Method for producing antibody populations

Abstract

The present invention relates to a method for producing an antibody population, and more specifically, to a method for effectively producing an antibody population of a desired quality, that is, a high-quality antibody population with excellent biological activity, by precisely adjusting culture conditions such as pH and culture temperature during the culture of recombinant cells that express antibodies. The present invention can effectively produce an antibody population having a target ratio of main active antibodies and heterologous antibodies, and an antibody population having a target glycan structure by adjusting pH, culture temperature, and / or lactic acid supply. In addition, the method of the present invention can produce high-quality antibodies with excellent biological activity that can reach the target therapeutic efficacy when producing therapeutic monoclonal antibodies, and in particular, in the production of biosimilar drugs, it is possible to effectively produce antibodies of the same or very similar quality as the original drug by precisely adjusting the culture conditions.

Description

[Technical Field]

[0001] The present invention relates to a method for producing an antibody population. Specifically, the present invention relates to a method for obtaining an antibody population of a desired quality by precisely controlling culture conditions during the cultivation of recombinant cells that express antibodies. [Background technology]

[0002] According to the Biopharmaceutical Industry Trends Report published by the Korea Biopharmaceutical Association in December 2020, the global biopharmaceutical market grew at an average annual rate of approximately 8.6% from 2012 to 2019, and is expected to grow at an average annual rate of approximately 10.1% in the future, reaching approximately $505 billion in 2026.

[0003] Korea's biopharmaceutical industry is also expected to continue expanding as the population ages and chronic diseases increase, and considering that the biopharmaceutical market grew 16.6% from KRW 2.23 trillion in 2018 to KRW 2.60 trillion in 2019, the scale of growth is expected to further expand. However, despite the increase in the size of the domestic biopharmaceutical market, it has been reported that domestic biopharmaceutical production and exports decreased by 2.8% and 12.8%, respectively, in 2019 compared to 2018. This calls for active research and development to improve the productivity and quality of biopharmaceuticals in response to the growth of the global biopharmaceutical market.

[0004] Antibody therapeutics, which account for 51.1% of global biopharmaceutical sales based on 2019 sales, are produced through a bioprocess that involves modifying mammalian cells, such as Chinese hamster ovary (CHO) cells, and culturing them in a suitable bioreactor. This process is sensitive to even small changes in culture conditions, such as medium, temperature, and pH, affecting not only antibody productivity but also the quality of drugs using the modified cells. Therefore, long-term research has been conducted to adjust various process parameters in culture conditions to maximize the yield of high-quality antibodies. However, even small changes in values ​​can have large effects, and significant differences in productivity and antibody quality (e.g., N-glycan analysis) can occur for each specific antibody under specific conditions, making it difficult to obtain satisfactory results.

[0005] In addition, in the case of biosimilar drugs, it is important to obtain products with the same quality as the original drug as much as possible, and therefore, the ratio of main active antibodies to heterologous antibodies in the obtained antibody population and the glycan structure are also important factors. Therefore, establishing optimized culture conditions taking these factors into consideration remains a technical challenge.

[0006] Patent documents related to research into adjusting the culture conditions of recombinant cells to improve product productivity, quality, etc. include Korean Patent Publication No. KR10-2021-0043618 (publication date: 2021.04.21) and Korean Patent Registration No. KR10-1724405 (registration date: 2017.04.03). However, no methods are known for maximizing antibody productivity and quality, and for appropriately adjusting the ratio of main active antibodies to heterologous antibodies in antibody populations, as well as the glycan structure. Summary of the Invention [Problem to be solved by the invention]

[0007] In order to solve the above-mentioned conventional problems, the main object of the present invention is to provide a method for producing an antibody population of a desired quality, that is, a high-quality antibody population with excellent biological activity, by precisely controlling the culture conditions during the culture of recombinant cells that express antibodies.

[0008] Specifically, an object of the present invention is to provide a method for producing an antibody population having a target ratio of primary and secondary antibodies by adjusting the pH, culture temperature, and / or lactic acid supply during the culture of recombinant cells that express the antibody.

[0009] Another object of the present invention is to provide a method for producing an antibody population having a target glycan structure by adjusting the pH, culture temperature, and / or lactic acid supply during the culture of recombinant cells that express the antibody.

[0010] The object of the present invention is not limited to the above description, but is provided for the purpose of all cases in which the present invention can be utilized to obtain appropriate effects. [Means for solving the problem]

[0011] The present inventors first demonstrated that in order to produce an antibody population of the desired quality, the ratio of the main active antibody and the heterologous antibody in the antibody population can be adjusted, and the glycan structure can be controlled, through precise control of the pH, culture temperature, and lactic acid supply during recombinant cell culture, thereby completing the present invention.

[0012] Specifically, the present invention provides a method for producing a recombinant antibody-expressing cell culture medium, comprising: (a) culturing the recombinant antibody-expressing cell culture medium at a first temperature of 36°C to 38°C and a pH of 7.0 to 7.1 for 4 to 6 days; and (b) a second temperature culture step of culturing the cultured recombinant cells in a medium for 6 to 8 days at a second culture temperature set to be 2 to 4°C lower than the first culture temperature in step (a) and to be 34°C or higher, and at a pH of 7.0 to 7.1.

[0013] The present invention also provides a method for producing an antibody population, wherein step (b) further comprises culturing the cells by adding 80 to 120 μl of 0.1 to 2 M lactic acid per 10 ml of medium every day from days 1 to 3 of culturing at the second culture temperature. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram illustrating the culture conditions according to Example 1 of the present invention. [Figure 2] FIG. 2 shows the results of IgG titer measurement according to Example 2 of the present invention. [Figure 3a] Figures 3a to 3c show the results of charge variant analysis using LapChip® and CE-HPLC equipment for the ratio of the main active antibody according to Example 3-1 of the present invention. Specifically, Figure 3a is a graph showing the results of charge variant analysis using LapChip® equipment, Figure 3b is a graph showing the results of charge variant analysis using CE-HPLC equipment, and Figure 3c is a graph comprehensively showing the analysis results for the ratio of the main active antibody as a function of pH and temperature. The names and colors of each column on the graphs in Figures 3a and 3b are the same as those shown in Figure 1, which illustrates the culture conditions. [Figure 3b] Same as above. [Figure 3c] Same as above. [Figure 4a] Figures 4a to 4c show the results of charge variant analysis using LapChip® and CE-HPLC equipment for the ratio of acidic isomeric antibodies according to Example 3-2 of the present invention. Specifically, Figure 4a is a graph showing the results of charge variant analysis using LapChip® equipment, Figure 4b is a graph showing the results of charge variant analysis using CE-HPLC equipment, and Figure 4c is a graph comprehensively showing the analysis results for the ratio of acidic isomeric antibodies as a function of pH and temperature. The names and colors of each column on the graphs in Figures 4a and 4b are the same as those shown in Figure 1, which illustrates the culture conditions. [Figure 4b] Same as above. [Figure 4c] Same as above. [Figure 5a] Figures 5a to 5c show the results of charge variant analysis of the ratio of basic isomer antibodies according to Example 3-3 of the present invention. Specifically, Figure 5a is a graph showing the results of charge variant analysis using LapChip® equipment, Figure 5b is a graph showing the results of charge variant analysis using CE-HPLC equipment, and Figure 5c is a graph comprehensively showing the analysis results of the ratio of basic isomer antibodies as a function of pH and temperature. The names and colors of each column on the graphs in Figures 5a and 5b are the same as those shown in Figure 1, which illustrates the culture conditions. [Figure 5b] Same as above. [Figure 5c] Same as above. [Figure 6a] 6a to 6c are graphs showing the results of charge variant analysis using LapChip® equipment for changes in the ratio of main active antibodies and heterologous antibodies due to lactic acid supply in Examples 3 and 4 of the present invention. Specifically, Fig. 6a is a graph showing changes in the ratio of main active antibodies, Fig. 6b is a graph showing changes in the ratio of acidic heterologous antibodies, and Fig. 6c is a graph showing changes in the ratio of basic antibodies. [Figure 6b] Same as above. [Figure 6c] Same as above. [Figure 7a] 7a and 7b are diagrams showing the results of N-glycan analysis according to Example 4 of the present invention. Specifically, Fig. 7a is a table showing the results of N-glycan analysis of the obtained antibody population and the results of a similarity analysis with a control drug (Humira), and Fig. 7b is a diagram showing the glycan structure used in the glycan analysis. [Figure 7b] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0015] The term "antibody" as used herein refers to a substance produced in the immune system in response to antigen stimulation, present in lymph and blood, and capable of specifically binding to a specific antigen to induce an antigen-antibody reaction. The type of antibody used in the present invention is not particularly limited and may include all antibodies commonly known in the art, commonly used therapeutic antibodies, etc. For purposes of the present invention, the antibody may be a monoclonal antibody, and a more specific example thereof may be adalimumab.

[0016] The term "monoclonal antibody" as used in the present invention refers to an antibody that reacts with only one antigenic determinant (epitope) and is produced from a single antibody-producing cell.

[0017] The term "adalimumab" used in the present invention refers to a monoclonal antibody against TNF-α, which is known to be effective in treating rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, hidradenitis suppurativa, uveitis, pediatric idiopathic arthritis, etc. Adalimumab is a component of the pharmaceutical product Humira (registered trademark) developed by AbbVie, Inc. in the United States, and is distributed as a therapeutic agent for autoimmune diseases, etc.

[0018] The term "recombinant cells capable of expressing an antibody" as used herein refers to cells that have been modified by transformation to produce a specific antibody, and the type of the cells is not particularly limited. Non-limiting examples of the recombinant cells include mammalian cells, more specifically, Chinese hamster ovary cells (CHO).

[0019] For the purposes of the present invention, the "recombinant cells expressing an antibody" may be recombinant cells expressing a monoclonal antibody, more specifically, recombinant cells expressing adalimumab. In one embodiment of the present invention, recombinant Chinese hamster ovary cells (CHO) expressing adalimumab were used.

[0020] The term "medium" as used herein broadly refers to a nutrient-containing solution that supplies nutrients to proliferating cells, and such a solution generally includes, but is not limited to, essential and non-essential amino acids, vitamins, carbon sources, lipids, and trace elements necessary for cell growth and / or survival. The medium is preferably prepared at a pH and salt concentration that is optimal for cell survival and proliferation depending on the type of cell to be cultured, and may further include substances widely used in the art as components that increase proliferation and / or survival, such as hormones and growth factors.

[0021] In the present invention, the method for producing an antibody population may include a first-temperature culture step and a second-temperature culture step, which are divided into two steps depending on the culture temperature conditions for culturing recombinant cells that express the antibody. The first-temperature culture step can be performed by culturing recombinant cells that express the antibody in a culture medium under conditions of pH 7.0 to 7.1 at a first culture temperature of 36°C to 38°C for 4 to 6 days. In order to produce monoclonal antibodies with excellent biological activity, for example, a high-quality adalimumab antibody population, in the first-temperature culture step, the pH range can be set to pH 7.0 to 7.1, more specifically, around pH 7.0, and even more specifically, pH 7.0; the first culture temperature range can be set to 36°C to 38°C, more specifically, 36.5°C to 37.5°C, and even more specifically, around 37°C; and the culture period can be set to 4 to 6 days, more specifically, 5 days.

[0022] The second-temperature culture step can be performed by culturing the recombinant cells cultured in the first-temperature culture step in a medium for 6 to 8 days at a second culture temperature set to 34°C or higher and 2°C to 4°C lower than the first culture temperature of the first-temperature culture step, under conditions of pH 7.0 to 7.1. In order to produce a monoclonal antibody with excellent biological activity, for example, a high-quality adalimumab antibody population, the second-temperature culture step can be performed at a pH range of 7.0 to 7.1, more specifically, around pH 7.0, and even more specifically, at pH 7.0; the second culture temperature can be set to 34°C to 36°C, more specifically, 34.5°C to 35.5°C, and even more specifically, around 35°C; and the culture period can be set to 6 to 8 days, more specifically, 7 days.

[0023] Furthermore, in the present invention, the second-temperature culture step can be performed by adding 80 to 120 μL of 0.1 to 2 M lactic acid per 10 ml of medium daily from days 1 to 3 of culture at the second temperature. When the second-temperature culture step is additionally performed, in order to produce a monoclonal antibody with excellent biological activity, for example, a high-quality adalimumab antibody population, the timing of adding lactic acid can be set from days 1 to 3 of culture at the second temperature, more specifically, from day 2 of culture, and more specifically, from day 7 of total culture, which is the second day of culture at the second temperature after five days of culture at the first temperature, and the amount of lactic acid added can be set to 80 to 120 μL daily, more specifically, 90 to 110 μL, and even more specifically, about 100 μL.

[0024] In the above-mentioned addition of lactic acid, the meaning of "per 10 ml of medium" means per 10 ml of working volume (WV) including the medium and culture solution in the culture vessel on days 1 to 3 of the second-temperature culture stage, and the meaning of "working volume (WV)" is something that would be obvious to a person of ordinary skill in the art.

[0025] The form of the lactic acid to be added is not particularly limited, and any form of lactic acid commonly used in the art can be used. As a non-limiting example, for the purposes of the present invention, lactic acid can be used in the form of a lactic acid solution, more specifically, an L-lactic acid solution, and the concentration can be 0.1 to 2 M, more specifically, 0.5 to 2 M, and even more specifically, about 1 M.

[0026] In accordance with one aspect of the present invention, in order to produce a monoclonal antibody having excellent biological activity, e.g., a high-quality adalimumab antibody population, in one embodiment, (a) a first temperature culture step in which recombinant cells expressing the adalimumab antibody are cultured in a culture medium at 37°C and pH 7.0 to 7.1 for 5 days; and (b) A second temperature culture step was performed in which the recombinant cells cultured for 5 days were cultured at 35°C and pH 7.0 to 7.1 for 7 days, and from the third day of culture, 100 μl of 1 M L-lactic acid solution was added to the medium every day. An antibody population was produced by this method.

[0027] In one example of the present invention, an adalimumab antibody population was produced using the above method, resulting in a high-quality antibody population containing 55% or more, more specifically 60% or more of the primary active antibody and 45% or less, more specifically 40% or less of heterologous antibody, specifically an antibody population of very similar quality to Humira, the control drug for adalimumab (Example 3). Furthermore, an adalimumab antibody population was produced using the above method, resulting in a high-quality antibody population having glycan structures including 65% to 80% GOF form, 3% to 6% GOF+GN form, and 3% to 6% GOF-N form, specifically an antibody population of very similar quality to Humira, the control drug for adalimumab (Example 4).

[0028] The term "antibody population" as used herein refers to a group of antibodies including primary active antibodies, heterologous antibodies, or both primary active antibodies and heterologous antibodies. For purposes of the present invention, the antibody population may include both primary active antibodies and heterologous antibodies, and the heterologous antibodies may include both acidic and basic isoantibodies.

[0029] The term "primary active antibody" as used in the present invention refers to an antibody with the highest biological activity among antibodies in an antibody population, in which amino acids within the antibody are not modified by deamination, oxidation, etc. The proportion of primary active antibodies is an important factor in determining the quality of antibody therapeutic agents.

[0030] The term "antibody" as used herein refers to an antibody in an antibody population in which some amino acids have been modified by deamination or oxidation, resulting in reduced biological activity. Antibodies include acidic and basic antibodies. There are slight differences in charge between active, acidic, and basic antibodies, and these are also called charge variants. Antibodies can be separated using these charge differences. Non-limiting examples of such antibodies include antibodies in which asparagine has been deaminated to form aspartate and antibodies in which methionine has been oxidized to form methionine sulfate. Furthermore, when glutamate is present at the N-terminus of the heavy chain, the glutamate forms a pentagonal ring structure, thereby including antibodies in which the glutamate has been modified to form pyroglutamate.

[0031] When antibodies are produced in host cells such as CHO cells, the heterologous antibodies are present in high proportions in the host cell culture medium and may be removed through processes such as chromatography and included in the antibody population at a desired proportion. To produce a high-quality antibody population from host cells transfected with a vector containing an antibody-encoding polynucleotide, the above-mentioned primary and heterologous antibodies must be present in the desired amounts.

[0032] These heterologous antibodies are antibodies in which some amino acids have been modified by deamination or oxidation, and it is known that each heterologous antibody has different biological activity. Therefore, maintaining a consistent distribution of the heterologous antibody content is important for maintaining consistent quality. In particular, in the manufacture of antibody biosimilar drugs, it is important to manufacture them so that they contain a corresponding or similar composition to the original drug (reference drug) and contain an adjusted ratio of the main active antibody and the heterologous antibody. The ratio of the main active antibody and the heterologous antibody can be an important criterion for quality comparison with the reference drug and verification of homogeneity.

[0033] An antibody population produced by the method for producing an antibody population according to the present invention may contain both primary and heterologous antibodies, or, for one purpose of the present invention, the antibody population may contain 50% or more primary antibodies and 50% or less heterologous antibodies. Furthermore, to produce an antibody population with superior biological activity, the method for producing an antibody population according to the present invention can produce an antibody population containing 55% or more primary antibodies and 45% or less heterologous antibodies, or an antibody population containing 60% or more primary antibodies and 40% or less heterologous antibodies.

[0034] According to one aspect of the present invention, an adalimumab antibody population can be produced. Using Humira (registered trademark), the original adalimumab drug, as a control, an antibody population having a ratio of primary active antibodies and heterologous antibodies similar to that of the control drug can be produced. To produce a biosimilar drug of similar quality to the control drug, the antibody population may be an antibody population with 55% or more primary active antibodies and 45% or less heterologous antibodies; an antibody population with 55% or more primary active antibodies, 15% or less acidic heterologous antibodies, and 30% or less basic heterologous antibodies; an antibody population with 55% or more primary active antibodies, 20% or less acidic heterologous antibodies, and 25% or less basic heterologous antibodies; an antibody population with 60% or more primary active antibodies and 40% or less heterologous antibodies; or an antibody population with 60% or more primary active antibodies, 20% or less acidic heterologous antibodies, and 20% or less basic heterologous antibodies. According to one embodiment of the present invention, the antibody population may be 60% or more primary active antibodies, 20% or less acidic heterologous antibodies, and 25% or less basic heterologous antibodies.

[0035] In order to obtain an antibody population with a higher purity, the method for producing an antibody population according to the present invention may further include a step of removing impurities such as host cell proteins (HCPs), host cell-derived DNA (HCD), factors for cell growth, etc. after obtaining a mixed sample containing the antibody population through culture. The method for removing the impurities is not particularly limited and can be performed by a method commonly used in the art.

[0036] When an antibody population is produced by the method for producing an antibody population according to the present invention, additional filtration and purification steps can be performed after the removal of impurities to further refine the target ratio of primary active antibody and heteroantibody. However, when produced by the method for producing an antibody population according to the present invention, an antibody population containing primary active antibody and heteroantibody at a ratio approaching that which maximizes the biological activity of the target antibody, e.g., a monoclonal antibody, more specifically, adalimumab, can be produced by adjusting the culture conditions by pH, culture temperature, and addition of lactic acid, and by achieving a synergistic effect, without additional filtration and purification steps other than the removal of impurities, as is commonly done in the art. Furthermore, an antibody population containing primary active antibody and heteroantibody at the same or very similar ratio as compared to a control drug, adalimumab, can be produced.

[0037] In the present invention, the method for measuring the ratio of the primary active antibody and the heterologous antibody in the antibody population is not particularly limited and can be performed by a method commonly used in the art. As a non-limiting example, in one embodiment of the present invention, the ratio of the primary active antibody and the heterologous antibody was measured using LapChip (registered trademark) and liquid chromatography (CE-HPLC).

[0038] The term "glycan structure" as used in the present invention refers to a sugar chain structure formed by fucosylation, afucosylation, galactosylation, etc. of the Fc region of an antibody, and the main forms of glycan structures include G0, G0F, G0F-N, G0F+GN, G1F, G1F+GN, G1, G2, and G2F (Figure 7b).

[0039] It is known that the biological activity of antibodies varies depending on the glycan structure, and for therapeutic antibodies, having an appropriate glycan structure is an important factor in the quality of the drug. In particular, in the production of antibody biosimilar drugs, it is important to adjust the production so that the glycan structure corresponds to or is similar to that of the original drug (reference drug). Glycan structure can be an important criterion for quality comparison with the reference drug and verification of homogeneity.

[0040] The method for producing an antibody population according to the present invention can produce antibody populations with a variety of glycan structures depending on the goal, specifically, the quality of the antibody population to be obtained. For one purpose, the present invention precisely controls the pH, culture temperature, and lactic acid supply to produce antibody populations with glycan structures containing 65% to 80% of the GOF form, antibody populations with glycan structures containing 3% to 6% of the GOF+GN form, and antibody populations with glycan structures containing 3% to 6% of the GOF-N form.

[0041] Furthermore, for one object of the present invention, an adalimumab antibody population can be produced, and an antibody population having a glycan structure similar to that of the original adalimumab drug, Humira (registered trademark), can be used as a control drug.

[0042] The term "fucosylation" as used herein refers to the attachment of fucose residues to N-glycans, O-glycans, and glycolipids. Regulation of fucosylation or afucosylation is known to have a significant effect on the function of monoclonal antibodies, and it is known that monoclonal antibodies with reduced fucose in the glycosylation pattern, i.e., afucosylated, exhibit higher antibody-dependent cell-mediated cytotoxicity (ADCC) than fucosylated antibodies.

[0043] The term "afucosylation" as used herein means the opposite of fucosylation and refers to the removal of fucose residues. It can be used interchangeably with "defucosylation," specifically, it refers to a reduction in the degree of fucosylation.

[0044] In the present invention, the method for analyzing the glycan structure of an antibody, i.e., the method for performing N-glycan analysis, is not particularly limited and can be performed by a method commonly used in the art, such as, for example, quadrupole time-of-flight (Qtof) mass spectrometry.

[0045] Terms not otherwise defined in the present invention are to be interpreted as having the meanings commonly used in the art. Furthermore, the term "or" used in the present invention can be interpreted as including "and" unless otherwise specified.

[0046] The scope of the present invention is not limited by the specific descriptions disclosed in the present invention, and each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. In other words, all possible combinations of the various elements disclosed in the present invention are considered to fall within the scope of the present invention. Furthermore, a person skilled in the art can recognize or ascertain many equivalents to specific embodiments of the present invention through routine experimentation, and such equivalents are considered to fall within the scope of the present invention.

[0047] [Effects of the invention] The method for producing an antibody population of the present invention involves precisely adjusting culture conditions, such as pH and culture temperature, during the cultivation of recombinant cells, and can effectively produce an antibody population of the desired quality, a high-quality antibody population with excellent biological activity.

[0048] Specifically, the present invention enables the effective production of antibody populations with a target ratio of primary and secondary antibodies by adjusting the pH, culture temperature, and / or lactic acid supply during the culture of recombinant cells expressing antibodies.

[0049] Furthermore, the present invention enables the effective production of antibody populations having the desired glycan structure by adjusting the pH, culture temperature, and / or lactic acid supply during the culture of recombinant cells expressing antibodies.

[0050] Furthermore, the method of the present invention enables the production of high-quality antibodies with excellent biological activity that can achieve the target therapeutic efficacy when producing monoclonal antibodies, particularly therapeutic monoclonal antibodies. In particular, in the production of biosimilar drugs, precise adjustment of pH, culture temperature, and / or lactic acid supply conditions allows the effective production of antibodies of the same or very similar quality as the original drug. [Example]

[0051] The present invention will be described in more detail below through specific examples. However, these examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way.

[0052] Example 1: Production of antibody populations by adjusting pH, temperature, and lactate supply Recombinant Chinese hamster ovary cells (CHO) expressing the adalimumab antibody were cultured in a medium, and 1.1 mL of the culture medium was inoculated into 24 15 mL disposable culture vessels. The vessels were cultured in a Sartorius Ambr® 15 bioreactor for a total of 12 days under the culture conditions shown in Table 1 below.

[0053] [Table 1]

[0054] Specifically, 12 of the 24 culture vessels were divided into a group in which temperature was changed during culture (CS1-1 to CS1-12), and the other 12 were divided into a group in which temperature was not changed (CS2-1 to CS2-12). Each group was further divided into three subgroups and cultured under pH 6.9, pH 7.0, and pH 7.1, respectively. Furthermore, one culture vessel per subgroup under each pH condition was cultured by injecting 100 μl of 1M L-lactic acid solution per 10 ml of working volume (WV) every day from days 7 to 11 of culture. The culture vessels under these conditions are shown diagrammatically in Figure 1.

[0055] After the culture was completed, the culture medium was purified using a protein A column, followed by IgG titer, LapChip (registered trademark), cation exchange high-performance liquid chromatography (CE-HPLC), and N-glycan analysis.

[0056] Example 2: Measurement of IgG titer The IgG titer of the antibody population obtained in Example 1 was measured, and the results are shown in FIG.

[0057] The measurement results confirmed that the IgG titer of the culture medium was significantly affected by both the culture temperature and pH conditions, with temperature having a greater effect than pH. Furthermore, within a certain range, the IgG titer tended to increase with lower temperatures and higher pH. Therefore, it was confirmed that a relatively high pH of 7.0 to 7.1 and a temperature change in which the culture temperature was lowered from an initial temperature of 37°C to 35°C were advantageous for increasing antibody productivity of recombinant cells (Figure 2).

[0058] Example 3: LapChip® and Cation Exchange High Performance Liquid Chromatography (CE-HPLC) Analysis Charge variant analysis of the antibody population obtained in Example 1 was performed using LapChip® and CE-HPLC equipment, and the results are shown in FIGS.

[0059] Example 3-1: Analysis results of main active antibody The results of charge variant analysis using LapChip® and CE-HPLC equipment are shown in Figure 3. Specifically, Figure 3a is a graph showing the results of charge variant analysis using LapChip® equipment, Figure 3b is a graph showing the results of charge variant analysis using CE-HPLC equipment, and Figure 3c is a graph comprehensively showing the analysis results for the ratio of major active antibodies as a function of pH and temperature. The names and colors of the columns on the graphs in Figures 3a and 3b are the same as those used to diagram the culture conditions in Example 1 and Figure 1. Specifically, the upper graphs in Figures 3a and 3b are for the CS1 group, which was cultured at 37°C from days 1 to 4 of culture and then shifted to 35°C from day 5 of culture, and the lower graphs are for the CS2 group, which was maintained at 37°C without any change in culture temperature. The orange columns on the graph are for the pH 6.9 condition; the green columns are for the pH 7.0 condition; and the blue columns are for the pH 7.1 condition. The three columns framed in yellow are for the condition in which lactic acid was supplied from the 7th to 11th day of culture.

[0060] Charge variant analysis using the LapChip® and CE-HPLC equipment showed that, when compared under the same pH conditions, the CS1 group, which was subjected to a temperature change from 37°C to 35°C on day 5 of culture, had a higher overall proportion of primary reactive antibodies than the CS2 group, which was not subjected to a temperature change. Furthermore, it was confirmed that the lower the pH within a certain range, the higher the proportion of primary reactive antibodies. Under the same pH conditions, the addition of lactic acid to the medium from day 7 of culture resulted in a higher proportion of primary reactive antibodies (Figures 3a and 3b).

[0061] The results of the overall effect of pH and temperature on the ratio of the major reactive antibodies are shown in Figure 3c. Specifically, it was shown that changes in pH and temperature have a significant effect on the ratio of the major reactive antibodies, with pH having a greater effect than temperature. Furthermore, it was confirmed that the ratio of the major reactive antibodies tends to be higher as the pH and temperature are lower within a certain range (Figure 3c).

[0062] Example 3-2: Analysis of acidic isomeric antibodies The results of charge variant analysis using LapChip® and CE-HPLC equipment are shown in Figure 4. Specifically, Figure 4a is a graph showing the results of charge variant analysis using LapChip® equipment, Figure 4b is a graph showing the results of charge variant analysis using CE-HPLC equipment, and Figure 4c is a graph showing the overall analysis results for the ratio of acidic isomeric antibodies depending on pH and temperature. The names and colors of each column on the graphs in Figures 4a and 4b are the same as those shown in Figure 1, which illustrates the culture conditions, and the specific details are the same as those described in Example 3-1.

[0063] Charge variant analysis using the LapChip® and CE-HPLC equipment showed that, when compared under the same pH conditions, the CS1 group, which was subjected to a temperature change from 37°C to 35°C on day 5 of culture, had an overall lower proportion of acidic isomeric antibodies than the CS2 group, which was not subjected to a temperature change (Figures 4a and 4b).

[0064] The results of the overall effect of pH and temperature on the ratio of acidic isomeric antibodies are shown in Figure 4c. Specifically, it was shown that temperature had a greater effect than pH on the ratio of acidic isomeric antibodies, and it was confirmed that the lower the temperature within a certain range, the lower the ratio of acidic isomeric antibodies (Figure 4c).

[0065] Example 3-3: Analysis results of basic isomeric antibodies The results of charge variant analysis using LapChip® and CE-HPLC equipment are shown in Figure 5. Specifically, Figure 5a is a graph showing the results of charge variant analysis using LapChip® equipment, Figure 5b is a graph showing the results of charge variant analysis using CE-HPLC equipment, and Figure 5c is a graph showing the overall analysis results for the ratio of basic isomeric antibodies depending on pH and temperature. The names and colors of each column on the graphs in Figures 5a and 5b are the same as those shown in Figure 1, which illustrates the culture conditions, and the specific details are the same as those described in Example 3-1.

[0066] Charge variant analysis using the LapChip® and CE-HPLC equipment showed that, when compared under the same pH conditions, the CS2 group, which was not exposed to a temperature change, had a lower overall proportion of basic antibodies than the CS1 group, which was exposed to a temperature change from 37°C to 35°C on day 5 of culture. It was also confirmed that the proportion of basic antibodies decreased as the pH decreased within a certain range, and that under the same pH conditions, the proportion of basic antibodies decreased when lactic acid was added to the medium from day 7 of culture (Figures 5a and 5b).

[0067] The results of the overall effect of pH and temperature on the ratio of basic antibodies are shown in Figure 5c. Specifically, it was shown that changes in pH and temperature have a significant effect on the ratio of basic antibodies, with pH having a greater effect than temperature. Furthermore, it was confirmed that the ratio of basic antibodies tends to decrease as the pH decreases and the temperature increases within a certain range (Figure 5c).

[0068] Example 3-4: Analysis of changes in the ratio of main active antibody and heterologous antibody due to the addition of lactic acid In Example 1, 100 μl of 1 M L-lactic acid solution was injected every day from day 7 to day 11 of culture. The changes in the ratio of the main active antibody and the heterologous antibody were analyzed in more detail using LapChip (registered trademark), and the results are shown in Figures 6a to 6c.

[0069] Specifically, in the case of the main active antibody, when lactic acid was added, an increase in the antibody ratio was observed in both the CS1 group, which had undergone a culture temperature change, and the CS2 group, which had not, and it was shown that the increase was approximately 1.5 to 6.6% depending on the pH conditions (Figure 6a).

[0070] In the case of acidic isomer antibodies, when lactic acid was added, an increase in the antibody ratio of approximately 0.6 to 3.1% was observed in the CS1 group, which was cultured at a different temperature, depending on the pH conditions. In the CS2 group, which was not cultured at a different temperature, an increase in the antibody ratio of approximately 0.9% or a decrease of approximately 0.8 to 2.1% was observed depending on the pH conditions (Figure 6b).

[0071] In the case of basic isomer antibodies, when lactic acid was added, the proportion of antibodies was observed to decrease in both the CS1 group, which was cultured at a different temperature, and the CS2 group, which was not, and it was shown that the decrease was approximately 2.1 to 4.7% depending on the pH conditions (Figure 6c).

[0072] Example 3-5: Comprehensive analysis of the ratio of primary and secondary antibodies Considering the results of charge variant analysis using LapChip (registered trademark) and CE-HPLC equipment in Examples 3-1 to 3-4, it was confirmed that advantageous conditions for obtaining an antibody population with similar quality (ratio of main active antibody and heterologous antibody) to the control drug were a pH of around 7.0, a temperature change in which the culture temperature was lowered from the initial temperature of 37°C to 35°C, and the addition of lactic acid.

[0073] Example 4: N-glycan analysis Among the antibody populations obtained in Example 1, N-glycan analysis was performed on the CS1 group, which was subjected to a temperature shift on day 5 of culture, and the results are shown in Figure 7. Specifically, Figure 7a is a table showing the results of N-glycan analysis and a similarity analysis with a control drug (Humira), and Figure 7b is a diagram showing the glycan structures used in the glycan analysis.

[0074] The N-glycan analysis results, focusing on the GOF, GOF-N, and GOF+GN forms of the glycan structures, showed that without lactic acid at pH 6.9, the glycan contained 60.87% GOF, 6.71% GOF-N, and 9.13% GOF+GN forms, while with lactic acid, the glycan contained 63.05% GOF, 6.73% GOF-N, and 8.38% GOF+GN forms. Afucosylation was analyzed to be 7.04% and 6.71%, respectively, and galactosylation was analyzed to be 80.70% and 82.31%, respectively.

[0075] At pH 7.0, without lactic acid, the sample contained 72.41% GOF, 4.51% GOF-N, and 4.37% GOF+GN forms, whereas with lactic acid, the sample contained 70.23% GOF, 5.71% GOF-N, and 4.39% GOF+GN forms. The afucosylation and galactosylation results were 4.72% and 4.87%, respectively, and 84.52% and 85.47%, respectively.

[0076] At pH 7.1, without lactic acid, the sample contained 71.25% GOF, 4.35% GOF-N, and 4.39% GOF+GN forms, whereas with lactic acid, the sample contained 76.07% GOF, 3.76% GOF-N, and 3.31% GOF+GN forms. The afucosylation and galactosylation results were 5.12% and 4.88%, respectively, and 83.39% and 86.60%, respectively.

[0077] The results were compared with the glycan structure of the control drug Humira ("HE-4" in Figure 7a). As a result, it was analyzed that the G0F, G0F-N, and G0F+GN forms were very similar to or slightly different from the control drug at pH 7.0 and 7.1, and the afucosylation and galactosylation results were also similar.

[0078] To summarise the results of Examples 1 to 4, when the IgG titer, charge variant and N-glycan analysis results of the obtained adalimumab antibody population were taken into account, it was determined that a pH of 7.0 to 7.1, particularly a pH of 7.0, a temperature change in which the culture temperature was lowered from the initial temperature of 37°C to 35°C, and the addition of lactic acid were advantageous for obtaining an antibody population of similar quality to the control drug.

[0079] This specification omits detailed descriptions of content that can be fully understood and inferred by a person having ordinary skill in the art of the present invention, and various modifications are possible within the scope of the technical idea and essential components of the present invention other than the specific examples described in this specification. Therefore, the present invention may be implemented in a manner different from that specifically explained and exemplified in this specification, and this is something that can be understood by a person having ordinary skill in the art of the present invention. (Addendum) The invention of the present disclosure includes the following aspects. <Item 1> (a) a first temperature incubation step in which recombinant cells expressing the antibody are cultured in a medium at a first incubation temperature of 36°C to 38°C and a pH of 7.0 to 7.1 for 4 to 6 days; and (b) a second temperature culture step in which the cultured recombinant cells are cultured in a medium at a second culture temperature set to be 2°C to 4°C lower than the first culture temperature in step (a) and to be at least 34°C, at a pH of 7.0 to 7.1, for 6 to 8 days. <Item 2> The method for producing an antibody population according to <Item 1>, wherein step (b) comprises culturing the cells by adding 80 to 120 μl of 0.1 to 2 M lactic acid per 10 ml of medium every day from the first to third days of culturing at the second culture temperature. <Item 3> The method for producing an antibody population according to <Item 1>, wherein the antibody is a monoclonal antibody. <Item 4> The method for producing an antibody population according to <Item 1>, wherein the antibody is adalimumab. <Item 5> The method for producing an antibody population according to <Item 1>, wherein the antibody population contains 55% or more of the main active antibody and 45% or less of the heterologous antibody. <Item 6> The method for producing an antibody population according to <Item 1>, wherein the antibody population contains 60% or more of the main active antibody and 40% or less of the heterologous antibody. <Item 7> The method for producing an antibody population according to <Item 1>, wherein the antibodies in the antibody population have glycan structures containing 65% to 80% of the GOF form. <Item 8> The method for producing an antibody population according to <Item 1>, wherein the antibodies in the antibody population have glycan structures containing 3% to 6% of G0F+GN forms. <Item 9> (a) a first temperature culture step in which recombinant cells expressing the antibody are cultured in a culture medium at 36.5°C to 37.5°C and pH 7.0 to 7.1 for 5 days; and (b) a second temperature culture step in which the recombinant cells cultured for 5 days are cultured for 7 days at a culture temperature of 34.5°C to 35.5°C and a pH of 7.0 to 7.1, and from the third day of culture, 95 to 105 μl of 0.5 to 2 M lactic acid per 10 ml of medium is added daily. <Item 10> The method for producing an antibody population according to <Item 9>, wherein the antibody is adalimumab.

Claims

1. (a) a first temperature culturing step in which recombinant cells expressing the antibody are cultured in a medium at a first culture temperature of 36°C to 38°C and a pH of 7.0 to 7.1 for 4 to 6 days; and (b) a second temperature culturing step in which the cultured recombinant cells are cultured in a medium at a second culture temperature set to be 2°C to 4°C lower than the first culture temperature of (a) and not lower than 34°C, and at a pH of 7.0 to 7.1 for 6 to 8 days, and from the first to third days of the culture, 80 to 120 μl of 0.1 to 2 M lactic acid per 10 ml of medium is added daily, the antibody is adalimumab; The antibody population comprises 55% or more of the primary active antibody and 45% or less of the heterologous antibody.

2. The method for producing an antibody population according to claim 1 , wherein the antibody population contains 60% or more of the main active antibody and 40% or less of the heterologous antibody.

3. The method for producing an antibody population according to claim 1, wherein the antibodies in the antibody population have glycan structures containing 65% to 80% of the G0F form.

4. The method for producing an antibody population according to claim 1, wherein the antibodies in the antibody population have glycan structures containing 3% to 6% of the G0F+GN form.

5. (a) a first temperature culture step in which recombinant cells expressing the antibody are cultured in a culture medium at 36.5°C to 37.5°C and pH 7.0 to 7.1 for 5 days; and (b) a second temperature culturing step of culturing the recombinant cells cultured for 5 days at a culture temperature of 34.5°C to 35.5°C and a pH of 7.0 to 7.1 for 7 days, and from the third day of culture, adding 95 to 105 μl of 0.5 to 2 M lactic acid per 10 ml of medium every day, wherein the antibody is adalimumab.

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