Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, USA.
Mol Ther. 2011 Feb;19(2):302-9. doi: 10.1038/mt.2010.239. Epub 2010 Nov 16.
Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.05 pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5 mg/l of recombinant fVIII at the rate of 9 pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies.
患有 A 型血友病的患者会出现自发性出血，有时甚至会出现危及生命的情况，这些情况需要使用血液凝血因子 VIII（fVIII）替代产品进行治疗。尽管这些产品有效，但由于供应限制和产品成本，在全球范围内的可用性有限，这些成本主要源于制造的复杂性。目前，哺乳动物细胞培养制造系统的重组 fVIII 产量约为 100µg/L，每个细胞的产量为 0.05pg/细胞/天，这代表与其他类似大小的重组蛋白（例如单克隆抗体）相比，产量低了约 10000 倍。人类 fVIII 的表达受到通过细胞分泌途径的低效运输的限制。最近，我们发现同源的猪 fVIII 具有两个独特的序列元件，可增强分泌运输效率。在此，我们描述了使用新型慢病毒驱动的重组蛋白制造平台开发生物工程 fVIII 产品的情况。这两种技术的综合应用产生了生产细胞系，可在每天 9pg/细胞的速度下生物合成超过 2.5mg/L 的重组 fVIII，这是迄今为止报道的重组 fVIII 产量的最高水平，从而验证了这两种技术的实用性。
