Characterization and viral safety validation study of a pasteurized therapeutic concentrate of antithrombin III obtained through affinity chromatography

Abstract

BACKGROUND AND OBJECTIVE: Antithrombin III (ATIII) concentrates are employed as therapy for congenital or acquired deficiencies. These concentrates are obtained from Cohn's fraction IV1. To improve yields, purity and safety, our group developed a procedure to obtain a pasteurized ATIII concentrate from the supernatant of Cohn's fraction II + III including a highly efficient heparin affinity chromatography purification and pasteurization as a viral inactivation step. DESIGN AND METHODS: Three steps of the manufacturing procedure (Cohn's fraction II + III precipitation, affinity chromatography and pasteurization) were selected to examine their efficacy in inactivating and/or removing the selected viruses. RESULTS: The industrial batches show a purity higher than 99% with approximately 95% native heparin binding ATIII. Only albumin and IgG could be detected at trace levels (0.07% and 0.16% of the total protein present, respectively). The specific activity of the product was approximately 6.65 IU/mg protein. Five viruses were spiked into the manufacturing starting materials and samples were collected at various points to determine the infection level of virus. The study showed a reduction factor (log 10) > or = 11.7 for HIV-1; > or = 8.1 for bovine herpes virus (analyzed as a model for herpes and hepatitis B viruses); > or = 8.1 for bovine diarrhea virus (model for hepatitis C and G) and > or = 6.0 for encephalomyocarditis virus (model for hepatitis A and other non-enveloped viruses). INTERPRETATION AND CONCLUSIONS: No biochemical alterations of the ATIII were detected in the final product. A high viral elimination capacity of the production process was demonstrated. So far, more than 32 million units of ATIII have been transfused in the form of this therapeutic concentrate without any detected seroconversion.