Near UV and Visible Light-Induced Degradation of IgG2: Photo-Fenton Reaction in Citrate Buffer.

PURPOSE: Evaluation of the stability of an IgG2 in citrate buffer, upon exposure to near-UV and visible light and in the presence of relevant trace amounts of iron.

METHODS

We monitored the oxidation of amino acid residues, the formation of protein aggregates, fragments and charge variants by SDS-PAGE and two-dimensional gel electrophoresis (2-DIGE). Degradation products in individual gel spots were isolated and characterized by HPLC-MS/MS.

RESULTS

We detected an increase in the formation of DOPA, protein aggregates, and fragments with increasing concentrations of added Fe3+. Light exposure resulted in the generation of more acidic products, evident from a shift of the protein pI detected by 2-DIGE. Oxidation products of Tyr, His, Cys, and Trp were detected. The addition of EDTA or DTPA showed a significant protection against degradation.

CONCLUSIONS

IgG2 was significantly modified by the photo-Fenton reaction in citrate buffer. We demonstrated the oxidation of Tyr, His, Cys, and Trp residues, the formation of aggregation and degradation products, as well as the formation of different charge variants.