Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes.
A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.
Dainiak
M B
MB
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, S-221 00, Sweden.
Muronetz
V I
VI
Izumrudov
V A
VA
Galaev
I Y
IY
Mattiasson
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D000941:Antigens / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007202:Indicators and Reagents; D018482:Muscle, Skeletal / Q000201:enzymology; D010206:Papain; D011145:Polyvinyls; D011817:Rabbits
D000911:Antibodies, Monoclonal; D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D011145:Polyvinyls; C027968:poly-4-vinyl-N-ethylpyridine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010206:Papain