METHOD FOR PURIFICATION OF IMIGLUCERASE Russian patent published in 2018 - IPC C07K1/12 C07K1/16 C12N9/24 

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and biotechnology and can be used in the pharmaceutical industry. Method for deglycosylating imiglucerase and purifying imiglucerase is proposed, comprising the step of deglycosylating imiglucerase. Method of deglycosylation involves the isolation of the imiglucerase obtained in CHO cells and its subsequent treatment with neuraminidase, β-1,4-galactosidase and β-N-acetylglucosaminidase in a single step. Ratio of neuraminidase enzymes: β-1,4-galactosidase: β-N-acetylglucosaminidase in the mixture is: 6 E:1 E:14 E per 1 g of protein. Method for purifying imiglucerase involves hydrophobic chromatography on Butyl-Sepharose, cation exchange on SP-Sepharose, metal-chelate chromatography on IMAC-Sepharose, hydrophobic chromatography on Phenyl-Sepharose combined with deglycosylation of the desired protein, filtration through Q-Sepharose and cation exchange on CM Sepharose.

EFFECT: invention allows to obtain a product of pharmacopoeial quality with a content of not less than 99 % of imiglucerase (by reverse-phase HPLC), no more than 4 % of related impurities (by HPLC exclusion), with a specific activity of at least 35 IU/mg and a residual protein content of the producing cell not more than 50 ng/mg.

4 cl, 1 dwg, 10 ex