A Highly Purified Antithrombin III Concentrate Prepared From Human Plasma Fraction IV-1 by Affinity Chromatography

Abstract

We describe an improved method for large-scale purification of antithrombin III (AT-III) from human plasma involving heparin affinity chromatography of redissolved fraction IV-1 paste, viral inactivation by heating, followed by a second heparin affinity column. The characteristics of a new heparin affinity resin and the ability to extrapolate process behavior from small-scale (20 ml) to large-scale (40 liter) columns are described. This supports the use of the small-scale column for process optimization and validation studies in compliance with current regulatory requirements for biological products. The process has been characterized by analytical techniques including sodium dodecyl sulfate (SDS), reducing SDS, and nondenaturing polyacrylamide gel electrophoresis; laser desorption time-of- flight mass spectroscopy, and electrospray 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.

Quantification of Antithrombin Isoform Proportions in Plasma Samples of Healthy Subjects, Sepsis Patients, and in Antithrombin Concentrates

“…In concentrate A, which is equivalent to the current WHO AT standard, ß-isoform contents were found to be close to the average plasma levels of healthy donors [33], whereby concentrate A contained significantly higher amounts of AT-ß than concentrates B, C and D. In concentrates B and D we found minimal AT-ß levels -close to 1% or even below -reflecting differences in the manufacturing process, which includes heparin adsorption as the main purification step. Adsorption and elution conditions influence the yields of the ß-isoform, as well as different plasma fractions used as raw material [34][35][36].…”

Separation of Native and Latent Forms of Human Antithrombin by Hydrophobic Interaction High-Performance Liquid Chromatography

“…The resolution between AT and L-AT in the HIC-HPLC method was high in the two buffer 1 To whom correspondence should be addressed. 2 Abbreviations used: Ac-Trp, acetyl tryptophan; AT, antithrombin; HIC, hydrophobic interaction chromatography; HPLC, highperformance liquid chromatography; L-AT, latent antithrombin. systems used, eluting with negative gradients consisting of ammonium sulfate or sodium chloride.…”