Serum-free culture medium suitable for large-scale suspension multiplication culture of CHO cells and preparation and application of serum-free culture medium

Abstract

The invention discloses a serum-free culture medium, a preparation method thereof and an application of the serum-free culture medium to large-scale suspension multiplication culture of CHO cells. The pH value of the serum-free culture medium is 6.9-7.0, and the serum-free culture medium comprises an amino acid composition, an inorganic salt composition, a vitamin composition, a trace element composition, a buffer system composition, a supplementary factor composition and water. On the basis of the serum-free culture medium, the amino acid composition comprises 1-2 mg/L of glutamine dipeptide GlutaMAX-I, the buffer system composition comprises 3-8 g/L of HEPES, 1-5 g/L of sodium bicarbonate, 100-300 mg/L of sodium dihydrogen phosphate, 100-300 mg/L of disodium hydrogen phosphate, 3-10 g/L of sodium chloride and 200-500 mg/L of potassium chloride, and the supplementary factor composition comprises 10-30 g/L of a soybean extract, 10-30 g/L of a yeast extract and 80-100 [mu]g/L of Long-R3-IGF-I. The preparation method comprises the following steps: weighing and mixing the components according to the formula, adjusting the pH value to 6.9-7.0, and performing filtering by using a filter membrane with the pore diameter of 0.22 [mu]m.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a serum-free medium suitable for large-scale suspension expansion culture of CHO cells and its preparation and application. Background technique [0002] The use of animal cell engineering to ferment and express protein drugs and antibodies is an important direction for the development of modern medicine. Nearly half of the recombinant drugs in the world are expressed in mammalian cells, and 70% of them are expressed in Chinese Hamsters Ovary (CHO) cells. Obtained, CHO cells are of great value as engineered cells for recombinant drug expression. [0003] In the process of culturing recombinant CHO cells in vitro, it is often necessary to add animal serum to the medium. Animal serum contains nutrients such as hormones, growth factors, carrier proteins, adhesion and diffusion factors, and trace elements, and is usually added to the medium at a content of up to 10%. Medium to ...

AI Technical Summary

Problems solved by technology

However, this medium does not contain L-glutamine, a nitrogen source necessary for cell growth, and contains 1.0-2.0 mg / L of phenol red. At the same time, phenol red may be harmful to the environment, especially water bodies, so it is not conducive to large-scale cultivation

The CHO cell serum-free medium (CN107460160A) developed by Song Guofang et al. is based on CHO-S-SFM II, and added 1mg / ml glutamine, 1mg / ml methionine, 1mg / ml proline, 1mg / ml tryptophan and 10mg / ml insulin, but CHO-S-SFM II itself is a serum-free medium, which is expensive and not conducive to large-scale cultivation. At the same time, insulin and L-glutamine are also easy to The problem of degradation, L-glutamine degradation products are cytotoxic and not conducive to cell growth

CHO cell serum-free medium (CN109337861A) developed by Wang Xiaoke, the main components include amino acids, inorganic salts, vitamins, trace elements, yeast hydrolyzate and albumin and other components, it only uses a buffer system, and the pH is only suitable for 7.0-7.4 range, and the shaker needs to use CO 2 Maintain pH, while high-density cell culture is prone to produce a large amount of metabolic waste in the later stage, the culture solution becomes acidic, and the medium with low buffer capacity is not conducive to high-density cell culture

[0007] To sum up, although many serum-free media have been developed on the market, each media is only suitable for the growth of one type of cell, or the added product is unstable, easy to degrade and produce cytotoxicity

At the same time, most serum-free media also contain macromolecular protein additives such as albumin, which seriously affects the separation and purification of later expression products and increases production costs.

Images