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论文类型：期刊论文

发表时间：2015-03-01

发表刊物：PROCESS BIOCHEMISTRY

收录刊物：SCIE、EI、Scopus

卷号：50

期号：3

页面范围：367-377

ISSN号：1359-5113

关键字：PEGylation; Recombinant hirudin; Mixed aqueous-organic solutions; Solvent effect; Reaction kinetics

摘要：Serious loss of polyethylene glycol (PEG) reagent is confronted during PEGylation of proteins in mildly acidic aqueous solution. In this study, a novel approach of PEGylation of recombinant hirudin variant-2 (HV2) with monomethoxy-PEG-succinimidyl carbonate (mPEG-SC) was developed in six mixed aqueous-organic solutions. The PEGylation efficiency in each mixed solutions was greater than that in pure aqueous solution. Structure analysis of HV2, hydrolysis kinetics of mPEG-SC, and PEGylation kinetics of HV2 in different solutions revealed that solvent effects occurred in mixed aqueous-organic solutions. According to the dominant solvent effect of PEGylation acceleration and mPEG-SC hydrolysis inhibition, the selected mixed aqueous-organic solutions could be divided into different types (PEGylation-driven, mPEG-SC hydrolysis-driven, both PEGylation and mPEG-SC hydrolysis-driven). In aqueous-DMSO solutions, the desired yield (approximately 50%) of mono-PEG-HV2 was achieved under the optimized reaction conditions. The optimal usage of PEG reagent could decrease 3-5 fold compared with that of PEGylation in pure aqueous solution. The mono-PEG-HV2 could restore its structure and bioactivity after being purified into aqueous solution. This study demonstrated that PEGylation of proteins in mixed aqueous-organic solutions could be used as an alternative method to PEGylation in pure aqueous solution. (C) 2015 Elsevier Ltd. All rights reserved.