Photolysis of Recombinant Human Insulin in the Solid State: Formation of a Dithiohemiacetal Product at the C-Terminal Disulfide Bond

Abstract

Exposure of protein pharmaceuticals to light can result in chemical and physical modifications, potentially leading to loss of potency, aggregation, and/or immunogenicity. To correlate these potential consequences with molecular changes, the nature of photoproducts and their mechanisms of formation must be characterized. The present study focuses on the photochemical degradation of insulin in the solid state. Solid insulin was characterized by solid-state NMR, polarized optical microscopy and scanning electron microscopy; various insulin preparations were exposed to UV light prior to product analysis by mass spectrometry. UV-exposure of solid human insulin results in photodissociation of the C-terminal intrachain disulfide bond, leading to formation of a CysS(•) thiyl radical pair which ultimately disproportionates into thiol and thioaldehyde species. The high reactivity of the thioaldehyde and proximity to the thiol allow the formation of a dithiohemiacetal structure. Dithiohemiacetal is formed during the UV-exposure of both crystalline and amorphous insulin. Dithiohemiacetals represent novel structures generated through the photochemical modification of disulfide bonds. This is the first time that such structure is identified during the photolysis of a protein in the solid state.