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MANUFACTURE OF ACTIVE HIGH-PHOSPHORILATED HUMAN N-ACETYLGALACTOSAMINE-6-SULPHATASE AND ITS APPLICATION
专利权人:
БАЙОМАРИН ФАРМАСЬЮТИКАЛ ИНК. (US)
发明人:
КОППАКА Виш (US),ВЕЛЛАР Мишель Клод (US),ОКХАМАФЕ Аугустус О. (US),АРАЙА Кидисти (US)
申请号:
RU2013107765/10
公开号:
RU2013107765A
申请日:
2011.07.22
申请国别(地区):
RU
年份:
2014
代理人:
摘要:
1. A method of purifying a recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme, wherein said GALNS enzyme comprises an amino acid sequence identical to at least 95% amino acids 27-522 of SEQ ID NO: 4, and: (i) has a purity of at least about 95% as determined by Coomassie blue staining for SDS-PAGE analysis under non-reducing conditions, (ii) has at least about 50% conversion of the cysteine residue at position 53 to C-formyl glycine (FGly), and (iii) optionally has from 0.5 to 0.8 bis-phosphoryl oligomannose chains per monomeric protein chain, and at least 97% of the indicated GALNS enzyme is presented in the form of a precursor, as determined by SDS-capillary gel electrophoresis (SDS-CGE), including: a) filtering the nutrient medium containing the GALNS enzyme secreted from a mammalian cell line expressing human sulfatase modification factor 1 (SUMF1) and recombinant human GALNS enzyme, ultrafiltration / diafiltration of the filtered nutrient medium, and filtration through activated carbon feeder ultrafiltration / diafiltration medium; b) loading the culture medium that has been activated carbon filtered, ultrafiltration / diafiltration from step a) into the recovery column, washing the recovery column under conditions under which the GALNS enzyme is retained in the recovery column, and eluting the GALNS enzyme from the retention column; c) optionally, filtering the eluate from the recovery column from step b) through a filter to remove viruses; d) adjusting the pH of the eluate from the recovery column from step b) or filtrate1. Способ очистки рекомбинантного фермента N-ацетилгалактозамин-6-сульфатазы (GALNS) человека, при этом указанный фермент GALNS включает аминокислотную последовательность, идентичную по меньшей мере на 95% аминокислотам 27-522 последовательности SEQ ID NO:4, и:(i) имеет чистоту по меньшей мере примерно 95%, как это определено окрашиванием Кумасси синим при проведении анализа SDS-PAGE в невосстанавливающих условиях,(ii) имеет по
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