School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore.
J Chromatogr A. 2012 Feb 3;1223:64-71. doi: 10.1016/j.chroma.2011.12.037. Epub 2011 Dec 16.
The Hepatitis B Virus X (HBx) protein is a potential therapeutic target for the treatment of hepatocellular carcinoma. However, consistent expression of the protein as insoluble inclusion bodies in bacteria host systems has largely hindered HBx manufacturing via economical biosynthesis routes, thereby impeding the development of anti-HBx therapeutic strategies. To eliminate this roadblock, this work reports the development of the first 'chromatography refolding'-based bioprocess for HBx using immobilised metal affinity chromatography (IMAC). This process enabled production of HBx at quantities and purity that facilitate their direct use in structural and molecular characterization studies. In line with the principles of quality by design (QbD), we used a statistical design of experiments (DoE) methodology to design the optimum process which delivered bioactive HBx at a productivity of 0.21 mg/ml/h at a refolding yield of 54% (at 10 mg/ml refolding concentration), which was 4.4-fold higher than that achieved in dilution refolding. The systematic DoE methodology adopted for this study enabled us to obtain important insights into the effect of different bioprocess parameters like the effect of buffer exchange gradients on HBx productivity and quality. Such a bioprocess design approach can play a pivotal role in developing intensified processes for other novel proteins, and hence helping to resolve validation and speed-to-market challenges faced by the biopharmaceutical industry today.
乙型肝炎病毒 X (HBx) 蛋白是治疗肝细胞癌的潜在治疗靶点。然而，由于该蛋白在细菌宿主系统中以不溶性包涵体的形式持续表达，因此在经济的生物合成途径中极大地阻碍了 HBx 的制造，从而阻碍了抗 HBx 治疗策略的发展。为了消除这一障碍，本工作报道了使用固定化金属亲和层析（IMAC）开发的第一个基于“层析复性”的 HBx 生物工艺。该工艺能够生产出大量和高纯度的 HBx，便于直接用于结构和分子特征研究。按照质量源于设计（QbD）的原则，我们使用统计实验设计（DoE）方法学来设计最优工艺，该工艺在复性浓度为 10mg/ml 时的复性产率为 54%（54%），复性产量为 0.21mg/ml/h，比稀释复性提高了 4.4 倍。本研究采用的系统 DoE 方法学使我们能够深入了解不同生物工艺参数的影响，如缓冲液交换梯度对 HBx 产率和质量的影响。这种生物工艺设计方法可以在为其他新型蛋白质开发强化工艺方面发挥关键作用，从而有助于解决当今生物制药行业面临的验证和上市速度挑战。
