Preparation method and product of recombinant human blood coagulation factor VIII

Abstract

The invention provides a preparation method of a recombinant human blood coagulation factor VIII and a product of the recombinant human blood coagulation factor VIII. The preparation method comprises the step of culturing a mammalian cell strain for expressing the recombinant human blood coagulation factor VIII in a serum-free culture medium of a compound containing copper ions and methionine sulfoximine. By adopting the preparation method provided by the invention, the purity of the recombinant human blood coagulation factor VIII product can be further improved on the basis of keeping the high activity and high yield of the recombinant human blood coagulation factor VIII product obtained by culture.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a method for preparing recombinant human coagulation factor VIII and its product. Background Technology

[0002] Coagulation factor VIII (FVIII), also known as antihemophilic factor, plays a crucial role in the intrinsic coagulation system and is a cofactor for activating coagulation factor IX. The human coagulation factor VIII gene is located at the terminal end of the long arm of the X chromosome (Xq28), is 186 kb in length, contains 25 introns and 26 exons, and encodes a mature protein composed of 2332 amino acids. The FVIII protein can be cleaved by thrombin (IIa) at Arg372, Arg740, and Arg1689, producing active FVIII (FVIIIa), which separates from von Willebrand factor (vWF). Natural FVIII always exists stably bound to vWF in a complex. FVIIIa is a cofactor for activated FIX (FIXa), and in Ca2+... 2+ In the presence of both FIXa and FVIIIa (PF3), FIXa and FVIIIa form the FVIII complex, which can activate coagulation factor X (FX) in the intrinsic coagulation pathway to produce activated FX (FXa). FXa can then be released into the coagulation pathway. 2+ When present with PF3, it binds with FV to form prothrombin activator, which further activates thrombin to participate in subsequent coagulation pathways to achieve coagulation.

[0003] Recombinant FVIII is produced by inserting a gene fragment encoding the FVIII protein into a plasmid vector using gene recombination technology, and then transferring the plasmid into mammalian cells capable of expressing the FVIII protein. The amino acid sequence of recombinant coagulation factor VIII is highly similar to that of natural coagulation factor VIII (amino acid sequence shown in SEQ ID No. 1). It is a glycoprotein containing 1438 amino acids and is a heterodimer secreted extracellularly, containing an 80 kDa light chain and a 90 kDa heavy chain.

[0004] Due to the complex structure of the FVIII protein, mammalian host cells must be selected as the expression vector for recombinant expression. Unlike bacterial or yeast cells, mammalian cells possess post-translational modification functions, such as protein cleavage, glycosylation, hydroxylation, and sulfation. These functions are essential for the formation of the secondary and tertiary structures of the active FVIII protein. However, because FVIII is a special protein with complex glycosylation and instability, its stability during culture is greatly affected by culture conditions such as temperature. Early recombinant human coagulation factor VIII products, such as Kogenate Bayer / Kogenate FS, utilized perfusion culture, adding human albumin and recombinant insulin to the culture medium to prevent factor VIII degradation. Second-generation products improved some purification processes to prepare serum-free and protein-free recombinant factor VIII products. Many patent documents report the addition of calcium chloride, sodium chloride, or histidine to the buffer used in the purification process.

[0005] In the large-scale production of recombinant factor VIII using cell culture, serum-containing culture media or co-transfection of FVIII and vWF genes in mammalian cells are used to stabilize factor VIII expression and increase its expression level. However, current methods for culturing recombinant human coagulation factor VIII suffer from low yield, activity, and purity (e.g., activity as low as 10–25 IU / ml). Therefore, there is an urgent need to develop a new culture method to express recombinant human coagulation factor VIII, enabling industrial-scale production of recombinant factor VIII under new culture conditions, increasing both yield and quality.

[0006] Methionine sulfoximine (MSX) is an effective inhibitor of glutamine synthetase activity and can be used as an additive in the seed cell expansion stage. Chinese patent application CN201710687334.4 discloses a method for culturing a CHO cell line that efficiently secretes and expresses classical swine fever E2 protein. In the scale-up stage, the serum-free medium used is a mixture of Dynamis and EX-CELL 302 medium containing 25 μM MSX, while in the fermentation stage, the serum-free medium used is a mixture of Dynamis and EX-CELL 302 medium without 25 μM MSX. Experiments show that the highest expression yield of classical swine fever E2 protein is achieved in the basal medium without MSX (25 μM). Summary of the Invention

[0007] The purpose of this invention is to provide a novel method for preparing recombinant human coagulation factor VIII and the recombinant human coagulation factor VIII product obtained by this method. Through extensive experimental research, this invention has found that adding a copper-containing compound and methionine sulfoxide imine to a serum-free culture medium can significantly improve the purity of the recombinant human coagulation factor VIII product while ensuring its high activity and high yield.

[0008] In this application, when weight ratios, volume ratios, molar ratios, and other numerical values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, they should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value and any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range "2 to 50" is disclosed, the described range should be interpreted as including ranges "2 to 49", "2 to 40", "2 to 35", "2 to 25", "5 to 50", etc. When numerical ranges are described in this application, unless otherwise stated, the range is intended to include its endpoints and all integer and fractional values ​​within that range.

[0009] The approximate terms used in this specification and claims are used to modify quantities, indicating that the invention is not limited to that specific quantity, but also includes acceptable modifications that are close to that quantity and do not alter the relevant essential function. In this specification and claims, scope definitions can be combined and / or interchanged, and unless otherwise stated, these scopes include all sub-scopes contained therein.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] On the one hand, the present invention provides a method for preparing recombinant human coagulation factor VIII, which includes culturing a mammalian cell line expressing recombinant human coagulation factor VIII in a serum-free culture medium containing a copper ion compound and methionine sulfoxide imine.

[0012] The inventors of this invention unexpectedly discovered that using a serum-free culture medium containing a combination of copper ions and MSX in both the cell expansion and fermentation stages of a mammalian cell line expressing recombinant human coagulation factor VIII, while ensuring product safety, can not only guarantee the final yield and activity of the product, but also significantly improve the purity of the product.

[0013] According to some embodiments of the present invention, the copper-containing compound is selected from one or more of copper sulfate, copper chloride, copper nitrate and their hydrates.

[0014] Preferably, the concentration of the copper ion-containing compound in the serum-free culture medium is 0.15–20 μmol / L, more preferably 0.25–10 μmol / L, and even more preferably 5–10 μmol / L.

[0015] According to some embodiments of the present invention, the molar ratio of the copper-containing compound to methionine sulfoxide imine is (0.15-20):(20-30), preferably (0.25-10):25; more preferably (5-10):25.

[0016] According to some embodiments of the present invention, the serum-free culture medium comprises a cell-based culture medium.

[0017] Preferably, the cell basal culture medium is ActiPro medium, CDM4PERMAb medium, CDM4MAb medium or CD OptiCHO medium; more preferably, it is ActiPro medium.

[0018] More preferably, the weight of the cell basal culture medium is 1.5 to 5% of the total weight of the serum-free culture medium, preferably 2 to 3%.

[0019] According to some embodiments of the invention, the serum-free culture medium further comprises a supplement containing hypoxanthine and / or thymidine.

[0020] Preferably, the molar ratio of hypoxanthine to thymine in the supplement containing hypoxanthine and thymine nucleoside is (8-12):(1.2-2), more preferably 10:1.6.

[0021] Preferably, the weight of the supplement containing hypoxanthine and thymidine is 0.3% to 2.5% of the total weight of the serum-free culture medium, more preferably 0.5% to 1%. Through extensive experiments, the inventors have found that when the weight of the supplement containing hypoxanthine and thymidine is 0.3% to 2.5% of the total weight of the serum-free culture medium, it has a significant effect on improving the quality of the recombinant human coagulation factor VIII product.

[0022] According to some embodiments of the present invention, the serum-free culture medium further comprises a glutamine synthase expression culture medium supplement.

[0023] Preferably, the weight of the glutamine synthase expression medium supplement is 0.5% to 4% of the total weight of the serum-free medium, more preferably 1% to 2%. During optimization experiments, the inventors found that the effect was better when the weight of the glutamine synthase expression medium supplement was 0.5% to 4% of the total weight of the serum-free medium.

[0024] In this invention, supplements containing hypoxanthine and thymidine are provided, but are not limited to, in the form of HT supplements, which are mixtures of hypoxanthine (10 mmol / L) and thymidine (1.6 mmol / L), dissolved in the culture medium in a specific ratio, and used as supplements for the DNA salvage synthesis pathway as additives and nutrients in hybridoma screening culture media to overcome the inhibitory effect of residual intracellular aminopterin on the classical DNA synthesis pathway.

[0025] In this invention, the glutamine synthase expression culture medium supplement can be provided in the form of a GS supplement.

[0026] According to some preferred embodiments of the present invention, the serum-free culture medium comprises a supplement containing hypoxanthine and thymidine nucleoside, as well as a supplement containing glutamine synthase expression medium.

[0027] Through extensive experimentation, the inventors further discovered that when the ratio of copper ions to the two supplements is within a certain range, it can better enhance sample activity. According to some preferred embodiments of the present invention, in each kilogram of the serum-free culture medium, the ratio of the copper-containing compound: the supplement containing inosine and thymidine: the glutamine synthetase expression medium supplement is (0.25–25) μmol: (0.5–2.5) w / w%HT: (0.5–2.5) w / w%GS, preferably (0.5–15) μmol: (0.5–1) w / w%HT: (1–2) w / w%GS, and even more preferably (5–10) μmol: 1 w / w%HT: 2 w / w%GS.

[0028] According to some embodiments of the present invention, the preparation method includes the following steps: inoculating the mammalian cell line expressing recombinant human coagulation factor VIII into the serum-free culture medium for sequential amplification culture and fermentation culture.

[0029] According to some embodiments of the present invention, during the fermentation process, a first feed medium is added daily at 1 to 3% of the initial culture weight, and a second feed medium is added daily at 0.1 to 0.5% of the initial culture weight.

[0030] Preferably, the first fed culture medium contains Cell Boost 7a. More preferably, the first fed culture medium contains 14-25% Cell Boost 7a by weight.

[0031] Preferably, the second feed medium contains Cell Boost 7b. More preferably, the second feed medium contains 6-13% Cell Boost 7b by weight.

[0032] Preferably, the temperature during the first to fourth days of fermentation is 36.0 to 38.0°C, more preferably 37°C, and the temperature during the fifth to 12th days is 32.0 to 34.0°C, more preferably 33°C.

[0033] In another aspect, the present invention provides a recombinant human coagulation factor VIII product obtained by the preparation method provided by the present invention.

[0034] In another aspect, the present invention provides the use of the recombinant human coagulation factor VIII product in the preparation of medicaments for the treatment and / or prevention of hemophilia.

[0035] Compared with the prior art, the preparation method and product provided by the present invention have the following beneficial effects:

[0036] 1. In this invention, serum-free culture medium supplemented with MSX and copper ions is selected and added throughout the entire process of seed amplification and fermentation culture, thereby promoting cell proliferation and obtaining a culture medium with high product activity.

[0037] 2. This invention also solves the problem of low purity in one-step affinity purification of recombinant human coagulation factor VIII by adding a small amount of copper ions to a serum-free culture medium containing MSX and cell basal medium.

[0038] 3. The present invention further optimized and screened the necessity and dosage of adding GS supplements and HT supplements, as well as the culture time, and optimized the culture conditions to obtain recombinant human coagulation factor VIII products with higher activity and yield.

[0039] In summary, the experimental results show that this invention achieves a significant improvement in the process for preparing recombinant human coagulation factor VIII by selecting and adding compounds containing copper ions and methionine sulfoxide imine in serum-free culture. Specifically, MSX and copper ions are introduced during both cell amplification and fermentation cultures, ensuring the final yield and activity of the recombinant human coagulation factor VIII product. At the same time, the purity of the recombinant human coagulation factor VIII product is significantly improved with the addition of a small amount of copper ions. Attached Figure Description

[0040] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0041] Figure 1This is a graph showing the changes in cell density and cell viability of each group during the fermentation culture stage in Example 3 of the present invention; where 01 to 10 represent the changes in the corresponding indicators of metal ions and MSX under different concentration ratios in groups 1-10, respectively.

[0042] Figure 2 This is a graph showing the changes in cell density and cell viability during the fermentation culture stages of combinations 01 to 07 in Example 4 of the present invention. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments, which will provide a better understanding of the invention. However, those skilled in the art should readily understand that the following embodiments are merely descriptive and do not imply that the invention is limited to these specific embodiments. Those skilled in the art should recognize that the present invention covers all improvements, alternatives, and equivalents included within the scope of the claims.

[0044] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods, and the experimental materials used are all purchased from biochemical reagent companies unless otherwise specified.

[0045] The cell culture medium described in this invention is ActiPro medium, CDM4PERMAb medium, CDM4MAb medium or CD OptiCHO medium, and in this embodiment, ActiPro medium is preferred.

[0046] The APTT method was used to detect the activity of factor VIII units. The cell basal culture medium (ActiPro), Cell Boost 7a (HyClone), and Cell Boost 7b (HyClone) used in the examples were all purchased from Cytiva. MSX and GS Supplement were purchased from Sigma-Aldrich, Inc. HT Supplement was purchased from Life Technologies Corporation. WFI represents water for injection.

[0047] The culture medium used in the following examples has the following composition:

[0048] Table 1 M01 culture medium

[0049]

[0050]

[0051]

[0052] Example 1 Preparation steps

[0053] 1. Seed revival

[0054] Take one CHO cell line expressing recombinant human FVIII factor (amino acid sequence as shown in SEQ ID No. 1) (the preparation method of this cell line is described in Example 2 of Chinese Patent CN111440820B), thaw it in a water bath, and centrifuge it at room temperature and 200g for 5 min. Take 5 mL of the cell suspension from 25 mL of M01 medium and carefully pipette it until a mixed suspension is formed. Transfer the mixed suspension to the remaining M01 medium, mix well, and then take a sample for analysis.

[0055] 2. Seed amplification

[0056] (1) Cell expansion was performed, and the cell viability of the first seed cell sap obtained was ≥90.0%, and the viable cell density was ≥2.00×10⁻⁶. 6 The amplification conditions were: 37.0℃, 5% carbon dioxide, shaker incubator, 70 rpm for 48 h.

[0057] (2) The above-mentioned live cell culture medium was transferred from the shake flask to a 37°C wave reactor containing MO1 medium for N-1 and N-th generations (the last expansion before the cell culture stage), resulting in a cell viability ≥90.0% and a live cell density ≥2.00×10⁻⁶. 6 The second and third daughter cell solutions at cells / mL.

[0058] 3. Fermentation culture

[0059] (1) Inoculation: After N-generation culture, the third seed cell culture was inoculated into MO1 medium. The temperature was 37℃ for days 1-4 after inoculation and 33℃ for days 5-12. The culture speed was 90 rpm, the pH was 6.75-7.25, the surface aeration rate was 0.1-2.0 L / min, and the bottom aeration rate was 0.1-5.0 L / min. The culture was continued until the cell viability was ≥90.0% and the viable cell density was ≥2.00×10⁶ cells / min. 6 cells / ml.

[0060] (2) Fed culture: From day 3 to day 12 of culture, the first feed medium was added daily at 2% of the initial culture weight, and the second feed medium was added daily at 0.2% of the initial culture weight. The culture was terminated after 12 days of fed culture or when the cell viability was less than 90%, and the cell stock solution was obtained.

[0061] Example 2: Examination of Adding Time Periods to MSX

[0062] When the seed culture reached the Nth generation (the last amplification before the cell culture stage), Nth generation amplification and fermentation culture were continued using serum-free medium containing 25 μMMSX to examine the expression of factor VIII in the presence of MSX. In the control group, except for the removal of MSX from the serum-free cell medium used for Nth generation amplification and fermentation culture, all other culture medium components, preparation steps, and culture conditions were the same as in Example 1, with a culture scale / shake flask scale of 10 L / 20 L.

[0063] In this embodiment, both the experimental and control groups used serum-free culture medium supplemented with MSX before generation N. From generation N onwards, the control group used serum-free culture medium without MSX, while the experimental group continued to use serum-free culture medium supplemented with MSX. The MSX addition and results are shown in Table 4 below:

[0064] Table 4 MSX addition status, sample expression level, and product purity

[0065] control group experimental group N generations ago + + Nth generation - + Cell fermentation culture stage - + Peak activity time D11 D11 Peak activity (IU / mL) 1392.5 1886.7 Lactic acid content at peak activity (mmol / L) 3.02 0.1 Cell viability (%) at peak activity 98 97 <![CDATA[Average cell density (10 6 cells / mL)]]> 14.4 10.2 Sample main peak (%) 67.86％ 72.49％

[0066] Note: The highest activity, lactic acid content at the highest activity, cell viability at the highest activity, average cell density, and main peak data of the control group and experimental group in Table 4 are all average values ​​obtained from the test data of the three groups of samples.

[0067] As shown in Table 4, although the control group achieved a higher density of viable cells compared to the experimental group, the activity and purity of factor VIII were lower, while the cell viability remained essentially the same. Other metabolic parameters, except for lactate, were also largely consistent. Lactate, which is crucial for the synthesis of human coagulation factor VIII, was consumed very little in the control group. In the experimental group, the accumulated lactate was completely consumed by day 10. Activity tests showed that the unit activity of human coagulation factor VIII continuously accumulated in the later stages of culture. The product activity on the last day of fermentation culture in the experimental group was 1886.7 IU / mL, while that in the control group was 1392.5 IU / mL.

[0068] Therefore, while using serum-free cell culture medium with MSX removed for N-generation amplification and cell fermentation can promote cell proliferation, it does not help improve the activity of human coagulation factor VIII. On the other hand, using serum-free culture medium containing MSX for all cell amplification and fermentation culture, although it slightly inhibits cell proliferation, can not only improve the activity of human coagulation factor VIII, but also improve product purity, which is more beneficial for the industrial production of factor VIII.

[0069] Example 3: Investigation of the addition of divalent metal ions

[0070] The experimental design in this embodiment investigated the effects of adding divalent metal ions, particularly different concentrations of divalent copper ions, to the culture medium in the presence of MSX during the fermentation culture stage on the activity and purity of recombinant factor VIII.

[0071] The compound containing divalent copper ions used in this invention can be any one of copper sulfate, copper chloride, copper nitrate and their hydrates. In this embodiment, divalent copper ions are added in the form of CuSO4·5H2O.

[0072] The fermentation culture temperature used was 37℃ / 33℃ (the culture temperature of 37℃ / 33℃ here corresponds to the temperature of 37℃ on days 1-4 and the temperature of 33℃ on days 5-12), and the inoculation density was 0.5×10⁻⁶. 6 The culture volume was 120 mL (500 mL shake flasks), and the addition of divalent metal ions in the serum-free medium is shown in Table 5 below. Starting from D2, 2% of the first feed medium and 0.2% of the second feed medium were added daily until D10. Other medium components, preparation steps, and culture conditions were the same as in Example 1.

[0073] Table 5. Sample expression levels and SEC-HPLC detection results of additive screening experiments.

[0074]

[0075]

[0076] Note: The highest expression level (D10) titer and sample main peak data in Table 5 are the average values ​​obtained from the detection data of the three groups of samples.

[0077] Samples were taken from the fermentation culture stage to test cell density and viability. The test results are as follows: Figure 1 As shown.

[0078] The final results are analyzed as follows:

[0079] 1. The results of cell density and cell viability tests showed that the cell growth trends of each combination were basically consistent, and the cell viability was all above 92%, indicating that the combination contained different concentrations of Cu. 2+ Serum-free culture media containing compounds, MnCl2·4H2O, CaCl2·2H2O, and MSX had no negative impact on cell growth. In contrast, the medium containing CaCl2·2H2O and MSX promoted cell growth less than the medium containing compounds containing copper or manganese ions.

[0080] 2. According to Table 5, the purity of Factor VIII in the combination of MnCl2·4H2O + MSX was equivalent to or lower than that in the control group, indicating that adding MnCl2·4H2O could not improve the purity of human coagulation factor VIII; while adding Cu... 2+ The purity of human coagulation factor VIII in the combination of compounds was higher than that in the control group.

[0081] The addition of 1.00 mM CaCl2·2H2O increased the purity of human coagulation factor VIII by 6.82%, but this effect was less than that of combinations containing copper ions (e.g., 1.00 μM Cu). 2+ (Increased by 10.48%); Moreover, with the increase of copper ion concentration, the purity of the sample was further improved, indicating that copper ions are the key component for improving the purity of human coagulation factor VIII. Among them, when 5.00 μM copper ions were added, the purity could be increased to 92.61%. When 10.00 μM copper ions were added, although the expression level was higher than that of 5.00 μM copper ions, the purity, cell density and viability were lower.

[0082] In summary, when the copper ions and MSX in the culture medium are at (0.25–10) μM Cu 2+ When the concentration ratio is within the range of 25 μM MSX, the purity, cell density, and activity of recombinant human coagulation factor VIII samples can all achieve a good effect.

[0083] Example 4

[0084] This study investigated the effects of adding different amounts of GS supplement (GS Supplement 50X in this example) and HT supplement (HT Supplement 100X in this example) to 1 kg of serum-free culture medium on the product. Specifically, the study investigated the ratio of divalent copper ions to GS supplement and HT supplement, setting different addition ratios to examine the effects of the culture medium on cell growth, expression and quality of human coagulation factor VIII.

[0085] The divalent copper ions of the present invention can be added in the form of any one of copper sulfate, copper chloride, copper nitrate and their hydrates. In this embodiment, the divalent copper ions are added in the form of CuSO4·5H2O.

[0086] The fed-batch culture protocol is as follows:

[0087] Starting on day 3 of culture, feed was administered daily with 2% first feed medium and 0.2% second feed medium until the day before the end of culture. The culture scale was 15 mL, and the reactor was Ambr15. When the theoretically calculated residual sugar content after feeding was lower than 4.0 g / L, sugar was added to 6.0 g / L.

[0088] The experimental design for this embodiment is shown in Table 6 below. All other culture medium components, preparation steps, and culture conditions are the same as in Example 1.

[0089] Table 6. Expression of samples with different culture medium combinations

[0090]

[0091] Note: The amount of basic culture medium used in the culture medium combination in Table 6 is the same as in Example 1; the highest activity enhancement rate and the main peak data of the sample are the average values ​​obtained from the detection data of three groups of samples.

[0092] Depend on Figure 2 The cell viability curves show that the cell viability of all combinations was above 92% on the day the fermentation culture ended; the cell density change graph and Table 6 show that there was no significant difference in purity among the groups.

[0093] The culture media containing combinations 02 to 04 with added HT supplements and / or GS supplements showed higher activity enhancement rates than other combinations. Combination 04 showed a maximum activity enhancement rate of 171.17% for human coagulation factor VIII units.

[0094] Specifically, when HT and / or GS supplements were added to combinations 02–04, the highest activity enhancement rate of the samples was higher than that of combination 01. In combinations 05–07, when the ratios of copper ions:GS:HT were 5 μM:1% GS:1% HT, 5 μM:2% GS:0.5% HT, and 5 μM:1% GS:0.5% HT, respectively, the activity of human coagulation factor VIII was significantly enhanced, but the highest activity enhancement rate was lower than that of combination 04 (25 μM copper ions:2% GS:1% HT). It is evident that the culture medium containing the HT and GS combination can further improve factor VIII activity, with the serum-free culture medium containing the ratio of 5 μM copper ions:2% GS:1% HT showing the highest enhancement rate of sample activity.

[0095] In summary, the preparation method provided in this application can not only maintain a high yield and activity of factor VIII, but also further improve the purity of the product, and has good value for industrialization and application.

[0096] While various aspects and embodiments of the invention have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The aspects and embodiments disclosed herein are for illustrative purposes only and not for limiting purposes. The scope and spirit of the invention are determined solely by the appended claims.

Claims

1. A method for preparing recombinant human coagulation factor VIII, characterized in that, This includes culturing mammalian cell lines expressing recombinant human coagulation factor VIII in a serum-free medium containing copper ions and methionine sulfoxide imine.

2. The preparation method according to claim 1, characterized in that, The copper-containing compound is selected from one or more of copper sulfate, copper chloride, copper nitrate and their hydrates; Preferably, the concentration of the copper ion-containing compound in the serum-free culture medium is 0.15–20 μmol / L, more preferably 0.25–10 μmol / L, and even more preferably 5–10 μmol / L.

3. The preparation method according to claim 1 or 2, characterized in that, The molar ratio of the copper-containing compound to methionine sulfoxide imine is (0.15–20):(20–30), preferably (0.25–10):25; more preferably (5–10):

25.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The serum-free culture medium includes a cell-based culture medium; Preferably, the cell culture medium is ActiPro medium, CDM4PERMAb medium, CDM4MAb medium, or CD OptiCHO medium; more preferably, it is ActiPro medium. More preferably, the weight of the cell basal culture medium is 1.5 to 5% of the total weight of the serum-free culture medium, preferably 2 to 3%.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The serum-free culture medium also contains supplements containing hypoxanthine and thymidine. Preferably, the molar ratio of hypoxanthine to thymine in the supplement containing hypoxanthine and thymine nucleoside is (8-12):(1.2-2), more preferably 10:1.6; Preferably, the weight of the supplement containing hypoxanthine and thymidine is 0.3% to 2.5% of the total weight of the serum-free culture medium, more preferably 0.5% to 1%.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The serum-free culture medium also contains a glutamine synthase expression culture medium supplement; Preferably, the weight of the glutamine synthase expression medium supplement is 0.5% to 4% of the total weight of the serum-free medium, more preferably 1% to 2%.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The serum-free culture medium contains supplements of hypoxanthine and thymidine, as well as a supplement of glutamine synthase expression medium. Preferably, in each kilogram of the serum-free culture medium, the ratio of the copper-containing compound: the supplement containing inosine and thymidine: the glutamine synthase expression culture medium supplement is (0.25–25) μmol: (0.5–2.5) w / w%: (0.5–2.5) w / w%, preferably (0.5–15) μmol: (0.5–1) w / w%: (1–2) w / w%, and more preferably (5–10) μmol: 1 w / w%: 2 w / w%.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The preparation method includes the following steps: the mammalian cell line expressing recombinant human coagulation factor VIII is seeded into the serum-free culture medium and then subjected to amplification culture and fermentation culture in sequence; Preferably, during the fermentation process, a first feed medium is added daily at 1-3% of the initial culture weight, and a second feed medium is added daily at 0.05-0.3% of the initial culture weight. Preferably, the first fed culture medium contains CellBoost 7a; Preferably, the second feed culture medium contains CellBoost 7b; Preferably, the temperature during the first to fourth days of fermentation is 36.0 to 38.0°C, and the temperature during the fifth to twelfth days is 32.0 to 34.0°C.

9. The recombinant human coagulation factor VIII product obtained by the preparation method according to any one of claims 1 to 8.

10. The use of the recombinant human coagulation factor VIII product of claim 9 in the preparation of a medicament for the treatment and / or prevention of hemophilia.

Images