A culture medium and a method for producing an anti-cgrp r antibody

Abstract

The application discloses a culture medium and a method for producing anti-CGRPR antibodies by using the culture medium, which comprises the following steps: culturing cells capable of expressing the anti-CGRPR antibodies in a fermentation culture medium, adding histidine and a galactose-containing sugar type regulator in the fermentation culture medium, and adding a mannose-containing feed medium in a fermentation process by feeding. The method can effectively control antibody charge heterogeneity and optimize antibody sugar types, effectively reduce the acid peak of the antibodies, improve the basic peak of the antibodies, and optimize high mannose and G0F sugar types by controlling the cell process flow.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a culture medium and a method for producing anti-CGRPR antibodies using the medium. Background Technology

[0002] Due to the complex molecular structure and large molecular weight of monoclonal antibodies, they undergo numerous modifications during production and storage, resulting in multiple isoforms. The combination of these isoforms leads to charge heterogeneity and glycosylation modification heterogeneity in monoclonal antibodies. Glycosylation level and type are crucial parameters for monoclonal antibody drugs. Currently, methods for altering antibody glycosylation primarily focus on the gene level to change antibody glycoform or glycosylation level; this method is time-consuming and lacks assurance regarding safety and stability. Charge heterogeneity is a critical quality attribute in monoclonal antibody manufacturing processes. Charge heterogeneity generates acid-base peaks, especially acid peaks, which severely impact the stability and biological function of monoclonal antibodies, consequently affecting their tissue penetration and pharmacokinetics. Controlling antibody charge heterogeneity and optimizing antibody glycoform through cellular process control presents significant challenges.

[0003] Calcitonin gene-related peptide (CGRP) is a neurotransmitter that plays an important role in the development of migraines. Migraine attacks can be treated and prevented by blocking CGRP or its receptors; therefore, CGRP is a popular target for migraine treatment, leading to the development of therapeutic small-molecule CGRP receptor antagonists and preventative CGRP monoclonal antibodies. Erenumab is an effective monoclonal antibody that targets and blocks typical CGRP receptors. Multiple clinical trials have demonstrated the preventative efficacy and good tolerability of Erenumab against episodic migraine (EM) and chronic migraine (CM).

[0004] However, there is currently no technical solution that provides an optimized method for preparing errinumab. Summary of the Invention

[0005] To address the lack of effective methods for optimizing the preparation of anti-CGRPR antibodies in existing technologies, this invention provides a culture medium and a method for producing anti-CGRPR antibodies using the medium. The method of this invention regulates the charge heterogeneity of the antibody and optimizes its glycoform by controlling the cell process flow, producing erenumab with quality similar to the original drug.

[0006] To achieve the above objectives:

[0007] The first aspect of the present invention provides a method for producing anti-CGRPR antibodies, wherein the method involves culturing cells capable of expressing the anti-CGRPR antibodies in a fermentation medium, adding histidine and a glycoform regulator containing galactose to the fermentation medium, and adding a feed medium containing mannose during the fermentation process.

[0008] In some embodiments of the present invention, the method includes one or more of the following conditions:

[0009] (1) The anti-CGRPR antibody is errinumab;

[0010] (2) The concentration of histidine is 2 g / L;

[0011] (3) The concentration of the mannose is 50 g / L;

[0012] (4) The concentration of the glycoform regulator is 2%; and / or, the glycoform regulator further includes manganese ions and / or uridine; wherein % is a volume percentage;

[0013] (5) If the total glucose in the method is less than 3 g / L, it should be supplemented to 4-6 g / L.

[0014] In some embodiments of the present invention, the cells are CHO cells, preferably selected from CHO-K1, CHOG44 and CHO-S cells, and more preferably CHO-K1 cells.

[0015] In some embodiments of the present invention, the fermentation medium is selected from one or more of OPM-CHO CD063 DPM, OPMCD07 DPM, Dynamis (Gibco), actipro (Hyclone), and CHO MaxD (Maibon).

[0016] In some embodiments of the present invention, the feeding is performed every other day starting from day 3; and / or, the feeding culture medium is selected from XF01 DPM, CDFS06, EfficientFeed TM C+, HyClone TM Cell Boost TM 7a. HyClone TM Cell Boost TM 7b, one or more of MAX FA and MAX FB; and / or, supplement with 0.5% of the sugar form regulator on day 11.

[0017] In some specific embodiments of the present invention, the supplemental culture medium comprises 50 g / L mannose;

[0018] Preferably, the feeding medium is XF01 DPM or CDFS06.

[0019] In some embodiments of the present invention, the amount of material replenished by the XF01 DPM each time is 4%, and the amount of material replenished by the CDFS06 each time is 0.4%, where % is a volume percentage.

[0020] In some embodiments of the present invention, the method includes one or more of the following conditions:

[0021] (a) The fermentation temperature is 37°C from day 1 to day 6, and then drops to 32-33°C from day 7 onwards;

[0022] (b) The pH of the fermentation is 7.0±0.2, preferably 6.9±0.1;

[0023] (c) The fermentation time is 15 to 16 days;

[0024] (d) During fermentation, the DO content is 40% and the air flow rate is 1.2 L / h.

[0025] In some embodiments of the present invention, the method includes one or more of the following conditions:

[0026] (i) The carbon dioxide concentration was 5% during seed amplification in shake flasks;

[0027] (ii) The shaking speed is 130 rpm;

[0028] (iii) The temperature is 37℃;

[0029] (iv) Inoculation density is 0.5–1.0 × 10⁻⁶. 6 cells / ml.

[0030] In some specific embodiments of the present invention, WAVE seed amplification is also included, wherein the WAVE seed amplification includes one or more of the following conditions:

[0031] 1) The pH level for WAVE seed amplification is between 6.7 and 7.2;

[0032] 2) The temperature is 37℃;

[0033] 3) The rotation speed for the first stage of seed amplification is 15 rpm, and the rotation speed for the second stage is 20 rpm;

[0034] 4) The inoculation density for the first stage of seed amplification is 0.5–1.0 × 10⁻⁶. 6 The second-stage seeding density was 1.0–2.0 × 10⁶ cells / ml. 6 cells / ml.

[0035] A second aspect of the present invention provides a culture medium for producing anti-CGRPR antibodies, the culture medium comprising a cell fermentation medium, 2 g / L histidine, 2% galactose-containing glycoform regulator, and 50 g / L mannose; wherein,

[0036] The cells are preferably CHO cells, and more preferably selected from CHO-K1, CHO DG44 and CHO-S cells.

[0037] In some specific embodiments of the present invention, the culture medium for the fermentation cells is OPM-CHO CD063 DPM, and / or the glycoform regulator further includes galactose, manganese ions and / or uridine, for example, the glycoform regulator is S081912-001; and / or the anti-CGRPR antibody is errinumab; wherein % is a volume percentage.

[0038] A third aspect of the present invention provides a kit for producing CGRPR antibodies, the kit comprising a culture medium for fermenting cells, a separate glycoform regulator, histidine, and mannose; wherein,

[0039] The cells are preferably CHO cells, and more preferably CHO cells are selected from CHO-K1, CHO DG44 and CHO-S cells.

[0040] In some specific embodiments of the present invention, the culture medium for the fermentation cells is OPM-CHO CD063 DPM; and / or, the glycoform regulator further includes manganese ions and / or uridine, for example, the glycoform regulator is S081912-001; and / or, the anti-CGRPR antibody is errinumab; wherein % is a volume percentage.

[0041] A fourth aspect of the present invention provides the use of the culture medium described in the second aspect and the kit described in the third aspect in the production of CGRPR antibodies.

[0042] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0043] The reagents and raw materials used in this invention are all commercially available.

[0044] The positive and progressive effects of this invention are as follows:

[0045] The method for producing anti-CGRPR antibodies provided by this invention can effectively control antibody charge heterogeneity and optimize antibody glycoform. By controlling the cell process flow, it effectively reduces the acidic peak and increases the basic peak of the antibody, while optimizing glycoforms such as high mannose and GOF. The method of this invention produces anti-CGRPR antibodies with many parameters similar to the original drug, providing a reliable biosimilar. Attached Figure Description

[0046] Figure 1 This is a comparison of antibody charge heterogeneity when cells are cultured at different pH and temperature. Process 1: pH 7.0±0.2, temperature reduced to 33℃ on day 7; Process 2: pH 7.0±0.2, temperature reduced to 32℃ on day 7; Process 3: pH 6.9±0.05, temperature reduced to 32℃ on day 7; Process 4: pH 6.9±0.05, temperature reduced to 34℃ on day 7.

[0047] Figure 2A and Figure 2B The effects of histidine, mannose, and glycoform modifiers on antibody charge heterogeneity and glycosylation were investigated. Figure 2A This is a comparison diagram of antibody charge heterogeneity; Figure 2B This is a comparison chart of antibody glycosylation. Process 1: basal culture medium + 2 g / L histidine + 2% glycoform regulator, XF01 DPM without mannose, 0.5% glycoform regulator added on D11; Process 2: basal culture medium + 2 g / L histidine + 2% glycoform regulator, XF01 DPM containing 50 g / L mannose, 0.5% glycoform regulator added on D11.

[0048] Figure 3A and Figure 3B This refers to the application of cell culture technology in a 150L cell culture tank. Figure 3A Comparison of antibody charge heterogeneity; Figure 3B This is a comparison chart of antibody glycosylation. Process 1: basal culture medium + 2 g / L histidine + 2% glycoform regulator, XF01 DPM without mannose, 0.5% glycoform regulator added on D11; Process 2: basal culture medium + 2 g / L histidine + 2% glycoform regulator, XF01 DPM containing 50 g / L mannose, 0.5% glycoform regulator added on D11. Detailed Implementation

[0049] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0050] Table 1: Experimental Materials

[0051]

[0052]

[0053] Table 2: Experimental Instruments

[0054] Experimental instruments model factory Four-compartment can DASGIP 3.8L Eppendorf 150L cell culture vessel 150pro NBS Carbon dioxide shaker ZCZY-BS8ES Knowing Chu Cell counter IC1000 Countstar Biosafety cabinet BSC-1300IIA2 Su Jingantai Blood gas analyzer 500 Siemens Osmometer SMC 30C-1 Tianjin Tianhe sterilization cabinet XG0.6G Shandong Xinhua

[0055] The test sample was diluted to 1 mg / ml using mobile phase A, and detected using a high-performance liquid chromatograph U3000 under the following chromatographic conditions:

[0056] Table 3: Detection of Charge Variants

[0057] name parameter liquid chromatograph Thermo U3000 High Performance Liquid Chromatography System chromatographic column Thermo CEX-MabPac Scx-10 4*250mm,10μm Detection wavelength Detection wavelength 215nm Flow rate 0.6 mL / min, gradient elution (see table below for details) Sample volume 75 μL (total 75 μg) Collection time 70min Column temperature 40℃ Sample tray temperature 20℃

[0058] After denaturation, the antibody was derivatized using Agilent's N-glycan kit. The sample was washed and collected, then mixed with acetonitrile at a 3:7 ratio and detected by UPLC under the following chromatographic conditions:

[0059] Table 4: Sugar Form Detection

[0060]

[0061]

[0062] Example 1: Cell culture in a 3.8L cell culture vessel

[0063] 1. Cell resuscitation and expansion

[0064] Remove the frozen cells from the liquid nitrogen tank and thaw them in a water bath at 37°C. Transfer the thawed cryopreservation solution to a centrifuge tube containing 5 mL of basal culture medium, mix thoroughly, and centrifuge at 1000 rpm for 5 min. Discard the supernatant. Resuspend the cell pellet in 5 mL of basal culture medium and centrifuge at 0.30 × 10⁻⁶. 6 Cells were seeded at a density of 0.5 × 10⁶ cells / mL into 125 mL Erlenmeyer flasks containing 25 mL of CD063 liquid medium. The flasks were incubated in a CO₂ shaker at 5% CO₂ concentration, 37°C, and 130 rpm. Cell counts were performed every three days at a density of 0.5 × 10⁶ cells / mL. 6 Inoculate fresh CD063 liquid medium at a density of cells / mL and amplify by successive passages until the total number of cells reaches the requirement for inoculation.

[0065] 2.3.8L cell tank fermentation

[0066] Table 5: Fermentation process in a 3.8L cell tank

[0067]

[0068]

[0069] The technical solution of this invention selects to reduce the temperature and pH value to reduce the antibody acidic peak:

[0070] Table 6: Cell culture processes to reduce acidity peaks

[0071] process 1 2 3 4 (Control) temperature D7 temperature drops to 33℃ D 7 temperature drops to 32℃ D 7 temperature drops to 32℃ D 7 temperature drops to 34℃ pH 7.0±0.2 7.0±0.2 6.9±0.05 6.9±0.05

[0072] Table 7: Antibody charge variants during cell culture at different temperatures and pH levels

[0073]

[0074] From Table 7 and Figure 1 It can be seen that, compared with the control group, lower culture temperatures (33℃, 32℃) can reduce the acid peak and increase the main peak and alkaline peak. At the same culture temperature (32℃), lower pH control (6.9±0.05) can also obtain a lower acid peak, which is consistent with the theory. Therefore, based on the above data, we chose to lower the temperature to 32℃ on day 7 and control the pH at 6.9±0.1 (considering that controlling the pH at 6.9±0.05 would lead to excessively high carbon dioxide partial pressure in pilot-scale production, we slightly relaxed it to 6.9±0.1).

[0075] The technical solution of this invention selects to add 2 g / L histidine to the basal culture medium to enhance the alkaline peak, add 50 g / L mannose to the feed to enhance MAN5, add 2% glycoform regulator to the basal culture medium and add 0.5% glycoform regulator on day 11 to reduce G0F, and use the combination without mannose as a control to verify the effect of mannose. The specific scheme is shown in the table below.

[0076] Table 8: Processes for improving antibody basic peak and optimizing glycoform

[0077] process Histidine Mannose Glycotype regulators 1 2g / L / D11，0.5％ 2 2g / L 50g / L D11，0.5％

[0078] Table 9: Effects of histidine, mannose, and glycoform modifiers on antibody charge heterogeneity and glycosylation

[0079]

[0080] As shown in Table 9, Figure 2A and Figure 2B As shown, histidine significantly enhances the alkaline peak, while the acid peaks are slightly lower than the original drug, which is beneficial for the subsequent purification process that causes the acid peak to rise and approach the original drug level. This experiment also shows that adding 50 g / L of mannose to the feed significantly increases MAN5 compared to the control (without mannose). Furthermore, different fermentation times also indicate that MAN5 levels increase significantly with prolonged fermentation, especially after culturing for more than 14 days, further confirming that extending the fermentation time to 15-16 days also benefits MAN5 enhancement.

[0081] Example 2: Cell culture in a 150L cell culture vessel

[0082] The culture protocol for the 150L cell culture vessel is shown in the table below. The shake flask seed culture is amplified in three stages: 30ml-120ml-600ml, and the wave seed culture is amplified in two stages: 3.5-8.5L. After these two stages, the seed culture is inoculated into the 150L cell culture vessel for cell fermentation.

[0083] Table 10: Cell Culture Process in a 150L Cell Tank

[0084]

[0085]

[0086] 1. Cell resuscitation and expansion

[0087] Remove the frozen cells from the liquid nitrogen tank and thaw them in a water bath at 37°C. Transfer the thawed cryopreservation solution to a centrifuge tube containing 5 mL of basal culture medium, mix thoroughly, and centrifuge at 1000 rpm for 5 min. Discard the supernatant. Resuspend the cell pellet in 5 mL of basal culture medium and centrifuge at 0.30 × 10⁻⁶. 6 Cells were seeded at a density of 0.5 × 10⁶ cells / mL into 125 mL Erlenmeyer flasks containing 25 mL of CD063 liquid medium. The flasks were incubated in a CO₂ shaker at 5% CO₂ concentration, 37°C, and 130 rpm. Cell counts were performed every three days at a density of 0.5 × 10⁶ cells / mL. 6 Inoculate fresh CD063 liquid medium at a density of cells / mL and amplify by successive passages until the total number of cells reaches the requirement for inoculation.

[0088] 2. Wave Seed Culture

[0089] Count the cells from the expanded shake flasks, ensuring a cell density of 4–8 × 10⁻⁶. 6 If the cell / ml ratio meets the requirements, inoculate the seed culture into a WAVE reaction bag containing 3L of CD063 liquid medium (inoculation density requirement: (0.8±0.2)×10⁻⁶ cells / ml). 6 (cells / ml). The WAVE reactor culture conditions were set as follows: 37℃, aeration rate of 0.2 L / min, rotation speed of 15 rpm, angle of 8°, and culture for 2–3 days. The pH was controlled between 6.7 and 7.2 using CO2. The first-stage WAVE cell seed cells were counted, requiring a cell density > 3.0 × 10⁻⁶ cells / ml. 6 If the cell / ml ratio meets the requirements, pump 5L of OPM-CHO CD063 DPM into the reaction bag to ensure an initial seeding density of 1–2 × 10⁻⁶ cells / ml for the second stage. 6 cells / ml. The WAVE reactor culture conditions were set as follows: 37℃, aeration rate of 0.2 L / min, rotation speed of 20 rpm, angle of 8°, dissolved oxygen controlled at 40% using O2, and pH controlled between 6.7 and 7.2 using CO2. Cultured for 2–3 days until the viable cell density reached 5.0 × 10⁶ cells / ml. 6 cells / ml.

[0090] 3.150L cell culture vessel

[0091] As shown in Table 11, Figure 3A and Figure 3B As shown, the charge variants and glycoforms are quite similar to the original drug. Therefore, it can be basically determined that the charge variants are regulated by adding 2 g / L histidine to the basal culture medium, the galactose ratio is regulated by adding 2% glycoform regulator to the basal culture medium and 0.5% glycoform regulator to D11, and the MAN5 is regulated by adding 50 g / L mannose to XF01 DPM. So far, the antibodies produced by cell culture using this combination are most similar to the original drug in glycoform and best meet the process requirements in terms of charge isomers.

[0092] Table 11: Application of Cell Culture Process in 150L Cell Tanks

[0093]

Claims

1. A method of producing an anti-CGRPR antibody, characterized by, Cells expressing the anti-CGRPR antibody are cultured in a fermentation medium containing histidine and a galactose-containing glycoform regulator, and a fed-batch medium containing mannose is added during fermentation; wherein: (1) The anti-CGRPR antibody is errinumab; (2) The concentration of histidine is 2 g / L; (3) The concentration of the mannose is 50 g / L; (4) The concentration of the glycoform regulator is 2%; and the glycoform regulator further includes manganese ions and / or uridine; where % is a volume percentage; (5) If the total glucose level is less than 3 g / L, supplement it to 4-6 g / L; (6) The fermentation temperature is 37°C from day 1 to day 6, and then drops to 32-33°C from day 7 onwards; (7) The pH of the fermentation is 7.0 ± 0.2; (8) The fermentation time is 15 to 16 days.

2. The method according to claim 1, wherein the cell is a CHO cell.

3. The method of claim 2, wherein, The CHO cells were selected from CHO-K1, CHO DG44 and CHO-S cells.

4. The method according to claim 3, characterized in that, The CHO cells are CHO-K1 cells.

5. The method of claim 1, wherein, The fermentation medium is selected from one or more of OPM-CHO CD063 DPM, OPM CD07 DPM, Dynamis, actipro, and CHO MaxD.

6. The method of claim 1, wherein, The feeding is performed every other day starting from day 3; and / or, the feeding medium is selected from XF01 DPM, CDFS06, Gibco. ® EfficientFeed TM One or more of C+, HyClone™ Cell Boost™ 7a, HyClone™ Cell Boost™ 7b, MAX FA, and MAX FB; and / or, supplementing with 0.5% of the glycoform regulator on day 11; The feed culture medium contains 50 g / L mannose.

7. The method of claim 6, wherein, The feeding medium is XF01 DPM or CDFS06.

8. The method according to claim 7, characterized in that, The amount of material replenished by the XF01 DPM each time is 4%, and the amount of material replenished by the CDFS06 each time is 0.4%, where % is a volume percentage.

9. The method of claim 1, wherein, During fermentation, the dissolved oxygen (DO) content is 40%, and the air flow rate is 1.2 L / h.

10. The method according to any one of claims 1 to 9, characterized in that, The method includes one or more of the following conditions: (i) The carbon dioxide concentration was 5% during seed amplification in shake flasks; (ii) The shaking speed is 130 rpm; (iii) The temperature is 37℃; (iv) Inoculation density is 0.5~1.0×10 6 cells / ml.

11. The method of claim 10, wherein, The method also includes WAVE seed amplification, which includes one or more of the following conditions: 1) The pH level for WAVE seed amplification is 6.7-7.2; 2) The temperature is 37℃; 3) The rotation speed for the first stage of seed amplification is 15 rpm, and the rotation speed for the second stage is 20 rpm; 4) The inoculation density for the first stage of seed amplification is 0.5~1.0×10⁻⁶. 6 The second-stage seeding density was 1.0~2.0×10⁶ cells / ml. 6 cells / ml.

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