Department of Chemical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India.
J Sep Sci. 2012 Nov;35(22):3160-9. doi: 10.1002/jssc.201200476. Epub 2012 Oct 22.
Production of biotech therapeutics in Escherichia coli involves protein expression as insoluble inclusion bodies that need to be denatured and the resulting protein refolded into the native structure. In this paper, we apply a Quality by Design approach using Design of Experiments for optimization of the refolding process for a recombinant biotech therapeutic, granulocyte colony stimulating factor. First, risk analysis was performed to identify process parameters that require experimental examination. Next, the chosen parameters were examined using a fractional factorial screening design. Based on the results of this study, parameters that have significant effect on refold yield and product quality were identified and examined using a full factorial Design of Experiments for their interactions. The final model was statistically significant and delivered a refolding yield of 77%. Further, kinetics of refolding was evaluated under optimal conditions and was found to be of first order with a rate constant of 0.132/min. Design space was established for the three parameters for a given permissible range of yield, protein concentration, and purity. The primary objective of this paper is to provide a roadmap for implementing Quality by Design for development of a protein refolding step.
在大肠杆菌中生产生物技术治疗药物涉及到以不溶性包涵体形式表达蛋白质，这些包涵体需要变性，然后将得到的蛋白质重新折叠成天然结构。在本文中，我们应用了质量源于设计（Quality by Design）方法，使用实验设计（Design of Experiments）对重组生物技术治疗药物——粒细胞集落刺激因子的复性过程进行了优化。首先，进行了风险分析，以确定需要实验检查的工艺参数。接下来，使用部分因子筛选设计对选定的参数进行了检查。基于这项研究的结果，确定了对复性产率和产品质量有显著影响的参数，并使用全因子实验设计对其相互作用进行了检查。最终模型具有统计学意义，复性产率达到 77%。此外，还在最佳条件下评估了复性动力学，发现其为一级反应，速率常数为 0.132/min。为给定的产率、蛋白质浓度和纯度允许范围，为三个参数建立了设计空间。本文的主要目的是为蛋白质复性步骤的开发实施质量源于设计提供路线图。
