KR-102962784-B1 - Cell culture medium containing keto acids

Abstract

The present invention relates to a cell culture medium containing alpha-keto acids. The poor solubility of some amino acids, such as isoleucine, leucine, and valine, can be overcome by substituting them with their respective alpha-keto acids.

Inventors

• 침머 알리네
• 자이벨 론야
• 슈미트 코린나
• 빌레 그레고어 프란츠 베르너
• 피셔 마르쿠스 클라우스 로베르트

Assignees

• 메르크 파텐트 게엠베하

Dates

Publication Date

20260508

Application Date

20200408

Priority Date

20190411

Claims (13)

1. Fed-batch process for culturing cells in a bioreactor according to the following - Step of filling the bioreactor with cells and aqueous cell culture medium - The step of incubating cells in a bioreactor - A step of adding cell culture medium, in this case feed medium, to the bioreactor continuously over the entire incubation time of the cells in the bioreactor, or once or multiple times within said incubation time. Here, the feed medium has a pH of less than 8.5 and contains at least one keto acid selected from the group of 4-methyl-2-oxopentanic acid, 3-methyl-2-oxopentanic acid and/or its salts.
2. A feed-batch process according to claim 1, wherein the feed medium comprises at least 4-methyl-2-oxopentanic acid, 3-methyl-2-oxopentanic acid, and/or its salt at a concentration of 12 to 600 mmol/l.
3. A feed-batch process according to claim 1, wherein the feed medium comprises 50 to 400 g/l of solid components dissolved in a solvent.
4. In claim 1, the feed medium added to the bioreactor continuously during incubation or once or multiple times within the said time always has the same composition, in a feed-batch process.
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Description

Cell culture medium containing keto acids The present invention relates to a cell culture medium containing alpha-keto acids. The poor solubility of some amino acids, such as isoleucine, leucine, and valine, can be overcome by substituting them with their respective alpha-keto acids. Cell culture media support and maintain the growth of cells in an artificial environment. Depending on the type of organism whose growth needs to be supported, cell culture media contain a complex mixture of components, sometimes exceeding one hundred different ingredients. Cell culture media required for the proliferation of mammalian, insect, or plant cells are typically much more complex than media that support the growth of bacteria and yeast. The first cell culture media developed consisted of unlimited components, such as plasma, serum, embryonic extracts, or other unlimited biological extracts or peptones. Thus, significant progress was made with the development of chemically limited media. Chemically limited media often contain, but are not exclusively limited to, amino acids, vitamins, metal salts, antioxidants, chelators, growth factors, buffers, hormones, and many more substances known to those skilled in the art. Some cell culture media are provided as sterile aqueous liquids. The disadvantages of liquid cell culture media are reduced shelf life and difficulties in transportation and storage. As a result, many cell culture media are now provided as finely ground dry powder mixtures. They are manufactured for the purpose of dissolving in water and/or aqueous solutions, and in the dissolved state, often along with other supplements, are designed to provide a substantial nutrient base for cell growth and/or to produce biopharmaceuticals from said cells. Many biopharmaceutical production platforms are based on fed-batch cell culture protocols. The objective is typically to develop high-potency cell culture processes that meet increasing market demand and reduce manufacturing costs. In addition to the use of high-performance recombinant cell lines, improvements in cell culture media and process parameters are required to realize maximum production potential. In the feed-batch process, the basal medium supports initial growth and production, while the feed medium prevents nutrient depletion and sustains the production phase. The medium is selected to accommodate unique metabolic requirements during different production phases. Process parameter settings—including feed strategies and control parameters—define the chemical and physical environment suitable for cell growth and protein production. Optimization of the feed badge is a major aspect of feed-batch process optimization. Typically, feed media are highly concentrated to avoid dilution of recombinant proteins in bioreactors. The controlled addition of nutrients directly affects the growth rate, viability, and potency of the cultures. However, in other cell culture processes, such as batch or perfusion processes, there is also a need for precisely formulated and often highly concentrated medium preparations. Particularly in perfusion processes, the continuous exchange of medium in bioreactors requires operators to prepare and handle large volumes of liquid medium. To reduce the space occupied by storing these volumes, concentration of the medium is required. A limiting factor in the preparation of cell culture media from dry powder is the poor solubility or stability of some components, particularly some amino acids. Therefore, it would be advantageous to find a method to provide a dry powder medium composition that is sufficiently soluble to produce a highly concentrated liquid medium composition. Instead of the amino acids isoleucine, leucine, valine, phenylalanine, and methionine, each alpha-keto acid has been found to be usable with no negative effects, and sometimes with positive effects on cell growth and improved solubility. Furthermore, it was revealed that these keto acids even have a stabilizing effect on liquid cell culture medium formulations. In a 1959 paper on the metabolism of amino acids, it was mentioned that some amino acids could be substituted with their keto acids (Eagle H: Amino acid metabolism in mammalian cell cultures. Science 1959, 130(3373):432-437.). However, since then, it has not been known that certain keto acids can be used as amino acid substitutes in high-performance cell cultures and are suitable for overcoming the solubility and stability problems of some amino acids. The present invention therefore relates to a dry powder or dry, granulated cell culture medium comprising at least one alpha-keto acid from the group consisting of 4-methyl-2-oxopentanic acid (Keto Leu), 3-methyl-2-oxopentanic acid (Keto Ile), alpha-ketoisovaleric acid (Keto Val), phenylpyruvic acid (Keto Phe), and alpha-keto gamma-methylthiobyric acid (Keto Met), and/or its derivative, in an amount such that the concentration of each keto acid and/or its derivative in