Photosensitizers Form in Histidine Buffer and Mediate the Photodegradation of a Monoclonal Antibody

Fluorescent light (FL) photodegradation of a monoclonal antibody (mAb) formulated in histidine buffer is mediated by histidine‐derived photosensitizers that accumulate and greatly increase with light exposure. Histidine‐derived photosensitizers are the primary mediators of Trp photooxidation. FL‐photodegradation requires light exposure and is pH dependent. It is significantly reduced or eliminated by buffer exchanges, by oxygen depletion, or at pH values greater than 7. Antibody‐fragment MS ion counts reveal that oxidation of a single light chain Trp in CDR1 correlates with binding loss. Multiple heavy chain methionines oxidize, but poorly correlate with binding loss. Photosensitizers extracted from photo‐aged histidine buffer are potent mediators of FL‐photodegradation including oxidation and, to a lesser degree, fragmentation and aggregation of the mAb. These photosensitizers absorb visible light and have neutral mass of 187.1- 386.1 Da. They are also fluorescent with ex/em at 360/450 nm. When spiked into histidine or MES buffered mAb formulations they produce a concentration dependent and pronounced increase in FL‐photodegradation; however, no oxidation or loss of antibody function occurs in the dark and hydrogen peroxide does not oxidize Trp. The major component is consistent with histidine oxidation to 6a‐hydroxy‐2‐oxo‐octahydro‐pyrollo[2,3‐d]imidazole‐5‐carboxylic acid. Photosensitizer levels measured in the formulation prior to light exposure, are linearly related to the FL‐photodegradation observed and can predict degradation in photostability testing.