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 ( T_g; 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.