A HIGHLY PURIFIED ANTITHROMBIN-III CONCENTRATE PREPARED FROM HUMAN PLASMA FRACTION IV-1 BY AFFINITY-CHROMATOGRAPHY

We describe an improved method for large-scale purification of antithr ombin III (AT-III) from human plasma involving heparin affinity chroma tography of redissolved fraction IV-1 paste, viral inactivation by hea ting, followed by a second heparin affinity column. The characteristic s of a new heparin affinity resin and the ability to extrapolate proce ss behavior from small-scale (20 ml) to large-scale (40 liter) columns are described. This supports the use of the small-scale column for pr ocess optimization and validation studies in compliance with current r egulatory requirements for biological products. The process has been c haracterized by analytical techniques including sodium dodecyl sulfate (SDS), reducing SDS, and nondenaturing polyacrylamide gel electrophor esis; laser desorption time-of-flight mass spectroscopy, and electrosp ray mass spectroscopy. These results demonstrate that greater than 95% of the protein in the final products is AT-III, which is greater than 95% active as defined by thrombin inhibition.