Determination of PEGylation homogeneity of polyethylene glycol‐modified canine uricase

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 pI 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.