Comparison of membrane adsorber (MA) based purification schemes for the down-stream processing of recombinant h-AT III.

Several multistage chromatographic separation schemes based on Membrane Adsorbers as stationary phases were designed for the isolation of recombinant human Antithrombin III from supernatants of baby hamster kidney cell cultures. The Antithrombin III concentration of the culture supernatants varied between 6 micrograms/ml and 14 micrograms/ml. The culture media contained 10% foetal calf serum. The concomitant overall protein concentration ranged from 5.4 mg/ml to 6.2 mg/ml, with bovine serum albumin constituting approximately 60%. Strong cation and anion exchanger, Heparin-, and Cibacron Blue Membrane Adsorber were used. While Heparin-Membrane Adsorber were found to isolate and concentrate the Antithrombin III efficiently, the removal of the major bovine serum proteins (albumin, transferrin, immunoglobulins) required a multistage process. By using a sequence of ultrafiltration, diafiltration, Cibacron Blue, anion exchanger, and Heparin-Membrane Adsorber, an electrophoretically pure Antithrombin III could be obtained. Subsequent high sensitivity gel immunoelectrophoresis proved the isolated protein free of bovine IgG and bovine transferrin, while approximately 2% serum albumin could still be discerned. A reduction of the serum content (3%) allowed the isolation of high purity Antithrombin III (> 99.9%), however, the product's specific activity was halved. The Down-Stream-process was designed and optimised for a mobile phase flow rate of 2 ml/min (0.12 l/h). With the exception of the final Heparin affinity step, however, flow rates of up to 4.8 l/h could be used without adverse effect on the final purity, with a concomitant increase of the throughput. Batches of up to 10 1 cell culture supernatant were processed in an automated procedure.