Method for modulating galactosylation of recombinant proteins by optimizing culture medium

Abstract

This invention relates to: a method for preparing a galactosylation-controlled target recombinant protein or a method for controlling the galactosylation of a target recombinant protein, comprising the step of increasing the osmotic pressure of the culture solution of animal cells expressing the target recombinant protein during animal cell culture; a method for preparing a galactosylation-controlled target recombinant protein or a method for controlling the galactosylation of a target recombinant protein, comprising the step of supplementing the culture solution of animal cells expressing the target recombinant protein with asparagine during animal cell culture; a method for preparing a galactosylation-controlled target recombinant protein or a method for controlling the galactosylation of a target recombinant protein, comprising the step of increasing the osmotic pressure of the culture solution of animal cells expressing the target recombinant protein and supplementing it with asparagine during animal cell culture; and a galactosylation-controlled target recombinant protein prepared by said method.

Description

Technical Field

[0001] This invention relates to a method for preparing a galactosylation-regulated target recombinant protein or a method for regulating the galactosylation of a target recombinant protein, comprising increasing the osmotic pressure of the culture solution of animal cells expressing the target recombinant protein during animal cell culture; a method for preparing a galactosylation-regulated target recombinant protein or a method for regulating the galactosylation of a target recombinant protein, comprising supplementing the culture solution of animal cells expressing the target recombinant protein with asparagine during animal cell culture; a method for preparing a galactosylation-regulated target recombinant protein or a method for regulating the galactosylation of a target recombinant protein, comprising increasing the osmotic pressure of the culture medium of animal cells expressing the target recombinant protein during animal cell culture and supplementing it with asparagine; and a galactosylation-regulated target recombinant protein prepared by said method. Background Technology

[0002] Antibodies are proteins that bind to antigens, thereby interfering with or removing them. Due to the known significant potential of the therapeutic antibody market, extensive research has been conducted on the efficient expression and large-scale production of antibodies. One of the key aspects of antibody production is the homogeneity of the prepared antibody population. Specifically, in the preparation of monoclonal antibodies, glycoform distribution is crucial for consistent and reproducible monoclonal antibody production. However, many different variables can affect the glycosylation of the resulting monoclonal antibodies; therefore, methods to consistently control the glycosylation and antibody isoform content of the prepared antibody population are needed.

[0003] One of the key challenges in the commercial production of monoclonal antibodies is consistently maintaining the quality of each batch produced. While many quality attributes exist in the production of monoclonal antibodies, galactosylation is perhaps one of the most critical. This is because galactosylation is known to influence complement-dependent cytotoxicity (CDC), the mode of action (MOA) of monoclonal antibodies. Galactosylation is established by using galactose as a building block in a galactosylation chain reaction, where galactose is linked to an adjacent N-acetylglucosamine sugar via galactosyltransferases. Examples of methods for controlling galactosylation include adding manganese or galactose to the culture solution (US Patent Application Publication No. 2012-0276631), but further development of methods to control antibody galactosylation remains necessary. Summary of the Invention

[0004] Technical issues

[0005] In these cases, the inventors made considerable efforts to develop a method for effectively controlling the galactosylation of recombinant proteins prepared during animal cell culture, wherein the animal cells produce the recombinant proteins. As a result, the inventors discovered that increasing the osmotic pressure of the animal cell culture solution during animal cell culture, or supplementing with asparagine at specific time points, effectively regulates the galactosylation of the recombinant proteins. Furthermore, the inventors discovered that by simultaneously increasing the osmotic pressure of the animal cell culture solution and supplementing with asparagine, galactosylation can be controlled without a decrease in the content of acidic antibody isomers with increasing osmotic pressure, thereby completing the present invention.

[0006] Technical solution

[0007] The purpose of this invention is to provide a method for preparing a galactosylated regulated target recombinant protein, comprising increasing the osmotic pressure of the animal cell culture solution expressing the target recombinant protein during the culture of animal cells.

[0008] Another object of the present invention is to provide a method for regulating the galactosylation of a target recombinant protein, comprising increasing the osmotic pressure of the culture solution of the animal cells expressing the target recombinant protein during the culture of animal cells.

[0009] Another object of the present invention is to provide a method for preparing a galactosylated regulated target recombinant protein, comprising supplementing the culture solution of animal cells expressing the target recombinant protein with asparagine during animal cell culture.

[0010] Another object of the present invention is to provide a method for regulating the galactosylation of a target recombinant protein, comprising supplementing the culture solution of animal cells expressing the target recombinant protein with asparagine during animal cell culture.

[0011] Another object of the present invention is to provide a method for preparing a galactosylated regulated target recombinant protein, comprising increasing the osmotic pressure of the animal cell culture solution expressing the target recombinant protein during animal cell culture and supplementing it with asparagine.

[0012] Another object of the present invention is to provide a method for regulating the galactosylation of a target recombinant protein, comprising increasing the osmotic pressure of the animal cell culture solution expressing the target recombinant protein during animal cell culture and supplementing it with asparagine.

[0013] Another object of the present invention is to provide a target recombinant protein prepared by the above method.

[0014] Advantages of the present invention

[0015] The advantage of the method of this invention is that it can effectively adjust the galactose content of the target recombinant protein to the desired range. Furthermore, the method of this invention, which simultaneously increases osmotic pressure and adds asparagine, has the effect of regulating the acidic antibody isoform (acidic variant) and the galactose content. Therefore, the method of this invention can be effectively used to prepare the desired antibody population. Brief description of the attached diagram

[0017] Figure 1 This is a schematic diagram of the expression vector for adalimumab biosimilars.

[0018] Figure 2 This shows the time point at which sodium chloride (NaCl) aqueous solution was injected during fed-batch culture.

[0019] Figure 3 This shows the timing of asparagine supplementation during fed-batch culture.

[0020] Best way

[0021] To achieve the above objectives, the present invention provides a method for preparing a galactosylated target recombinant protein, comprising increasing the osmotic pressure of the culture solution of the animal cells expressing the target recombinant protein during the culture of animal cells.

[0022] Specifically, the above method is a method for preparing galactosylation-regulated target recombinant proteins, which includes increasing the osmotic pressure of the culture solution of animal cells expressing the target recombinant protein at specific time points during the culture of animal cells.

[0023] In this invention, the culture is a fed-batch culture, but it is not limited to this.

[0024] As used in this article, “feed-batch culture” refers to a cell culture in which initial cell culture begins with basal or production medium, and feed medium is added to the culture continuously or discontinuously at any time during the cell growth phase or antibody production phase while the culture continues.

[0025] As used herein, the term "cell culture medium" or "culture medium" refers to a nutrient solution used to maintain, grow, proliferate, or expand cells in an artificial in vitro environment (outside a multicellular organism or tissue). Cell culture media can be optimized for culturing specific cells, such as basal media prepared to support cell growth, production media prepared to promote monoclonal antibody production, and concentrated media prepared by concentrating nutrients at high concentrations. Nutrients and culture medium components refer to the components that constitute the cell culture medium, and they are used interchangeably in this invention.

[0026] Specifically, "basal culture medium" or "basal medium" refers to the minimum amount of culture medium that can support cell growth. Basal media not only provide standard inorganic salts such as zinc, iron, magnesium, calcium, and potassium, but also trace elements, vitamins, energy sources, buffers, and amino acids. Examples of basal media include, but are not limited to, Dulbecco's modified Eagle medium (DMEM), Eagle basal medium (BME), RPMI 1640, F-10, and F-12.

[0027] Furthermore, specifically, the term "cell culture production medium" or "production medium" refers to the culture medium used in bioreactors to optimize the expression of monoclonal antibodies. The composition of the production medium may be the same as or different from the basal medium, and when it is different, the production medium can be prepared by concentrating the basal medium or by adding specific components to the basal medium.

[0028] The terms "feeding medium" and "added medium" used in this article can refer to a medium composed of specific nutrients or multiple nutrients; both are concentrated components of the basal medium. Different feed medium components and concentrations can be prepared according to the cells to be cultured.

[0029] In the above culture process, the cell growth phase refers to the period of rapid cell growth after inoculation. Taking Chinese hamster ovary (CHO) cells as an example, it is known that the cell number increase is most active at a temperature of 35°C to 37°C and a pH of 7.0 to 7.3. In this invention, the culture parameters during the cell growth phase are 36.5°C and pH 7.1 or pH 7.0.

[0030] The term "seeding" refers to seeding cells into a culture medium provided for cell culture. Specifically, the culture medium can be provided before cell transfer or simultaneously with cell transfer to a cell bioreactor. In large-scale animal cell culture, the conventional practice is to pre-provide the culture medium, maintain the temperature and oxygen saturation within predetermined ranges, and then transfer the cells into the bioreactor.

[0031] The term "antibody production period" refers to a specific time during which process changes are applied to optimize the production of monoclonal antibodies. Specifically, process changes to optimize production can include all of the following: lowering the temperature, altering dissolved oxygen levels, and changing the culture medium.

[0032] This invention confirms that the degree of galactosylation alteration in recombinant proteins produced in animal cells expressing recombinant proteins can vary with the time points at which the osmotic pressure of the culture medium increases. Specifically, when the osmotic pressure gradually increases during culture, the degree of galactosylation is not significantly different compared to the group where the osmotic pressure does not increase. Conversely, when the osmotic pressure increases at specific time points during culture, the G0F content increases significantly, thus demonstrating that galactosylation can be modulated, and in particular, that galactosylation can be prevented or reduced.

[0033] Furthermore, in this invention, it is preferable to perform a step of increasing osmotic pressure during the master culture of animal cells expressing the target recombinant protein, but it is not limited thereto.

[0034] As used in this article, the term "master culture" refers to the culture step in which the actual production of monoclonal antibodies is completed. It is the final step in a culture process that begins with a single vial of cells, after which the resulting monoclonal antibodies are purified. Master culture can be distinguished from so-called "seed culture," the latter of which aims to gradually increase the culture volume.

[0035] The step of increasing osmotic pressure can be performed at a specific culture time point in the master culture process of animal cells; specifically, using day 0 as the master culture start date, any culture day in the master culture process from day 1 to day 10; more specifically, using day 0 as the master culture start date, any culture day among day 1, day 4, day 7, and day 10; more specifically, using day 0 as the master culture start date, day 4 or day 7, but not limited to these.

[0036] Furthermore, during the culture of animal cells expressing the target recombinant protein, the step of increasing osmotic pressure can be performed once.

[0037] Furthermore, steps can be taken to increase the osmotic pressure, thereby bringing the final osmotic pressure of the culture solution to within the range of 460 mOsm / kg to 500 mOsm / kg, more specifically, within the range of 460 mOsm / kg to 480 mOsm / kg, but not limited thereto. The final osmotic pressure in the culture solution refers to the osmotic pressure present at the end of the culture period, but is not specifically limited thereto.

[0038] Furthermore, the step of increasing osmotic pressure can be achieved in the culture solution by increasing the target osmotic pressure in the range of 400 mOsm / kg to 440 mOsm / kg, more specifically, in the range of 430 mOsm / kg to 440 mOsm / kg, but not limited thereto. The target osmotic pressure increase in the culture solution refers to the osmotic pressure level to be achieved at the point in time when the osmotic pressure step is performed, but is not specifically limited thereto.

[0039] Alternatively, an increase in osmotic pressure can be achieved by at least one method selected from the group consisting of: adding sodium chloride or potassium chloride to the animal cell culture solution and adding glucose to the culture solution, more specifically, adding sodium chloride to the culture solution, but not limited thereto.

[0040] Sodium chloride can be added to the culture solution on a specific day during fed-batch culture to increase the osmotic pressure to a certain level, but this is not the only method. When the osmotic pressure in the culture solution, and even further in the cells, is increased by adding sodium chloride solution, it leads to an increase in β-galactosidase activity, which may therefore inhibit the galactosylation of monoclonal antibodies.

[0041] As used herein, the terms "culture broth" or "culture medium" refer to a liquid containing cultured cells, contained in a shake flask or bioreactor. Culture broth and culture medium are used interchangeably. Furthermore, the presence of animal cells can distinguish between culture solutions and culture media.

[0042] The “recombinant protein” used in this article can be an antibody, preferably a monoclonal antibody.

[0043] As used in this article, "monoclonal antibody" refers to an antibody that can be formed by cells containing antibody-coding sequences and recognize a specific antigen.

[0044] The antibodies of the present invention may preferably include all therapeutic antibodies conventionally used in the art, particularly adalimumab, but are not limited thereto.

[0045] Furthermore, the concept of antibodies mentioned above encompasses both full-length antibodies and antibody fragments. Examples of antibody fragments include all Fv, Fab, Fab', F(ab')2, Fd, and so on. Fv includes both disulfide-bonded stable Fv (dsFv) and single-chain Fv (scFv). Fd refers to the heavy chain component contained within Fab.

[0046] Animal cells capable of expressing the target recombinant protein may include, but are not limited to, native or transfected cells capable of expressing the recombinant protein. For the purposes of this invention, animal cells may express recombinant proteins that are the object of regulation of galactose content, such as, but not limited to, Chinese hamster ovary cell lines (CHO) or mouse myeloma cell lines (NSO).

[0047] In addition, to regulate the galactosylation of monoclonal antibodies, one can optimize culture parameters, the concentration of the basal medium, and the culture medium for further feed optimization.

[0048] That is, the method of adjusting the galactosylation of the above-mentioned monoclonal antibody according to the degree of galactosylation modification required can be applied simultaneously after determining the desired galactosylation range of the monoclonal antibody and analyzing the galactosylation of the monoclonal antibody expressed in the treated cell line.

[0049] Specifically, examples of modification can be embodied in operating a bioreactor to produce galactosylated regulated monoclonal antibodies. These modifications may include optimizing culture parameters such as dissolved oxygen (DO), acidity (pH), culture temperature, agitation, etc. Specifically, in existing methods, where cell growth is carried out at 36.5°C with 30% dissolved oxygen and pH 7.0, and antibody production is carried out at 30°C with 30% dissolved oxygen and pH 7.0; if the temperature is changed to 28°C, the dissolved oxygen to 20%, and the acidity to pH 6.9 respectively before culturing, it has been confirmed that regulating the galactosylation of monoclonal antibodies is possible.

[0050] Furthermore, to regulate galactosylation, the basal medium to be used can be concentrated. Specifically, a medium free of animal-derived components and a basal medium optimized for fed-batch culture can be used to regulate the galactosylation of monoclonal antibodies. Specifically, the galactosylation of monoclonal antibodies may be regulated when using a medium prepared by concentrating the basal medium, for example, at a concentration of 0.8-fold or 1.4-fold, in the master culture.

[0051] Furthermore, the feed medium can be optimized to regulate galactosylation. In this invention, galactosylation is reduced by adjusting the concentration of the feed medium and optimizing the concentration of the metal components added to the feed medium.

[0052] The galactosylation of the target recombinant protein prepared by the above method can be detected by Q-TOF or UPLC devices through N-glycan distribution analysis. N-glycan distribution analysis provides various information, including GOF content, galactosylation index (GI), non-glycosylated heavy chain (NGHC), afucosylation %%, high mannose content, etc. In this invention, the change in GOF content is mainly mentioned.

[0053] Alternatively, the method of the present invention can be used to prepare antibody populations with reduced galactosylation. That is, it is a method applicable to the preparation of antibody populations in which the galactosylation level is lower than the analytical value after analysis of the galactosylation of monoclonal antibodies expressed by treated cell lines.

[0054] As used herein, the term "antibody cluster" refers to a group of antibodies that may have various glycan contents. For the purposes of this invention, an antibody cluster refers to a group of galactosylated antibodies, including antibodies with a target proportion of galactosylation.

[0055] Another aspect of the present invention provides a method for regulating the galactosylation of a target recombinant protein, comprising increasing the osmotic pressure of the animal cell culture solution expressing the target recombinant protein during animal cell culture.

[0056] The method and terminology are the same as explained above.

[0057] Specifically, the method may include increasing the osmotic pressure of the animal cell culture solution expressing the target recombinant protein on a specific day during the animal cell culture process, with the same details as explained above.

[0058] Another aspect of the present invention provides a method for preparing a galactosylated regulated target recombinant protein, comprising supplementing the culture solution of animal cells expressing the target recombinant protein with asparagine during the culture of animal cells.

[0059] The terminology used is the same as explained above.

[0060] In this invention, it can be confirmed that when asparagine is added to the culture solution of animal cells expressing the target recombinant protein during the culture of animal cells, the galactosylation of the target recombinant protein can be regulated.

[0061] Specifically, asparagine can be added in the form of asparagine concentrate or asparagine-containing culture medium.

[0062] Furthermore, asparagine can be supplemented multiple times at specific time points during the culture of animal cells expressing the target recombinant protein. Specifically, taking day 0 as the master culture start date, asparagine can be supplemented from day 4 to day 10; more specifically, taking day 0 as the master culture start date, asparagine can be supplemented on days 4, 7, and 10, thereby gradually increasing the concentration of asparagine in the culture solution.

[0063] When asparagine is supplemented in a single dose, the following drawback exists: the NH4+ in the culture solution... + The concentration of asparagine increases rapidly, thereby delaying cell growth and ultimately leading to a decrease in the final protein expression level. However, when asparagine is added in fractional doses to the feed according to the present invention, the advantage is that the required amount of protein can be produced, while galactosylation can be regulated without the aforementioned disadvantages.

[0064] Asparagine can be supplemented, so that the final concentration of asparagine in the animal cell culture medium can be in the range of 27.6 mM to 33.6 mM.

[0065] Specifically, asparagine can be supplemented to achieve a final concentration of 33.6 mM in the animal cell culture solution; for example, asparagine can be supplemented to further increase the final concentration of asparagine in the animal cell culture solution by 6 mM to 18 mM; more specifically, asparagine can be supplemented three times by sequentially adding asparagine at concentrations of 6 mM, 12 mM and 18 mM, but this is not the only possible method.

[0066] When this method is applied to animal cells, it induces ammonium ions (NH4+). + The production of β-galactosyltransferase increases the concentration of ammonium ions in cells. Consequently, the pH of the trans-Golgi network increases and the activity of β-galactosyltransferase decreases, thereby inhibiting the galactosylation of monoclonal antibodies.

[0067] In addition, as mentioned above, regulating the galactosylation of monoclonal antibodies may include optimizing culture parameters, the concentration of the basal medium, and further feed-optimized culture media.

[0068] That is, the method of adjusting the galactosylation of the above-mentioned monoclonal antibody according to the degree of galactosylation modification required can be applied simultaneously after determining the desired galactosylation range of the monoclonal antibody and analyzing the galactosylation of the monoclonal antibody expressed in the cell line.

[0069] Another aspect of the present invention provides a method for regulating the galactosylation of a target recombinant protein, comprising supplementing the culture solution of animal cells expressing the target recombinant protein during the culture of animal cells.

[0070] The methods and terminology used are the same as those explained above.

[0071] Another aspect of the present invention provides a method for preparing a galactosylated regulated target recombinant protein, comprising increasing the osmotic pressure of the culture solution of the animal cells expressing the target recombinant protein and supplementing the culture solution with asparagine during the culture of animal cells.

[0072] The terminology used is the same as explained above.

[0073] In this invention, it was confirmed that during the culture of animal cells, when asparagine is added to the culture solution of animal cells expressing the target recombinant protein, and the osmotic pressure of the culture solution is increased, the content of acidic antibody isomers (which can decrease with increasing osmotic pressure) increases with the decrease in galactosylation caused by the addition of asparagine.

[0074] In the above methods, the increase in osmotic pressure and the supplementation of asparagine can be performed simultaneously or sequentially. For example, the osmotic pressure in the culture solution can be increased by, for instance, by supplementing sodium chloride, followed by the supplementation of asparagine; or, asparagine can be supplemented first, and then the osmotic pressure in the culture solution can be increased by adding sodium chloride or its analogue; or the supplementation of asparagine and the increase in osmotic pressure in the culture solution can be performed simultaneously.

[0075] In particular, the above methods can be used to increase osmotic pressure.

[0076] It can increase the osmotic pressure, thereby bringing the final osmotic pressure in the culture solution to the range of 460 mOsm / kg to 500 mOsm / kg. It can also supplement asparagine, thereby adjusting the final concentration of asparagine in the animal cell culture solution to the range of 27.6 mM to 33.6 mM.

[0077] Furthermore, the recombinant protein can be an antibody, particularly a monoclonal antibody. The content of acidic antibody isoforms in the antibody group prepared by the above method can be regulated, and the galactosylation of the target recombinant antibody can be regulated by the method of the present invention. That is, galactosylation can decrease with increasing osmotic pressure, while the content of acidic antibody isoforms can be increased by adding asparagine, which can decrease with increasing osmotic pressure.

[0078] Therefore, the advantage of the above method is that it can regulate the galactosylation of the target antibody group and at the same time regulate the content of the acidic antibody isomers of the target antibody group.

[0079] An acidic antibody isomer is a type of antibody isomer. An antibody isomer is an antibody in which a portion of the amino acids possessing the primary activity is modified through deamination or oxidation, including acidic and basic antibody isomers. Examples include antibody isomers in which asparagine in the amino acid is converted to aspartic acid through deamination, and antibody isomers in which methionine is converted to methionine sulfate through oxidation, etc. Additionally, antibody isomers in which glutamate is converted to pyroglutamic acid through the formation of a pentagonal ring when glutamate is present at the N-terminus of the heavy chain are included.

[0080] These antibody isomers can be analyzed using methods such as chromatography; in this invention, cation exchange resin chromatography can be used. Due to the inherent characteristics of monoclonal antibodies, the content of acidic, predominant, and basic antibody isomers varies considerably. The content of charged antibody isomers (charged variants) of a monoclonal antibody can vary depending on the culture conditions (culture parameters, production medium, etc.) of the cell line expressing the monoclonal antibody. Therefore, the advantage of the above method is that it can adjust the required galactose content while simultaneously adjusting the content of acidic antibody isomers to the desired range. Invention Details

[0082] The invention will now be described in more detail with reference to the following embodiments. However, these embodiments are for illustrative purposes only, and the invention should not be limited to them.

[0083] Example 1: Regulation of galactosylation by artificially increasing osmotic pressure

[0084] Various methods exist for increasing the osmotic pressure of the culture solution during fed-batch culture, such as adding excess glucose or an aqueous sodium chloride solution. In this invention, a representative method for increasing osmotic pressure is the addition of a 4M aqueous sodium chloride solution to the culture solution.

[0085] Explanation of preparation method and culture medium composition

[0086] In this invention, the monoclonal antibody used to increase the osmotic pressure of the culture solution or to add asparagine is an adalimumab biosimilar antibody. Adalimumab biosimilars are therapeutic agents developed by Abbott Laboratories for the treatment of rheumatoid arthritis and Crohn's disease. Referring to the amino acid sequences of the heavy and light chains of the adalimumab antibody disclosed in US Patent 6,090,382, adalimumab biosimilar DNA was generated by PCR amplification. pCB-Am2.77_v5.4 was prepared using the promoter of the vector dpGL3CUCBin developed by LG Life Sciences Ltd. Figure 1 The adalimumab biosimilar was then transfected into the CHO-DXB11 cell line to prepare a cell line capable of expressing the biosimilar. pCUCBin, developed by LG Life Sciences, is one of the vectors disclosed in Korean Patent No. 10-1038126 (Novel Heterozygous Promoter and Recombinant Vector Containing the Promoter).

[0087] The culture media used in this invention are of three different types: basal culture medium, production culture medium (main culture medium), and supplemental culture medium (additional culture medium).

[0088] The basal medium is a culture medium used for seed culture purposes. The production medium is a culture medium used after seed culture for the production of the primary antibody (master culture), and can be prepared by concentrating one or more specific components of the basal medium or by adding one or more new components. In this invention, a 1.4-fold concentrated basal medium is used as the production medium. The feed medium is a culture medium added to promote cell growth and increase antibody expression during culture. In this invention, a 3.3-fold concentrated basal medium is used as the feed medium. The basal medium, production medium, and feed medium are all modified versions of the Iscove modified Dulbecco medium (IMDM), and their compositions are shown in Table 1 below.

[0089] [Table 1] Composition of basal culture medium

[0090]

[0091] The antibody production method used in this invention is a method of culturing animal cells using a bioreactor. Animal cell culture involves growing animal cells in a culture medium and then subjecting them to specific treatments (e.g., lowering the temperature) to induce antibody expression. Various culture methods exist, including batch culture, fed-batch culture, continuous culture, perfusion culture, etc. The culture method used in this invention for culturing adalimum biosimilar cell lines is fed-batch culture. Fed-batch culture is a culture method that proceeds in such a manner that additional culture medium is added at least once or twice during specific time periods of the culture, while production culture is carried out simultaneously.

[0092] The fed-batch culture method used in the examples typically involves adding additional culture medium four times on days 1, 4, 7, and 10 of the culture period, with the amount added corresponding to 5% of the current culture solution volume.

[0093] Example 1.1: Changes in galactosylation caused by an increase in osmotic pressure at certain time points

[0094] Fed-batch cultures were performed in 250 mL shake flasks, representing a true 30 mL scale. The feeding strategy and timing of adding sodium chloride solution in the fed-batch culture were discussed. Figure 2The results are the same as shown. The initial osmotic pressure of the culture broth was 320 mOsm / kg, which was artificially increased to 430 mOsm / kg by feeding with 4M sodium chloride (NaCl) aqueous solution, resulting in a final osmotic pressure of 450 mOsm / kg. Regarding the supplemental culture medium, the first feeding was performed on day 1 of culture, followed by further feedings every 3 days, for a total of 4 feedings. During these 4 feedings, 4M sodium chloride aqueous solution was added once or in separate doses four times (with a gradual increase in osmotic pressure during culture) to increase the osmotic pressure of the culture medium. After culture, the expression levels and galactosylation of the monoclonal antibodies were analyzed, and the results are listed in Table 2 below.

[0095] Table 2. Changes in antibody quality at different time points due to increased osmotic pressure

[0096]

[0097] *G0F(%)=G0F / (G0F+G1F+G2F)

[0098] Therefore, as shown in Table 2, the galactosylation of monoclonal antibodies differed at different time points depending on the feeding time of 4M sodium chloride aqueous solution. In shake-flask culture, dissolved oxygen (DO) and acidity (pH) are not easily adjusted directly during the culture process; therefore, the G0F (Gross Oxidation Factor) may not vary significantly between different experimental conditions. However, when cultured in a bioreactor, dissolved oxygen and acidity can be directly adjusted, thus maximizing the variation in G0F.

[0099] Example 1.2: Changes in galactosylation of monoclonal antibodies due to variations in osmotic pressure range

[0100] Example 1.1 demonstrates that the galactosylation of monoclonal antibodies can be altered based on the timing of increases in osmotic pressure of the culture medium. G0F was maximized when sodium chloride aqueous solution was added during the second feed (day 4) and third feed (day 7) of the supplemental culture medium. Furthermore, secondary batch cultures using shake flasks were performed to examine the effect of varying levels of osmotic pressure increase on the galactosylation of monoclonal antibodies. The initial osmotic pressure was 320 mOsm / kg. Following the target osmotic pressure increase, various volumes of 4M sodium chloride aqueous solution were added to the culture medium on days 4 or 7, similar to the initial shake flask culture, and the expression levels and galactosylation of monoclonal antibodies were analyzed.

[0101] Table 3. Changes in antibody quality due to differences in the range of osmotic pressure increase

[0102]

[0103] *G0F(%)=G0F / (G0F+G1F+G2F)

[0104] The galactosylation of monoclonal antibodies appears to change according to the level of increase in osmotic pressure of the culture solution. When the osmotic pressure of the culture broth increases to 480 mOsm / kg or higher during culture, the GOF content actually decreases. That is, there is an appropriate range of osmotic pressure (increased to a maximum of 400 mOsm / kg to 480 mOsm / kg during culture) to maximize the GOF content of monoclonal antibodies.

[0105] Example 1.3: Validation Experiment in a Bioreactor

[0106] Based on the results of primary and secondary shake-flask cultures, confirmatory experiments conducted in a bioreactor demonstrated that increased osmotic pressure led to changes in the galactosylation of monoclonal antibodies.

[0107] During the second feeding (day 4 of cultivation), the osmotic pressure was intentionally increased to 440 mOsm / kg, 500 mOsm / kg and 523 mOsm / kg respectively by adding 4M sodium chloride (NaCl) aqueous solution to the culture medium in a 1.4L bioreactor with a working volume of 1L before cultivation.

[0108] Table 4. Changes in antibody quality due to differences in the range of osmotic pressure increase in bioreactors

[0109]

[0110] *G0F(%)=G0F / (G0F+G1F+G2F)

[0111] When the osmotic pressure increased to a higher level of 500 mOsm / kg during the second feed (final osmotic pressures were 549 mOsm / kg and 567 mOsm / kg), the G0F content actually decreased. In the bioreactor experiment, where the osmotic pressure increased to 440 mOsm / kg during the second feed, the G0F content was 71.8%.

[0112] Example 2: The addition of asparagine to the culture solution produces excess ammonium ions.

[0113] According to the experimental results (Table 5), when excess asparagine is added to the culture solution, the ammonium ions (NH4+) in the culture solution... + The concentration of ammonium ions in the culture solution increases significantly. Galactosylation of monoclonal antibodies can be indirectly regulated by adding an excess of asparagine to increase the concentration of ammonium ions in the culture solution.

[0114] Table 5. Increase in ammonium levels caused by the addition of asparagine to the culture medium.

[0115]

[0116] Example 2.1: Feed-and-separation culture with added asparagine concentrate

[0117] Fed-batch cultures were performed using 250 mL shake flasks, with an actual scale of 30 mL. The feeding strategy and timing of adding sodium chloride aqueous solution in the fed-batch culture were discussed. Figure 3 The same applies as shown. Asparagine concentrate was prepared to a concentration of 200 mM. Supplemental culture medium was added on days 1, 4, 7, and 10 of culture, with asparagine concentrate added at each subsequent feed starting from the second feed point (day 4 of culture) (a total of 3 times). After culture, expression levels and galactosylation of the monoclonal antibody were analyzed.

[0118] Table 6. Changes in galactosylation of monoclonal antibodies caused by the addition of asparagine.

[0119]

[0120] *G0F(%)=G0F / (G0F+G1F+G2F)

[0121] As asparagine was added to the culture solution via fed-batch addition, the GOF content increased. With increasing asparagine concentration, the final NH4+ concentration in the culture solution also increased. The increase in GOF content induced by asparagine injection in 1X production medium was greater than that in 1.4X production medium. When asparagine was added all at once at the start of culture, the NH4+ concentration became higher than at the start of culture, thus delaying cell growth and ultimately reducing the final antibody expression level. Therefore, a method of adding asparagine in fractional doses during fed-batch addition was adopted.

[0122] Example 2.2: Feeding with supplemental culture medium containing excess asparagine

[0123] In fed-batch culture using a bioreactor, unlike shake-flask fed-batch culture, asparagine concentrate was not prepared separately but included in an additional culture medium. Based on a protocol that adds an extra 6 mM asparagine during feeding, an additional culture medium containing 120 mM asparagine was prepared and used for feeding. Cultures were performed in a 1.4 L bioreactor with a working volume of 1 L, and the expression levels and galactosylation of monoclonal antibodies were analyzed.

[0124] Table 7. Bioreactor experiments using supplementary culture medium with added excess asparagine.

[0125]

[0126] Batch cultured with an additional medium containing excess asparagine showed a 10.2% increase in G0F content. This is likely due to the presence of NH4+ in the culture solution containing excess asparagine. + The concentration increased by 9.9 mM, confirming that the decrease in galactosylation of the monoclonal antibody was due to NH4+ in the culture solution.+ Changes in concentration. The osmotic concentration in the culture solution with added excess asparagine increased by 58 mOsm / kg.

[0127] Example 3: Combination of increased osmotic pressure and addition of asparagine

[0128] During fed-batch culture, the GOF of monoclonal antibodies can be increased by intentionally increasing osmotic pressure or adding asparagine. The effects of increasing osmotic pressure and adding asparagine in combination on the galactosylation of monoclonal antibodies and the formation of charged antibody isoforms (charged variants) were investigated.

[0129] Example 3.1: Experiment on simultaneously increasing osmotic pressure and adding asparagine in a bioreactor

[0130] Fed-batch culture was conducted in a 1.4L bioreactor with a working volume of 1L by simultaneously increasing osmotic pressure and adding asparagine. In the case of bioreactor with simultaneous osmotic pressure increase and asparagine addition, considering the effect of asparagine addition on increasing osmotic pressure (120mM asparagine added to the supplementary medium), the osmotic pressure of the culture medium increased to a maximum of 423 mOsm / kg. After culture, the expression level and galactosylation status of monoclonal antibodies were analyzed.

[0131] Table 8. Changes in galactosylation induced by simultaneous increase in osmotic pressure and addition of asparagine in a bioreactor

[0132]

[0133] Between two different scenarios—one involving osmotic pressure alone and the other involving a combination of osmotic pressure and asparagine—the difference in G0F was not significant, but the G0F content increased further. When asparagine was added to the culture medium, the percentage of acidic antibody isoforms (acidic variants) decreased compared to the culture medium without asparagine (Tables 6 and 7). However, when a combination of osmotic pressure and asparagine was added to the culture broth, the percentage of acidic antibody isoforms (acidic variants) actually increased.

[0134] To increase GOF content, methods such as increasing osmotic pressure, adding asparagine, and simultaneously increasing osmotic pressure while adding asparagine can be employed. In particular, the strategy of simultaneously increasing osmotic pressure and adding asparagine appears to be more useful in order to increase the percentage of acidic antibody isoforms (acidic variants) in the charge (antibody) distribution and the GOF content.

[0135] Those skilled in the art will understand that the invention may be implemented in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered in all respects as illustrative rather than restrictive. Therefore, the scope of the invention is indicated by the appended claims rather than the foregoing description. All modifications falling within the meaning and scope of the equivalents of the claims should be included within the scope of the invention.

Claims

1. A method for preparing adalimumab with reduced galactosylation, comprising increasing the osmotic pressure of the animal cell culture solution expressing adalimumab, characterized in that, An increase in osmotic pressure is achieved by adding sodium chloride or potassium chloride to the culture solution of animal cells; Increase osmotic pressure to adjust the final osmotic pressure of the culture solution to the range of 460 mOsm / kg to 500 mOsm / kg; The main culture begins on day 0, with an increase in osmotic pressure performed on either day 4 or day 7; and The animal cells mentioned are CHO cells.

2. The method as described in claim 1, characterized in that, The culture is carried out by fed-batch culture, batch culture, continuous culture, or perfusion culture.

3. The method as described in claim 1, characterized in that, Sodium chloride or potassium chloride is supplemented by adding a feed medium containing sodium chloride or potassium chloride.

4. The method as described in claim 1, characterized in that, The method described is for preparing adalimumab groups with reduced galactosylation.

5. A method for reducing the galactosylation of adalimumab, comprising increasing the osmotic pressure of the animal cell culture solution expressing adalimumab, characterized in that, An increase in osmotic pressure is achieved by adding sodium chloride or potassium chloride to the culture solution of animal cells; Increase osmotic pressure to adjust the final osmotic pressure of the culture solution to the range of 460 mOsm / kg to 500 mOsm / kg; The main culture begins on day 0, with an increase in osmotic pressure performed on either day 4 or day 7; and The animal cells mentioned are CHO cells.

6. A method for preparing adalimumab with reduced galactosylation, comprising: (i) Increase the osmotic pressure of the animal cell culture solution expressing adalimumab, and (ii) During animal cell culture, asparagine is added to the culture solution. One method of increasing osmotic pressure is to add sodium chloride or potassium chloride to the culture solution of animal cells. This involves increasing the osmotic pressure, thereby adjusting the final osmotic pressure of the culture solution to be in the range of 460 mOsm / kg to 500 mOsm / kg; The main culture begins on day 0, with an increase in osmotic pressure performed on either day 4 or day 7; and The animal cells mentioned are CHO cells. The method described herein is intended to reduce the galactosylation of adalimumab and to regulate the content of acidic antibody isoforms (acidic variants) in the adalimumab family prepared by the method.

7. The method as described in claim 6, characterized in that, Simultaneously or sequentially, osmotic pressure is increased and asparagine is supplemented.

8. The method as described in claim 6, characterized in that, Asparagine supplementation is to replenish asparagine, thereby adjusting the final concentration of asparagine in the animal cell culture solution to the range of 27.6 mM to 33.6 mM.

Images