LC-MS characterization of proteolytic cleavage sites in therapeutic monoclonal antibodies

摘要

IgG2 monoclonal antibody was sampled before the last purification step to allow a slightly elevated level of host cell proteins (HCPs). The IgG2 antibody was incubated for 8 weeks at 37°C in pH 5 formulation, which resulted in cleavage on (Fab)2, CH2 and CH3 fragments. Main cleavage sites were at F241/L242 and S337/K338 with additional ladder cleavages at the termini. The IgG2 cleavage pattern (after bulky hydrophobic residues and serine) is consistent with the specificity of pepsin family of enzymes including Cathepsin D. Pepsin A and Cathepsin D were spiked. Pepsin A produced very similar cleavage pattern. Commercially available Cathepsin D showed low activity and different sources will be tried. Minor aspartate/proline (D/P) clips were caused by acid hydrolysis on the reversed-phase LC column at pH 2 and 75°C. The accurate mass and terminal ladder cleavages allowed reliable mass spec identification of the cleavage sites and were utilized to confirm enzyme identity through cleavage specificity.