Semi-preparative purification and validation of monoclonal antibodies for immunotherapy in mice.
A number of rat hybridomas were adapted to grow in RPMI containing either 5% IgG-depleted FCS or 1% serum-free Nutridoma. Alternatively, protein-free Ultradoma PF was used. Growth in these media allowed purification procedures to be used that are based on tangential ultrafiltration in combination with affinity chromatography on gels linked to protein G or anti-rat L chain coupled antibodies. The isolated antibody preparations were found to be pure and to consist of monomeric intact IgG. The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endotoxin contamination. Whereas during isolation endotoxin contamination increased, the endotoxin content per mg purified protein did not. Affinity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations obtained were found to be of sufficient purity, activity and low endotoxin content to permit their in vivo use in animal models of immunotherapy.
Savelkoul
H F
HF
Department of Immunology, Erasmus University, Rotterdam, Netherlands.
Vossen
A C
AC
Breedland
E G
EG
Tibbe
G J
GJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification* / Q000627:therapeutic use; D004340:Drug Contamination / Q000517:prevention & control; D004731:Endotoxins / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas / Q000276:immunology*; D007074:Immunoglobulin G / Q000302:isolation & purification; D008033:Limulus Test; D051379:Mice; D051381:Rats
D000911:Antibodies, Monoclonal; D004731:Endotoxins; D007074:Immunoglobulin G