Design Space identification of hydrolysis conditions for amino acid analysis applying Quality by Design approach

Amino acid analysis (AAA) is an assay for the determination of the protein concentrations in drug substances, performed by the digestion of the intact proteins into free amino acids constituting the primary structure and by the chromatographic separation and determination of the free amino acid molecules. The hydrolysis procedure plays a fundamental role and should be carefully optimized, as it should allow the release of all the amino acids of the protein avoiding their chemical degradation. Quality by Design approach was applied in the development of the proteolysis procedure for AAA method. Preliminary experiments led to select the RP-UPLC technology, and AccQ•Tag Ultra method was employed for the protein content quantitation in the concentrated bulk of Neisseria Meningitidis antigens and Bovine Serum Albumin. Suitable experimental designs were used for investigating both gas- and liquid- proteolysis. Contour plots were drawn and with the aid of Monte Carlo simulation allowed the identification of the design space, where the critical method requirements were fulfilled with a selected probability, and of the final working conditions. Validation of the final AAA method was performed according to ICH guidelines and GSK internal policies, and demonstrated the method reliability.