A method of producing a PD-1 antibody

Abstract

This invention provides a method for producing PD-1 antibodies with high protein expression levels, high antibody activity, and / or high galactosyl levels. This method involves culturing cells in a culture medium until the target protein is obtained. The culture temperature is 37°C, which is then lowered to 33-34°C on the 5th day of culture. The culture medium also contains additives, namely galactose and manganese chloride. The concentrations of galactose and manganese chloride added are 0.05-6 mM and 0.05-3 μM, respectively. The culture medium consists of a basal medium and a feed medium. The basal medium is Star CHO, and the feed medium is Star CHO Feed and CDFS36. The basal medium also contains 1 g / L poloxamer. The Star CHO Feed feed is added at 4% / 5% / 6% / 6% / 5% / 4% of the initial culture volume each time, and the CDFS36 feed is added at 0.4% / 0.5% / 0.6% / 0.6% / 0.5% / 0.4% of the initial culture volume each time. The additives galactose and manganese chloride, as well as the Star CHO feed feed... Feed and CDFS36 were added every 2 days starting from day 3 of culture; the dissolved oxygen in the culture system was 40%, and the pH was 7.0 ± 0.1; the rotation speed of the culture system was 240 rpm.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for producing PD-1 antibodies. Background Technology

[0002] In the field of biopharmaceuticals, antibody drugs are widely used in the treatment of diseases, and therapeutic antibodies are the fastest-growing products in the biopharmaceutical market.

[0003] Antibody (including Fc fusion protein) drugs exert their important role in targeting and eliminating target cells (such as tumor cells) through two main mechanisms (effective functions): ADCC (antibody-dependent, cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). CDC involves specific antibodies binding to corresponding antigens on the cell membrane surface, forming a complex that activates the classical complement pathway. The resulting membrane-attack complex exerts a lytic effect on the target cell.

[0004] The literature (Jason Hodoniczky, “Control of Recombinant Monoclonal Antibody Effector Functions by Fc N-Glycan Remodeling in Vitro”, Biotechnol. Prog. 2005, 21, 1644-1652) reports that cleaving the galactose residues of monoclonal antibodies with galactosidase reduces the complement-dependent cytotoxicity (CDC) activity of the antibody by 25%, while highly galactosylated monoclonal antibodies can increase CDC activity by 50%. This demonstrates that galactosylation is extremely important for the CDC activity of monoclonal antibodies. PD-1 antibodies are anti-tumor antibodies, and enhancing the CDC effect is particularly important for killing tumor cells and improving anti-tumor efficacy.

[0005] In addition, protein expression levels and antibody activity are also important monitoring indicators in the antibody production process. Protein expression refers to the process of converting exogenous genes into proteins. In antibody preparation, protein expression is one of the crucial steps. Through protein expression, large quantities of specific proteins can be obtained, thereby producing high-quality antibodies. Activity assays determine the effective components and content of the drug, as well as its potency, and are important quality control indicators to ensure the effectiveness of antibody drugs.

[0006] There is no existing technology that discloses a method for producing PD-1 antibodies that can simultaneously achieve high levels of protein expression, antibody activity, and galactosyl content. Summary of the Invention

[0007] The purpose of this invention is to provide a method for producing PD-1 antibodies with good protein expression levels, high antibody activity, and / or high galactosyl levels.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for producing PD-1 antibody, characterized by comprising the following steps: culturing cells in a culture medium until the target protein is obtained; the culture temperature of the culture system is 37°C, and the temperature is lowered to 33-34°C on the 5th day of culture.

[0010] The culture medium of the present invention also contains additives, namely galactose and manganese chloride.

[0011] The concentrations of galactose and manganese chloride added in this invention are 0.05-6 mM and 0.05-3 μM, respectively; preferably 0.2-0.8 mM and 0.1-0.5 μM; more preferably 0.5-0.8 mM and 0.3-0.5 μM.

[0012] The culture media described in this invention are a basal culture medium and a supplemental culture medium, wherein the basal culture medium is StarCHO and the supplemental culture medium is Star CHO Feed and CDFS36.

[0013] The additives galactose and manganese chloride, as well as the supplemental culture medium Star CHO Feed and CDFS36, are added every two days starting from day 3 of culture.

[0014] The feeding medium described in this invention is Star CHO Feed, and the amount added each time is 4% / 5% / 6% / 6% / 5% / 4% of the initial culture volume.

[0015] The feeding medium described in this invention is CDFS36, and the amount added each time is 0.4% / 0.5% / 0.6% / 0.6% / 0.5% / 0.4% of the initial culture volume.

[0016] The basic culture medium of the present invention also contains 1 g / L of surfactant, preferably poloxamer.

[0017] The dissolved oxygen content of the culture system described in this invention is 40%; the pH value of the culture system is 7.0 ± 0.1.

[0018] The rotation speed of the culture system described in this invention is 180-280 rpm; preferably, the rotation speed of the culture system is 240 rpm.

[0019] This invention provides a method for producing PD-1 antibodies, comprising the following steps: culturing cells in a culture medium until the target protein is obtained; the culture temperature of the culture system is 37°C, and the temperature is lowered to 33-34°C on the 5th day of culture; the culture medium consists of a basal medium and a feed medium, wherein the basal medium is Star CHO, the feed medium consists of Star CHO Feed and CDFS36, the basal medium also contains 1 g / L poloxamer, the feed medium is Star CHO Feed added at 4% / 5% / 6% / 6% / 5% / 4% of the initial culture volume each time, and the feed medium is CDFS36 added at 0.4% / 0.5% / 0.6% / 0.6% / 0.5% / 0.4% of the initial culture volume each time; the culture medium also contains additives, wherein the additives are galactose and manganese chloride added at concentrations of 0.5-0.8 mM and 0.3-0.5 μM, respectively; the additives galactose and manganese chloride, and the feed medium Star CHO... Feed and CDFS36 were added every 2 days starting from day 3 of culture; the dissolved oxygen in the culture system was 40%, and the pH was 7.0 ± 0.1; the rotation speed of the culture system was 240 rpm.

[0020] The PD-1 antibody described in this invention is selected from one or more of pembrolizumab, nivolumab, sintilimab, toripalimab, and camrelizumab; preferably pembrolizumab.

[0021] The method for producing PD-1 antibodies provided by the present invention can achieve a protein expression level of 3.9 g / L or higher, a galactose level of 20% or higher, and a relative binding activity of 80% or higher; furthermore, the method for producing PD-1 antibodies provided by the present invention can achieve a protein expression level of 4 g / L or higher, a galactose level of 20% or higher, and a relative binding activity of 95% or higher; even further, the method for producing PD-1 antibodies provided by the present invention can achieve a protein expression level of 4 g / L or higher, a galactose level of 25% or higher, and a relative binding activity of 95% or higher. Detailed Implementation

[0022] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application. Those skilled in the art can make various changes and modifications to the invention based on the disclosed content, and such changes should also fall within the scope of protection claimed in this application.

[0023] Unless otherwise specified, all chemical reagents used in the embodiments of this invention are obtained through conventional commercial means or by preparing them according to existing technology, and do not limit the invention.

[0024] Main experimental equipment

[0025]

[0026] Main experimental materials

[0027]

[0028] Star CHO DPM: A basal medium with a defined composition, free of hydrolysates, proteins, and any animal-derived chemicals. CDFS36: A fully chemically defined, ultra-concentrated supplemental feed, free of hydrolysates, growth factors, and any animal-derived components.

[0029] Star CHO Feed: A chemically defined feed medium that contains no hydrolysates or any animal-derived ingredients.

[0030] CHO cells expressing monoclonal antibodies against PD-1 can be obtained through conventional methods, such as the following: Host cells CHO-K1 are used to construct expression vectors encoding the light and heavy chains of the PD-1 antibody, which are then transfected into CHO-K1 cells. The transfected cells are cultured, and clones with high expression levels and good antibody quality are selected as seed cells for upstream cell culture processes.

[0031] Antibody purity can be obtained through conventional methods, such as the following: SEC-HPLC is used for detection. The sample is diluted and injected directly. After elution, an elution chromatogram is obtained, and the antibody purity is calculated using the following formula:

[0032] The content of monomers was calculated using the area normalization method.

[0033] Monomer = (Single peak area / Total protein peak area) × 100%

[0034] Polymer = (Polymer peak area / Total protein peak area) × 100%

[0035] Low molecular weight substances = peak area of ​​low molecular weight components / total peak area of ​​proteins × 100%.

[0036] Antibody galactosylation can be obtained through conventional methods, such as enzymatic digestion with PNGase enzyme according to the instructions of the reagent kit, followed by detection of galactose glycosylation level using HILIC-UPLC (manufacturer: Agilent Technologies, USA, catalog number: Agilent 1290).

[0037] Antibody binding activity can be obtained through conventional methods, such as the following: using a competitive ELISA method, the absorbance of the sample is measured at 450 nm / 630 nm using a microplate reader. The reference / test sample concentration is plotted on the x-axis, and the OD value is plotted on the y-axis. The EC50 of the reference / test sample is then calculated using analytical software. 50 EC of reference material 50 Value divided by EC of the test sample 50 The relative binding activity of the test sample is expressed as a percentage of the value.

[0038] Cell resuscitation:

[0039] After removing the cells from the liquid nitrogen container, quickly transfer them to a 37°C water bath for thawing. After thawing, transfer the supernatant to 9 mL of fresh, preheated culture medium and centrifuge at 200 x g for 5 min. Resuspend the cells in a 125 mL shake flask and adjust the cell density to (0.5 ± 0.1) × 10⁻⁶ cells / mL. 6 The cells / mL were cultured on a shaker under the following conditions: 170 rpm (25 mm), 37.0 °C, 5.0% CO2, and 80% humidity.

[0040] Cell passage:

[0041] The cells were passaged and amplified every 3 ± 1 days, with a passage density of (0.5 ± 0.1) × 10⁻⁶. 6 The cells / mL were cultured on a shaker under the following conditions: 170 rpm (25 mm), 37.0 °C, 5.0% CO2, and 80% humidity.

[0042] Cell culture fermentation:

[0043] Once the cells have been revived and passaged to a sufficient quantity, they are inoculated into shake flasks or reactors for culture.

[0044] Example 1

[0045] In this embodiment, the bioreactor culture volume was 2L, and the inoculation density was (0.5±0.1)×10⁻⁶. 6Cells / ml, initial culture volume 1.5L, air circulation 0.01 vvm. For groups 1-5, aeration exceeded 0.2 vvm on day 8, and the rotation speed was increased to 220 rpm. On day 9, aeration did not decrease significantly, and the rotation speed was increased to 280 rpm. On day 10, cell viability decreased significantly, and the rotation speed was adjusted to 240 rpm. When glucose concentration fell below 4 g / L, it was supplemented to 6 g / L. Star CHO was used as the basal medium, and Star CHO Feed and CDFS36 were used as feed media. Feeding: Day 0 was considered the day of inoculation. Each addition of feed medium was a percentage of the initial culture volume, as shown in Table 1. Galactose and manganese chloride were added with the feed medium at the same time and frequency as the feed medium, and the concentrations are shown in Table 1. Cells were harvested on day 15, and the expression level of the harvested cells was tested. After further affinity chromatography purification, the purity of the target protein, galactose, and binding activity were detected, and the results are shown in Table 2.

[0046] Table 1

[0047]

[0048] Table 2

[0049] Grouping Protein expression level (g / L) SEC-HPLC% Galactose % Relative binding activity % Group 1 4.31 99.6 30.2 90.1 Group 2 4.23 99.3 33.1 86.2 Group 3 4.25 99.2 33.6 84.6 Group 4 4.21 99.5 35.7 86.2 Group 5 4.14 99.5 37.9 84.3 Group 6 2.03 99.6 38.6 79.5 Group 7 3.31 99.5 31.2 76.3

[0050] Example 2

[0051] In this embodiment, the bioreactor culture volume was 2L, and the inoculation density was (0.5±0.1)×10⁻⁶. 6 Cells / ml, initial culture volume 1.5L, air circulated at 0.01 vvm; Star CHO basal medium, Star CHO Feed and CDFS36 as feed media. Feeding: Day 0 was considered the day of inoculation. Feed media were added as a percentage of the initial culture volume each time, as shown in Table 3. When the glucose concentration was below 4 g / L, it was added to 6 g / L. Galactose and manganese chloride were added with the feed media at the same time and frequency as the feed media, and the concentrations are shown in Table 3. Cells were harvested on day 15, and the expression level of the harvested cells was tested. After further affinity chromatography purification, the purity of the target protein, galactose, and binding activity were detected. The results are shown in Table 4.

[0052] Table 3

[0053]

[0054] Table 4

[0055] Grouping Protein expression level (g / L) SEC-HPLC% Galactose % Relative binding activity % Group 1 4.43 99.5 21.8 98.0 Group 2 4.34 99.6 29.0 97.7 Group 3 4.32 99.7 27.9 96.2 Group 4 3.94 99.6 29.3 93.7 Group 5 4.24 99.6 26.5 86.7 Group 6 4.38 99.6 27.8 97.8 Group 7 4.35 99.5 13.6 98.0

[0056] Example 3

[0057] In this embodiment, the bioreactor culture volume was 2L, and the inoculation density was (0.5±0.1)×10⁻⁶. 6 The reactor was set with cells / ml and a volume of 1.5L. Initial culture conditions were: temperature 37.0℃, cooled to 33.0℃ on Day 5, rotation speed 240 rpm, constant air circulation (0.01 vvm), pH 7.00 (dead zone 0.10), DO 40.0%. When glucose concentration fell below 4 g / L, it was replenished to 6 g / L. Star CHO was used as the basal medium, with 1 g / L P188 added. Star CHO Feed and CDFS36 were used as feed media. Feeding was performed on the day of inoculation, considered day 0. Each addition of feed medium was a percentage of the initial culture volume. Feeding times were: Days 3 / 5 / 7 / 9 / 11 / 13, Star CHO... Feed: 4% / 5% / 6% / 6% / 5% / 4%, CDFS36: 0.4% / 0.5% / 0.6% / 0.6% / 0.5% / 0.4%; Galactose and manganese chloride were added along with the feed medium at the same time and frequency, with the following concentrations each time: 0.5 mM galactose and 0.5 μM manganese chloride. Cells were harvested on day 15, and the expression level of the harvested cells was tested. After further affinity chromatography purification, the purity of the target protein, galactose, and binding activity were detected. The results are shown in Table 5.

[0058] Table 5

[0059] Protein expression level (g / L) SEC-HPLC% Galactose % Relative binding activity % 4.25 99.6 25.8 98

[0060] Example 4

[0061] In this embodiment, the bioreactor culture volume was 2L, and the inoculation density was (0.5±0.1)×10⁻⁶. 6The reactor was set with cells / ml and a volume of 1.5L. Initial culture conditions were: temperature 37.0℃, cooled to 34.0℃ on Day 5, rotation speed 240 rpm, constant air circulation (0.01 vvm), pH 7.00 (dead zone 0.10), DO 40.0%. When glucose concentration fell below 4 g / L, it was replenished to 6 g / L. Star CHO was used as the basal medium, with 1 g / L P188 added. Star CHO Feed and CDFS36 were used as feed media. Feeding was performed on the day of inoculation, considered day 0. Each addition of feed medium was a percentage of the initial culture volume. Feeding times were: Days 3 / 5 / 7 / 9 / 11 / 13, Star CHO... Feed: 4% / 5% / 6% / 6% / 5% / 4%, CDFS36: 0.4% / 0.5% / 0.6% / 0.6% / 0.5% / 0.4%; galactose and manganese chloride were added along with the feed medium at the same time and frequency, with the following concentrations each time: galactose 0.5 mM, manganese chloride 0.5 μM. Cells were harvested on day 15, and the expression level of the harvested cells was tested. After further affinity chromatography purification, the purity of the target protein, galactose, and binding activity were detected. The results are shown in Table 6.

[0062] Table 6

[0063] Protein expression level (g / L) SEC-HPLC% Galactose % Relative binding activity % 4.56 99.6 26.8 101.1

Claims

1. A method for producing PD-1 antibodies, characterized in that, The process includes the following steps: culturing cells in a culture medium until the target protein is obtained; the culture temperature of the culture system is 37°C, and the temperature is lowered to 33-34°C on the 5th day of culture.

2. The method according to claim 1, characterized in that, The culture medium also contains additives, namely galactose and manganese chloride.

3. The method according to claim 2, characterized in that, The concentrations of galactose and manganese chloride added are 0.05-6 mM and 0.05-3 μM, respectively; preferably 0.2-0.8 mM and 0.1-0.5 μM; more preferably 0.5-0.8 mM and 0.3-0.5 μM.

4. The method according to claim 1, characterized in that, The culture media are a basal culture medium and a supplemental culture medium, wherein the basal culture medium is Star CHO and the supplemental culture medium is Star CHO Feed and CDFS36.

5. The method according to any one of claims 2-4, characterized in that, The additives galactose and manganese chloride, as well as the feed medium Star CHO Feed and CDFS36, were added every two days starting from day 3 of culture.

6. The method according to claim 4, characterized in that, The feed medium is Star CHO Feed, and the amount added each time is 4% / 5% / 6% / 6% / 5% / 4% of the initial culture volume.

7. The method according to claim 4, characterized in that, The feeding medium is CDFS36, and the amount added each time is 0.4% / 0.5% / 0.6% / 0.6% / 0.5% / 0.4% of the initial culture volume.

8. The method according to claim 4, characterized in that, The basal culture medium also contains 1 g / L of surfactant; preferably poloxamer.

9. The method according to claim 1, characterized in that, The dissolved oxygen content of the culture system is 40%; the pH of the culture system is 7.0 ± 0.

1.

10. The method according to claim 1, characterized in that, The rotation speed of the culture system is 180-280 rpm; preferably, the rotation speed of the culture system is 240 rpm.

11. The method according to any one of claims 1-10, characterized in that, A method for producing PD-1 antibodies includes the following steps: culturing cells in a culture medium until the target protein is obtained; the culture temperature is 37°C, and the temperature is lowered to 33-34°C on the 5th day of culture; the culture medium consists of a basal medium and a feed medium, wherein the basal medium is Star CHO, the feed medium is Star CHO Feed and CDFS36, the basal medium is supplemented with 1 g / L poloxamer, the feed medium is Star CHO Feed added at 4% / 5% / 6% / 6% / 5% / 4% of the initial culture volume each time, and the feed medium is CDFS36 added at 0.4% / 0.5% / 0.6% / 0.6% / 0.5% / 0.4% of the initial culture volume each time; the culture medium also contains additives, namely galactose and manganese chloride at concentrations of 0.5-0.8 mM and 0.3-0.5 μM, respectively; the additives galactose and manganese chloride, and the feed medium Star CHO Feed and CDFS36 are also present in the culture medium. CHOFeed and CDFS36 were added every 2 days starting from day 3 of culture; the dissolved oxygen in the culture system was 40%, and the pH was 7±0.1; the rotation speed of the culture system was 240 rpm.

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