Determination of the origin of the N-terminal pyro-glutamate variation in monoclonal antibodies using model peptides.

The purpose of this work is to determine the cause of the cyclization of the N-terminal glutamine in recombinant proteins and monoclonal antibodies. This cyclization reaction commonly occurs on the N-terminal of light and/or heavy chains of antibodies and leads to heterogeneity of the final product. Two model peptides and an antibody containing an N-terminal glutamine were used to investigate the formation of N-terminal pyro- glutamic acid under various experimental conditions and different stages of the biosynthetic process. LC-MS analysis was used to separate and quantify the N-terminal variants. Experiments prove that the cyclization reaction is spontaneous and highly dependent on temperature and buffer composition and less dependent on pH. The conditions presented in most biopharmaceutical processes accelerate the formation of this variant. The majority of the near complete conversion (>95%) of N-terminal glutamine to pyro-glutamic acid commonly observed for antibodies appears to occur inside the bioreactor with only a small contribution from purification, formulation, and analytical preparation.