Analytical characterization of a novel degradation product in a PEGylated recombinant protein

We report the identification and characterization of a novel degradation product associated with PEGylation of a recombinant protein. After several months of storage at 2°C-8°C, an unexpected increase was observed in the proportion of an impurity that eluted with the native unPEGylated protein by size exclusion chromatography‐-from less than 0.01% at the start of storage to more than 0.25% at 12 months. An investigation into the nature of the impurity determined the presence of an N‐terminal adduction with a mass increase of +58 Da over the native unPEGylated protein species, demonstrating that this impurity was the result of degradation. The impurity was subjected to thorough analytical characterization using orthogonal methods to establish its identity, and a mechanistic model proposed for its formation. The data implicate the presence of a monomethoxy polyethylene glycol (mPEG)-acetal aldehyde impurity in the mPEG-aldehyde raw material, indicating the need for diligent raw material testing prior to use. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4607-4616, 2011