High-performance capillary electrophoresis for in-process control in the production of antithrombin III and human clotting factor IX.

Antithrombin III (ATIII) and factor IX (FIX), two proteins from the clotting cascade, were investigated in parallel experiments, using capillary gel electrophoresis and capillary isoelectric focusing. The results from these experiments were compared with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and slab gel isoelectric focusing. In the case of ATIII, capillary gel sieving showed comparable results to SDS-PAGE with the added advantage of the shorter time required for analysis. By optimizing capillary isoelectric focusing (cIEF), a separation of the ATIII isoforms was achieved. In the case of FIX, capillary gel electrophoresis (SDS-CE) of a FIX preparation gave similar results to those obtained by size-exclusion high-performance liquid chromatography and SDS-PAGE, but turned out to be less sensitive in detecting protein impurities at low concentrations. The microheterogeneity of this protein was shown by using cIEF.