MEMBRANE CHROMATOGRAPHY FOR RAPID PURIFICATION OF RECOMBINANT ANTITHROMBIN-III AND MONOCLONAL-ANTIBODIES FROM CELL-CULTURE SUPERNATANT

The task of purifying monoclonal antibodies (MAbs) and human recombina nt antithrombin Ill (rATIII) from cell culture supernatant was carried out using two different approaches, both based on the use of membrane ous matrices. The first approach employed a strongly acidic and a stro ngly basic membrane ion exchanger, which were evaluated for their abil ity to purify monoclonal antibodies and the human active recombinant a ntithrombin III from cell culture supernatant. Within minutes gram amo unts of product could be purified in a high-flux system, specially dev eloped for this purpose, achieving purities of 80% for MAbs and 75% fo r rATIII, respectively. The capacity of the acidic membrane ion exchan ger for MAbs was found to be 1 mg/cm2 with recoveries up to 96% and th at of the basic membrane ion exchanger for rATIII was 0.15 mg/cm2 with recoveries up to 91%. The second approach consisted of using heparin, a mucopolysaccharide with a strong affinity towards ATIII, coupled to amine-modified or epoxy-activated membranes by reductive amination, f or the purification of rATIII. The ATIII binding capacities of the mem branes were found to be 91 mug/cm2 for the amine-modified and 39 mug/c m2 for the epoxy-activated membrane, achieving purities of 75%. The co upling proved to be fairly stable over a period of 5 months and the me mbranes remained operable even after steam sterilization and treatment with sodium dodecyl sulphate. Final purification in both instances wa s carried out by gel filtration.