Cell culture method for regulating charge heterogeneity and glycotype of antibody

Abstract

The invention discloses a cell culture method for producing trastuzumab, which can effectively reduce the acid peak of an antibody, improve the CE-SDS main peak of the antibody and reduce G0F and other glycoform modifications by adjusting the ingredients and culture temperature of supplemented materials without changing other key quality attributes of trastuzumab. The culture method is simple to operate and suitable for large-scale production.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a cell culture process for regulating charge heterogeneity and glycoforms of trastuzumab biosimilars. Background Art

[0002] Antibodies have complex molecular structures and large molecular weights. They can undergo a large number of modifications during production and storage, resulting in multiple isomers of antibodies. The combination of these isomers leads to charge heterogeneity, glycosylation modification heterogeneity, etc.

[0003] Glycosylation modification may be affected by a variety of process parameters, and thus is prone to heterogeneity, and antibody glycosylation is closely related to antibody half-life, immunogenicity, ADCC, CDC, etc. Studies have shown that the main glycoforms of trastuzumab are: G0F, G1F, and G2F, among which G0F is the most. High G0F modification can promote the complement pathway and accelerate the clearance rate. Therefore, in the production process of trastuzumab generics, it is necessary to control the glycoform modification of trastuzumab, especially the proportion of G0F modification.

[0004] Charge heterogeneity is caused by the post-translational modification and degradation of antibody molecules, which leads to changes in surface charge characteristics such as isoelectric point or spatial charge distribution, and then leads to the generation of charge variants. The occurrence of heterogeneity often causes changes in antibody structure and function such as antibody biological activity, pharmacokinetics, thermal stability, and immunogenicity, and is also a key quality attribute of antibody drugs. Although many in vitro chemical mechanisms that cause charge variants are relatively clear, the root cause of the formation of charge variants in cell culture is still unclear due to the involvement of multiple factors, especially various culture medium components and their interactions. Therefore, for each generic drug, it is necessary to further explore the ultimate impact of various components on antibodies.

[0005] Replacing cell lines, screening of culture medium components or targeted development are huge challenges in terms of time and cost, so optimizing the cell culture process is the preferred strategy for controlling charge heterogeneity and sugar modification. Fed-batch culture is the most widely used animal cell culture process, in which process parameters such as temperature, DO, pH, accumulation of metabolic waste during culture, and culture time may affect charge heterogeneity. At present, the mechanism of the influence of these parameters on charge heterogeneity is not fully understood, and the regulatory effects on different products are not completely consistent, making it difficult to form a control strategy with a wide range of applicability.

[0006] Therefore, for specific antibodies or cell lines, it is still necessary to study the matching cell culture process. Summary of the invention

[0007] The present disclosure provides a cell culture process suitable for producing trastuzumab, which adjusts the charge heterogeneity of trastuzumab (effectively reducing the acidic peak of the antibody) and sugar modification, especially G0F modification, by optimizing the composition, concentration and culture temperature of the feed, while increasing the CE-SDS main peak content of the antibody. And it does not change other key quality attributes of trastuzumab. The culture method of the present invention is simple to operate and suitable for large-scale production.

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

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

[0010] b. Feeding:

[0011] i) supplementing a feed medium, wherein the feed medium comprises Maxfeed 401, wherein the concentration of the Maxfeed 401 is 9.0 g / L to 50 g / L; ii) supplementing amino acids, wherein the amino acids are tyrosine, cysteine ​​and asparagine, and the amino acids are supplemented simultaneously with the feed medium;

[0012] c. Harvesting 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 point 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 schemes, the concentration of Maxfeed 401 is 9.51 g / L. In some embodiments, the concentration of Maxfeed 401 is 19.02 g / L.

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

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

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

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

[0018] In some embodiments, during the fed-batch culture phase, galactose is also supplemented, and the galactose is supplemented simultaneously with the feed 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 the present disclosure, the concentrations of Maxfeed 401, tyrosine, cysteine, asparagine and galactose are calculated based on the feed volume.

[0021] In some embodiments, the cell culture time in the feeding phase is 10 to 14 days. In some embodiments, the cell culture time in the feeding phase is 12 days.

[0022] In some embodiments, the feeding time is the 3rd day, the 6th day and the 9th day after the start of the fed-batch culture phase.

[0023] In some embodiments, the feed amount is 8% to 12% (v / v) of the initial culture volume. In some embodiments, the feed amount 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 cells / mL~1.8×10 6 In some embodiments, the cell seeding density in step a is 1.09×10 6 cells / mL~1.5×10 6In some embodiments, the cell seeding density in step a is 1.09×10 6 cells / mL.

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

[0026] In some embodiments, the cell culture medium can be a chemically defined medium in which all chemical components are known. As will be appreciated by those skilled in the art, a defined medium can be prepared without undue experimentation, provided that it is suitable for the specific cells being cultured.

[0027] Commercially available culture media may be used, including but not limited to: Maxpro 301 of Hyclone, Maxpro303 of Hyclone, EX-CELL Advanced CHO basal medium of Millipore, ExpiCHO basal medium of Gibco, CHO CD04 of Zhongshan Kangsheng, CD OptiCHO, Hycell, DN Feed1 of Shanghai Duoning, CHO Feed 02 of Zhongshan Kangsheng, GrowthA, Dynamis, Iscove's Modified Dulbecco's Medium, GMEM with glutamine, or any other culture media prepared for specific cell types known to those skilled in the art. Supplementary components or ingredients, including optional components, may be added to the above exemplary culture media at appropriate concentrations or amounts as needed or as needed and as known and implemented by those skilled in the art using routine skills.

[0028] In some embodiments, the basal medium is Maxpro 301, Maxpro 303, AdvancedCHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DN Feed 1, CHO Feed 02, GrowthA or Dynamis.

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

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

[0031] In some embodiments, the pH in the fed-batch culture stage is 6.8 to 7.0. In some embodiments, the dissolved oxygen in the fed-batch culture stage is 20% to 60%. In some embodiments, the dissolved oxygen is 40%.

[0032] In some embodiments, the cell culture method comprises a seed culture stage.

[0033] In some embodiments, the culture medium in 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 embodiments, the culture medium in the seed culture stage is Maxgrow 201.

[0035] In some embodiments, the cell seeding density in the seed culture stage is 0.35×10 6 ~0.8×10 6 In some embodiments, the cell seeding density in the seed culture stage is 0.35×10 6 cells / mL.

[0036] In some embodiments, the culture temperature in the seed culture stage is 37°C and the culture time is 2 to 3 days. In some embodiments, in the seed culture stage, when the cell density reaches 1.5×10 6 ~3.0×10 6 cells / mL; in some embodiments, continuous subculturing is performed at a ratio of 1:4. In some embodiments, in the seed culture stage, when the cell density reaches 2.0×10 6 ~3.6×10 6 When the concentration of 404 K was 1.547 W / mL, it was mixed with the production medium in an equal volume of 1:1 to start the fed-batch culture and enter the fed-batch culture phase. The day was called day 0 of the fed-batch culture phase.

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

[0038] In some embodiments, the culture conditions in the seed culture stage 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 was 0.35×10 6 ~0.8×10 6 cells / mL;

[0041] iii) pH 6.8 to 7.0; and

[0042] iv) Cultivate at a constant temperature of 36.5°C to 37.5°C for 2 to 3 days.

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

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

[0045] b. Feeding:

[0046] i) supplementing feed medium, wherein the feed medium is Maxfeed 401, wherein the concentration of Maxfeed 401 is 9.5 g / L to 20 g / L; ii) supplementing 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 supplemented simultaneously with the feed medium, and the concentration of the amino acids is calculated based on the feed volume; iii) supplementing 45 g / L to 50 g / L galactose, wherein the galactose is supplemented simultaneously with the feed medium, and the concentration of the galactose is calculated based on the feed volume;

[0047] c. The feeding culture time is 10 to 14 days, and the antibodies are harvested.

[0048] The present invention uses a cell culture method using a CHO-K1 expression system, optimizes feed ingredients and feed culture temperature, and cultures antibody-expressing cell lines. The method can achieve the purpose of optimizing antibody G0F glycoform modification and increasing CE-SDS main peak while reducing the acidic peak ratio, so that the acidic peak and glycosylation level of the antibody are consistent with the original drug standard, thereby ensuring the efficacy of the antibody. DETAILED DESCRIPTION

[0049] The mammalian cell expression system involved in the present disclosure is a cell line that can stably express IgG1 monoclonal antibodies, which is derived from transfecting CHO-K1 cells with a single expression vector containing the trastuzumab coding gene. The specific operation is as follows: the single expression vector contains the gene sequences encoding the light chain and heavy chain of trastuzumab and different screening marker genes, and then is transfected into CHO-K1 cells by electroporation in a certain proportion, and then passaged in a culture medium containing screening pressure. Monoclones are further obtained by 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 the present invention, whose trade name is Herceptin, has the CDR regions defined in US Pat. No. 5,821,337. Its full-length sequence can be obtained by referring to FIG. 4A (light chain) and FIG. 4B (heavy chain) in CN106163558A.

[0051] definition

[0052] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operating procedures used herein are all conventional procedures widely used in the corresponding fields.

[0053] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising", "having", "including", etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to". Unless otherwise specified, "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 of time. Typically, a cell culture medium comprises the following components: an energy source, which will typically be a carbohydrate, preferably glucose, amino acids, preferably amino acids of the basic group, 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" is a medium used to culture cells, which medium itself is used directly to culture cells and is not used as an additive to other culture media, but various components can be added to the basal medium. For example, if CHO cells are cultured in DMEM (a well-known, commercially available mammalian cell culture medium) and regularly fed with glucose or other nutrients, DMEM would be considered a basal medium.

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

[0057] The term "inoculation" refers to the seeding of cells into a culture medium provided for cell culture. Specifically, the culture medium can be provided before the cells are transferred, or can be provided while the cells are transferred to a cell bioreactor. In large-scale animal cell culture, the conventional practice is to provide the culture medium in advance, and after maintaining the temperature and oxygen saturation within a predetermined range, the cells are transferred 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 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 therebetween. The internal conditions of the bioreactor are usually controlled during the culture period, including but not limited to pH, dissolved oxygen and temperature. A bioreactor can be composed of any material (including glass, plastic or metal) suitable for accommodating a mammalian cell culture suspended in the culture medium under the culture conditions of the present invention.

[0060] The term "fed batch culture" or "fed culture" refers to a method of culturing cells in which additional components are provided to the culture once or more after the start of the culture process. The components so provided generally include nutritional components of the cells that are consumed during the culture process. In the present disclosure, after the seed culture stage is completed, the cells are inoculated into the basal medium, and the fed culture stage is entered, and the day of inoculation of the cells is taken as day 0 of the fed culture.

[0061] The term "initial culture volume" is the culture volume after inoculating cells in the basal medium.

[0062] The present invention will be further described below by specific examples, but the scope of the present invention is not limited thereto. Unless otherwise specified, the reagents and instruments used in the present invention are conventional reagents and instruments, which can be obtained commercially; the methods used are conventional techniques, and those skilled in the art can complete the experiment and obtain the corresponding results without any doubt according to the contents recorded in the specification.

[0063] Reagents

[0064] The reagents and sources used in the present invention are as follows:

[0065] Table 1. Main reagents

[0066] Culture medium / reagents factory use Maxgrow 201 Hyclone Seed culture medium Maxpro 301 Hyclone Basic culture medium Maxfeed 401 Hyclone Feed medium L-Glutamine Sigma Feed L-Cysteine ​​hydrochloride monohydrate SIAL Feed L-Tyrosine disodium salt dihydrate Sigma Feed L-Asparagine (monohydrate) Applichem Feed D-Galactose Sigma Feed Herceptin Roche Reference drug

[0067] Determination and detection methods

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

[0069] (2) Purification of trastuzumab:

[0070] The antibody was purified by one-step affinity chromatography, the steps of which were as follows: the cell culture supernatant of the stably transfected recombinant plasmid was collected by centrifugation (12000g for 10 min), purified using a ChromaFlow600 affinity chromatography column (MabSelect SuRe filler) on a protein purification chromatography system (Cytiva, AKTA process), and eluted with a pH 2.8 acetic acid solution.

[0071] (3) Determination of trastuzumab concentration:

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

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

[0074] Determined by HPLC: chromatographic column: Propac WCX-10, 4.0*250nm, injection volume 50μL, flow rate 0.8mL / min, detection wavelength 214nm, sample plate temperature 4℃, column temperature 25℃, running time 48min.

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

[0076] Determined by HPLC: During detection, the chromatographic column is first balanced, that is, 100% mobile phase is used to balance the system at a flow rate of 0.5 mL / min until the baseline is stable, and then the sample is injected and the sample is measured. The chromatographic conditions are: chromatographic column: Propac WCX-10, 4.0*250 nm, injection volume is 25 μL, flow rate is 0.5 mL / min, detection wavelength is 280 nm, column temperature is 25°C, and running time is 30 min.

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

[0078] Capillary electrophoresis was used for detection. 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 sample was filled with running buffer at 4.0 bar for 10 min. The capillary was 33 cm long, with an effective length of 8.5 cm, a detection wavelength of 214 nm, a column temperature of 20 °C, and a sample tray of 15 °C.

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

[0080] Chromatographic conditions (chromatographic 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 plate temperature 10℃;

[0081] Mass spectrometry conditions: instrument mode Auto MS / MS, ion mode Dual AJS ESI source, positive ion, drying gas N2, drying gas flow rate (temperature) 8 L / min, 325°C; collision voltage 150 V; capillary voltage (VCap) 3500 V; jacket gas temperature (flow rate): 350°C, 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 ingredients on trastuzumab

[0083] 1.1 Cell culture

[0084] The recombinant CHO-K1 cells were revived in seed culture medium until the cell density reached 1.5×10 6 ~3.0×10 6 When the cell density reaches 2.0×106 ~3.6×10 6 cells / mL, and mixed with basal medium in equal volumes at a ratio of 1:1 (i.e., inoculation density of 1.0 to 1.8×10 6 cells / mL), with an initial culture volume of 20 mL. The culture was carried out in Bioreactor 50 (i.e., fed-batch culture). The glucose concentration was controlled at 4 g / L according to the results of the biochemical analyzer. The culture conditions were: 37° C., 125 rpm, and 5% CO 2 .

[0085] On the 3rd, 6th and 9th day of culture, 10% (v / v) of the initial culture volume of feed was added to explore 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 and purity of trastuzumab, and the culture was harvested on the 12th day. The specific composition of the feed medium is shown in Table 2

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

[0087] Table 2. Feed composition

[0088]

[0089] Note: The concentrations of the above ingredients are the concentrations in the feed, that is, they are calculated based on the feed volume.

[0090] 1.2 Results Analysis

[0091] At the end of the 12th day of culture, the cell viability of each group was above 85%, and the cell density was 4.5×10 6 In the above, the antibody expression levels were all above 2 g / L. The amount of feed added had an effect on the antibody expression level, but there was no obvious pattern.

[0092] Table 3. Effect of feeding on the expression of trastuzumab

[0093] Technology Craft 5 Process 6 Craft 7 Craft 11 Craft 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 Maxfeed401 dry powder ratios in the feed had no effect on the SEC-HPLC test results of the antibody, and the monomer content of SEC-HPLC was above 99%. However, it did affect the main peak content detected by CE-SDS (NR), among which the main peak content of antibodies produced by process feeds No. 5, No. 7 and No. 11 was low, and was not suitable for the production of trastuzumab.

[0097] The charge variants and glycoforms of trastuzumab produced by feed processes No. 6 and No. 14 were further tested, and the results are shown in Table 5. The IEC-HPLC test results showed that the main peaks of trastuzumab produced by processes No. 6 and No. 14 were close to the original research, but lower than the original research, and the acidic peak was higher than the original research. The proportion of G0F glycoform modification was significantly higher than that of the original research, and the culture process needed to be further optimized.

[0098] Table 5. Effect of feed on the quality of trastuzumab

[0099]

[0100]

[0101] Example 2: Optimization of glycoforms of trastuzumab

[0102] Based on the processes No. 6 and No. 14 in Example 1, galactose was further added to the feed, and the composition of the feed is shown in Table 6 below. The cells were cultured according to the cell culture scheme in Example 1. After the culture, the supernatant was harvested to analyze the expression level, and the supernatant was purified by affinity chromatography to detect the glycoform ratio. The results are shown in Table 7.

[0103] Table 6. Feed composition and concentration

[0104]

[0105] Note: The concentrations of the above ingredients are the concentrations in the feed, that is, they are calculated based on the feed volume.

[0106] Table 7. Glycoform analysis results

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

[0108] From the data in the above table, we know that adding galactose to the feed 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] In order to make the antibody expression quality consistent with Herceptin, it is necessary to further increase the main peak content of the charge variant of the anti-antibody. This example explores the effect of temperature on the quality of the antibody by changing the culture temperature and the time of changing the temperature. The culture was carried out according to the culture conditions in Example 1. The specific culture temperature and feed medium composition are shown in Table 8.

[0111] 1.0~1.8×10 6 The cells were inoculated into the basal medium at an inoculation density of cells / mL. On the 3rd, 6th and 9th days of culture, 10% of different Maxfeed401 dry powders and amino acids were added, among which the concentration of L-tyrosine disodium salt hydrate was 0.85g / L, the concentration of L-cysteine ​​hydrochloride monohydrate was 0.75g / L; the concentration of L-asparagine was 1.12g / L. During the culture process, the glucose concentration in the fermentation broth was detected. If it was lower than 4g / L, glucose was supplemented to 4g / L. The culture temperature and feed culture scheme are shown in 8. After the culture was completed, the supernatant was harvested for expression analysis, and the supernatant was subjected to quality analysis after affinity purification. The results are shown in Table 9.

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

[0113]

[0114]

[0115] Note: The concentrations of the above components are the concentrations in the feed, that is, calculated based on the feed volume. C3 represents the third day of fed-batch culture, and the rest are analogous.

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

[0117]

[0118] The results show that different cooling methods affect the charge heterogeneity of trastuzumab. Compared with cooling methods on the 3rd, 6th and 9th days of culture, directly lowering the culture temperature to 35°C is more helpful to increase the content of the main peak in the charge variant and reduce the content of the acidic peak: the main peak ratio increased from 60.44% (#12) before optimization to 64.13%, and the acidic peak ratio decreased from 28.79% before optimization to 23.89%. At the same time, directly lowering the culture temperature to 35°C can also increase the main peak content of CE-SDS.

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

[0120] According to 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 process

[0122]

[0123]

[0124] After testing, on the 12th day of culture, the cell viability was 88.6%, the cell density was about 6.76E+6, and the expression level of the antibody was 2.1g / L.

[0125] SEC-HPLC detection showed that the main peak content was as high as 99.11%. The glycoforms of trastuzumab were further examined, and the results showed that the proportion of G0F glycoforms was 42.73%, which was close to the glycoform of Herceptin 46.0%. This shows 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, wherein the fed-batch culture stage comprises the following steps: a. Inoculate cells in basal medium and culture at 35°C; b. Feeding: i) supplementing a feed medium, wherein the feed medium comprises Maxfeed 401, wherein the concentration of the Maxfeed 401 is 9.0 g / L to 50 g / L; ii) supplementing amino acids, wherein the amino acids are tyrosine, cysteine ​​and asparagine, and the amino acids are supplemented simultaneously with the feed medium; c. Harvesting antibodies.

2. The cell culture method according to claim 1, wherein: The concentration of Maxfeed 401 is 9.0 g / L to 25 g / L; The tyrosine concentration is 0.5 g / L to 1.7 g / L, the cysteine ​​concentration is 0.5 g / L to 1.5 g / L, and / or the asparagine concentration is 0.8 / L to 2.5 g / L, and the amino acid concentrations are calculated based on the feed volume; preferably, The concentration of Maxfeed 401 is 19.02 g / L; The tyrosine concentration is 0.85 g / L, the cysteine ​​concentration is 0.75 g / L, and / or the asparagine concentration is 1.12 g / L.

3. The cell culture method according to claim 1 or 2, further comprising supplementing galactose during the fed culture stage, wherein the galactose is added simultaneously with the feed medium; preferably, the concentration of the galactose is 21 g / L to 55 g / L, and the concentration of the galactose is calculated based on the feed volume; more preferably, the concentration of the galactose is 45 g / L to 50 g / L, and most preferably 48 g / L.

4. The cell culture method according to any one of claims 1 to 3, wherein the culture time of the fed-batch culture stage is 10 to 14 days; preferably, the feed is supplemented on the 3rd, 6th and 9th days of the fed-batch culture stage.

5. The cell culture method according to any one of claims 1 to 4, wherein the feed amount is 8% to 12% (v / v) of the initial culture volume, preferably 10%.

6. The cell culture method according to any one of claims 1 to 5, wherein the cell seeding density in step a is 1.0×10 6 ~1.8×10 6 cells / mL.

7. The cell culture method according to any one of claims 1 to 6, 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; preferably, in the fed-batch culture stage, the concentration of glucose in the medium is 3.5 g / L-4.5 g / L, more preferably 4.0 g / L, and the concentration of glucose is calculated based on the initial culture volume.

8. The cell culture method according to any one of claims 1 to 7, wherein the pH of the fed-batch culture stage is 6.8-7.0, and the dissolved oxygen is 20%-60%; preferably, the dissolved oxygen is 40%.

9. The cell culture method according to any one of claims 1 to 8, wherein the cells are CHO cells; preferably, the CHO cells are selected from CHO-K1, CHO DG44 and CHO-S cells, more preferably CHO-K1 cells.

10. The cell culture method according to any one of claims 1 to 9, wherein the culture conditions in the seed culture stage include one or more of the following conditions: i) the culture medium was Maxgrow 201, Advanced CHO, ExpiCHO, CHO CD04, CD OptiCHO, Hycell, DNFeed1, CHO Feed 02, GrowthA or Dynamis; ii) The cell seeding density was 0.35×10 6 ~0.8×10 6 cells / mL; iii) pH 6.8 to 7.0; and iv) Cultivate at a constant temperature of 36.5°C to 37.5°C for 2 to 3 days.

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