Volkov G L, Andrianov S I, Horoshnykova T V, Borysevych O S, Gavryliuk O S
Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv.
Ukr Biokhim Zh (1999). 2000 Jul-Oct;72(4-5):109-21.
We have examined the technology for an industrial chromatographic production highly purified factor VIII concentrate intended for therapy of the hemophilia A and characterized this factor VIII. The final product has been prepared from cryoprecipitate of pooled human plasma using a large-scale procedure combining three conventional chromatographic steps based on AEM and CEM ion exchange and SPG or SHR gel filtration chromatography. The specific activity of the product was 459 +/- 19 IU factor VIII/mg protein (n = 10), corresponding to a purification factor of about 15,000. The concentrate was free of the fibrinogen, alpha-2-macroglobulin, alpha-1-acidglycoprotein, haptoglobin. Only three contaminants could be detected: fibronectin, immunoglobulins A and G (about 0.020, 0.004 and 0.034 microgram/IU factor VIII, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Another examination was concern to the technology for an industrial chromatographic production highly purified factor IX concentrate intended for therapy of the hemophilia B and characterized this factor IX. The final product has been prepared from pooled human plasma using a large-scale procedure combining four conventional chromatographic steps based on AEM ion exchange, AFM affinity and SGS gel filtration chromatography. The specific activity of the product was 149 +/- 10 IU factor IX/mg protein (n = 10), corresponding to a purification factor of about 9000. The concentrate was free of the vitamin K-dependent clotting factors II, VII and X and of proteins C and S. Most of possible contaminants were absent in this new product. High-molecular-weight kininogen, factor VIII, XI, XII or prekallikrein were not detected. There were no activated factors, such as factors IXa and Xa, no thrombin and no phospholipids. Only two contaminants could be detected: C4 and inter-alpha-trypsin inhibitor (about 0.8 and 1.2 mg/IU factor IX, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Thrombogenicity tests in rabbits revealed that the high purified factor IX by Institute of Biochemistry technology tested had a lower thrombogenic power than the commercial factors IX tested. The concentrate has been subjected to a special solvent--detergent treatment for definite time and temperature during its production to virus inactivation (it will be describe in following special examination). These data demonstrate that a highly purified therapeutic clotting factor VIII and IX concentrates can be prepared from human plasma by conventional chromatographic methods developed by Institute of Biochemistry of NAS of Ukraine and Combio Ltd.
我们研究了用于工业色谱生产高纯度凝血因子 VIII 浓缩物的技术，该浓缩物用于治疗甲型血友病，并对该凝血因子 VIII 进行了表征。最终产品是由混合人血浆的冷沉淀制备而成，采用了大规模程序，该程序结合了基于 AEM 和 CEM 离子交换以及 SPG 或 SHR 凝胶过滤色谱的三个传统色谱步骤。该产品的比活性为 459±19 IU 凝血因子 VIII/毫克蛋白质（n = 10），对应于约 15,000 的纯化因子。该浓缩物不含纤维蛋白原、α-2-巨球蛋白、α-1-酸性糖蛋白、触珠蛋白。仅检测到三种污染物：纤连蛋白、免疫球蛋白 A 和 G（分别约为 0.020、0.004 和 0.034 微克/IU 凝血因子 VIII）。最终产品的纯度通过 SDS 聚丙烯酰胺凝胶电泳、醋酸纤维素电泳、格拉巴尔 - 威廉姆斯免疫电泳和双向免疫电泳得以确认。另一项研究涉及用于工业色谱生产高纯度凝血因子 IX 浓缩物的技术，该浓缩物用于治疗乙型血友病，并对该凝血因子 IX 进行了表征。最终产品是由混合人血浆制备而成，采用了大规模程序，该程序结合了基于 AEM 离子交换、AFM 亲和以及 SGS 凝胶过滤色谱的四个传统色谱步骤。该产品的比活性为 149±10 IU 凝血因子 IX/毫克蛋白质（n = 10），对应于约 9000 的纯化因子。该浓缩物不含维生素 K 依赖性凝血因子 II、VII 和 X 以及蛋白 C 和 S。该新产品中不存在大多数可能的污染物。未检测到高分子量激肽原、凝血因子 VIII、XI、XII 或前激肽释放酶。不存在活化因子，如因子 IXa 和 Xa，不存在凝血酶，也不存在磷脂。仅检测到两种污染物：C4 和α-胰蛋白酶抑制剂（分别约为 0.8 和 1.2 毫克/IU 凝血因子 IX）。最终产品的纯度通过 SDS 聚丙烯酰胺凝胶电泳、醋酸纤维素电泳、格拉巴尔 - 威廉姆斯免疫电泳和双向免疫电泳得以确认。在兔子身上进行的血栓形成性测试表明，乌克兰国家科学院生物化学研究所技术制备的高纯度凝血因子 IX 的血栓形成能力低于所测试的市售凝血因子 IX。该浓缩物在生产过程中经过了特定时间和温度的特殊溶剂 - 去污剂处理以进行病毒灭活（将在后续的特殊检查中描述）。这些数据表明，通过乌克兰国家科学院生物化学研究所和 Combio 有限公司开发的传统色谱方法，可以从人血浆中制备高纯度的治疗性凝血因子 VIII 和 IX 浓缩物。
