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')2 mu' 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')2 mu 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')2 mu fragments was homogeneous (purity higher than 97%) by both sodium dodecyl sulfate-polyacrylamide 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')2 mu fragments. The molecular mass of F(ab')2 mu was estimated to be 144-146 kDa. In comparison with IgM, F(ab')2 mu lost entirely the complement C1q 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')2 mu, suggesting that the fragments are useful for immunological application.
Inouye
K
K
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Morimoto
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007075:Immunoglobulin M / Q000737:chemistry*; D007148:Immunoglobulin mu-Chains / Q000737:chemistry; D051379:Mice; D008970:Molecular Weight; D010434:Pepsin A; D011108:Polymers; D012995:Solubility; D014867:Water / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M; D007148:Immunoglobulin mu-Chains; D011108:Polymers; D014867:Water; C080942:TSKgel ether-5PW; D010434:Pepsin A