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Culture medium comprising dipeptides

A culture medium and cell culture technology, applied in the direction of cell culture medium, culture process, tissue culture, etc., can solve the problem that the culture medium is not optimal

Active Publication Date: 2019-10-25
EVONIK OPERATIONS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] While the cell culture media described in the above prior art provide satisfactory performance and properties for certain cell culture processes, the prior art media are still not optimal

Method used

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  • Culture medium comprising dipeptides
  • Culture medium comprising dipeptides
  • Culture medium comprising dipeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1: Synthesis of L-glutaminyl-L-asparagine

[0105] 27.8 g of triethylamine was added to 70 g of benzyloxycarbonyl-L-glutamine (CAS registry number 2650-64-8, available from Peptides International, Inc., USA) in 350 ml of acetone. The solution was cooled to -10°C and 31.6 g of pivaloyl chloride (Sigma) were added. The mixture was stirred for 10 minutes and then added to a solution of 39.6 g L-asparagine, 19.0 g potassium hydroxide 85% and 41.3 g potassium carbonate in 350 ml water. Warm to room temperature (20°C) and wash with 300ml H 2 O dilution. HCl (conc.) was added to pH 2. The resulting solid was filtered, washed with water and acetone, and dried in vacuo. 65.2 g of benzyloxycarbonyl-L-glutaminyl-L-asparagine were obtained in the form of colorless crystals. The melting point is 202-204°C.

[0106] 1 H-NMR(DMSO):1.69(m,1H),1.92(m,1H),2.14(m,2H),2.54(m,2H),4.02(m,1H),4.52(m,1H),5.03 (s, 2H), 6.64 (d, 2H), 7.28 (d, 2H), 7.37 (m, 5H), 7.43m, 1H), 8.09 (...

Embodiment 2

[0109] Embodiment 2: Effect of Gln-Asn on cell culture

[0110] The effect of Gln-Asn dipeptide on antibody-producing Chinese hamster ovary (CHO) cells (subclone DG44; Life Technologies Corporation, Carlsbad, USA) was studied using one-way first-time (OFAT) analysis.

[0111] The analysis is based on a comparison of viable cell density and cell viability in a series of cell cultures performed under essentially the same culture conditions, however, with increasing amounts of Gln-Asn dipeptide in the medium. In order to keep the total amount of Asn and Gln units (molar basis) constant in each medium, the molar concentration of free glutamine and free asparagine was decreased according to the molar amount of dipeptide added. A series of media with increasing amounts of Gln-Asn dipeptide was prepared by mixing different amounts of basal media (containing free Asn and Gln) and test media (containing Asn-Gln dipeptide). A commercially available defined cell culture medium (TC-42, T...

Embodiment 3

[0125] Example 3: Preparation of an improved DMEM medium comprising Asn-containing dipeptides

[0126] Dulbecco's Modified Eagle's Medium (DMEM) is a widely used basal medium to support the growth of many different mammalian cells. Cells successfully cultured in DMEM include primary fibroblasts, neurons, glial cells, HUVECs, and smooth muscle cells, as well as cell lines such as HeLa, 293, Cos-7, and PC-12. By adding Gln-Asn to commercially available DMEM medium (Gibco (R) DMEM, High Glucose, SKU#41965-039, Life Technologies Corp., Carlsbad, CA, USA) to prepare the supplemented DMEM medium according to the present invention. The final concentration of dipeptide in the medium was 4.5 mM.

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Abstract

The present invention relates to a culture medium, preferably a cell culture medium, comprising at least one dipeptide, the dipeptide comprising asparagine (Asn) and glutamine (Gln). The invention further relates to the use of a culture medium of the invention for culturing cells, preferably plant cells, animal cells or mammalian cells. Another aspect of the invention relates to a method of manufacturing a cell culture product comprising the steps of (i) providing a cell capable of producing said cell culture product; (ii) contacting said cell with a culture medium according to the invention;and (iii) obtaining said cell culture product from said culture medium or from said cell.

Description

[0001] field of invention [0002] The present invention relates to biotechnological production methods. More specifically, the present invention relates to improved media for use in biotechnological production processes, methods using such improved media and products obtained from such methods using improved media. [0003] Background of the invention [0004] Biotechnological methods are widely used in the production of biological products. These methods generally involve culturing the cells in a culture medium under conditions that permit growth of the cultured cells and product formation. Cells useful in biotechnological production are bacterial cells, fungal cells, yeast cells and cells of animal or plant origin. [0005] Animal cell cultures have long been used to produce biological products such as therapeutic proteins, polypeptides, oligopeptides or other biomolecules such as therapeutic polysaccharides. In this method of cell culture, animal cells, usually genetical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00
CPCC12N5/0018C12N5/0031C12N2500/32C12N2510/02C12N5/005C12N2500/99
Inventor F·梅茨A·卡劳T·赫尔曼G·纳奥普
Owner EVONIK OPERATIONS GMBH