Tryptophan Oxidation of a Monoclonal Antibody Under Diverse Oxidative Stress Conditions: Distinct Oxidative Pathways Favor Specific Tryptophan Residues

Abstract

Tryptophan oxidation can play an important role in selecting therapeutic monoclonal antibodies for commercialization. Monoclonal antibodies that harbor particularly sensitive tryptophan residues are typically discarded in favor of oxidation resistant antibodies. The susceptibility of any individual tryptophan residue to oxidation is typically evaluated through forced degradation studies during the molecule development process. We compared the results of several common forced degradation "stress tests" for each tryptophan residue in a monoclonal antibody and found that high-stress oxidation conditions consistently provide a different ranking of oxidative sensitivity across the individual tryptophan residues compared to long-term thermal stability or low-stress conditions. We subsequently determined that this difference in ranking is largely due to an overabundance of double oxidation (i.e. detected as +32 Da) of specific tryptophan residues under high stress conditions compared to single oxidation (i.e. +16 Da). We posit that this double oxidation is in fact mechanistically distinct from the observed single oxidation and that high stress conditions favor the double oxidation mechanism (and double oxidation sensitive tryptophan residues) while single oxidation appears to be the primary mode of oxidation under H₂O₂ stress and long-term thermal stability and favors different tryptophan residues which are more susceptible to the single oxidation mechanism.