Development of human thrombin production at National Blood Centre, Thai Red Cross Society

Abstract

Abstract:

Introduction: Human thrombin (HT) has been widely used in hemostatic products and used as an active ingredient to produce fibrin sealant or fibrin glue. Fibrin sealant was produced by National Blood Centre, Thai Red Cross Society (NBC, TRCS) since 1997 using imported HT. It was clinically applied in many surgeries such as dental and heart procedures. However, since 2010, the manufacturer has denied to supply the product. As a result, fibrin sealant production was terminated. The development of HT from local plasma was initiated in phase I during 2016-2020 in order to utilize proteins in plasma to maximize their benefits in patient treatment. Objective: This study aims to develop a process for producing HT from plasma at the NBC, TRCS instead of importing from abroad which is expensive. Materials and Methods: The production of thrombin started with prothrombin complex (PTC) separation, where pooled cryo-removed plasma (CRP) was processed with DEAE Sephadex resin and eluted to obtain crude PTC before converting PTC to thrombin by glycine, calcium chloride, and pH control. Solvent detergent (SD) treatment was performed to ensure viral safety before thrombin purification using SP Sephadex column chromatography and nanofiltration. The prothrombin and purified thrombin were analyzed for activity and purity using coagulation assays, spectrophotometry, gas chromatography, and SDS-PAGE. Results: HT production from three 20 L of CRP batches yielded titers of 12,665, 11,143 and 12,727 IU/mL, with specific activities of 1,550, 1,450, and 1,509 IU/mg, respectively. SD residues were within acceptable limits in 2 batches, and SDS-PAGE showed no other protein bands post-purification. Scaling up to 40 L of CRP for two lots produced results consistent with the 20 L batches in terms of thrombin titer, specific activity, and SD residues. Conclusion: NBC, TRCS developed scalable HT from plasma production meeting the criteria for medical and research applications.