全层析法在由低温乙醇法组分Ⅲ沉淀制备人纤维蛋白溶酶原中的应用  

Feasibility of preparing human plasminogen by full chromatography from precipitation of FractionⅢin low temperature ethanol method

作　　者：张金 岳胜兰 朱晨 彭焱 周雁翔 林连珍 陈克金 冯璐 胡勇 周志军 ZHANG Jin;YUE Shenglan;ZHU Chen;PENG Yan;ZHOU Yanxiang;LIN Lianzhen;CHEN Kejin;FENG Lu;HU Yong;ZHOU Zhijun(Sinopharm Wuhan Bio-Pharmaceutical Co.,LTD.,Wuhan 430207,China)

机构地区：[1]国药集团武汉生物制药有限公司,湖北武汉430207

出　　处：《中国输血杂志》2024年第11期1293-1300,共8页Chinese Journal of Blood Transfusion

摘　　要：目的确定以低温乙醇工艺的组分Ⅲ沉淀(以下简称FⅢ-P)为原料,用全层析工艺制备人纤维蛋白溶酶原(plasminogen,Pg)(以下简称FⅢ-P工艺)的可行性。方法用溶解缓冲液以不同稀释倍数和搅拌时间溶解FⅢ-P沉淀,检测溶解物中Pg的效价和抗原浓度,确定FⅢ-P的溶解和澄清条件;对FⅢ-P的溶解物进行前处理和亲和层析的预实验,通过观察层析过程判断FⅢ-P原料对亲和层析进行是否有影响,通过检测过程样品的血浆蛋白抗原浓度和Pg效价并计算Pg抗原回收率来评估亲和层析能否达到纯化Pg的目的。进行2批FⅢ-P全层析工艺(Pg亲和层析+SP层析,以下简称FⅢ-P工艺)的逐级放大研究,计算Pg比活性、步骤及总回收率、每吨血浆Pg产出量,通过与以血浆为原料的全层析工艺(以下简称血浆工艺)的数据进行比较来评估FⅢ-P工艺制备Pg的可行性。结果FⅢ-P用溶解缓冲液稀释10倍,搅拌1 h,室温10000×g离心15 min,上清用筛网初滤后再用8/0.8μm滤器澄清,最后用0.45/0.2μm滤器过滤后上样。预实验显示,以澄清过滤为起点到Pg亲和层析,Pg活性和抗原的步骤回收率分别为39.51%和108.64%,亲和层析后Pg的抗原浓度提高31.16倍,活性提高11.39倍,达到亲和层析纯化Pg的效果。两批逐级放大FⅢ-P工艺结果显示FⅢ-P工艺从血浆到SP层析的Pg抗原和活性总回收率分别为(45.76±1.10)%和(24.15±0.59)%,与血浆工艺相比总计损失了约1/3的抗原和约2/3的活性;SP层析洗脱液的Pg比活性为(4.68±0.25)U/mg,约为血浆工艺的一半,但是达到>4 U/mg的企业内部标准。FⅢ-P工艺的每吨血浆Pg抗原产出量为血浆工艺的68.73%,每吨血浆Pg活性产出量为血浆工艺的29.82%,基本达到FⅢ-P废物利用的目的。结论用全层析工艺从FⅢ-P中制备Pg的技术路线可行。Objective To determine the feasibility of preparing plasminogen(Pg)with FractionⅢprecipitation(hereinafter 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 determined.Pre-treatment of loading sample and pre-experiment of affinity chromatography were carried out on the FⅢ-P dissolution sample to judge whether the loading sample had an impact on the chromatography by observing the performance of the affinity chromatography column and to evaluate whether the affinity chromatography could achieve the purpose of purifying Pg by detecting the plasma protein antigen concentration and Pg potency of the samples in the process.Two batches of FⅢ-P process were studied step by step,and the specific activity,steps and total recovery,and the output of Pg per ton of 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 8/0.8μm filter,and finally filtered with a 0.45/0.2μm 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 purification of Pg.The results of 2 batches of step-by-step scale-up FⅢ-P process showed that the total recoverie

关 键 词：纤维蛋白溶酶原 组分Ⅲ沉淀 活性回收率 抗原回收率 比活性 

分 类 号：R331.141[医药卫生—人体生理学] O658.1[医药卫生—基础医学]