One-step purification of monoclonal IgG antibodies from mouse ascites. An evaluation of different adsorption techniques using high performance liquid chromatography

Abstract

Monoclonal antibodies were purified in one step from mouse ascites by four different high performance liquid chromatography (HPLC) adsorption techniques. The antibodies were of the IgG1, IgG2a, IgG2b and IgG3 subtypes, with isoelectric points between 6.4 and 7.6. Similarly pretreated samples were applied to columns for anion exchange (Mono Q), cation exchange (Mono S), chromatofocusing (Mono P) and hydrophobic interaction chromatography (Alkyl Superose), respectively. Highly purified IgG fractions, as judged by electrophoresis, were obtained in all these techniques. The yields of immunoreactive material were above 80%. A strategy for single step HPLC purification of monoclonal IgG antibodies from mouse ascites by adsorption techniques is proposed, as a complement to the well-established technique of affinity chromatography on immobilized protein A. The strategy involves (i) cation exchange chromatography as a first choice for antibodies with high isoelectric points (greater than 7.2), and (ii) hydrophobic interaction chromatography as a first choice when the isoelectric point is below 7.2.