Identification of D-Amino Acids in Light Exposed mAb Formulations

We previously demonstrated that D-amino acids can form as a result of photo-irradiation of a monoclonal antibody (mAb) at both λ = 254 nm and λ > 295 nm (λ_max = 305 nm), likely via reversible hydrogen transfer reactions of intermediary thiyl radicals. Here, we investigate the role of various excipients (sucrose, glucose, L-Arg, L-Met and L-Leu) on D-amino acid formation, and specifically the distribution of D-amino acids in mAb monomers and aggregates present after light exposure.

The mAb-containing formulations were photo-irradiated at λ = 254 nm and λ_max = 305 nm, followed by fractionation of aggregate and monomer fractions using size exclusion chromatography. These aggregate and monomer fractions were subjected to hydrolysis and subsequent amino acid analysis.

Both aggregate and monomer fractions collected from all formulations showed the formation of D-Glu and D-Val, whereas the formation of D-Ala was limited to the aggregate fraction collected from an L-Arg-containing formulation. Interestingly, quantitative analysis revealed higher yields of D-amino acids in the L-Arg-containing formulation.

Generally, D-amino acids accumulated to similar extents in monomers and aggregates.