SINGLE-STEP PURIFICATION OF F(AB')2-MU FRAGMENTS OF MOUSE MONOCLONAL-ANTIBODIES (IMMUNOGLOBULINS M) BY HYDROPHOBIC INTERACTION HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING TSKGEL ETHER-5PW

A procedure is described for preparation and single-step purification of F(ab')2 fragments, herein designated as F(ab')2mu' from mouse monoclonal antibodies of the IgM class. Hydrophobic interaction high-performance liquid chromatography (HPLC) using TSKgel Ether-5PW was well applicable to the purification. The IgM was digested with pepsin at the pepsin-to-IgM ratio of 1:200 (w/w) in 100 mM citrate buffer (pH 4.2) at 37-degrees-C for 2 h. The digests were applied to the gel equilibrated with the buffer containing 1 M ammonium sulfate. F(ab')2mu fragments were adsorbed onto the gel with the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2mu fragments was homogeneous (purity higher than 97%) by both sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and gel-filtration HPLC. The recovery of the antigen-binding site was 55-72%. The cycle time of the Ether-5PW HPLC was 40 min, and up to 98 mg F(ab')2mu was purified in a cycle. This method could be suitable especially for large-scale purification of F(ab')2mu fragments. The molecular mass of F(ab')2mu was estimated to be 144-146 kDa. In comparison with IgM, F(ab')2mu lost entirely the complement Clq binding activity, and the sugar content was greatly reduced. The binding of IgM with non-specific proteins turned to be negligible, when IgM was converted to F(ab')2mu, suggesting that the fragments are useful for immunological application.