Glucuronylation as a new acidic post-translational modification on therapeutic monoclonal antibodies

A method for identifying glucuronylation of a protein drug product comprising

• deglycosylating the protein drug product;
• treating the deglycosylated protein drug product to produce one Fc* fragment comprising two identical Fc/2 fragments bound together through non-covalent interactions, and one Fab2 fragment comprising one Fab2 fragment;
• separating the Fc* and Fab2 complexes using ion exchange chromatography into acidic fractions of Fc* and Fab2;
• drying and denaturing the acidic fractions;
• alkylating the acidic fractions;
• digesting the acidic fractions with trypsin to form a non-reduced sample;
• incubating a portion of the non-reduced sample with NaBH4 to form a reduced sample;
• and
• subjecting the reduced sample to reverse phase liquid chromatography/mass spectroscopy analysis to identify glucuronylation of the protein drug product.