Wr. Lebing et al., A HIGHLY PURIFIED ANTITHROMBIN-III CONCENTRATE PREPARED FROM HUMAN PLASMA FRACTION IV-1 BY AFFINITY-CHROMATOGRAPHY, Vox sanguinis, 67(2), 1994, pp. 117-124
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.