Traceless pH-Sensitive Antibody Conjugation Inspired by Citraconic Anhydride

We introduce a pH-sensitive amide bond, inspired by citraconic anhydride, for the reversible conjugation of polymers to the lysine residues of proteins and antibodies. The pH sensitivity arises from a conformation lock at the end of the polymer, which we introduce by means of a Diels-Alder reaction, that positions a carboxylic acid close to the amide after conjugation occurs. The amide is stable over weeks at pH 7.4 but sensitive to hydrolysis at pH 5.5 and below, returning the amine to its original state. The pH sensitivity can be tuned by positioning secondary amide groups nearby. We use this approach to PEGylate an antibody to human serum albumin at high dilution and demonstrate successful recovery of the activity after hydrolysis at pH 5.5. These results offer a convenient and traceless approach to protein and antibody functionalization.