Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW.
Hydrophobic interaction high performance liquid chromatography (HPLC) using TSKgel Phenyl-5PW was applicable to single-step purification of F(ab')2 fragments from pepsin digests of mouse monoclonal antibodies of IgG1 class. The digests were applied to the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate. F(ab')2 fragments were adsorbed onto the gel using the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 fragments was homogeneous (purity: higher than 98%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration HPLC. The recovery of the antigen binding site was 42-58%. The cycle time of the Phenyl-5PW HPLC was 45 min, and F(ab')2 of up to 2200 mg was purified in a cycle. This method could be useful especially for large scale purification of F(ab')2 fragments.
Morimoto
K
K
Biotechnology Research Laboratories, TOSOH Corporation, Kanagawa, Japan.
Inouye
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004983:Ethanolamines; D005782:Gels; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry*; D007202:Indicators and Reagents; D007475:Ion Exchange Resins; D051379:Mice; D008807:Mice, Inbred BALB C; D011108:Polymers
D000911:Antibodies, Monoclonal; D004983:Ethanolamines; D005782:Gels; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D007475:Ion Exchange Resins; D011108:Polymers; C007369:2-diethylaminoethanol