Release Time:2019-03-09  Hits:

Indexed by: Journal Article

Date of Publication: 2015-03-01

Journal: PROCESS BIOCHEMISTRY

Included Journals: Scopus、EI、SCIE

Volume: 50

Issue: 3

Page Number: 367-377

ISSN: 1359-5113

Key Words: PEGylation; Recombinant hirudin; Mixed aqueous-organic solutions; Solvent effect; Reaction kinetics

Abstract: 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.