Direct Quantification of PEGylation for Intact Bioconjugates and Nanoparticles by the Colorimetric Barium/Iodide Assay

Several methods are available to determine the average number of methoxy poly(ethylene glycol) (mPEG) chains grafted to a protein or peptide, referred to as the degree of PEGylation. Nevertheless, the development of simple, versatile, and multiplexable methods for determining PEGylation remains desirable. Childs and Kurfürst have respectfully reported the quantitative and qualitative use of a colored 'barium-iodide-PEG' complex for the titration of PEG in solutions and to stain PEG-containing bands on electrophoresis gels. Remarkably, this assay has yet to be employed to directly determine the extent of PEGylation of protein bioconjugates or intact nanoparticles. This study validates this assay for these purposes, via libraries of 54 mPEG-protein conjugates and 10 polymeric nanoparticles. The effect of mPEG molecular weight, terminal functional groups, and architecture were analyzed, among other parameters. Practical details and known artifacts are discussed to enhance the accuracy and reproducibility of the assay.