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Determination of PEGylation homogeneity of polyethylene glycol‐modified canine uricase
Author(s) -
Li Haigang,
Huo JingJing,
Sun Dan,
Guo Yong,
Jiang Liang,
Zhang Haijuan,
Shi Xiaowei,
Zhao Zhilong,
Zhou Jinchuan,
Hu Chunlan,
Zhang Chun
Publication year - 2021
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.202000268
Subject(s) - pegylation , polyethylene glycol , peg ratio , monomer , chromatography , conjugated system , chemistry , polymer , biochemistry , organic chemistry , finance , economics
Polyethylene glycol‐modified canine uricase (PEG‐UHC) was prepared by modifying the ε‐amino group of lysine residues on the canine uricase (UHC) protein to near‐saturation with 5 kDa monomethoxyl‐polyethylene glycol succinimide (mPEG‐SPA‐5k). In order to accurately determine the PEGylation uniformity of PEG‐UHC, CZE, 3–8% gradient gel SDS‐PAGE, and imaging CIEF (iCIEF) analyses were compared. CZE could not effectively separate PEG‐UHC proteins with different degrees of modification, 3–8% gradient gel SDS‐PAGE could separate PEG‐UHC into seven gel bands; however, most of the gel bands were smeared or blurred, and the separation of PEG‐UHC samples by iCIEF was significantly better than that by 3–8% gradient gel SDS‐PAGE. Under denatured conditions, iCIEF separated 12 p I peaks, and could also accurately quantify the relative monomer PEG‐UHC content. More than 85% of the total monomeric PEG‐UHC was conjugated with 7–12 PEG molecules; of this 85%, approximately 40% was conjugated with 9–10 PEG molecules. These results demonstrated that iCIEF exhibits good potential for determining the PEGylation homogeneity of PEGylated protein drugs.