Precise and comparative pegylation analysis by microfluidics and mass spectrometry

Standard SDS–PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1–12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent confirmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins.