Specification
Name of invention: Method for regulating galactosylation of recombinant protein through optimization of culture medium
Technology field
[One]
The present invention comprises the step of increasing the osmotic pressure of the animal cell culture medium during the process of culturing animal cells expressing the desired recombinant protein, the method for producing the desired recombinant protein or the method for producing the desired recombinant protein, wherein the galactosylation is regulated. A method for regulating galactosylation of proteins; In the process of culturing animal cells expressing the desired recombinant protein, comprising the step of replenishing the animal cell culture medium with asparagine, the method for producing galactose is regulated, the method for preparing the desired recombinant protein, or the galactosylation of the desired recombinant protein is controlled. How to; During the cultivation process of animal cells expressing the desired recombinant protein, the method comprising the steps of raising the osmotic pressure of the animal cell culture solution and replenishing asparagine, wherein the galactose is regulated, the method for producing the desired recombinant protein or the desired recombinant protein To regulate galactosylation of; And a target recombinant protein produced by the above method, wherein the galactosylation is regulated.
[2]
Background
[3]
Antibodies are proteins that can bind antigens and interfere with the action of antigens or eliminate antigens. As the therapeutic antibody market has been found to have considerable potential, research for efficient expression and mass production of antibodies has been actively conducted. have. One of the important points in the production of such antibodies is the homogeneity of the population of antibodies produced. In particular, in the production of monoclonal antibodies, the constant and reproducible monoclonal antibody glycoform profile is important. However, during the cell culture process, various variables may affect the glycosylation profile of the monoclonal antibody produced. Accordingly, there has been a demand for development of a method capable of constantly controlling the glycosylation of the prepared antibody population and the content of isomer antibodies.
[4]
[5]
One of the important tasks in the commercial production process of monoclonal antibodies is to consistently maintain the quality of the monoclonal antibodies produced per batch. The quality to be checked during the production process of monoclonal antibodies is numerous, but galactosylation of the antibody can be considered as one of the most important. This is because galactosylation is known to affect the process of CDC (complement dependent cytotoxicity), one of the largest activity mechanisms (MOAs) of monoclonal antibodies. The galactosylation is performed by using galactose as a constituent unit of a glycosylation chain reaction, and then attaching it to the N-acetylglucosamine sugar through galactosyltransferase. As a method for regulating such galactosylation, a method of adding manganese or galactose to a culture solution has been proposed (US Publication No. 2012-0276631), but the development of a method for regulating galactosylation of antibodies is still required. do.
[6]
Detailed description of the invention
Technical challenges
[7]
Under this background, the present inventors tried hard to develop a method capable of effectively controlling the galactosylation of the recombinant protein produced in the process of culturing animal cells producing recombinant protein, and during the cultivation process of animal cells expressing the recombinant protein. When raising the osmotic pressure of the animal cell culture at a specific culture time point or supplementing asparagine, it was confirmed that galactosylation of the recombinant protein produced can be effectively produced. In addition, when supplementing asparagine with an increase in osmotic pressure of the animal cell culture, it was confirmed that it is possible to control galactosylation without reducing the content of the acidic isomer antibody due to the increase in osmotic pressure, thereby completing the present invention.
[8]
Task resolution
[9]
One object of the present invention comprises the step of raising the osmotic pressure of the animal cell culture medium during the cultivation process of the animal cell expressing the desired recombinant protein, galactosylation (galactosylation) is regulated, the method for producing the desired recombinant protein Is to provide
[10]
Another object of the present invention is to provide a method for regulating galactosylation of a desired recombinant protein, comprising the step of raising the osmotic pressure of the animal cell culture medium during the cultivation process of the animal cell expressing the desired recombinant protein.
[11]
Another object of the present invention, comprising the step of supplementing asparagine to the animal cell culture during the process of culturing animal cells expressing the desired recombinant protein, galactosylation is regulated, to provide a method for producing a desired recombinant protein will be.
[12]
Another object of the present invention is to provide a method for regulating galactosylation of a desired recombinant protein, comprising the step of supplementing asparagine with the animal cell culture medium during the process of culturing animal cells expressing the desired recombinant protein. .
[13]
Another object of the present invention includes the step of raising the osmotic pressure of the animal cell culture medium and supplementing asparagine during the cultivation process of the animal cell expressing the desired recombinant protein. It is to provide a manufacturing method.
[14]
Another object of the present invention to increase the osmotic pressure of the animal cell culture medium during the process of culturing animal cells expressing the desired recombinant protein, and supplementing asparagine, to regulate the galactosylation of the desired recombinant protein Is to provide a way.
[15]
Another object of the present invention is to provide a desired recombinant protein prepared by the above method.
[16]
Effects of the Invention
[17]
The method of the present invention has the advantage of effectively controlling the galactose content of the desired recombinant protein to a desired range. In addition, since the method of the present invention using both the increase in osmotic pressure and the addition of asparagine has an effect of controlling not only galactose but also the content of acidic isomer antibodies, it can be effectively used in the production of the desired antibody population.
[18]
Brief description of drawings
[19]
1 shows the expression vector of adalimumab biosimilar.
[20]
Figure 2 shows the injection time of sodium chloride aqueous solution during the fed-batch culture period.
[21]
Figure 3 shows the injection time of asparagine during the fed-batch culture period.
[22]
Best mode for carrying out the invention
[23]
One aspect of the present invention for achieving the above object comprises the step of raising the osmotic pressure of the animal cell culture medium during the cultivation process of the animal cell expressing the desired recombinant protein, galactosylation is regulated, the desired recombinant protein It is a manufacturing method.
[24]
[25]
Specifically, the method is a method for producing a desired recombinant protein, wherein galactosylation is regulated, including the step of raising the osmotic pressure of the animal cell culture medium in a specific culture day during the process of culturing animal cells expressing the desired recombinant protein. .
[26]
[27]
In the present invention, the culture may be fed-batch culture, but is not limited thereto.
[28]
In the present invention, the term "fed-batch culture (fed-batch culture)" is to start the culture with a base medium or production medium and then continuously or discontinuously feeding the feeding medium to the culture at any time during the cell growth phase or antibody production phase. Refers to ongoing cell culture.
[29]
In the present invention, "cell culture medium" or "culture medium" refers to a nutrient solution for maintenance, growth, proliferation or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. The cell culture medium may be optimized for specific cell culture, for example, a basic culture medium prepared to support cell growth, or a cell culture production medium prepared to promote the production of monoclonal antibodies, and concentrated by nutrient concentration. There is a concentrated medium. The terms nutrient and medium components refer to the components constituting the cell culture medium, and are used interchangeably herein.
[30]
Specifically, the term "basic culture medium" or "basic medium" means a medium capable of supporting minimal cell growth. The basic medium supplies standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as trace elements, vitamins, energy sources, buffer systems and amino acids. Dulbecco's Modified Eagle's Medium (DMEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12 medium are included, but are not limited to.
[31]
Also, specifically, the term "cell culture production medium" or "production medium" refers to a medium used for the purpose of maximizing the expression of a monoclonal antibody in a bioreactor. The production medium may be the same as or different from the basic medium, and if so, it may be produced by concentrating the basic medium itself or by adding specific ingredients to the basic medium.
[32]
The terms "feeding medium" and "additional culture medium" used in the present invention are medium composed of a specific nutrient or a plurality of nutrients, and may be all concentrated products of a basic medium. Depending on the cell being cultured, various components and concentrations of the feeding medium can be produced.
[33]
[34]
In the culturing process, the cell growth phase refers to a time when cell growth rapidly progresses after inoculation. In the case of Chinese hamster ovary (CHO) cells, the temperature is known to increase most actively in the range of 35 to 37 ° C and pH of 7.0 to 7.3. In the present invention, the cell growth phase culture parameter was a temperature of 36.5 ° C and a pH of 7.1 or 7.0.
[35]
The term "inoculation" refers to injecting a medium to start cell culture and injecting cells into the injected medium. Here, the medium may be pre-injected prior to injecting the cells or may be injected into the cell reactor simultaneously with the cells. In a large-scale animal cell culture, the medium can be injected in advance, and the cells can be injected after maintaining the temperature and oxygen saturation within a predetermined range.
[36]
The term "antibody production phase" refers to the time when the change of the culture process is applied to maximize the production of monoclonal antibodies. Here, changes in the process for maximizing production include changing the medium or lowering the dissolved oxygen.
[37]
[38]
In the present invention, it was confirmed that the degree of change in galactosylation of the recombinant protein produced in animal cells expressing the recombinant protein may vary depending on the point of time when the osmotic pressure is increased. That is, when the osmotic pressure was gradually increased during the culture period, the degree of galactosylation did not significantly differ from the group that did not increase the osmotic pressure, whereas when the osmotic pressure was increased at a specific culture time point during the culture period, the G0F content increased significantly. By indicating, it was confirmed that it is possible to control galactosylation, specifically to inhibit or reduce galactosylation.
[39]
[40]
In addition, the step of raising the osmotic pressure in the present invention is preferably performed during the main culture process of animal cells expressing the desired recombinant protein, but is not limited thereto.
[41]
As used herein, the term "main culture" refers to a culture step in which actual production of a monoclonal antibody is performed. As the last step of the culture process started with one vial of cells, the purification of the produced monoclonal antibody is continued when the main culture is completed. It can be distinguished from the term seed culture for the purpose of gradually increasing the culture volume prior to the main culture.
[42]
[43]
The step of raising the osmotic pressure may be performed at a specific culture time point during the main culture process of animal cells, and specifically, performed on any one of the culture days from the 1st to the 10th day of the main culture based on the 0th day of the start of the main culture. It may be, and more specifically, may be performed on any one of the 1st, 4th, 7th and 10th culture days based on the 0th day of the start of the main culture, and more specifically, the 0th day of the start of the main culture. It may be performed on the 4th or 7th as a standard, but is not limited thereto.
[44]
In addition, the step of raising the osmotic pressure may be performed in a single step during the culture process of animal cells expressing the desired recombinant protein.
[45]
[46]
In addition, the step of raising the osmotic pressure is not particularly limited, but may be performed such that the final osmotic pressure in the culture medium is 460 to 500 mOsm / kg, and more specifically, the increase in the osmotic pressure is 460 to the final osmotic pressure in the culture medium. It may be performed to be 480 mOsm / kg, but is not limited thereto. The final osmotic pressure in the culture medium refers to the osmotic pressure appearing at the end of the culture, but is not particularly limited.
[47]
In addition, the step of raising the osmotic pressure may be an osmotic pressure increase target in the culture medium of 400 to 440 mOsm / kg, more specifically, 430 to 440 mOm / kg, but is not limited thereto. The osmotic pressure increase target in the culture medium refers to the degree of osmotic pressure to be increased at the time of performing the step of raising the osmotic pressure, but is not particularly limited thereto.
[48]
In addition, the osmotic pressure increase may be performed by one or more methods selected from the group consisting of a method of supplementing sodium chloride or potassium chloride to the culture medium of the animal cell and a method of adding glucose to the culture medium, and more specifically, supplementing the culture medium with sodium chloride. It may be performed in a method, but is not limited thereto.
[49]
The method of replenishing the sodium chloride to the culture solution may be performed by adding a 4M aqueous sodium chloride solution to the culture solution on a specific day during the fed-batch culture process to increase the osmotic pressure to a certain level. It is not limited to this. When the osmotic pressure in the culture medium, and even the cell, is increased through the addition of an aqueous sodium chloride solution, the activity of beta-galactosidase increases, so that the galactosylation of the monoclonal antibody can be suppressed.
[50]
In the present invention, the term "culture" or "culture medium" refers to a liquid containing cells, contained in a shaker flask or bioreactor in culture. The culture and the culture medium can be used interchangeably. In addition, the culture medium and the culture medium can be distinguished according to the presence or absence of animal cells.
[51]
[52]
In the present invention, the recombinant protein may be an antibody, preferably a monoclonal antibody.
[53]
As used herein, the term "monoclonal antibody" refers to an antibody that can be produced by a single antibody-forming cell, and recognizes one antigenic determinant.
[54]
The antibody of the present invention is not limited thereto, but may preferably include all therapeutic antibodies commonly used in the art, and may specifically be adalimumab.
[55]
In addition, the antibody is a concept that includes both the full-length antibody and the form of the antibody fragment, and the type of the antibody fragment includes Fv, Fab, Fab ', F (ab') 2 , Fd, and the like. The Fv includes both double disulfide Fv (dsFv) and short chain Fv (scFv) forms. Fd refers to the heavy chain portion contained in the Fab fragment.
[56]
Animal cells expressing the desired recombinant protein include, without limitation, natural or transformed cells expressing the recombinant protein. For the purposes of the present invention, it may be an animal cell capable of expressing a recombinant protein targeted for regulating galactose content, and may be, for example, a Chinese hamster ovary cell line (CHO) or a mouse myeloma cell line (NSO), but is not limited thereto. no.
[57]
[58]
In addition, in order to control the galactosylation of the monoclonal antibody, optimization of the culture parameters, concentration of the basic culture medium, and feeding of the optimized additional culture medium may be included.
[59]
That is, after setting the galactosylation range of the desired monoclonal antibody, analyzing the galactosylation of the monoclonal antibody expressed by the retained cell line, the method of controlling the galactosylation of the monoclonal antibody specified above according to the degree of change in galactosylation is also applied. You can.
[60]
Specifically, examples of changes that can be implemented in the operation of the incubator for the production of monoclonal antibodies with regulated galactosylation are optimization of culture parameters such as dissolved oxygen (DO), acidity (pH), culture temperature, and agitation. . Specifically, in the cell growth phase, the temperature was 36.5 ° C, dissolved oxygen was 30%, and the acidity was 7.0. In the antibody production phase, the temperature was 30 ° C, dissolved oxygen was 30%, and the acidity was 7.0. In the method, it was confirmed that it is possible to control the galactosylation of the monoclonal antibody when cultured by changing the temperature in the antibody production machine to 28 ° C, dissolved oxygen to 20%, and acidity to 6.9.
[61]
In addition, the basic culture medium may be concentrated and used to control galactose. Specifically, a culture medium without animal-derived protein can be used, and the basic medium developed for fed-batch culture can be optimized to change galactosylation of monoclonal antibodies. Specifically, when the medium in which the basic culture medium was concentrated, specifically 0.8 or 1.4 times concentrated in this culture was used, it was possible to control the galactosylation of the monoclonal antibody.
[62]
In addition, it is possible to optimize the additional culture medium for the control of galactosylation. In the present invention, by adjusting the concentration of the additional culture medium, the concentration of metal components added to the additional culture medium was optimized to reduce galactose.
[63]
[64]
Galactosylation of the desired recombinant protein prepared through the above method can be measured by N-glycan profile analysis using Q-TOF or UPLC devices. The information provided by N-glycan profile analysis varies with G0F content, GI (Galactosylation Index), NGHC (non-glycosylated heavy chain), percent afucosylation, and high mannose contents, but the present invention The major changes in G0F content were noted in.
[65]
[66]
In addition, the method of the present invention may be for preparing a population of antibodies with reduced galactosylation. That is, after analyzing the galactosylation of the monoclonal antibody expressed by the cell line, it is a method that can be applied to prepare a population of antibodies having a lower level of galactosylation than the analyzed value.
[67]
[68]
In the present invention, the term, "antibody population" refers to a group of antibodies comprising antibodies that may vary in sugar chain content, and for the purposes of the present invention, the antibody population comprises galactosylated antibodies in the desired ratio, galactosylation Means a group of regulated antibodies.
[69]
[70]
Another aspect of the present invention is a method of controlling galactosylation of a desired recombinant protein, comprising the step of raising the osmotic pressure of the animal cell culture medium during the process of culturing the animal cell expressing the desired recombinant protein.
[71]
The method and each term are as described above.
[72]
Specifically, the method may include the step of raising the osmotic pressure of the animal cell culture solution on a specific culture day during the cultivation process of the animal cell expressing the desired recombinant protein, as described above in detail.
[73]
[74]
Another aspect of the present invention is a method for producing a desired recombinant protein, wherein galactosylation is regulated, comprising the step of supplementing asparagine in the animal cell culture medium during the cultivation process of the animal cell expressing the desired recombinant protein.
[75]
The terms described above are as described above.
[76]
In the present invention, it was confirmed that when the animal cell culture medium is supplemented with asparagine during the cultivation process of animal cells expressing the desired recombinant protein, galactosylation of the desired recombinant protein can be controlled.
[77]
[78]
Specifically, the asparagine may be added in the form of a culture medium containing asparagine concentrate or asparagine.
[79]
In addition, the supplementation of the asparagine may be performed multiple times at a specific culture point in the process of culturing animal cells expressing the desired recombinant protein. Here, the supplementation of the asparagine can be performed from 4 days to 10 days of culture based on the 0th day of the main culture, and specifically, performed on 4th, 7th and 10th days of culture on the 0th day of the main culture. Thus, the concentration of asparagine in the culture medium can be gradually increased.
[80]
When asparagine is replenished in a single step, there is a disadvantage in that the concentration of NH 4 + in the culture medium is rapidly increased, resulting in a delay in cell growth and a decrease in the final protein expression level. In this case, there is an advantage of achieving the desired amount of protein production while controlling galactosylation without these disadvantages.
[81]
[82]
The supplementation of the asparagine may be performed so that the final concentration of asparagine in the culture of animal cells is 27.6 to 33.6 mM.
[83]
Specifically, asparagine may be supplemented so that the final concentration of asparagine in the culture of animal cells is 33.6 mM, for example, asparagine may be supplemented such that the concentration of asparagine in the culture of animal cells is additionally increased by 6 to 18 mM, As a more specific example, asparagine may be supplemented three times by sequentially adding asparagine concentrations in animal cells in 6mM, 12mM, and 18mM increments, but is not limited thereto.
[84]
When this method is applied to animal cells, ammonium ion (NH 4 + ) induction is induced to increase the concentration of ammonium ion in the cell, and accordingly, the pH of the trans-Golgi body increases to increase the activity of beta-galactosyltransferase. Falling may inhibit the galactosylation of monoclonal antibodies.
[85]
[86]
In addition, as described above, in order to control the galactosylation of the monoclonal antibody, optimization of the culture parameters, concentration of the basic culture medium, and feeding of the optimized additional culture medium may be included.
[87]
That is, after setting the galactosylation range of the desired monoclonal antibody, analyzing the galactosylation of the monoclonal antibody expressed by the retained cell line, the method of controlling the galactosylation of the monoclonal antibody specified above according to the degree of change in galactosylation is also applied. You can.
[88]
[89]
Another aspect of the present invention is a method of controlling galactosylation of a desired recombinant protein, comprising the step of supplementing asparagine with the animal cell culture medium during the cultivation process of the animal cell expressing the desired recombinant protein.
[90]
The method and each term are as described above.
[91]
[92]
Another aspect of the present invention includes the step of increasing the osmotic pressure of the animal cell culture medium and supplementing asparagine during the cultivation process of the animal cell expressing the desired recombinant protein, wherein the desired recombinant protein is regulated with galactosylation. It is a manufacturing method.
[93]
The terms described above are as described above.
[94]
In the present invention, when adding asparagine while increasing the osmotic pressure of the animal cell culture medium during the cultivation process of the animal cell expressing the desired recombinant protein, monoclonal antibody, at the same time as the decrease in galactosylation due to the increase in osmotic pressure, decreases as the osmotic pressure increases It was confirmed that the content of the acidic isomer antibody that can be increased by the addition of asparagine.
[95]
[96]
In the above method, the increase in osmotic pressure and supplementation of asparagine may be performed simultaneously or sequentially. For example, the osmotic pressure in the culture medium is increased by a method of supplementing sodium chloride, and the like, supplemented with asparagine, or supplemented with asparagine, and then the osmotic pressure in the culture medium is increased by adding sodium chloride, etc. The osmotic pressure rise in the culture can be applied simultaneously.
[97]
Here, the osmotic pressure may be increased by applying the above-described method.
[98]
The increase in osmotic pressure may be performed so that the final osmotic pressure in the culture medium is 460 to 500 mOsm / kg, and supplementation of the asparagine may supplement asparagine so that the final concentration of asparagine in the culture of animal cells is 27.6 to 33.6 mM. have.
[99]
[100]
In addition, the recombinant protein may be an antibody, specifically a monoclonal antibody, and the content of the acidic isomer antibody of the antibody population produced by the method at the same time as controlling the galactosylation of the desired recombinant antibody through the method of the present invention as described above. Can be adjusted. That is, it is possible to decrease galactosylation with increasing osmotic pressure, and at the same time, increase the content of acidic isomer antibodies that can decrease with increasing osmotic pressure as the addition of asparagine.
[101]
Therefore, using the above method has the advantage of being able to control the degree of galactosylation of the desired antibody population and at the same time the content of the acidic isomer antibody of the desired antibody population.
[102]
The acidic isomer is an isomer. An isomer antibody refers to an antibody in which some amino acids of the main active antibody are modified by deamine or oxidation, and includes acidic and basic isomer antibodies. Examples include isomers of asparagine from amino acids that have been deaminated to become aspartates, isomers of amino acids with methionine oxidized to become methionine sulfate, and the like. In addition, when glutamate is present at the N-terminal of the heavy chain, the glutamate forms a pentagonal ring structure to include an isomer antibody modified with pyruglutamate.
[103]
Analysis of the isomer antibody may be performed using chromatography or the like, and in the present invention, it is performed by cation exchange resin chromatography analysis. Depending on the properties of the monoclonal antibody, the contents of acidic, main, and basic isomers vary widely. Depending on the culture conditions (culture parameters, production medium, etc.) of the cell line expressing the monoclonal antibody, the charge isomer antibody content of the monoclonal antibody may be changed. Thus, the method has the advantage of controlling the content of the acidic isomer antibody to the desired range at the same time as controlling the desired galactose content.
[104]
Mode for carrying out the invention
[105]
Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.
[106]
[107]
Example 1: Regulation of galactosylation due to artificial increase in osmotic pressure
[108]
[109]
There are various methods to increase the osmotic pressure of the culture medium during the fed-batch culture. There is a method of adding excess glucose to the culture solution, a method of adding potassium chloride or sodium chloride aqueous solution to the culture solution, and the like. In the present invention, as a representative method for increasing the osmotic pressure, a method of adding 4M sodium chloride aqueous solution to the culture solution was applied.
[110]
[111]
Description of manufacturing method and medium composition
[112]
[113]
In the present invention, the monoclonal antibody to which the osmotic pressure rise or asparagine addition to the culture medium is applied is an adalimumab biosimilar antibody, and adalimumab is a therapeutic agent for rheumatoid arthritis and Crohn's disease developed by abbott. After amplifying and generating adalimumab biosimilar DNA through polymerase chain reaction with reference to the amino acid sequences of the heavy and light chains of adalimumab antibody of US 6,090,382, pCB using the promoter of pGL3 CUCBin, a vector developed by the company -Am2.77_v5.4 (FIG. 1) was produced and transformed into a CHO-DXB11 cell line to produce an adalimumab biosimilar expressing cell line. PCUCBin developed by the company is one of the vectors mentioned in the Korean patent (a new fusion promoter and a recombinant vector containing the same, registration number 10-1038126).
[114]
[115]
The culture medium used in the present invention is three types: a basic culture medium, a production medium (this culture medium), and an additional culture medium (feeding medium).
[116]
The basic culture medium is a medium used for passage. The production medium is a medium used for culture (main culture) performed for full-scale antibody production after subculture, and may be prepared by strengthening a specific component in a basic culture medium or adding a new component. In the present invention, a 1.4-fold enhanced basic culture medium was used as a production medium. The additional culture medium is a culture medium added for the purpose of promoting cell growth and increasing the amount of antibody expression during the culture. In the present invention, a 3.3-fold enhanced basic culture medium was used as an additional culture medium. The basic culture medium, the production medium, and the additional culture medium are mediums modified in IMDM (Iscove's Modified Dulbecco's Medium), and the media components are shown in Table 1.
[117]
Table 1 [Table 1] Basic culture medium composition
division ingredient
IMDM modified medium Merck 100131
Buffer HEPES
Buffer Sodium bicarbonate
Carbon source Glucose
Nitriogen source Glutamine
Etc Sodium chloride
Growth hormone Insulin
Etc MTX
Vitamin L-ascorbic acid
Vitamin Biotin
Vitamin Choline chloride
Vitamin Folic acid
Peptone Sheff CHO plus pf acf (Kerry)
Metal Boric acid
Metal Cobalt Chloride Hexahydrate
Metal Copper Sulfate Pentahydrate
Metal Ferric Citrate
Metal Magnesium chloride anhydrous
Metal Manganese sulfate monohydrate
Metal Potassium nitrate
Metal Sodium selenite pentahydrate
Metal Zinc sulfate heptahydrate
[118]
[119]
The method for preparing the antibody used in the present invention is an animal cell culture through a bioreactor, and the animal cell culture is a process of growing animal cells in a culture medium and expressing the antibody through special measures (eg, lowering the temperature). The cultivation method is various such as batch culture, fed-batch culture, continuous culture, medium exchange culture, etc., but the culture method of the adalimumab biosimilar cell line used in the present invention is fed-batch culture. The fed-batch culture is a culture method in which the culture is performed with the production medium while the additional culture medium is added once or twice on a specific day of culture.
[120]
[121]
The fed-batch culture method used in the examples is to add additional culture medium in an amount corresponding to 5% of the current culture medium volume of the production medium four times on the 1st, 4th, and 7th day of culture.
[122]
[123]
Example 1.1: Change in galactosylation with increasing osmotic pressure
[124]
[125]
Using a 250 mL shaker flask, fed-batch culture was performed at an actual 30 mL scale. Feeding strategy of fed-batch culture and the addition time of sodium chloride aqueous solution were as shown in FIG. 2. Osmotic pressure was artificially raised to 430 mOms / kg by artificially raising the osmotic pressure to 430 mOms / kg by feeding 4M sodium chloride (NaCl) aqueous solution to the culture starting with 320 mOsm / kg. The additional culture medium is subjected to primary feeding on the first day of culture, and after every 3 days, additional feeding is performed to perform a total of 4 feedings, divided into once or 4 times (the osmotic pressure gradually increases during the culture period) 4 After adding M sodium chloride (NaCl) aqueous solution to increase the osmotic pressure of the culture and proceed with the culture, the expression level and galactosylation of the monoclonal antibody were analyzed, and the results are shown in Table 2 below.
[126]
Table 2 [Table 2] Change in antibody quality according to osmotic pressure rise
Culture conditions Antibody expression level (mg / L) G0F (%) * Acidic (%) Final osmotic pressure (mOsm / kg)
Osmotic pressure gradually increases during the culture period 1392.0 60.4 20.1 453.0
During the first feeding (1 day of culture), the osmotic pressure is increased. 1215.5 66.1 17.6 445.0
Increases the osmotic pressure during the second feeding (4 days of culture) 1268.3 68.8 16.7 451.0
3rd feeding (7 days of culture) increases osmotic pressure 1438.7 67.1 16.4 451.0
Increases the osmotic pressure during the 4th feeding (10 days of culture) 1580.1 66.2 17.4 454.0
Does not increase osmotic pressure 1771.8 63.5 18.5 364.0
[127]
* G0F (%) = G0F / (G0F + G1F + G2F)
[128]
[129]
As a result, as shown in Table 2, there was a difference in galactosylation of the monoclonal antibody according to the timing of feeding the 4M sodium chloride (NaCl) aqueous solution. In the case of the shaker flask culture, it is not easy to directly control the dissolved oxygen (DO) and the acidity (pH) during the culture process, so the difference in G0F between experimental conditions may not be significant. However, in bioreactor culture, the dissolved oxygen and the degree of acidification can be controlled directly, so the difference in G0F can be maximized.
[130]
[131]
Example 1.2: Change in galactosylation of monoclonal antibody according to the difference in the range of osmotic pressure increase
[132]
[133]
Example 1.1 showed that the galactosylation of the monoclonal antibody may vary depending on the point of time in which the osmotic pressure of the culture is raised. When the sodium chloride aqueous solution was fed at the second feeding (4 days of culture) and the 3rd feeding (7 days of culture) of the additional feeding medium, the G0F was increased to the maximum. Additionally, a secondary shaker flask fed-batch culture was performed to investigate the effect of the difference in the osmotic pressure increase level of the culture on galactosylation of the monoclonal antibody. The osmotic pressure at the beginning of the culture is 320 mOsm / kg, and different amounts of 4 M sodium chloride (NaCl) aqueous solution are added to the culture solution on the 4th or 7th day of the culture, similar to the primary shaker flask culture, depending on the osmotic pressure increase target. Then, the expression level and galactosylation of the monoclonal antibody were analyzed.
[134]
Table 3 [Table 3] Change in antibody quality according to the difference in the range of osmotic pressure increase
Culture conditions Osmotic pressure increase target (mOsm / kg) Antibody expression level (mg / L) G0F (%) * Acidic (%) Final osmotic pressure (mOsm / kg)
Osmotic pressure rise X X 1136.2 67.4 15.6 365.0
Increases the osmotic pressure during the second feeding (4 days of culture) Up to 400 948.9 70.4 15.0 424.0
Up to 440 824.1 71.0 14.6 460.0
Up to 480 573.7 67.9 14.5 505.0
Up to 520 543.8 66.5 13.9 539.0
3rd feeding (7 days of culture) increases osmotic pressure Up to 400 1053.4 70.5 14.9 416.0
Up to 440 927.4 69.5 14.5 454.0
Up to 480 851.1 68.5 14.3 497.0
Up to 520 775.1 66.9 14.2 533.0
[135]
* G0F (%) = G0F / (G0F + G1F + G2F)
[136]
[137]
Depending on the level of osmotic pressure increase in the culture medium, galactosylation of the monoclonal antibody was varied. When the osmotic pressure of the culture was increased to 480 mOsm / kg or more during the cultivation, the G0F content was rather decreased. That is, there was an appropriate range of osmotic pressure to increase the G0F content of the monoclonal antibody to the maximum (up to 400 ~ 480 mOsm / kg during the culture).
[138]
[139]
Example 1.3: Bioreactor (Bioreactor) verification experiment
[140]
[141]
As a result of performing the verification experiment in the bioreactor based on the primary and secondary shaker flask culture results, it was again possible to confirm the change in galactosylation of the monoclonal antibody according to the rising conditions of osmotic pressure.
[142]
In the actual 1 L scale in a 1.4 L bioreactor, upon secondary feeding (4 days of culture), 4 M sodium chloride aqueous solution was added to the culture to artificially increase the osmotic pressure to 440, 500, 523 mOsm / kg and then culture. Proceeded.
[143]
Table 4 [Table 4] Change of antibody quality according to the range of osmotic pressure increase in bioreactor
Osmotic pressure (mOsm / kg) rising condition during the 2nd feeding Expression level (mg / L) G0F (%) * Acidic (%) NH 4 + concentration (mM) Final osmotic pressure (mOsm / kg)
Do not raise 1980.6 63.7 26.5 3.6 321
Raises to 440 1740.2 71.8 18.4 7.7 479
To 500 1361.6 58.3 18.4 7.3 549
Raises to 523 1361.7 54.8 19.3 5.6 567
[144]
* G0F (%) = G0F / (G0F + G1F + G2F)
[145]
[146]
When the osmotic pressure of the culture medium was increased by 500 mOsm / kg or more during the second feeding (final osmotic pressure 549, 567 mOsm / kg), the G0F content decreased. In the case of the bioreactor experiment in which the osmotic pressure was increased to 440 mOsm / kg during the second feeding, the content of G0F was 71.8%.
[147]
[148]
Example 2: Excess ammonium production in culture medium by adding asparagine
[149]
[150]
According to the results of the applicant's culture (Table 4), when an excess of asparagine was added to the culture, the concentration of ammonium ion (NH 4 + ) in the culture increased dramatically. Galactosylation of monoclonal antibodies can be controlled by indirectly increasing the concentration of NH 4 + in the culture medium by adding an excess of asparagine .
[151]
Table 5 [Table 5] Increase of ammonium by adding asparagine to culture
Culture conditions Added asparagine concentration (mM) Final ammonium ion (mM)
No asparagine added. 0 3.4
Asparagine added 18.9 8.1
[152]
[153]
Example 2.1: fed-batch culture adding asparagine concentrated solution
[154]
[155]
Using a 250 mL shaker flask, fed-batch culture was performed at an actual 30 mL scale. Feeding strategies of fed-batch culture and the timing of injecting asparagine were as shown in FIG. 3. The asparagine concentrated solution was prepared at 200 mM. The time of injecting additional culture medium is 1, 4, 7, and 10 days of culture, and the addition of the asparagine concentrate was performed for each feeding from the second feeding time (4 days of culture) (total 3 times). After incubation, the expression level of the monoclonal antibody and galactosylation were analyzed.
[156]
Table 6 [Table 6] Change of galactosylation of monoclonal antibody according to the addition of asparagine
Culture conditions Antibody expression level (mg / L) G0F (%) * Acidic (%) Final NH 4 + (mM)
Production badge Asparagine concentration added in one feeding (mM) Total asparagine concentration added (mM)
1 X 0 0 1738.5 56.3 25.9 4.1
1 X One 3 1705.8 59.9 21.7 5.2
1 X 2 6 1535.2 61.4 19.2 6.7
1 X 4 12 1500.0 64.3 19.3 8.8
1.4 X 0 0 1729.9 60.2 21.1 5.6
1.4 X One 3 1721.0 61.0 21.3 7.4
1.4 X 2 6 1588.5 61.5 20.1 7.3
1.4 X 4 12 1563.7 61.9 19.3 9.0
[157]
* G0F (%) = G0F / (G0F + G1F + G2F)
[158]
[159]
The G0F content increased as the asparagine was added to the culture medium by feeding. As the concentration of asparagine added increased, the final NH 4 + concentration in the culture medium increased. The increase in G0F content for asparagine input was greater in the 1X production medium compared to the 1.4X production medium. When asparagine was added at the beginning of culture at a time, NH 4 + concentration increased from the beginning of culture, cell growth was delayed, and eventually the final antibody expression was lowered.
[160]
[161]
Example 2.2. : Feeding of additional culture medium containing excess asparagine
[162]
[163]
Unlike the shaker flask fed-batch culture, in the bioreactor fed-batch culture, an asparagine concentrated solution was not separately made and included in an additional culture medium. An additional 6 mM of asparagine was planned for each feeding, and an additional culture medium to which 120 mM asparagine was added was prepared and then fed. After the culture was performed in an actual 1 L scale in a 1.4 L bioreactor, the expression level and galactosylation of the monoclonal antibody were analyzed.
[164]
Table 7 [Table 7] Bioreactor experiment using additional culture medium with an excess of asparagine
Culture conditions Antibody expression level (mg / L) G0F (%) Acidic (%) NH 4 + (mM) Final osmotic pressure (mOsm / kg)
No asparagine added 1980.6 63.7 26.5 3.6 321.
From 2nd feeding, 6 mM asparagine is added per feeding (total of 18 mM) 1281.8 73.9 20.6 13.5 379
[165]
The G0F content was increased by 10.2% in a batch using an additional culture medium in which asparagine was added in excess. The asparagine NH excess of culture medium was added 4 + , given that the concentration is increased 9.9 mM, the galactose Chemistry reduction of the mAb in the culture solution NH 4 + can be seen that due to the concentration change. Osmotic pressure was increased by 58 mOsm / kg in the culture medium in which asparagine was added in excess.
[166]
[167]
Example 3: Combination of artificial osmotic pressure elevation and asparagine addition
[168]
[169]
During the fed-batch culture, artificial elevation of osmotic pressure or addition of asparagine was applied to increase the G0F content of the monoclonal antibody. To investigate the effect of monoclonal antibodies on galactosylation and charge isomerism (charge variants), a combination of artificial osmotic pressure elevation and asparagine addition was applied.
[170]
[171]
Example 3.1: Experiment of bioreactor with simultaneous increase in osmotic pressure and addition of asparagine
[172]
[173]
In a 1.4 L bioreactor, an artificial increase in osmotic pressure or an increase in osmotic pressure and addition of asparagine were applied at the same time to a fed-batch culture. In the case of the bio-cultivator in which the osmotic pressure increase and the addition of asparagine were simultaneously applied, the osmotic pressure of the culture was increased to 423 mOsm / kg in consideration of the effect of increasing the osmotic pressure by adding asparagine (adding 120 mM asparagine to the additional culture medium). After incubation, the expression level of the monoclonal antibody and galactosylation were analyzed.
[174]
Table 8 [Table 8] Change in galactosylation by simultaneously applying osmotic pressure and adding asparagine in a bioreactor
Culture conditions Antibody Expression (mg / L) G0F (%) Acidic (%) NH 4 + (mM) Final osmotic pressure (mOsm / kg)
Raises the osmotic pressure to 450 1511.6 73.6 20.6 8.80 502.0
Raises the osmotic pressure to 423 + adds asparagine 1548.1 75.4 23.1 10.5 490.0
[175]
[176]
[177]
Although the difference in G0F between the two conditions applied to the process by combining the osmotic pressure only with the osmotic pressure and asparagine was not great, the G0F content increased further. In the case of a culture in which asparagine was added, the specific gravity of the acidic variant decreased compared to the culture in which no addition was added (Tables 6 and 7), but as a result of applying a combination of osmotic pressure and asparagine, the acidic isomer antibody increased. Did.
[178]
[179]
In order to increase the G0F content, a method of artificially increasing the osmotic pressure, adding asparagine, and artificially increasing the osmotic pressure can be applied, and the method of adding asparagine can be applied. In order to increase the specific gravity of the solution, a strategy to simultaneously apply osmotic pressure increase and asparagine addition will be useful.
[180]
[181]
From the above description, those skilled in the art to which the present invention pertains may understand that the present invention may be implemented in other specific forms without changing its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts, rather than the above detailed description, are included in the scope of the present invention.
Claim
[Claim 1]
During the cultivation process of the animal cells expressing the desired recombinant protein, comprising the step of increasing the osmotic pressure of the animal cell culture solution on a specific culture day, galactosylation (galactosylation) is regulated, a method for producing a desired recombinant protein.
[Claim 2]
The method of claim 1, wherein the culture is fed-batch culture.
[Claim 3]
The method according to claim 1, wherein the step of raising the osmotic pressure is performed on any one of the culture days from the 1st to the 10th day of the main culture based on the 0th day from the start of the main culture.
[Claim 4]
The method of claim 1, wherein the step of raising the osmotic pressure is performed on any one of the 1st, 4th, 7th, and 10th cultivation days based on 0 days from the start of the main culture.
[Claim 5]
The method of claim 4, wherein the step of raising the osmotic pressure is performed on the 4th or 7th day based on the 0th day of the start of the main culture.
[Claim 6]
The method of claim 1, wherein the increase in osmotic pressure is performed such that the final osmotic pressure in the culture medium is 460 to 500 mOsm / kg.
[Claim 7]
The method of claim 1, wherein the increase in osmotic pressure is performed by one or more methods selected from the group consisting of a method of supplementing sodium chloride or potassium chloride with the culture medium of the animal cell and adding glucose to the culture medium.
[Claim 8]
The method of claim 1, wherein the recombinant protein is an antibody.
[Claim 9]
The method of claim 8, wherein the method is for preparing a population of antibodies with reduced galactosylation.
[Claim 10]
The method of claim 1, wherein the step of raising the osmotic pressure is performed in a single step during the culturing of animal cells expressing the desired recombinant protein.
[Claim 11]
A method of regulating galactosylation of a desired recombinant protein, comprising raising the osmotic pressure of the animal cell culture solution on a specific culture day during the process of culturing the animal cell expressing the desired recombinant protein.
[Claim 12]
A method of producing a desired recombinant protein, wherein galactosylation is regulated, comprising the step of replenishing the animal cell culture medium with asparagine during the cultivation process of the animal cell expressing the desired recombinant protein.
[Claim 13]
The method of claim 12, wherein the asparagine is in the form of an asparagine concentrate or a culture medium containing asparagine.
[Claim 14]
The method of claim 12, wherein the supplementation of the asparagine is performed multiple times at a specific culture time point during the cultivation process of animal cells expressing the desired recombinant protein.
[Claim 15]
15. The method of claim 14, wherein the supplementation of asparagine is performed for 10 days from 4 days of culture based on 0 days from the start of the main culture.
[Claim 16]
15. The method of claim 14, wherein supplementation of the asparagine is carried out on the 4th, 7th and 10th day of culture on the 0th day of the start of the main culture.
[Claim 17]
The method of claim 12, wherein the method is supplemented with asparagine so that the final concentration of asparagine in the culture of animal cells is 27.6 to 33.6 mM.
[Claim 18]
The method of claim 17, wherein the method is supplemented with asparagine so that the final concentration of asparagine in the culture of animal cells is 33.6 mM.
[Claim 19]
The method of claim 12 or 17, wherein the method is supplemented with asparagine three times such that the concentrations of asparagine in the culture of animal cells are sequentially added by 6 mM, 12 mM, and 18 mM.
[Claim 20]
A method of regulating galactosylation of a desired recombinant protein, comprising the step of replenishing the animal cell culture medium with asparagine during the process of culturing the animal cell expressing the desired recombinant protein.
[Claim 21]
In the process of culturing animal cells expressing the desired recombinant protein, the method comprising the step of raising the osmotic pressure of the animal cell culture solution and replenishing asparagine, wherein the galactosylation is regulated, and the method for producing the desired recombinant protein.
[Claim 22]
The method of claim 21, wherein the increase in osmotic pressure and supplementation of asparagine are performed simultaneously or sequentially.
[Claim 23]
The method of claim 21, wherein the increase in osmotic pressure is performed by one or more methods selected from the group consisting of a method of supplementing sodium chloride or potassium chloride with a culture medium of the animal cell and adding glucose to the culture medium.
[Claim 24]
The method of claim 21, wherein the increase in osmotic pressure is performed such that the final osmotic pressure in the culture medium is 460 to 500 mOsm / kg.
[Claim 25]
The method of claim 21, wherein the supplementation of asparagine is to supplement the asparagine so that the final concentration of asparagine in the culture of animal cells is 27.6 to 33.6 mM.
[Claim 26]
The method of claim 21, wherein the recombinant protein is an antibody.
[Claim 27]
27. The method of claim 26, wherein the method is for regulating the galactosylation of the desired recombinant antibody and the content of the acidic isomer antibody in the antibody population prepared by the method.