CONTROL OF THE CULTIVATION PROCESS OF ANTITHROMBIN-III AND ITS CHARACTERIZATION BY CAPILLARY ELECTROPHORESIS

The production by baby hamster kidney cells of recombinant antithrombi n III (r-AT III), the main inhibitor of thrombin, factor Xa and other proteases of the clotting cascade, was monitored by capillary isotacho phoresis using mixtures of continuous spacers. The results were compar ed with those obtained by capillary zone electrophoresis (CZE). The do wnstream process, which incorporated anion-exchange and heparin affini ty chromatography, was monitored by CZE under acidic conditions and vo ltage ramping. The purified product was characterized by its isoelectr ic point and molecular mass. Isoelectric points of the three major and three minor isoforms of AT III were evaluated by capillary isoelectri c focusing using a pH range of 4-6 and various mobilization procedures . The molecular mass of AT III was investigated by capillary gel elect rophoresis (CGE), applying removable dextran gels. Both parameters cou ld be determined within 30 min using only one coated capillary. The re sults showed an excellent correspondence with those achieved with conv entional slab gels. The affinity complex between AT III and thrombin c ould also be detected by CGE and the heparin dependence of the affinit y reaction could be investigated.