Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl- l -tryptophan Amide

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λ max of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N -acetyl- l -tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λ max of ca. 450 nm and UV–vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) and 1 H, 13 C, and 1 H– 13 C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N -(2,4-dihydrocyclopenta[ b ]indol-2-yl)acetamide (structure P3a ) or a tautomer ( P3b – d ).