Facile F(ab′) 2 Manufacturing: Strategies for the Production of Snake Antivenoms

Abstract

Asimple process for the manufacture of polyclonal F(ab′)2 fragments that might be adopted for the facile preparation of antivenoms is described. The production of polyclonal F(ab′)2 fragments from serum commonly involves the initial purification of IgG's prior to their proteolytic cleavage and further purification. To reduce the number of processing steps the authors have compared the digestion of whole serum by free and immobilized pepsin with that of pure IgG. It was observed that with equal units of pepsin activity, caprylic acid pre-purifi ed IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was signifi cantly greater in the latter case. The effi ciencies of whole serum digestion by the same total units of free and immobilized pepsin was also compared. IgG digestion was complete after 12 hours, as was serum digestion. About 80% of the antigen binding activity was retained in either case after 24 hours though the immobilized pepsin lost about 40% of its proteolytic activity after first use. The purifi cation of F(ab′)2 on two ion exchange adsorbents (StreamlineTMSP and paramagnetic CM beads) on paramagnetic thiophilic beads and on a hydrophobic custom assembled guanidine agarose was studied. The binding capacity on CM beads was too low to be useful for F(ab′)2 preparation. Most of the F(ab′)2 bound to the Streamline SP at low pH (pH 4.0 and 3.0), as did significant amounts of low molecular weight digestion products. The F(ab′)2 fragments were eluted completely with 0.3M NaCl whilst the low molecular weight digestion products required 1M NaCl to elute them. F(ab′)2 also bound to the thiophilic beads, free from the low molecular weight digestion products, and could be eluted with low ionic strength solutions. The guanidine agarose beads showed a lower capacity than the thiophilic beads but their selectivity was comparable. Overall the thiophilic and hydrophobic matrices showed better selectivity than the cation exchange matrices.