[0090] 1.2 Results Analysis

[0091] At the end of day 12 of culture, the cell viability in all groups was above 85%, and the cell density was 4.5 × 10⁻⁶. 6 The antibody expression levels were all above 2 g / L. The amount of feed added affected the antibody expression level, but there was no obvious pattern.

[0092] Table 3. Effect of feed addition on trastuzumab expression levels

[0093] process Process 5 Process 6 Process 7 Process 11 Process 14 Expression level (g / L) 2.69 2.14 2.20 2.77 2.41

[0094] Table 4. Quality test results of trastuzumab

[0095]

[0096] The results showed that different concentrations of amino acids and the proportion of Maxfeed401 dry powder in the feed had no effect on the SEC-HPLC detection results of the antibody, and the monomer content in the SEC-HPLC was all above 99%. However, it did affect the content of the main peak in the CE-SDS (NR) detection. The main peak content of the antibody produced by the feeds in processes 5, 7, and 11 was lower, making them unsuitable for the production of trastuzumab.

[0097] Further analysis of the charge variants and glycoforms of trastuzumab produced by feed processes 6 and 14 was conducted, and the results are shown in Table 5. IEC-HPLC results showed that the main peaks of trastuzumab produced by processes 6 and 14 were close to those of the original drug, but lower, while the acid peaks were higher. The proportion of G0F glycoform modification was significantly higher than that of the original drug, indicating a need for further optimization of the culture process.

[0098] Table 5. Results of the effect of feed addition on the quality of trastuzumab

[0099]

[0100]

[0101] Example 2: Glycoform optimization of trastuzumab

[0102] Based on processes 6 and 14 in Example 1, galactose was further added to the feed. The composition of the feed is shown in Table 6 below. Cells were cultured according to the cell culture protocol in Example 1. After culture, the supernatant was harvested for expression level analysis. The supernatant was purified by affinity chromatography and the glycoform ratio was determined. The results are shown in Table 7.

[0103] Table 6. Feed composition and concentration

[0104]

[0105] Note: The concentrations of the above components are the concentrations in the feed, i.e., calculated based on the feed volume.

[0106] Table 7. Results of glycoform analysis

[0107] process G2F (%) G1F (%) G0F(%) Process 6 4.6 23.4 56.6 Process 7 4.0 21.2 59.6 Process 8 6.2 25.8 52.5 Process 9 4.8 24.2 55.6 Herceptin 5.8 32.3 48.6

[0108] The data in the table above show that adding galactose to the fed culture medium can reduce the proportion of G0F modification. When the galactose concentration is 48 g / L, the proportion of G0F sugar modification can be significantly reduced.

[0109] Example 3: Further optimization for charge variants

[0110] To ensure that the antibody expression quality is consistent with that of Herceptin, it is necessary to further increase the peak content of the charge variant of the anti-antibody. This example investigates the effect of temperature on antibody quality by varying the culture temperature and the time spent at that temperature. The culture was conducted according to the conditions described in Example 1; specific culture temperatures and feed medium components are shown in Table 8.

[0111] With 1.0~1.8×10 6 Cells were seeded into basal medium at a seeding density of cells / mL. On days 3, 6, and 9, 10% of different Maxfeed 401 dry powders and amino acids were added, with the concentrations of L-tyrosine disodium hydrate (0.85 g / L), L-cysteine ​​hydrochloride monohydrate (0.75 g / L), and L-asparagine (1.12 g / L). During culture, the glucose concentration in the fermentation broth was monitored; if it fell below 4 g / L, glucose was added to bring it up to 4 g / L. The culture temperature and fed-batch culture protocol are detailed in Table 8. After culture, the supernatant was harvested for expression level analysis. The supernatant was then purified by affinity chromatography and subjected to quality analysis; the results are shown in Table 9.

[0112] Table 8. Optimization schemes for different culture temperatures

[0113]

[0114]

[0115] Note: The concentrations of the above components are in the fed culture, i.e., calculated based on the fed culture volume. C3 indicates the third day of fed culture, and so on.

[0116] Table 9. Analysis of optimization results for different culture temperatures

[0117]

[0118] The results show that different cooling methods affect the charge heterogeneity of trastuzumab. Compared with cooling on days 3, 6, and 9 of culture, directly lowering the culture temperature to 35℃ is more effective in increasing the content of the main peak in the charge variant and reducing the content of the acidic peak: the proportion of the main peak increased from 60.44% (#12) before optimization to 64.13%, and the proportion of the acidic peak decreased from 28.79% before optimization to 23.89%. Simultaneously, directly lowering the culture temperature to 35℃ also increases the content of the main peak of CE-SDS.

[0119] Example 4: Scale-up of trastuzumab cell culture process

[0120] Following the feeding process 1 in Example 3, the effect of the final process on the glycoform of trastuzumab was investigated in a 3L bioreactor. The specific process is shown in Table 10.

[0121] Table 10. Cell Culture Processes

[0122]

[0123]

[0124] On day 12 of culture, the cell viability was 88.6%, and the cell density was approximately 6.76E+6. The antibody expression level was 2.1 g / L.

[0125] SEC-HPLC analysis showed a main peak content as high as 99.11%. Further examination of the trastuzumab glycoform revealed that the G0F glycoform accounted for 42.73%, close to the 46.0% glycoform content of Herceptin. This indicates that the above culture process can be used for the production of trastuzumab biosimilars.

Claims

1. A cell culture method for producing trastuzumab, the method comprising a seed culture stage and a fed-batch culture stage, the fed-batch culture stage comprising the following steps: a. inoculating cells in a basal medium, incubating at 35°C; b. feeding: i) supplementing a feed medium, the feed medium comprising Maxfeed 401; ii) supplementing amino acids, wherein the amino acids are tyrosine, cysteine and asparagine, the amino acids being supplemented simultaneously with the feed medium; c. harvesting the antibody; wherein the concentration of Maxfeed 401 is 9.0 g / L to 25 g / L; the concentration of tyrosine is 0.85 g / L, the concentration of cysteine is 0.75 g / L, and the concentration of asparagine is 1.12 g / L, the concentrations of the amino acids being calculated based on the volume of the feed; the culture time of the fed-batch culture stage is 10 days to 14 days; the feeding is supplemented on the 3rd day, the 6th day and the 9th day of the fed-batch culture stage; and the feeding amount is 8% to 12% (v / v) of the initial culture volume.

2. The cell culture method of claim 1, wherein the concentration of Maxfeed 401 is 19.02 g / L.

3. The cell culture method of claim 1 or 2, wherein, in the fed-batch culture stage, galactose is further supplemented, the galactose being supplemented simultaneously with the feed medium.

4. The cell culture method of claim 3, wherein the concentration of galactose is 21 g / L to 55 g / L, the concentration of galactose being calculated based on the volume of the feed.

5. The cell culture method of claim 3, wherein the concentration of galactose is 45 g / L to 50 g / L, the concentration of galactose being calculated based on the volume of the feed. wherein 6. The cell culture method of claim 3, wherein the concentration of galactose is 48 g / L, the concentration of galactose being calculated based on the volume of the feed.

7. The cell culture method of claim 1, wherein the feeding amount is 10% (v / v) of the initial culture volume.

9. The cell culture method of any one of claims 1 to 2, 4 to 8, wherein the basal medium is selected from Maxpro 301, Maxpro 303, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DNFeed1, CHO Feed 02, GrowthA and Dynamis.

10. The cell culture method of any one of claims 1 to 2, 4 to 8, wherein the concentration of glucose in the medium during the fed-batch culture stage is 3.5 g / L to 4.5 g / L, the concentration of glucose being calculated based on the initial culture volume.

2. The cell culture method of claim 1, wherein, 11. The cell culture method of any one of claims 1 to 2, 4 to 8, wherein the concentration of glucose in the medium during the fed-batch culture stage is 4.0 g / L, the concentration of glucose being calculated based on the initial culture volume.

12. The cell culture method of any one of claims 1 to 2, 4 to 8, 10 to 11, wherein the pH of the fed-batch culture stage is 6.8 to 7.0, and the dissolved oxygen is 20% to 60%.

4. The cell culture method of claim 3, wherein, 13. The cell culture method of claim 12, wherein the dissolved oxygen is 40%.

5. The cell culture method of claim 3, wherein, 14. The cell culture method of any one of claims 1 to 2, 4 to 8, 10 to 11, 13, wherein the cells are CHO cells.

6. The cell culture method of claim 3, wherein, ​ ​ 8. The cell culture method of claim 1, wherein the cell seeding density in step a is 1.0 x 10 6 ~1.8 x 10 6 cells / mL. ​ 10. The cell culture method of claim 9, wherein, ​ 11. The cell culturing method of claim 9, wherein, ​ ​ ​ ​ 15. The cell culture method according to claim 14, wherein, The CHO cells are selected from the group consisting of CHO K1, CHO DG44 and CHO S cells.

16. The cell culture method according to claim 14, wherein, The CHO cell is a CHO K1 cells.

17. The cell culture method of any one of claims 1 to 2, 4 to 8, 10 to 11, 13, 15 to 16, wherein the culture conditions of the seed culture stage comprise one or more of the following conditions: i) the culture medium is Maxgrow 201, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DNFeedl, CHO Feed 02, GrowthA, or Dynamis; ii) a cell seeding density of 0.35 x 10 6 ~0.8 x 10 6 cells / mL; iii) the pH is 6.8 to 7.0; and iv) incubation at a constant temperature of 36.5 °C to 37.5 °C for 2 to 3 days.

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