Cyclization of N-Terminal Glutamic Acid to pyro-Glutamic Acid Impacts Monoclonal Antibody Charge Heterogeneity despite Its Appearance as a Neutral Transformation

Abstract Pyroglutamic acid (pyroGlu) is commonly observed at the N-terminus of therapeutic monoclonal antibodies. Notably, the term “pyroGlu” refers to a single product that could originate from the cyclization of either an N-terminal glutamine or an N-terminal glutamic acid. This is an important and easily overlooked distinction that has major implications on the charge variant nature of a pyroGlu relative to its uncyclized form. Cyclization of an N-terminal glutamine for instance clearly produces an acidic variant with a lower isoelectric point owing to the loss of the positively charged N-terminal amine. In this report, we demonstrate that cyclization of an N-terminal glutamic acid on the other hand produces a basic variant with a higher isoelectric point contrary to the typical assumption that the simultaneous loss of the N-terminal amine and the carboxylic acid side-chain would negate the formation of a charge variant. The results of our investigation demonstrate the need to consider the relative strengths of the acidic and basic functional groups which are altered when assessing whether the product will be a charge variant. This study also adds new knowledge and experimental evidence to understand charge heterogeneity in monoclonal antibodies.