Photolytic Labeling To Quantify Peptide–Water Interactions in Lyophilized Solids

Interactions of a lyophilized peptide with water and excipients in a solid matrix were explored using photolytic labeling. A model peptide "KLQ" (Ac–QELHKLQ–NHCH3) was covalently labeled with NHS–diazirine (succinimidyl 4,4′-azipentanoate), and the labeled peptide (KLQ–SDA) was formulated and exposed to UV light in both solution and lyophilized solids. Solid samples contained the following excipients at a 1:400 molar ratio: sucrose, trehalose, mannitol, histidine, or arginine. Prior to UV exposure, the lyophilized solids were exposed to various relative humidity (RH) environments (8, 13, 33, 45, and 78%), and the resulting solid moisture content (Karl Fischer titration) and glass transition temperature (Tg; differential scanning calorimetry, DSC) were measured. To initiate photolytic labeling, solution and solid samples were exposed to UV light at 365 nm for 30 min. Photolytic-labeling products were quantified using reversed-phase high-performance liquid chromatography (rp-HPLC) and mass spectrometry (MS). In lyophilized solids, studies excluding oxygen and using H218O confirmed that the source of oxygen in KLQ adducts with a mass increase of 18 amu are attributable to reaction with water, while those with a mass increase of 16 amu are not attributable to reaction with either water or molecular oxygen. In solids containing sucrose or trehalose, peptide–excipient adducts decreased with increasing solid moisture content, while peptide–water adducts increased only at lower RH exposure and then plateaued, in partial agreement with the water replacement hypothesis.