Production of a human monoclonal IgM directed against human cardiac myosin in a hollow-fiber bioreactor for membrane anion exchange chromatography one-step purification.
Purification of human IgM monoclonal antibodies (MAbs) has proved to be difficult. Since IgM Mabs tend to bind strongly to a variety of resin support surfaces, the number of chromatographic steps used in the purification of these biomolecules should be minimized. Here we describe procedures developed for the optimal production and purification of the human monoclonal IgM B7, which specifically binds to the myosin heavy chain of human ventricular myocardium. This property makes this antibody potentially useful for the diagnosis of myocardial necrosis. Several chromatographic techniques were evaluated (size exclusion, ion exchange, affinity chromatography). The best results were obtained with anion exchange membrane chromatography using Sartobind Q15 (98% purity, 30% recovery). IgM production was improved by the hollow fiber technology which permitted the use of serum-reduced medium and an increase in antibody concentration to an average production of 300-400 microg/ml, compared to 20 microg/ml in flask culture. Several flow-rates were also evaluated, the optimal being 20 ml/minute for 30% of recovery. Importantly, the purified IgM molecule was able to bind to human myosin in ELISA and Western-blotting, thus allowing the IgM to be kept intact for further radiolabeling.
Jacobin
Marie-Josée
MJ
CNRS UMR 5533, Hôpital Cardiologique, 33604 Pessac, France.
Santarelli
Xavier
X
Laroche-Traineau
Jeanny
J
Clofent-Sanchez
Gisèle
G
D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D019149:Bioreactors; D015153:Blotting, Western; D024722:Cardiac Myosins / Q000276:immunology*; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007075:Immunoglobulin M / Q000096:biosynthesis* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D024722:Cardiac Myosins