Fermentation, Purification And Quality Control Study Of Anti-thrombin Protein EH

We mainly studied the fermentation and purification of protein EH. Both FXa and FXIa respectively recognized the site of EPR, which was linked to the N-terminal of hirudin for blocking its anticoagulation temporarily. After enough protein was got, we established the way of quality control for EH. The results are as follows:To establish the high-density fermentation and purification process of hirudin derivative, EH. EH was expressed in Pichia pastoris GS115 under a high-density fermentation. After fermentation, the fermentation broth was centrifuged and the supernatant was collected and purified by ultrafiltration and two-step ion-exchange chromatography. The purity of the purified production was determined by SDS-PAGE and RP-HPLC. Antithrombin activity of EH was measured by clot method. After 80 hours cultivation under the high-density fermentation, high-throughput production was achieved:410 OD600 in biomass and 420g/L in wet weight. In the purification technique, the protein recovery rate was 43%. After purification, the purity of EH, determined by RP-HPLC, was over 97%. The results of clot method showed that the N-terminus of H V2 in EH was blocked by linker peptide EPR, before cleavaged, EH don't have antithrombin activity, after cleavaged the antithrombin activity of EH was 256～512 ATU/mg. The production process of EH is simple, high efficiency, and the process is easy to scale up.When got enough EH, the quality control methods of EH was established. According to the third pharmacopoeias of the People's Republic of China(2010) and the relevant directions about biological products, the detection methods of the following items were established:the protein concentration was monitored with Lowry method; the detection of ultraviolet absorption spectrum; the purity was detected with RP-HPLC; antithrombin activity of EH was analyzed with chromogenic substrate method. The protein concentration of EH was determined three times by Lowry methods, and the result are 20.02mg/mL,20.08mg/mL and 20.14mg/mL, respectively, accord with the standard of>20mg/mL. EH was diluted to 2mg/mL using ultra-pure water. The maximum absorption at 275nm was 0.52. Definited the UV absorption peak of EH is (275±3)nm; RP-HPLC assay results of EH were 96.24%,96.93% and 97.01%, which all in line with the standard of>95%. The optimal cleavage conditions for EH with FXa in vitro is that EH was reacted with FXa at 37℃for 6 hours in saline solution with the molar ratio (1:180) of FXa and EH. The antithrombin activity of EH were 364,396, 410ATU/mg, we defined the antithrombin activity of EH was 400×(1±30%) ATU/mg. The evaluation methods of protein concentration, purity, ultra-violet absorption spectrum and antithrombin activity of EH were established in the study. And these methods established a standard way of the quality control for EH. The experiment provided a foundation for EH in drug registration.