Production of a human monoclonal IgM directed against human cardiac myosin in a hollow-fiber bioreactor for Membrane Anion Exchange Chromatography one-step purification

Article type: Research Article

Authors: Jacobin, Marie-Josée^a | Santarelli, Xavier^c | Laroche-Traineau, Jeanny^a | Clofent-Sanchez, Gisèle^{a; b; *}

Affiliations: [a] CNRS UMR 5533, Hôpital Cardiologique, 33604 Pessac, France | [b] CNRS UMR 5536, Université Victor Segalen Bordeaux 2 33076 Bordeaux Cedex, France | [c] Ecole Supérieure de Technologies des Biomolécules de Bordeaux (ESTBB), Université Victor Segalen Bordeaux 2 33076 Bordeaux Cedex, France

Correspondence: [*] Corresponding author. Tel.: +33 5 57 57 11 75; Fax: +33 5 57 57 45 56; E-mail: [email protected]

Abstract: 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 μg/ml, compared to 20 μg/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.

Keywords: human monoclonal antibody, IgM Purification, membrane ion exchangers, myosin

DOI: 10.3233/HAB-2004-13303

Journal: Human Antibodies, vol. 13, no. 3, pp. 69-79, 2004