Purification method of bacterial capsular polysaccharide

Abstract

The invention discloses a method for purifying bacterial capsular polysaccharide. The method comprises the following steps: treating a streptococcus pneumonia fermentation culture solution with an inactivator, adding acid for regulating the pH value to be less than or equal to 6.5, precipitating impurities, centrifuging, and carrying out microfiltration for removing precipitates, so that polysaccharide clarified liquor is obtained; carrying out ultrafiltration liquid change on the polysaccharide clarified liquor by adopting a membrane with the molecular weight cut-off of 10-100KDa and concentrating, and carrying out chromatography on the obtained capsular polysaccharide solution by combining compound type ion exchange chromatography and hydroxyl phosphate grey salt chromatography, so that the purified capsular polysaccharide solution is obtained; and concentrating and carrying out ultrafiltration on the capsular polysaccharide solution, and carrying out sterile filtration and storing. By combining the compound type ion exchange chromatography and the hydroxyl phosphate grey salt chromatography, the contents of protein and nucleic acid impurities can be effectively reduced to be lower than 1%, the quality of a purified polysaccharide product is higher than European Union pharmacopoeia standard requirements, and the recovery rate is more than 60%. The method disclosed by the invention has the advantages that the technology is simple and rapid, the enlargement is easy, and industrial production can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for purifying crude bacterial capsular polysaccharides. Background technique [0002] Streptococcus pneumoniae is a Gram-positive bacterium with more than 90 serotypes, most of which are coated with a layer of capsular polysaccharide. Capsular polysaccharides can evade recognition by the immune system and prevent phagocytosis by immune cells, thus allowing bacteria to multiply in the body and cause disease. Years of research and clinical studies have proved that the capsular polysaccharide of Streptococcus pneumoniae as a vaccine can induce specific antibodies and has a good immune protection against the corresponding Streptococcus pneumoniae. The composition and structure of the capsular polysaccharides of different serotypes of Streptococcus pneumoniae are different, so it is usually necessary to use a combination of polysaccharides of different serotypes ...

AI Technical Summary

Problems solved by technology

[0005] Ethanol precipitation is usually effective in removing protein contaminants from polysaccharides, but it is difficult to meet the purity requirements of vaccines for injection

In addition, the use of ethanol can also bring the following problems: 1) the use of ethanol requires fire prevention facilities, and the cost of designing such facilities is very high; Waste liquid treatment is very difficult; 3) It is difficult to redissolve the polysaccharide after treatment, and steps such as freeze-drying are required

Since some serotype capsular polysaccharides have phosphate ions (such as 6A, 6B, 19F, etc.), the filler will bind a large number of such polysaccharide molecules during chromatography, resulting in a significant reduction in the polysaccharides that need to be recovered in the effluent

Patent CN200610048875 uses Sepharose 4FF gel to purify bacterial polysaccharides, but due to the nature of the selected filler, the volume of the treated solution is obviously limited, which is not suitable for scale-up production

Ji Yu et al. (Chinese Journal of New Drugs, Vol. 25, No. 10, 2016) used DOE design to establish a pneumonia polysaccharide purification process based on Capto Adhere, but there are still a small number of proteins that cannot be effectively removed, and the control requirements for chromatographic purification parameters are very strict. , usually the content of protein and nucleic acid is difficult to reach the standard, which is not conducive to actual production

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