Purification Of A Human Monoclonal IgM Antibody From Bioreactor Supernatant Using A Combination Of Cation And Anion Exchange Chromatography

Few protocols are currently available for the purification of antibodies of IgM class. Here, we describe a novel procedure to purify a human IgM monoclonal antibody, SK- 1.45, with specificity for an adenocarcinoma associated antigen. SK-1.45 is produced by a hybridoma cell line in bioreactors using protein-free tissue culture media. SK-1.45 binds to both anion and cation exchange supports when applied at a neutral pH. It is therefore possible to attain a high degree of purity through a two step procedure involving cation and anion exchange chromatography. Initially, bioreactor supernatant is buffer exchanged into 50 mM Na , pH 7.2 via gel permeation chromatography on Sephadex ® G-25 (Pharmacia) and fractionated using cation exchange chromatography, Macro- Prep ® high S support (Bio-Rad Laboratories). Partially purified IgM is eluted with 50 mM Na , 100 mM NaCl, pH 7.2, diluted 1:1 with water and loaded onto an anion exchange column, Macro-Prep high Q support (Bio-Rad Laboratories). Purified IgM is eluted with 30 mM Na , 250 mM NaCl, pH 7.0. For SK-1.45, this procedure resulted in a final product which was > 98% pure as determined by SDS-PAGE and a yield of 60% as determined by ELISA. DNA levels were reduced by a factor of > 5,000 from the levels found in the bioreactor supernatant, and biological activity, binding to pure recombinant antigen, was fully retained. This protocol was used to purify gram quantities of human IgM under GMP for use in clinical trials. P P P