An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization

Abstract

Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

Probing Protein Conformation Destabilization in Sterile Liquid Formulations through the Formation of 3,4-Dihydroxyphenylalanine

“…After AAPH/O 2 oxidation, the aliquoted oxidized samples were ABS tagged and digested. As reported in previous publications, 24,29 the ABS tagging reaction of DOPA containing peptides generates three major products corresponding to an increase of the mass of Tyr residues by either 181, 196, or 366 Da (Figure S3). The three ABS products were taken into account during the MS/MS search for oxidized and ABS-tagged peptide 20.…”

“…Tyr residues that were oxidized in the above samples yield 3,4-dihydroxyphenylalanine (DOPA). The latter were further reacted with ABS using a modified protocol published by Bommana et al 24 In brief, 20 μg of proteins (or 50 μL of the original AAPH/O 2 oxidized protein solutions) were mixed with 300 μL of PBS (100 mM, pH 9.2) in the presence of K 3 Fe(CN) 6 and ABS at concentrations of 500 μM and 10 mM, respectively. K 3 Fe(CN) 6 and ABS stock solutions were prepared freshly in PBS (100 mM, pH 9.2) at concentrations of 10 and 100 mM, respectively.…”