CN107460221B

CN107460221B - Cell culture method for reducing protein polymer in anti-PD-L1 antibody

Info

Publication number: CN107460221B
Application number: CN201610392283.8A
Authority: CN
Inventors: 张哲文; 吕海丽; 程艳菊; 赵伟; 张喜全; 王善春
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority date: 2016-06-02
Filing date: 2016-06-02
Publication date: 2021-01-15
Anticipated expiration: 2036-06-02
Also published as: CN107460221A

Abstract

The invention belongs to the field of cell culture, and provides a cell culture method for reducing protein polymers in an anti-PD-L1 antibody, which comprises the steps of feeding lysine, arginine and calcium chloride under the cell culture condition that the anti-PD-L1 antibody is allowed to generate, so that the content of the antibody polymers is obviously reduced by 28.5%, the pressure for subsequent purification of the anti-PD-L1 antibody is reduced, and meanwhile, the method is simple and convenient to operate and easy to implement, and the quality of the antibody is effectively improved.

Description

Cell culture method for reducing protein polymer in anti-PD-L1 antibody

Technical Field

The present invention relates to the field of cell culture. More specifically, the invention relates to a method for reducing protein polymers in expressed anti-PD-L1 antibodies by optimizing cell culture conditions.

Background

PD-L1(Programmed death-ligand 1), also known as CD247 and B7-H1, is a ligand for Programmed death molecule 1 (PD-1). PD-L1 is highly expressed on the surface of various tumor cells, and the malignancy degree and poor prognosis of tumors are closely related to the expression level of PD-L1. In a tumor microenvironment, PD-L1 on the surface of a cancer cell inhibits the activation and proliferation of T cells, promotes effector T cells to enter a failure or non-response state, induces the apoptosis of the T cells, stimulates the differentiation of helper T cells into regulatory T cells through the combination with PD-1 or CD80 on the surface of the T cells, thereby preventing the killing effect of the T cells on the tumor cells. The PD-L1 antibody can block the interaction of PD-L1 with PD-1 and CD80, so that related negative regulation signals can not be started and conducted, the activity of effector T cells in a tumor microenvironment is prevented from being inhibited, and the T cells can play the functions of killing and inhibiting the tumor cells. The PD-L1 antibody can directly act on tumor tissues, so that the PD-L1 antibody has higher specificity and safety. At present, the main PD-L1 monoclonal antibody drug products in the world comprise Atezolizumab of Roche and Durvalumab of Asricon, which are in the late stage of clinical research, and no PD-L1 monoclonal antibody drug is produced in China. WO2016022630 discloses a novel anti-PD-L1 antibody, which has high affinity for PD-L1, can obviously inhibit the interaction between PD-L1 and PD-1 on the cell surface, and obviously promotes T cells to secrete IL-2 and IFN-gamma.

The large-scale culture of host cells which are genetically modified has been widely used for producing monoclonal antibodies, immune regulatory factors and various gene recombinant protein drugs. However, the phenomenon of protein aggregation during cell culture has been widely reported, and in fact, the level of aggregation for some monoclonal antibodies expressed in mammalian cell culture is as high as 30% (Kramarczyk JF, et al.2008.high-throughput screening of chromatographic separations: II. hydrophic interaction. Biotechnol Bioeng 100(4): 707-720). Protein aggregation may form during cell culture (i) when intracellular proteins are expressed and (ii) when the proteins are secreted into the cell culture medium following expression. During expression, massive Protein aggregation with large peptide net negative charge, which accumulates inside the cell, is caused by interactions of unfolded Protein molecules or by inefficient recognition of nascent polypeptide chains by chaperones responsible for correct folding (Zhang YB, et al 2004.Protein aggregation reduction overexpression by peptide extensions with large peptide net negative charge. Protein expression 36(2): 207; 216). After secretion into the culture medium after expression of the protein, the secreted protein may be exposed to conditions that are unfavorable for protein stability, which may also be responsible for the production of protein aggregation.

The presence of protein polymers greatly increases the immunogenicity of the antibody, thereby affecting the safety and effectiveness of the antibody. The polymer is easy to cause immune reaction in vivo, directly causes a series of clinical adverse reactions, and comprises the generation of neutralizing antibodies (ADA) inhibiting the therapeutic effect of the drug; even cross-reacting with endogenous proteins, causing severe allergic reactions (Maria VR et al 2011.Aggregates in Monoclonal Antibody Manufacturing processes Biotechnology and Bioengineering,108(7): 1494-1508). Although some types of biological product aggregates can function properly, maintaining product quality consistency remains important because product consistency is a prerequisite for regulatory approval.

There are many factors that influence the amount of Antibody protein aggregation during cell culture, including the selection of optimized cell lines and optimized cell culture conditions, such as medium composition, feeding methods, temperature and pH (Maria VR et al 2011.Aggregates in Monoclonal Antibody Manufacturing processes Biotechnology and Bioengineering,108(7): 1494. sup. 1508). Adjusting pH and temperature is an effective means, but may affect cell growth and the amount and quality of antibody expression. There are currently less studies on the reduction of antibody polymers by optimization of cell culture conditions.

Disclosure of Invention

In order to solve the problem that the content of the protein polymer of the antibody obtained by the original cell culture method is high, the invention provides a cell culture method for reducing the content of the protein polymer in the anti-PD-L1 antibody, which comprises the step of feeding one or more amino acids to a culture medium in equal amount once or more times in a culture period under the cell culture condition that the anti-PD-L1 antibody is allowed to generate.

Wherein the additional amino acids are selected from arginine, lysine, or combinations thereof;

preferably, arginine and lysine are supplemented at the same time;

in a specific embodiment, the total amount of arginine supplemented is 2-8g/L medium and the total amount of lysine is 2-10g/L medium;

preferably, the total amount of the supplemented arginine is 4-6g/L of the culture medium, and the total amount of the lysine is 3-8g/L of the culture medium;

preferably, the total amount of arginine supplemented is 4g/L of medium and the total amount of lysine supplemented is 6g/L of medium.

Wherein, the amino acid is supplemented for 2 to 4 times in the culture period;

in a specific embodiment, the amino acid is supplemented 2 times on any of

days

4, 6, 8 and 10 of the culture cycle;

in another embodiment, the amino acid is supplemented 3 times on any of

days

4, 6, 8, and 10 of the culture cycle;

in a specific embodiment, 4g/L arginine and 6g/L lysine were supplemented at equal amounts on

days

4, 6, 8 and 10 of the culture cycle.

In one embodiment, the amino acids are supplemented with calcium chloride in a total amount of 0.055-0.33g/L of medium.

Preferably, the anti-PD-L1 antibody provided by the present invention comprises the following amino acid sequence: a heavy chain CDR1 region having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology to the amino acid sequence set forth in SEQ ID NO 1 or SEQ ID NO 4; a heavy chain CDR2 region having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology to the amino acid sequence set forth in SEQ ID NO 2 or SEQ ID NO 5; a heavy chain CDR3 region having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology to the amino acid sequence set forth in SEQ ID NO 3 or SEQ ID NO 6; a light chain CDR1 region having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology to the amino acid sequence set forth in SEQ ID NO 7 or SEQ ID NO 10; a light chain CDR2 region having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology to the amino acid sequence set forth in SEQ ID NO 8 or SEQ ID NO 11; a light chain CDR3 region having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology to the amino acid sequence set forth in SEQ ID NO 9 or SEQ ID NO 12.

In one embodiment, the anti-PD-L1 antibody provided by the present invention comprises the amino acid sequence: a heavy chain CDR1 region selected from SEQ ID NO. 1 or SEQ ID NO. 4; a heavy chain CDR2 region selected from SEQ ID NO 2 or SEQ ID NO 5; a heavy chain CDR3 region selected from SEQ ID NO 3 or SEQ ID NO 6; a light chain CDR1 region selected from SEQ ID NO. 7 or SEQ ID NO. 10; a light chain CDR2 region selected from SEQ ID NO 8 or SEQ ID NO 11; a light chain CDR3 region selected from SEQ ID NO 9 or SEQ ID NO 12.

Preferably, the anti-PD-L1 antibody provided by the present invention comprises the following amino acid sequence: a heavy chain variable region having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) homology to the amino acid sequence set forth in SEQ ID NO 13 or SEQ ID NO 14; a light chain variable region that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) homologous to the amino acid sequence set forth in SEQ ID NO 15 or SEQ ID NO 16.

In one embodiment, the anti-PD-L1 antibody provided by the invention comprises the following amino acid sequence, such as the heavy chain variable region shown in SEQ ID NO: 13; the light chain variable region shown as SEQ ID NO. 15.

In another embodiment, the invention provides an anti-PD-L1 antibody comprising an amino acid sequence as shown in SEQ ID NO. 14 in the heavy chain variable region; the variable region of the light chain as shown in SEQ ID NO 16.

In one embodiment, the anti-PD-L1 antibodies provided herein bind to PD-L1 with binding constants in the range of about 0.001nM to 100nM, about 0.002nM to 50nM, about 0.005nM to 5nM, and about 0.01nM to 1 nM. In another specific embodiment, the anti-PD-L1 antibody binds PD-L1 with a binding constant of less than about 10nM, less than about 5nM, less than about 1nM, less than about 0.9nM, less than about 0.8nM, less than about 0.5nM, less than about 0.4nM, less than about 0.3nM, less than about 0.2nM, less than about 0.1nM, less than about 0.09nM, less than about 0.08nM, less than about 0.07nM, less than about 0.06nM, less than about 0.05nM, less than about 0.04nM, less than about 0.03nM, less than about 0.02nM, less than about 0.01 nM.

The present invention further provides an anti-PD-L1 antibody obtained by any one of the cell culture methods described above.

Noun term

anti-PD-L1 antibody: the antibody of the present invention is a binding protein having at least one antigen binding domain, and may be a whole antibody or a fragment thereof, including a monoclonal antibody or a fragment thereof, an antibody variable region or a fragment thereof, and an immune copolymer. Antibody fragments include Fab fragments, Fab 'fragments, F (ab)' fragments, Fv fragments, individual CDR regions, single chain Fv fragments, and other antibody fragments known to those skilled in the art. The anti-PD-L1 antibody of the invention refers to an antibody or antigen binding fragment capable of binding to PD-L1, and the antibody or fragment can bind to PD-L1 including but not limited to the antibodies selected from human, murine and cynomolgus monkey. The anti-PD-L1 antibody referred to herein may be of an isotype selected from IgG1, IgG2, IgG3 or IgG4, and may be selected from murine, chimeric and humanized antibodies. In one embodiment of the invention, the anti-PD-L1 antibody is a humanized IgG1 isotype antibody.

Protein polymer: the antibody polymer of the present invention refers to a dimer, multimer or fragmented protein aggregate produced by an antibody due to aggregation tendency, and the dimer may exist as a small dimer or fragment at the initial stage of formation and then gradually develop into a larger structure, such as a sub-visible or visible particle.

The invention has the beneficial effects that: according to the cell culture condition, the amino acid (such as lysine and arginine) and calcium chloride are added in a supplementing manner, so that the polymer content of the antibody is obviously reduced by 28.5%, the pressure of subsequent purification of the anti-PD-L1 antibody is reduced, and meanwhile, the method is simple and convenient to operate and easy to implement, and the quality of the antibody is effectively improved.

Drawings

FIG. 1 is a graph of the growth of cells expressing anti-PD-L1 antibody for different feed culture conditions over the culture period.

FIG. 2 comparison of polymer content under different feed culture conditions.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1: cell culture expression of anti-PD-L1 antibody

One cell of the anti-PD-L1 monoclonal antibody working library (self-made, 1ml) was quickly thawed in a water bath at 37 ℃, transferred to a 125ml cell culture flask containing 20ml of seed culture medium Dynamis (Life technologies), and placed at 37 ℃ in an 8% CO atmosphere₂Culturing at 110-130 rpm in carbon dioxide constant temperature incubator (Thermo Co., Ltd.)And (5) nourishing. Observing cell state every day, sampling for cell counting and detecting cell viability (trypan blue method), cell density is about 3.0-4.0 × 10⁶Subculturing cells/ml, the subculturing density is about 0.8 +/-0.2X 10⁶cells/ml, obtaining 100ml seed solution after 3-4 passages, inoculating the seed solution into a 1L glass reactor (Sadolis Biostat B), wherein the inoculation density is 0.8 +/-0.2 multiplied by 10⁶cells/ml, culture volume 500ml, culture temperature 36.5 deg.C, pH 6.90 + -0.20, DO 40%, stirring 200 rpm. Dissolved oxygen from O₂Cascade control, pH by NaHCO₃Or NaOH and CO₂And (5) controlling. The pH set point was changed to 6.80. + -. 0.05 by culture to day 4 and the temperature was reduced to 35 ℃ by culture to day 9. Setting 4 groups of experiments, and supplementing arginine, lysine and CaCl on the 4 th, 6 th, 8 th and 10 th days of culture₂The medicine is supplemented for 4 times, the supplement amount of each time is the same, and the total supplement amount of 4 times is shown in table 1. And (5) culturing to the 14 th day, finishing culturing, and harvesting the feed liquid.

TABLE 1 four sets of experimental designs

Determination of the Polymer: after the culture is finished, the cell harvest liquid is subjected to one-step purification, and then the purity of the antibody is detected by using a size exclusion chromatography (SEC-HPLC), and the aggregation degree of the antibody is reflected. The SEC-HPLC method refers to molecular exclusion chromatography of 0514 in the third part of the pharmacopoeia 2015 edition of China.

The experimental results are as follows:

as a result, arginine, lysine and CaCl were added at different concentrations₂Has small influence on cell density, the highest cell density and cell density at harvest in the culture process are shown in Table 2, the cell growth curve is shown in FIG. 1, arginine, lysine and CaCl with different concentrations are added₂The polymer content can be reduced, the specific protein polymer content is shown in Table 2, the content of the high-concentration amino acid combination group (Arg 2 g; Lys 3g) is reduced by 26.2 percent compared with the content of the protein polymer of a control group, and the high-concentration amino acid combination group plus salt group (Arg 2 g; Lys 3 g; CaCl)₂0.078g) was reduced by 28.5% compared to the control histone polymer content, as detailed in FIG. 2, indicating fractionationThe addition of arginine and lysine in batches unexpectedly reduces the content of protein polymer in the cell harvest solution, and in addition, the addition of calcium chloride can also reduce the content of protein polymer to a certain extent.

TABLE 2 highest cell density, cell density at harvest and protein Polymer content during culture

Claims

1.A cell culture method for reducing the protein polymer content of an anti-PD-L1 antibody, comprising the feeding of 2-4 equal amounts of arginine in a total amount of 2-8g/L medium and lysine in a total amount of 2-10g/L medium to the medium during a culture cycle under cell culture conditions that allow the production of the anti-PD-L1 antibody, said anti-PD-L1 antibody comprising the amino acid sequence: a heavy chain CDR1 region selected from SEQ ID NO. 1 or SEQ ID NO. 4; a heavy chain CDR2 region selected from SEQ ID NO 2 or SEQ ID NO 5; a heavy chain CDR3 region selected from SEQ ID NO 3 or SEQ ID NO 6; a light chain CDR1 region selected from SEQ ID NO. 7 or SEQ ID NO. 10; a light chain CDR2 region selected from SEQ ID NO 8 or SEQ ID NO 11; a light chain CDR3 region selected from SEQ ID NO 9 or SEQ ID NO 12.

2. The cell culture method of claim 1, wherein the total amount of arginine fed to the medium is 4-6g/L and the total amount of lysine is 3-8 g/L.

3. The cell culture method of claim 2, wherein the total amount of arginine fed to the medium is 4g/L and the total amount of lysine fed to the medium is 6 g/L.

4. The method for cell culture according to claim 1, wherein the amino acid is supplemented 2 times on any of days 4, 6, 8 and 10 of the culture cycle.

5. The method for cell culture according to claim 1, wherein the amino acid is supplemented 3 times on any of days 4, 6, 8 and 10 of the culture cycle.

6. The method of claim 1, wherein the medium is supplemented with equal amounts of arginine at 4, 6, 8 and 10 days of the culture cycle and lysine at 6 g/L.

7. The cell culture method of claim 6, further supplemented with calcium chloride in a total amount of 0.055-0.33g/L medium.

8. The cell culture method of any one of claims 1-7, wherein the anti-PD-L1 antibody comprises the amino acid sequence shown as SEQ ID NO 13 for the heavy chain variable region; the light chain variable region shown as SEQ ID NO. 15.

9. The cell culture method of claim 8, wherein the anti-PD-L1 antibody comprises an amino acid sequence as set forth in SEQ ID NO:14 for the heavy chain variable region; the variable region of the light chain as shown in SEQ ID NO 16.