Separation of active and inactive forms of human antithrombin by heparin affinity chromatography.
During the manufacturing of an antithrombin preparation, it is necessary to define all steps that may damage or alter the target molecule, and thus decrease the biological activity of the inhibitor in blood coagulation. Pasteurization, commonly used procedure for viral inactivation of plasma derived antithrombin concentrates, was shown to partially alter the conformation of the active native antithrombin to an inactive latent form. To study intensively the different forms of inactive antithrombin that are formed upon heat treatment, human alpha-antithrombin, human beta-antithrombin and an equimolar mixture of the two isoforms were incubated at 60 degrees C for 15 h in the presence of citrate as stabilizing agent. Using two subsequent heparin affinity chromatography steps, three different inactive fractions were separated. By comparison of the heparin binding capacities, isoelectric points and unfolding characteristics of these inactive forms, the alpha-latent and beta-latent antithrombin isoforms could be identified. It was also shown that additional inactive forms such as proteinase cleaved and/or oxidized forms of antithrombin are formed during the heat treatment process. In four commercially available antithrombin preparations, all produced by pasteurization, the amount of inactive protein varied between 0.5% and 9.5%.
Research and Development, Octapharma Pharmazeutika Produktionsges m b H, Oberlaaer Strasse 235, A-1100 Vienna, Austria. andrea.heger@octapharma.at
Andrea
A
Heger
Tom
T
Grunert
Petra
P
Schulz
Djuro
D
Josic
Andrea
A
Buchacher
D000990:Antithrombin III / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002863:Chromogenic Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007526:Isoelectric Point; D009842:Oligopeptides / Q000187:drug effects; D010084:Oxidation-Reduction; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism
D002863:Chromogenic Compounds; D009842:Oligopeptides; D020033:Protein Isoforms; C015608:benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide; D000990:Antithrombin III; D006493:Heparin