Feasibility of preparing human plasminogen by full chromatography from precipitation of Fraction Ⅲ in low temperature ethanol method
Objective To determine the feasibility of preparing plasminogen(Pg) with Fraction Ⅲ precipitation(here-inafter referred to as FⅢ-P) from low-temperature ethanol process by full chromatography(hereinafter referred to as FⅢ-P process). Methods The FⅢ-P was diluted with dissolution buffer at different dilution times and stirring time. The potency and antigen concentration of Pg in dissolution sample were detected and the dissolution and clarification conditions were de-termined. Pre-treatment of loading sample and pre-experiment of affinity chromatography were carried out on the FⅢ-P dis-solution sample to judge whether the loading sample had an impact on the chromatography by observing the performance of the affinity plasma were calculated. The feasibility of preparing Pg by FⅢ-P process was evaluated by comparing with the data of full chromatography process using plasma as raw material (hereinafter referred to as plasma process). Results The FⅢ-P was dissolved with 10 times of dissolution buffer,stirred for 1 hour,centrifuged at room temperature of 10000×g for 15 minutes. The supernatant was first filtered with a screen,then clarified with an 8/0.8 μm filter,and finally filtered with a 0.45/0.2 μm filter and loaded. Pre-test showed that from clarification and filtration to Pg affinity chromatography,the step recovery of activity and antigen was 39.51％ and 108.64％,respectively,the antigen concentration of Pg increased by 31.16 times and the activity increased by 11.39 times after affinity chromatography,which reaching the effect of affinity chromatography pur-ification of Pg. The results of 2 batches of step-by-step scale-up FⅢ-P process showed that the total recoveries of antigen and activity from plasma to SP chromatography of FⅢ-P process were (45.76±1.10)％ and (24.15±0.59)％,respective-ly,which had a total loss of about 1/3 of antigen and about 2/3 of activity compared to the plasma process. The Pg specific activity of SP chromatography eluent was (4.68±0.25)U/mg,which was about half of that of plasma process,but meeted the internal standard of>4 U/mg.The output of Pg antigen per ton of plasma in the FⅢ-P process was 68.73％ of that in the plasma process,and the output of Pg activity per ton of plasma in the plasma process was 29.82％ of that in the plasma process,which basically achieved the purpose of waste utilization of FⅢ-P. Conclusion The technical route of preparing Pg from FⅢ-P by full chromatography is feasible.
万方数据
维普
