Department of Environmental Sciences, Tuscia University, Viterbo, Italy.
Electrophoresis. 2011 Nov;32(21):2941-50. doi: 10.1002/elps.201100246. Epub 2011 Oct 14.
In this study, we investigated the heterogeneity and the purity grade of three commercially available plasma-derived clotting factor VIII (FVIII) concentrates, which highly differ with regard to purification strategies, relative concentrations of stabilizers (von Willebrand factor, with or without albumin) and virus inactivation strategies (solvent/detergent and/or heat/pasteurization treatments). Western blot analyses were used to evaluate product-specific variations from Emoclot(®) , Alphanate(®) and Haemate(®) both in the presence and absence of reducing agents (dithiotreithol). All the plasma-derived concentrates showed a strong heterogeneity, as they all included a significant amount of truncated forms of the full-length (FL) clotting FVIII protein. The intact protein accounted for the 38% of the total FVIII proteins in Haemate(®) and 29 and 23% in Alphanate(®) and Emoclot(®) , respectively. Lower intact FVIII amounts in Emoclot might be mainly due to the low von Willebrand factor dosage and the absence of albumin. Upon addition of thrombin, both the FL and truncated forms of the FVIII protein were almost completely digested. Indeed, after thrombin activation, we could still observe a mixture of B-domain truncated forms of the FL protein along with biologically active digested-A1 forms. Batch-to-batch variation was tested with no evident changes appearing among different batches. Despite the variables in manufacturing processes, inter-product comparisons yielded similar results for all the plasma-derived FVIII considered in this study. However, we could individuate in Emoclot a band that was not digested by thrombin, which we could characterize as the 200 kDa FVIII heavy chain. This investigation prompts new concerns about the strong heterogeneity observed upon thrombin digestion of plasma-derived FVIII, which might contribute to the development of inhibitory antibodies at an early stage of therapy, and to which extent these untoward phenomena could be avoided through direct intervention on routine manufacturing processes.
在这项研究中，我们研究了三种市售的血浆源性凝血因子 VIII (FVIII) 浓缩物的异质性和纯度等级，它们在纯化策略、稳定剂（血管性血友病因子，有或没有白蛋白）的相对浓度以及病毒灭活策略（溶剂/去污剂和/或热/巴氏消毒处理）方面有很大的不同。使用 Western blot 分析评估了 Emoclot ® 、 Alphanate ® 和 Haemate ® 在存在和不存在还原剂（二硫苏糖醇）的情况下的产品特异性变化。所有的血浆源性浓缩物都显示出很强的异质性，因为它们都包含大量全长（FL）凝血因子 VIII 蛋白的截断形式。完整的蛋白质占 Haemate ® 中总 FVIII 蛋白的 38%，Alphanate ® 和 Emoclot ® 中的分别为 29%和 23%。Emoclot 中的完整 FVIII 量较低可能主要是由于低血管性血友病因子剂量和缺乏白蛋白。加入凝血酶后，FL 和截断形式的 FVIII 蛋白几乎完全被消化。事实上，在凝血酶激活后，我们仍然可以观察到 FL 蛋白 B 结构域截断形式与具有生物活性的消化 A1 形式的混合物。未发现不同批次之间出现明显变化，因此进行了批次间变异测试。尽管制造过程存在变量，但对本研究中考虑的所有血浆源性 FVIII 进行的产品间比较产生了相似的结果。然而，我们可以在 Emoclot 中鉴定出一种不被凝血酶消化的条带，我们可以将其特征化为 200 kDa 的 FVIII 重链。这项研究促使人们对血浆源性 FVIII 在凝血酶消化过程中观察到的强烈异质性产生了新的关注，这可能导致在治疗的早期阶段产生抑制性抗体，以及在多大程度上可以通过直接干预常规制造过程来避免这些不良现象。
