On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice.
Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000276:immunology; D002478:Cells, Cultured; D007126:Immunoglobulin Allotypes / Q000032:analysis / Q000302:isolation & purification*; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000032:analysis / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007126:Immunoglobulin Allotypes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010447:Peptide Hydrolases