Photoinduced aggregation of a model antibody-drug conjugate.

Abstract

During synthesis, purification, and especially storage, antibody-drug conjugates (ADCs) may be exposed to various types of light. Several of the drugs commonly conjugated to antibodies contain photosensitive functional groups. Exposure to light could generate an excited state of the drug that subsequently triggers drug and/or protein degradation. To mimic and study photoinduced ADC degradation, we designed a model ADC in which the monoclonal antibody (mAb) trastuzumab was treated with the amine-reactive probe eosin-5-isothiocyanate to yield an antibody-eosin conjugate (T-EO). Photoinduced degradation was monitored by size exclusion chromatography (SEC), dynamic light scattering (DLS), SDS-PAGE under reducing and nonreducing conditions, and MS/MS analysis. SEC analysis of the model ADC showed the formation of higher molecular weight species directly following a 20 W-hr/m(2) exposure of UVA light. DLS analysis of these samples showed the formation of larger soluble particles, and precipitate was observed 24 h post light exposure. These results were not seen in control samples of the model ADC that were shielded from light. Furthermore, these results were not seen in control samples containing mAb alone, suggesting that aggregation was the result of light exposure of the conjugate. Importantly, when eosin-5-isothiocyanate was added separately to solutions containing mAb (i.e., without conjugation), the extent of photoinduced aggregation was substantially less, indicating that the conjugation of the photosensitizer to the mAb specifically promoted photoinduced aggregation. Reducing and nonreducing SDS-PAGE suggested that photoinduced interchain covalent cross-linking occurred through a mechanism other than disulfide formation. Using peptide mapping and MS/MS analysis, we identified key peptides in the T-EO sequence that undergo photodegradation. Finally, we also show that cross-linking products formed in as little as 1 h of exposure to ambient light. These findings suggest that precautions should be taken to ensure minimal exposure to light during the synthesis, purification, and storage of ADCs containing photosensitive drugs.

DOI: 10.1021/mp5006799

Cite this paper

@article{Cockrell2015PhotoinducedAO, title={Photoinduced aggregation of a model antibody-drug conjugate.}, author={Gregory M Cockrell and Michael S Wolfe and Janet Wolfe and Christian Sch{\"{o}neich}, journal={Molecular pharmaceutics}, year={2015}, volume={12 6}, pages={1784-97} }