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-interf eron-alpha 2A pegylation, we have found that this heterogeneity includes tw o classes of pegylation site chemistry, the relative proportions of which c an 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 characte rized by peptide mapping, chemical stability to neutral hydroxylamine, and biologic activity. Results. Reactions at basic pH levels produced a mixture of products pegyla ted at lysine residues as has been observed elsewhere. However, the dominan t product of reactions at mildly acidic levels of pH showed distinct chemis try 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 involv ing carboxyalkylated histidines. This assay showed that histidine was pegyl ated preferentially at low pH levels with another protein, rh-Interleukin-1 0. Conclusions. Reaction pH can be used to select the preferred pegylation sit e chemistry.