Purification of monoclonal antibodies with light-chain heterogeneity produced by mouse hybridomas raised with NS-1 myelomas: application of hydrophobic interaction high-performance liquid chromatography.
Monoclonal antibodies (mAbs) of IgG1 class produced by hybridomas raised with NS-1 myelomas, which were purified homogeneously by anion-exchange high-performance liquid chromatography (HPLC), contained two types of immunoglobulin light (kappa) chain. Since NS-1 myeloma synthesizes the light (kappa) chain, the mAb was presumed to be the mixture of hybrid mAbs formed by the random association of heavy (gamma l) and light chains from antigen-immunized spleen cells and light chain from NS-1 cells. Hydrophobic interaction HPLC using TSKgel Phenyl-5PW was applicable to separate 3 species of hybrid mAb from mAb fractions obtained by anion-exchange HPLC. mAbs in the fractions were adsorbed onto the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate and eluted by reducing the concentration to 0 M. The hybrid mAbs were purified separately. The hydrophobic interaction HPLC could discriminate a small difference in hydrophobicity between kappa chains from spleen and NS-1 cells. The immunoreactivities of hybrid mAb bearing light chains only from spleen cells and that bearing those from both spleen and NS-1 cells were almost the same, and hybrid mAb bearing light chains derived only from NS-1 cells showed a relatively lower immunoreactivity than the others. The method described here could be useful for purification of hybrid mAbs.
Abe
N
N
Biotechnology Research Laboratory, Tosoh Corporation, Kanagawa, Japan.
Inouye
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000941:Antigens / Q000276:immunology / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D009101:Multiple Myeloma / Q000276:immunology* / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D012967:Sodium Dodecyl Sulfate