Antigenic (Immunogenic) Profiling of Bovine Thrombin and its Purified Forms

Abstract

purified and further processed by ultrafiltration. Analytical studies have shown that the current manufacturing processes are capable of removing significant amounts of extraneous proteins and result in a reduction of factor Va light chain content levels below the limit of detection of the Western blot assay (less than 92 ng/mL, when reconstituted as directed). However, the clinical significance of this reduction of factor Va–related antigen is unknown. The US FDA approval of the revised labeling regarding the purity of Thrombin JMI is a testimonial for the advanced methods that can be used to purify biologicals, such as bovine thrombin, to remove contaminants, which may lead to the generation of antibodies or may trigger other adverse reactions. Although the removal of bovine factor V, factor Va and/or their fragments from the product has been shown, it is highly likely that other potential contaminants and carryover products are also removed in this additional purification step. Therefore, a comparison of the crude thrombin and the 2 purified versions for other potential contaminants, including prothrombin, factors VII and X, may also be helpful because bovine plasma is used as the starting material for the preparation of the crude thrombin. The purified Thrombin JMI will likely show a relatively improved safety profile with much lesser immunogenic potential. Despite the fact that the reported hemostatic abnormalities with the use of this product since the 1940s are relatively rare, 2-6 the improved product may show an even lesser prevalence of these reported adverse reactions. It should remain clear that despite improved purification processes, some biological products may retain inherent immunologic properties leading to the generation of antibodies, as has been the case with many of the highly purified drugs such as recombinant hirudin, aprotinin, and recombinant Bovine thrombin is widely used as a topical hemostatic agent in various surgical procedures. To date, more than 10 million individuals have been exposed to this product. Rare case reports of adverse reactions such as antigenic reactions and apparent generation of antibodies leading to hemostatic disruption have been reported. Isolated reports on the presence of factor Va–related antigen and other contaminant proteins have also been made available. Additionally, case reports have commented on the generation of antibodies to other human coagulation factors. However, bovine thrombin has been remarkably safe than other biological products, such as aprotinin, unfractionated heparin, and erythropoietin. The article by Terrab, et al in this issue of Clinical and Applied Thrombosis/Hemostasis reports on the inclusion of a membrane-filtration step in the manufacturing process, which is performed to remove viruses from chromatographically purified bovine thrombin. It has been found that this step also results in the effective removal of extraneous protein contaminants, in particular factor V, factor Va and/or their fragments from the product. On review of the data summated for this purification process, the US Food and Drug Administration (FDA) has approved a revised labeling for Thrombin JMI (Thrombin, topical bovine, United States Pharmacopenia) to recognize the reduced factor Va light chain content (considered to be a factor for the immunogenic response). The revised labeling states that Thrombin JMI has been chromatographically