Invented by Shawn Barrett, Scott JACOBIA, Life Technologies Corp
The use of cell culture medium containing small peptides has become increasingly popular in recent years due to its ability to support the growth of a wide range of cell types. This medium is typically composed of a mixture of amino acids, vitamins, minerals, and other nutrients that are essential for cell growth and survival.
One of the key benefits of using cell culture medium containing small peptides is its ability to support the growth of cells in a controlled environment. This allows researchers to study the behavior of cells under different conditions and to develop new drugs and therapies that target specific cellular pathways.
Another benefit of using cell culture medium containing small peptides is its ability to reduce the risk of contamination. This medium is typically sterilized before use, which helps to prevent the growth of bacteria and other microorganisms that can interfere with cell growth and development.
The market for cell culture medium containing small peptides is expected to continue to grow in the coming years. This is due in part to the increasing demand for new drugs and therapies that target specific cellular pathways, as well as the growing use of cell-based assays in drug discovery and development.
In addition, advances in biotechnology and genetic engineering are expected to drive the development of new and more complex cell culture media in the coming years. This will likely lead to the development of more specialized and targeted cell culture media that are tailored to specific cell types and applications.
Overall, the market for cell culture medium containing small peptides is a promising area of growth in the biotechnology industry. As researchers continue to develop new drugs and therapies that target specific cellular pathways, the demand for specialized cell culture media is expected to continue to grow.
The Life Technologies Corp invention works as follows
Cell culture media, concentrated feeds and media, methods for manufacturing cell culture feeds and media, and methods for culturing are described. Cell culture media are enhanced with one or more dipeptides and small peptides.
Background for Cell culture medium containing small peptides
Cell culture media are the necessary nutrients to grow and maintain cells in an artificial, controlled environment. The nutrient formulations, pH and osmolality in cell culture media can vary depending on parameters like cell type, density and culture system.
Media formulations were used to cultivate animal, plant and bacteria cells. Cultivated cell cultures have a variety of uses, including the study and production of useful biological compounds. Polypeptides such as monoclonal antibody, hormones and growth factors are examples of useful products. These products are used in many therapeutic and commercial applications. With the introduction of recombinant-DNA technology, it is possible to engineer cells to produce large amounts of these products. Cells are routinely cultured to isolate, identify and grow viruses that can be used in vaccines or vectors. The ability to culture cells in vitro not only is important for studying cell physiology but also for producing useful substances that may not be otherwise obtained through cost-effective methods.
Cell culture media formulations are well documented and commercially available. Early cell culture media were developed based on chemical compositions and physicochemical characteristics (e.g. pH, osmolality). The ‘physiological solutions’ were made from blood. Ringer, S., J. Physiol. Waymouth, C. In: Cells and tissues in culture, Vol. Academic Press, London. pp. 99-142 (1965); Waymouth, C., In Vitro 6:109-127 (1970). Cells in mammalian tissues are exposed to varying microenvironments in terms of oxygen/carbon dioxide partial pressurization and the concentrations of vitamins, trace elements and nutrients. Therefore, different media formulations will be required for successful in vitro cultures of different cell types. Cell culture media typically contain amino acids, organic salts and inorganic minerals, vitamins, trace metals and sugars.
Glutamine has become a standard ingredient in cell culture media, as it is a proven energy source for cells. Eagle demonstrated in 1959 that the glutamine required for mammalian cells cultures to grow optimally is three to ten times more than other amino acids. Eagle et al., Science 130:432-37 (1959). However, glutamine can be toxic for certain cells when it is in an aqueous or high temperature solution. Roth et al., In Vitro Cellular & Developmental Biology 24(7):696-98 (1988). “Glutamate is usually added to the cell culture medium just before use.
Roth et. al., In Vitro Cellular & Developmental Biology, 24(7):696-988 (1988). It has been found that glutamine can be substituted with Glutamate to reduce ammonia accumulation in cell culture media. See Cell Culture Technology For Pharmaceutical and Cell-Based Therapies 52, Sadettin Ozturk and Wei-Shou Hu eds. Taylor and Francis Group, (2006).
Others have proposed acylating dipeptides, such as alanyl-glutamine, to increase the stability of dipeptides under heat sterilization. U.S. Pat. No. No. 5,534,538 describes N acyl dipeptides that are used in enteral and parenteral nutrition. The N acyl group delays the splitting of the dipeptide until the kidneys reach the N acyl group. U.S. Pat. No. No. above). U.S. Pat. No. No. Thus, U.S. Pat. No. No.
Applicants found that certain amino acid, such as tyrosine have a limited solubility in the concentrations required for maximum cell growth or production of protein. Other amino acids like cysteine are also unstable and susceptible to precipitation in aqueous medium for cell culture, especially concentrated medium. Because cysteine contains a thiol, it is susceptible for an oxidation process in which two cysteine molecules are linked by a disulfide to form cystine. Cystine is not soluble in water, and it precipitates easily out of solution. It has been impossible to create a liquid cell culture medium that is shelf-stable and contains the concentrations of cysteine and tyrosine necessary for maximum cell growth or protein production.
Applicants have also attempted to solve this problem by using a lower concentration of tyrosine and cysteine than desired in the aqueous cellular culture medium. This allows for a shelf-life acceptable for aqueous products. This is achieved at the cost of optimal viral, protein or cell growth. “In other words, cell cultures with lower concentrations tyrosine or cysteine support reduced cell growth as well as protein production compared to cells culture media with optimal concentrations tyrosine or cysteine.
There is a need today for a medium or concentrate medium that contains sufficient amounts of tyrosine (or cysteine) to support maximum cell growth, protein production, or viral production, while avoiding problems resulting from the limited solubility or limited stability of cysteine. This includes the tendency of the cysteine to precipitate over time. A concentrated feed supplement is also needed so that it does not add a lot of volume to the cell culture system. It is also necessary that, when adding such nutrient-based feeds, pH and osmolality are automatically balanced. The medium or concentrated feed should be available either in liquid or dry form. There is also a requirement for a concentrated feed supplement that contains sufficient amounts of cysteine or tyrosine to support maximum cell growth, protein production and/or high-quality expressed proteins.
The compositions of this invention are directed in part to cell culture media or concentrated feed supplements that contain concentrations of cysteine or tyrosine. These compounds support maximum cell growth, protein or viral production, while avoiding problems associated with their limited solubility or stability. In one aspect, media, concentrated feeds, or concentrated media can be composed without serum. The compositions can include human serum components like human serum albumin. In another aspect, human serum albumin can be recombinant (r-human sera albumin) and derived from recombinant source, such as plant sources (rice, corn, wheat or potato), or fungal sources (yeast or equivalent microorganisms known to be used by the art). In another aspect, media, concentrated feed supplement, or concentrated media can be composed of protein-free compounds. In yet another aspect, the media, concentrated feed supplements, or concentrated media may be protein-hydrolysate free compositions, and further, may be free of fractions of protein-hydrolysates. In one aspect, media, concentrated feeds, or concentrated media can be composed of serum-free, non-protein, and non-protein hydrolysate compositions. These compositions can be characterized as serum-free, non-protein, and free from any protein hydrolysates or fractions. In some embodiments, cell culture mediums, concentrated media or cell culture feed supplements that contain one or two small peptides or dipeptides do not include any of the following components: lipids or hydrolysates, or fractions thereof.
The invention also relates to methods of analyzing compositions for the presence or lack of a short polypeptide containing cysteine or Tyrosine. The media can be analyzed by any method known in the art. For example, mass spectrometry, capillary electrophoresis, or HPLC.
In an embodiment of the invention the concentrations of tyrosine, as contained in the small peptide, in solution in a medium or feed, or in supplements, are greater than those concentrations of tyrosine which would remain soluble if tyrosine was present in monomer form (that is, adding tyrosine to a small polypeptide can make the solution “supersaturated” for tyrosine). In one embodiment, a solution has a soluble tyrosine content between 1 and 100 times or 1 and 25 times that of a monomer. “For example, in some cell cultures, concentrated feeds, or supplements, the concentration of dipeptides and small peptides may be between 1 and at most about 100 times (or about 25 times) the soluble concentration.
In an embodiment of the invention the concentrations of cysteine, as contained in the small peptide, will be higher in the solution (than the concentrations of cysteine which would remain soluble if the monomer was present) than the concentrations of cysteine, that would remain in an identical solution when the cysteine is present (that’s to say, cysteine within a small polypeptide can make the solution “supersaturated with cysteine”). In one embodiment the solubility (or lack thereof), of cysteine, would include, for instance, loss of soluble caused by conversion from cysteine into cystine. In one embodiment, a solution will contain at least between 1 and 25 times the concentration of soluble cysteine that is capable with the monomer. In some cell culture media or concentrated feeds, for example, small peptides and dipeptides may be present at concentrations of 1-5 times the soluble cysteine as the monomer would allow. In one embodiment, both tyrosine (tyrosine) and cysteine (cysteine) are supersaturated in the solution.
In one embodiment the solution is supersaturated with both tyrosine, and cysteine, in the same ranges as described for either tyrosine, or cysteine, alone.
In one embodiment, adding the media, feeds or supplements described above to an existing culture system (e.g. a fed-batch) could reduce the amount of supplementation. The culture can be ongoing for a short time, or even a couple of days. In another embodiment, increasing solubility of cysteine, tyrosine, and small peptides within feeds can allow for the design single-part concentrated feeds that may be pH neutral. This is part of invention. The invention is directed to the preparation of concentrated feeds and media, and concentrated supplements, which contain cysteine and/or tyrosine within a small, peptide to increase their stability and solubility within the composition.
The cell-culture medium, concentrated feed, or cell-culture supplement comprising one or two small peptides or dipeptides may optionally comprise one or all of the following elements: a carbohydrate or sugar, a vitamin or salt, an organic element or buffering agent, an amino acid or salt thereof. In one embodiment, the carbohydrate comprises hexose. Pentose or hexose or derivatives thereof or equivalents can be used as an alternative. In certain instances, oligosaccharides and their derivatives can be used. Or, the hexose can be selected from the group of glucose, galactose fructose or maltose. In one embodiment, the carbohydrate used is glucose.
The amino acid or salt of the amino acid is one or more from arginine (or asparagine), cysteine (or aspartic acid), glutamic acid or glutamine, isoleucine (or leucine), lysine (or methionine), phenylalanine (or proline), serine, tryptophan and valine.
The amino acid or salt thereof may include one or more of arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The amino acid or salt thereof may include one or more of the following: arginine (or aspartic acid), asparagine (or aspartic acid), cysteine (or glutamic acid), histidine, leucine (or isoleucine), lysine (or methionine), phenylalanine (or proline), serine, tryptophan, valine, or aspartic acid.
In one embodiment, cell culture medium, concentrated food or cell culture supplements have one or two small peptides or dipeptides suitable for culturing an insect cell such as Drosophila, Spodoptera, Trichoplusia, or C. elegans. Other examples include a nematode (e.g. BHK, AE-1, SP2/0, L5.1, hybridoma, or human cells) and a mammalian (e.g.
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