Protein PEGylation can extend serum half-life and reduce immunogenicity and toxicity, which are appealing attributes for therapeutic proteins. This covalent modification, commonly targeting the e-amino group of exposed lysine sites, has been applied in approved PEGylated protein drugs with many others in development. Monitoring protein PEGylation provides unique analytical challenges. Protein heterogeneity is magnified by the potential for multiple individual PEGylation sites, depending on the amino acid sequence, as well as an increasing degree of PEGylation depending on reaction conditions. Cation exchange chromatography can be a useful tool for evaluating the PEGylation degree and the positional isomers present. In this work, a method has been developed to monitor the PEGylation of the model proteins lysozyme and ribonuclease A as a function of reaction time and reactant molar ratio. This method shows good separation between the native unmodified protein as well as multiple positional isomers, allowing detailed protein characterization.

Monitoring protein PEGylation with ion exchange chromatography

245th National Meeting (2013)

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