A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as model. evaluation focused on purification fragments through nonaffinity-based process centrifugal ultrafiltration technique and mild reduction conditions for intact production F(ab’) with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, defined chemical composition were successfully obtained via proteolytic digestion followed controlled reaction maintaining integrity binding sites. appears be suitable removal digestive enzyme but inefficient Fc fragments, thus requiring additional processing. A analytical strategy developed, allowing us demonstrate reformation bridges between two reduced cysteines within fragments.