Mouse IgG2b monoclonal antibody fragmentation. Preparation and purification of Fab, Fc and Fab/c fragments.
Mouse IgG2b fragmentation has been poorly described in the literature because of the sensitivity of this subclass to proteases and the inability to produce F(ab')2 fragments. The fragments obtained include both Fc and Fab fragments and an intermediate of degradation, the Fab/c fragment, consisting of the Fc region and one Fab arm, which was first described by Parham (1983). Optimised pepsin digestion led to the formation of Fab/c in yields of up to 30% depending on the IgG2b antibodies susceptibility. DEAE-cellulose chromatography of the digested antibodies allowed, in all cases, separation of Fab fragments from Fc bearing fragments. Depending on the differences in pI between the fragments, Fab/c fragment purification was achieved either by CM-cellulose chromatography or by recycling HPLC gel filtration chromatography. Both procedures gave 97.5% purity Fab/c fragments. Fc fragments were purified by HPLC gel filtration chromatography. In cancer therapy the monovalent Fab/c fragments could be useful for drug targeting or for immunotherapy providing it retains a good affinity.
Demignot
S
S
Cancer Research Campaign Laboratories, University of Nottingham, U.K.
Garnett
M C
MC
Baldwin
R W
RW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D002848:Chromatography, DEAE-Cellulose; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000032:analysis*; D051379:Mice; D010434:Pepsin A / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D010434:Pepsin A