A method for preparing a recombinant human anti-VEGF antibody fusion protein

Abstract

This invention discloses a method for preparing recombinant human anti-VEGF antibody fusion protein, comprising basal culture and fed-batch culture. This method, by combining a specific basal culture medium with a specific fed-batch culture process, effectively reduces the content of the basic peak in the fusion protein while increasing protein expression, thereby enhancing the efficacy of the recombinant human antibody fusion protein and possessing practical application value.

Description

Technical Field

[0001] This invention specifically relates to a method for preparing a recombinant human anti-VEGF antibody fusion protein. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a highly specific pro-vascular endothelial cell growth factor that promotes increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis. The VEGF family has seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PlGF). Its receptors include tyrosine kinase receptors (VEGFR-1, VEGFR-2, VEGFR-3) and non-tyrosine kinase receptors (NRP-1, NRP-2). Among them, VEGF-A has the greatest impact on angiogenesis, primarily exerting its effects by binding to VEGFR-1 and VEGFR-2. The second immunoglobulin-like domain (VEGFR-1D2) and the third immunoglobulin-like domain (VEGFR-2D3) of the extracellular region of VEGFR-1 are linked to the gene of the human immunoglobulin IgG1 Fc fragment to recombinantly express the fusion protein rhVEGFR-Fc. Alternatively, the Fc fragment of IgG1 can be replaced with the Fc fragment of IgG2, IgG3, or IgG4. These fusion proteins can bind very well to VEGF family members, competitively inhibiting their binding to VEGF family receptors, preventing the activation of family receptors, and effectively controlling angiogenesis. This can achieve therapeutic effects on wet AMD, diabetic macular edema, pathological myopia, retinal vein occlusion, and other related diseases.

[0003] Currently, therapeutic fusion proteins used for ophthalmic diseases mainly include aflibercept and conbercept, which are proteins formed by fusing human vascular endothelial growth factor VEGFR-1 and VEGFR-2 with the Fc fragment of human immunoglobulin. With increasingly stringent requirements for the production of these therapeutic fusion proteins, culture and preparation methods have been improved to varying degrees. These improvements include refining cell selection, optimizing culture media, controlling the culture process, and purification techniques to enhance cell growth and viability, as well as protein yield and quality.

[0004] The production and purification of recombinant proteins involve numerous modifications and degradation processes, such as oxidative deamidation, glycosylation, C-terminal lysine removal, N-terminal pyroglutamic acid cyclization, disulfide bond modification, and sequence variation. The combination of these heterogeneous processes can lead to a large number of isomers in recombinant proteins. When the number or structure of charged groups changes, it can cause heterogeneity in the charge distribution of recombinant proteins, thereby affecting their biological activity, pharmacokinetics, immunogenicity, and structural stability. This can have a significant impact on the safety and efficacy of drugs in clinical use. Therefore, controlling the charge heterogeneity of recombinant protein products is a key quality attribute of the entire production process.

[0005] Existing technology CN114480492A involves supplementing NH4 starting on day 4 or 5 of the cell culture stage after transfection with a recombinant human antibody fusion protein expression vector. + This reduces the proportion of low-acid peaks in the protein and improves fusion quality. Existing technology CN111606968A uses anion exchange chromatography combined with flow-through mode purification to reduce the content of acidic charge isomers in Fc fusion proteins, thereby ensuring the quality of the Fc fusion protein. These methods all reduce acidic charge isomers; very few existing technologies provide methods that can simultaneously control both acidic and basic charge isomers. Summary of the Invention

[0006] To address the above problems, this invention provides a method for preparing a recombinant human anti-VEGF antibody fusion protein, comprising the following steps:

[0007] 1) Basic culture: Take the basic culture medium OPM CDP3 CHO and inoculate it into cells transfected with the recombinant human anti-VEGF antibody fusion protein expression vector;

[0008] 2) Fed culture: Starting from day 4 of the basic culture, add fed culture medium every other day until day 11 or until the cell viability is not less than 80%;

[0009] 3) Harvest the culture medium to obtain the product;

[0010] The feeding medium is VegaCHO Feed and / or CDFS36.

[0011] Furthermore, VegaCHO was added every other day at a volume of 3-5% of the basal culture medium.

[0012] Furthermore, VegaCHOFeed was added to the basal culture medium every other day at volumes of 4%, 4%, 5%, and 5%.

[0013] Furthermore, CDFS36 was added every other day at a volume of 0.3–0.5% of the basal culture medium.

[0014] Furthermore, CDFS36 was added every other day at volumes of 0.4%, 0.4%, 0.5%, and 0.5% of the basal culture medium.

[0015] Furthermore, the fed-batch culture also includes adding 1-5 μM of MnCl2, preferably 2 μM, to the basal culture up to day 4.

[0016] Furthermore, the fed-batch culture also includes the addition of 5-8 mM N-acetyl-D-mannosamine to the basal culture up to day 4.

[0017] Furthermore, the culture conditions are as follows: inoculation density 0.3 × 10⁻⁶. 6 cells / ml; culture temperature 37℃; culture medium pH 7.0±0.15, dissolved oxygen 40%.

[0018] Furthermore, the cells are CHO DG44 cell lines transfected with a recombinant human anti-VEGF antibody fusion protein expression vector.

[0019] Furthermore, the human anti-VEGF antibody fusion protein is an Fc fusion protein of VEGFR1 and VEGFR2, and its amino acid sequence is shown in SEQ ID No:1.

[0020] The present invention provides a method for preparing recombinant human anti-VEGF antibody fusion protein. By combining a specific basal culture medium and a specific fed culture process, the method effectively reduces the content of the basic peak in the fusion protein, while increasing the expression level of the protein and improving the efficacy of the recombinant human antibody fusion protein. This method has practical application value.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 The iCIEF detection spectrum of Example 2;

[0024] Figure 2 The iCIEF detection spectrum of Example 3;

[0025] Figure 3 The iCIEF detection spectrum of Experiment Example 1;

[0026] Figure 4 The iCIEF detection spectrum is shown in Comparative Example 1;

[0027] Figure 5 The iCIEF detection spectrum is shown in Comparative Example 2.

[0028] Figure 6 This is a typical spectrum for detecting the recombinant human antibody fusion protein iCIEF of this invention. Detailed Implementation

[0029] Raw material sources: Unless otherwise specified, all raw materials used in this invention are commercially available, and all culture media are commercially available culture media.

[0030] iCIEF assay: Dilute the reference and test samples to 20 mg / ml with ultrapure water. Add 5 μL each of the blank sample, diluted reference, and test sample to a sample tube containing 95 μL of premixed solution. Centrifuge, then transfer to a sample tray and continue centrifugation. Add the corresponding buffer solutions to the cartridge and system tray, place in a capillary electrophoresis apparatus, edit the batch, set the detection method, and run the batch for detection. Calculate the amounts of the acidic peak, main peak, and basic peak based on the spectrum, and compare with a typical spectrum (the typical spectrum is obtained using aflibercept for iCIEF assay). Figure 6 By comparison, peaks M1-M6 in the spectrum that are consistent with the typical spectrum are identified as main peaks, peaks A1-A3 in the typical spectrum are identified as acidic peaks, and peaks B1-B5 in the typical spectrum are identified as alkaline peaks.

[0031] Example 1

[0032] This embodiment details the fermentation and culture process of recombinant human antibody fusion protein using the VEGFR fusion protein as an example.

[0033] The recombinant human antibody fusion protein is a VEGFR (vascular endothelial growth factor receptor) fusion protein, specifically an Fc fusion protein of VEGFR1 and VEGFR2. It is a fusion protein of the Fc fragment of IgG1, meaning it is a recombinant protein in which the VEGFR1 and VEGFR2 genes are linked to the Fc fragment gene of immunoglobulin IgG1 at the gene level and expressed in a eukaryotic expression system. The protein has the amino acid sequence shown in SEQ ID No:1.

[0034] I. Construction of engineered cell lines for recombinant human antibody fusion protein gene

[0035] The pCHO 1.0 vector was double-digested with AvrII (CCTAGG) and PacI (TTAATTAA), then ligated with the recombinant human antibody fusion protein gene fragment. The ligation product was transformed into DH5α competent E. coli cells and plated on LB agar plates containing ampicillin. Single colony transformants were obtained by antibiotic selection. Recombinant plasmids were extracted from selected single colonies after culturing. The extracted recombinant plasmids were double-digested with AvrII / PacI and sequenced. The sequencing results of the recombinant plasmids were consistent with the expected sequence.

[0036] The correctly cloned recombinant plasmid Pvu I was digested and transformed into CHO-DG44 host cells (purchased from Invitrogen) in logarithmic growth phase to obtain an engineered strain. The engineered strain was inoculated into a medium containing MTX (methopterin, 500 nM / L) and cultured until the logarithmic growth phase. The engineered cell line for the recombinant human antibody fusion protein product of this embodiment was selected based on cell state and expression level. The selected cell line had an expression level greater than 3.0 g / L, significantly higher than the expression levels in existing CHO-K1 and CHO-S cells. In situ hybridization analysis confirmed that the recombinant human antibody fusion protein expression vector had been completely integrated into the chromosome of the engineered cell line. The amino acid sequence of the recombinant human antibody fusion protein is as shown in SEQ ID No.:1.

[0037] SEQ ID No:1 amino acid sequence:

[0038] SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDG

[0039] KRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVL

[0040] SPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLK

[0041] TQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKD

[0042] KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

[0043] VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0044] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

[0045] GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[0046] NVFSCSVMHEALHNHYTQKSLSLSPG

[0047] Establishment of Level II and Level III Cell Banks

[0048] Revive engineered cell lines and establish a three-tiered cell bank.

[0049] III. Cultivation

[0050] Thaw WCB in a 37.0℃ water bath and inoculate it into a shake flask containing C / B seed culture medium. The cell viability after recovery is >85%. After cell recovery, perform shake flask seed amplification and WAVE amplification in sequence. After 3 days of WAVE amplification culture, inoculate it into a 200L reactor and obtain recombinant human antibody fusion protein through basal culture and fed culture.

[0051] Basic culture: The culture medium was OPM CDP3 CHO;

[0052] Feeding culture: This includes adding 3 / 3 / 4 / 4% of the basal culture medium volume of VegaCHO Feed on Days 4 / 6 / 8 / 10 of the basal culture, respectively; adding 0.3 / 0.3 / 0.4 / 0.4% of the basal culture medium volume of CDFS36 on Days 4 / 6 / 8 / 10 of the basal culture, respectively; adding 2 μM MnCl2 on Day 4 of the basal culture (i.e., the final concentration of MnCl2 in the culture medium is 2 μM); and adding 5 mM N-acetyl-D-mannosamine on Day 4 of the basal culture (i.e., the final concentration of N-acetyl-D-mannosamine in the culture medium is 5 mM).

[0053] The culture parameters are set as follows:

[0054] pH: 7.00±0.15;

[0055] T: 37.0℃;

[0056] Dissolved oxygen (DO): 40%;

[0057] Speed: 95 rpm;

[0058] Air velocity: 1.50 lpm;

[0059] Surface ventilation: 2.00 lpm;

[0060] The culture lasted for 11 days, after which it was completed.

[0061] Examples 2-3, based on the conditions of Example 1, respectively added 5 μM and 2 μM MnCl2 during the fed-batch culture stage. Specific process parameters are shown in Table 1.

[0062] Table 1. Process flow of Examples 2-3.

[0063]

[0064] Protein iCIEF detection maps obtained in Examples 2-3 Figure 1 and Figure 2 Referring to typical spectra, the acid, base and main peak regions in the spectra were delineated, and the peak area normalization method was used to quantitatively calculate each peak. The results are summarized in Table 2.

[0065] Table 2. Results of protein quantity and expression level in Examples 2-3

[0066]

[0067] The beneficial effects of this invention are further illustrated by the following experimental examples and comparative examples.

[0068] Experimental Example 1

[0069] Based on Example 2, this experimental example does not add MnCl2 during fed-batch culture. Specific process parameters are shown in Table 3:

[0070] Table 3. Process of Experiment Example 1

[0071]

[0072] Protein iCIEF detection map of Experiment Example 1 Figure 3 Referring to typical spectra, the acid, base and main peak regions in the spectra were delineated, and quantitative calculations were performed on each peak. The results are summarized in Table 4.

[0073] Table 4 Results of protein quality and expression levels in Experiment Example 1

[0074]

[0075] Results Analysis: As shown in Tables 2 and 4, the following culture process effectively controlled the acid and basic peak contents of the fusion protein within the ideal range: the basal medium was OPM CDP3 CHO; the feed was supplemented with 4 / 4 / 5 / 5% VegaCHO feed on Day 4 / 6 / 8 / 10 respectively; the feed was supplemented with 0.4 / 0.4 / 0.5 / 0.5% CDFS36 on Day 4 / 6 / 8 / 10 respectively; and the culture process was supplemented with 5mM N-acetyl-D-mannosamine on Day 4 and 1-5uM MnCl2 on Day 4. This improved the quality of the fusion protein.

[0076] Experimental Example 2-5

[0077] Using the feeding process described in Example 1 of this invention, the effects of different basal culture media on the protein basic peak were investigated.

[0078] Table 5. Process of Experiment 2-5

[0079]

[0080] Table 6 Results of protein quantity and expression level in Experiments 2-5

[0081]

[0082] Results analysis: The results of Experiments 2-5 show that, based on the fed culture medium of the present invention, when other basic culture media are used, with or without the addition of MnCl2 during the culture process, the content of the protein basic peak is greater than 40%, which is much greater than the content of the protein basic peak obtained under the basic culture and fed culture processes of the present invention.

[0083] Experimental Examples 6-9

[0084] Using the basal culture medium of Example 1 of this invention, the effect of different feeding processes on the protein basic peak was investigated.

[0085] Table 7 Process of Experiment Examples 6-9

[0086]

[0087] Table 8 Results of protein quantity and expression level in Experiments 6-9

[0088]

[0089] Results analysis: The results of Experiments 6-9 show that, based on the basic culture medium of this invention, when other different feeding processes are used for culture, with or without the addition of MnCl2 during the culture process, the content of the protein basic peak is greater than 40%, which is much greater than the content of the protein basic peak obtained under the basic culture and feeding culture processes of this invention, and the protein expression level is not significantly changed.

[0090] Comparative Examples 1-2

[0091] Comparative Examples 1 and 2 were designed. Comparative Example 2 is the production process of the anti-VEGF antibody fusion protein in CN114480492A. Comparative Example 1 is based on Comparative Example 2, with 2 μM of MnCl2 added on Day 4 of the feed process.

[0092] Table 9 Comparative Example 1-2 Process Flowchart

[0093]

[0094] iCIEF detection spectra of Comparative Examples 1-2 Figure 4 and Figure 5 Referring to typical spectra, the acid, base and main peak regions in the spectra were delineated, and the peak area normalization method was used to quantitatively calculate each peak. The results are statistically presented in Table 10.

[0095] Table 10 Results of protein mass and expression levels in Comparative Examples 1-2

[0096]

[0097] Results analysis: As shown in Table 10, based on the CN114480492A process, simply adding MnCl2 has no effect on the basic peak content and protein expression level of the fusion protein.

[0098] Meanwhile, the comparative experiment also examined the feeding regimen with lower alkaline peak content in CN114480492A, which was based on advanced + FortiCHO mixed medium supplemented with S66 and C5. Although the fusion protein obtained by this regimen had a lower alkaline peak content, the protein expression level was also very low, only 0.745 g / L.

[0099] Based on the results of the embodiments, experimental examples and comparative examples of the present invention, it can be seen that the specific combination process of the basal culture medium and feeding process of the present invention, combined with the specific MnCl2 supplementation process on Day 4 of culture, can effectively reduce the content of the basic peak of the fusion protein and at the same time increase the expression level of the protein.

Claims

1. A method for preparing a recombinant human anti-VEGF antibody fusion protein, characterized in that: It includes the following steps: 1) Basic culture: Take the basic culture medium OPM CDP3 CHO and inoculate it into cells transfected with the recombinant human anti-VEGF antibody fusion protein expression vector; 2) Feed culture: Add 1-5 μM MnCl2 to the basal culture on day 4, and from day 4 onwards, add feed culture medium every other day until day 11 or the cell viability is not less than 80%; 3) Harvest the culture medium to obtain the product; The feeding medium consisted of VegaCHO Feed and CDFS36; VegaCHO was added every other day at 3-5% of the basal medium volume; CDFS36 was added every other day at 0.3-0.5% of the basal medium volume. The human anti-VEGF antibody fusion protein is an Fc fusion protein of VEGFR1 and VEGFR2, and its amino acid sequence is shown in SEQ ID No:1; The fed-batch culture also includes the addition of 5-8 mM N-acetyl-D-mannosamine to the basal culture up to day 4.

2. The preparation method according to claim 1, characterized in that: VegaCHO Feed was added every other day at volumes of 4%, 4%, 5%, and 5% of the basal culture medium.

3. The preparation method according to claim 1, characterized in that: The basal culture medium was supplemented every other day with 0.4%, 0.4%, 0.5%, and 0.5% of CDFS36, respectively.

4. The preparation method according to claim 1, characterized in that: The fed-batch culture also includes the addition of 2 μM MnCl2 on day 4 of the basal culture.

5. The preparation method according to claim 1, characterized in that: The culture conditions were as follows: inoculation density 0.3 × 10⁻⁶. 6 cells / ml; culture temperature 37℃; culture medium pH 7.0±0.

15.

6. The preparation method according to claim 1, characterized in that: The cells in question are the CHO DG44 cell line, which has been transfected with a recombinant human anti-VEGF antibody fusion protein expression vector.

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