Table 2. Characterization data for papain-generated fragments.

Papain-generated fragment monitored by SEC	Approximate molar mass by SEC-MALLS (g/mol)	Observed MW by LC-MS (Da)	Assigned SEC Peak Identity
Peak 1	100 x 103 g/mol	48206 Da, 47605 Da a (mass of Fab: LC + HC 1–230 or LC + HC 1–225)	Fab/Fab non-covalent dimer
Peak 2	55 x 103 g/mol	52818 Da (mass of intact disulfide-linked Fc)	Intact disulfide-linked Fc
Peak 3	51 x 103 g/mol	48204 Da, 47607 Da a (mass of Fab: LC + HC 1–230 or LC + HC 1–225)	Fab

Peaks 1, 2, and 3 were prominently observed in the papain digests of both the dimer and monomer samples, though there is more pre-existing Peak 1 in the dimer sample prior to papain-driven in situ Fab dimerization. ^aNon-specific cleavage of the heavy chain hinge region by papain generates Fab species with two heavy chain fragments differentiated by five residues at their C-terminus. Either Fab form is presumed to be involved in the observed in situ dimerization.