title: Antigen binding properties of highly purified bispecific antibodies.
abstract: A panel of three bispecific monoclonal antibodies (bsMAbs) binding to follitropin (FSH) and to beta-galactosidase have been prepared by fusion of hybridoma cell lines resistant to oubain and neomycin. One of these bispecific antibodies contains heavy chains of the same IgG subclass, and two are composed of heavy chains of different IgG subclasses. We have investigated methods for the purification of bispecific antibodies from hybrid hybridoma supernatant fluids grown in serum-free medium. Following ammonium sulfate precipitation, bispecific antibodies can be purified in a single step by mixed mode ion-exchange HPLC on Bakerbond Abx columns. In one case, three species were resolved by ion-exchange HPLC and functional analysis showed that two peaks contained parental antibodies, and the third contained the bispecific. Ion-exchange HPLC purification of serum-free preparations from two other hybrid hybridomas resolved seven protein-containing peaks, only one of which was active in a bispecific ELISA. The equilibrium affinity constants for each of the parental antibodies for both FSH and beta-galactosidase were determined and found to be similar to those of the purified bsMAbs. Further, the association of FSH to one binding site on a bispecific antibody was shown to have no effect on the equilibrium binding constant for beta-galactosidase binding to the other site. Our results suggest that bsMAbs can be readily purified from hybrid hybridomas by a simple and rapid method, and the binding of antigen to one binding site on a bsMAb is independent of antigen binding to the second site.
authors.affiliation: E.I. DuPont de Nemours, Medical Products Department, Glasgow, DE 19702.
authors.forename: W J
authors.initials: WJ
authors.lastname: Allard
authors.forename: C A
authors.initials: CA
authors.lastname: Moran
authors.forename: E
authors.initials: E
authors.lastname: Nagel
authors.forename: G
authors.initials: G
authors.lastname: Collins
authors.forename: M T
authors.initials: MT
authors.lastname: Largen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology*; D001666:Binding Sites, Antibody; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005640:Follicle Stimulating Hormone / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000032:analysis; D016130:Immunophenotyping; D051379:Mice; D001616:beta-Galactosidase / Q000276:immunology*
chemical_list: D000906:Antibodies; D000941:Antigens; D007074:Immunoglobulin G; D005640:Follicle Stimulating Hormone; D001616:beta-Galactosidase