Quantitative Analysis of Peptide-Matrix Interactions in Lyophilized Solids Using Photolytic Labeling.

Peptide-matrix interactions in lyophilized solids were explored using photolytic labeling with reversed phase high performance liquid chromatography (rp-HPLC) and mass spectrometric (MS) analysis. A model peptide (Ac-QELHKLQ-NHCH) derived from salmon calcitonin was first labeled with a heterobifunctional cross-linker NHS-diazirine (succinimidyl 4,4'-azipentanoate; SDA) at Lys5 in solution, with ∼100% conversion. The SDA labeled peptide was then formulated with the following excipients at a 1:400 molar ratio and lyophilized: sucrose, trehalose, mannitol, histidine, arginine, urea, and NaCl. The lyophilized samples and corresponding solution controls were exposed to UV at 365 nm to induce photolytic labeling, and the products were identified by MS and quantified with rp-HPLC or MS. Peptide-excipient adducts were detected in the lyophilized solids except the NaCl formulation. With the exception of the histidine formulation, peptide-excipient adducts were not detected in solution and the fractional conversion to peptide-water adducts in solution was significantly greater than in lyophilized solids, as expected. In lyophilized solids, the fractional conversion to peptide-water adducts was poorly correlated with bulk moisture content, suggesting that the local water content near the labeled lysine residue differs from the measured bulk average. In lyophilized solids, the fractional conversion to peptide-excipient adducts was assessed using MS extracted ion chromatograms (EIC); subject to the assumption of equal ionization efficiencies, the fractional conversion to excipient adducts varied with excipient type. The results demonstrate that the local environment near the lysine residue of the peptide in the lyophilized solids can be quantitatively probed with a photolytic labeling method.