JPH04299992A - Produciton of antibody - Google Patents

Abstract

PURPOSE:To provide a process for the production of an antibody suitable for the production of antibody by cell culture and having improved production efficiency. CONSTITUTION:The subject cell-culture process for the production of antibody is characterized by the control of the culture temperature to 29-36 deg.C which is lower than the standardized conventional culture temperature (37 deg.C). The yield of antibody is remarkably increased simply by controlling the culture temperature within the above range.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing antibodies by cell culture. More specifically, the present invention relates to a method for producing antibodies with good yield by controlling the culture temperature to a constant temperature during cell culture.

0002

BACKGROUND OF THE INVENTION Antibodies are in increasing demand as various diagnostic agents and pharmaceuticals, and there is a need for the development of inexpensive and simple manufacturing methods. In order to meet this demand, methods for producing antibodies using cell culture methods are being considered, and development of culture methods such as serum-free media, perfusion culture, and strain culture, and various culture devices are being developed. (For example, New Biochemistry Experiment Course, Volume 18, Cell Culture Technology, edited by the Japanese Biochemical Society; BIOTEC
HNOLOGY, Vol. 6b, 249 page H
．． J. (Rehm, ed.) In animal cell culture,
As culture conditions, a culture temperature of 37°C and a carbon dioxide concentration of 5% in the culture apparatus are widely and uniformly used, and there are no examples in the prior art literature that focus on the relationship between antibody yield, culture temperature, and carbon dioxide concentration. It is listed.

0003

[Problems to be Solved by the Invention] Although conventional antibody production methods have achieved their intended purpose, there is still room for improvement, and further improvements are needed to improve the productivity of antibody production using cell culture methods. There continues to be a need to provide a manufacturing method that includes the following: Therefore, the purpose of the present invention is to
Disclose culture conditions suitable for antibody production by cell culture method,
The purpose of the present invention is to provide a more efficient method for producing antibodies.

0004

[Means for Solving the Problems] As a result of extensive research in order to improve the productivity of antibody production using cell culture methods, the present inventors have discovered that the culture temperature and carbon dioxide gas in the culture apparatus, which had been overlooked in the past, We found that concentration is a factor that greatly influences antibody yield. Furthermore, the present invention was completed by confirming that antibodies can be obtained in unexpectedly high yields by controlling these conditions within a certain range. That is, the above-mentioned problem is a method for producing antibodies in which cells capable of producing the antibody of the present invention are cultured in a nutrient medium and antibodies are recovered from the culture, and the culture temperature is controlled within the range of 29°C to 36°C. This can be solved by a method that includes:

[0005] The present invention will be explained in detail below. The method of the present invention can be universally used in conventionally known cell culture methods, and as long as the desired effect is obtained under the controlled culture conditions of the present invention, the antibody production ability described in more detail below can be improved. Cells and nutrient media known per se can be used.

[0006] Cells capable of producing antibodies used in the present invention include antibody-producing cells and antibody-producing cells extracted from immunized animals using commonly practiced methods (for example, see Monoclonal Antibodies by Tatsuo Iwasaki et al.). Both hybridoma cells, which are a cell fusion of myeloma cells and myeloma cells,
can be used. As the nutrient medium, a cell culture medium known in the art can be used as it is, or a medium modified according to the characteristics of the cells to be cultured. These media are described in conventional technical literature, and some of them are commercially available and may be used as is. Preferred examples include Dulbecco's modified Eagle's medium, RPMI1640 medium (Gibco), ASF103 medium, ASF104 medium (
Examples include Ajinomoto Co.).

[0007] When performing a cell culture method using such cells and a nutrient medium, the present invention particularly controls the culture temperature and, if necessary, the carbon dioxide concentration in the culture atmosphere. It is essential to set the culture temperature at a temperature lower than 37°C, which has conventionally been uniformly used as described above, and in particular, to control it within the range of 29°C to 36°C. Furthermore, the carbon dioxide concentration in the culture atmosphere is lower than the conventionally uniformly used concentration of 5%, for example, an optimum concentration exists around 3%. The effect is achieved without any limitations. That is, the desired effect can be obtained if the carbon dioxide concentration is within the range of 3% to 7%. Note that the carbon dioxide concentration under the culture atmosphere here refers to the carbon dioxide concentration within the culture apparatus (or culture tank).

[0008] As a means for controlling the culture conditions as described above, any suitable method can be used as long as the culture temperature and the carbon dioxide concentration in the gas phase (inside the culture apparatus) in contact with the culture solution can be controlled. A type of culture device may also be used. Specific culture apparatuses that can be used include, but are not limited to, tissue culture flasks, spinner flasks, multi-trays, carbon dioxide gas culture apparatuses, jar fermenters, hollow fiber type culture apparatuses, and the like.

[0009] The antibodies thus produced by culturing for a certain period of time may be polyclonal or monoclonal. The produced antibodies can be recovered from the culture by appropriately applying commonly used protein separation and purification methods that take advantage of the various properties of antibodies. For example, highly pure antibodies can be obtained by removing cells from the culture medium by centrifugation, concentrating the antibodies with ammonium sulfate, crudely fractionating them, desalting them by dialysis, and purifying them using various types of chromatography.

0010

EXAMPLES The present invention will be explained in more detail below through the cultivation of hybridoma cells capable of producing monoclonal antibodies against human type IV collagen, but the scope of the present invention is not intended to be limited thereto. Example 1 Preparation of antibody-producing cells JP-A-63-78067 and JP-A-63-246396
According to the method described in the publication, monoclonal antibody JK-199 (immunoglobulin class IgG2a) against human type IV collagen was obtained from splenocytes obtained from BALB/C mice immunized with human type IV collagen and mouse myeloma cells SP2/O. The producing mouse hybridoma cells were formed and then selected to become the JK199 strain.
By subculturing the JK199 strain while gradually decreasing the concentration of serum added to the medium, the JK199 strain was finally grown in serum-free medium ASF104.
It was possible to acclimate to the culture medium (manufactured by Ajinomoto Co.). Example 2 Effect of culture temperature on antibody yield JK199 strain stored in a liquid nitrogen cell preserver was thawed.
The cells were placed in a 75 cm2 tissue culture flask (Costar) containing 50 mL of ASF104 medium supplemented with 0.3% polypeptone S (Daigo Nutrient Chemical Co., Ltd.) so that the cell concentration was approximately 105 cells/ml. The cells were inoculated and cultured stationary in a carbon dioxide gas culture device in which the temperature was maintained at 37° C. and the carbon dioxide gas concentration was maintained at 5%. After culturing for 3 days, this seed culture was inoculated into eight 75 cm2 tissue culture flasks containing 45 mL of the above medium with 5 mL each, and the culture temperature was maintained at 28 °C, 31 °C, 34 °C, 37 °C, and 40 °C, respectively. Two cells each were placed and cultured in a carbon dioxide culture device. The carbon dioxide concentration in the apparatus was all set to 5%. After culturing for 14 days, the total number of cells in the culture solution was counted using a hemocytometer. The antibody concentration in the culture medium was determined using a microplate coated with human type IV collagen, a biotinylated anti-mouse IgG2
It was measured by an enzyme-labeled immunoassay method using an a antibody, avidin peroxidase, and a chromogenic substrate. Table 1 shows the culture results controlled at each culture temperature. The antibody concentration is expressed as a relative value with the culture temperature at 37°C set as 100.

[0011]

[Table 1]

Example 3 Effect of carbon dioxide concentration in culture vessel on antibody yield The JK199 strain stored in a liquid nitrogen cell preserver was thawed, and the cells were transferred to ASF104 medium with polypeptone S.
Medium 50 supplemented with 0.3% concentration of (manufactured by Daigo Nutrient Chemical Co., Ltd.)
mL into a 75 cm2 tissue culture flask (Costar) at a cell concentration of approximately 105 cells/ml, and let stand in a carbon dioxide gas culture device maintained at a temperature of 37°C and a carbon dioxide gas concentration of 5%. Cultured. After culturing for 3 days, this seed culture was inoculated into 10 75 cm2 tissue culture flasks containing 45 mL of the above medium, 5 mL each, and the carbon dioxide concentration was maintained at 1%, 3%, 5%, 7%, and 9%, respectively. Two cells each were placed and cultured in a carbon dioxide culture device. The culture temperature was 37°C in all cases. After culturing for 14 days, the number of cells and antibody concentration in the culture were measured by the same method as in Example 2. Table 2 shows the culture results at each culture temperature. The antibody concentration is expressed as a relative value, with the carbon dioxide concentration being 5% as 100.

[0013]

[Table 2]

Example 4 Culture using a multi-tray The JK199 strain stored in a liquid nitrogen cell preserver was thawed, and the cells were placed in 50 mL of ASF104 medium supplemented with polypeptone S at a concentration of 0.3%. 75cm2
In three tissue culture flasks, the cell concentration was approximately 105 cells/
ml of each, and the culture temperature was set at 37°C.
The cells were statically cultured in a carbon dioxide culture device in which the carbon dioxide concentration was maintained at 5%. After culturing for 3 days, a multi-tray containing 1650 mL of the above medium (trade name: Cell Factory, 10 stages,
This seed culture (total volume: 150 mL) was inoculated into a Nunc Co., Ltd., culture temperature was 34℃, and the carbon dioxide concentration in the culture device was 34℃.
The cells were statically cultured in a carbon dioxide culture device maintained at %. After culturing for 14 days, cells were removed from the culture medium by centrifugation,
The culture supernatant, whose pH was adjusted to 8.0 with sodium hydroxide, was passed through a Protein A Sepharose (Pharmacia) affinity column equilibrated with 0.1 M phosphate buffer (pH 8.0) to convert the antibody to Protein A. Combined. 0
．． After thoroughly washing the column with 1M phosphate buffer (pH 8.0), the antibody was eluted with 8M urea aqueous solution (pH 7.0).
Dialysis was performed against 0.1M phosphate buffer (pH 7.0) using a dialysis membrane to obtain a purified antibody. The purified antibody concentration was calculated from the absorbance at 280 nm (OD280).

As a comparative example, culture was carried out using a multi-tray in a carbon dioxide gas culture device in which the culture temperature was maintained at 37° C. and the carbon dioxide gas concentration in the culture device was maintained at 5%, and after culturing for 14 days, the same method as above was carried out. Antibodies were collected from the culture solution. As is clear from Table 3, when the culture temperature was maintained at 34°C and the carbon dioxide concentration in the culture apparatus was maintained at 3%, the antibody yield increased dramatically compared to the comparative example, reaching 150 mg per liter of culture solution.
reached.

[0016]

[Table 3]

[0017]

Effects of the Invention According to the present invention, antibodies can be produced in which the antibody yield is dramatically improved by simply controlling the culture temperature of 37°C, which has conventionally been uniformly set, to a lower temperature. A method is provided.

Claims (1)

[Claims] Claim 1: A method for producing antibodies, which comprises culturing cells capable of producing antibodies in a nutrient medium and recovering antibodies from the culture, the method comprising controlling the culture temperature within the range of 29°C to 36°C. How to become.