Carboxyalkylated histidine is a pH-dependent product of pegylation with SC-PEG

Purpose. Pegylation of therapeutic protein usually results in a mixture of monopegylated proteins with differing sites of modification. With rh-interferon-alpha 2A pegylation, we have found that this heterogeneity includes two classes of pegylation site chemistry, the relative proportions of which can be adjusted by reaction pH. Methods. The effect of pegylation reaction pH on the relative proportion of three peaks produced was investigated. Products were purified and characterized by peptide mapping, chemical stability to neutral hydroxylamine, and biologic activity. Results. Reactions at basic pH levels produced a mixture of products pegylated at lysine residues as has been observed elsewhere. However, the dominant product of reactions at mildly acidic levels of pH showed distinct chemistry and higher cytopathic effect activity. The primary site of modification at this pH was His34. We developed a quantitative assay using sensitivity to neutral hydroxylamine to measure the proportion of urethane bonds involving carboxyalkylated histidines. This assay showed that histidine was pegylated preferentially at low pH levels with another protein, rh-Interleukin-10. Conclusions. Reaction pH can be used to select the preferred pegylation site chemistry.