Leong Susanna S J, Middelberg Anton P J
Centre for Biomolecular Engineering, Division of Chemical Engineering, The University of Queensland, St. Lucia, Queensland 4072, Australia.
Biotechnol Bioeng. 2007 May 1;97(1):99-117. doi: 10.1002/bit.21271.
A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies.
本文展示了一种简单有效的大肠杆菌生物工艺，用于制备重组人甲胎蛋白（rhAFP），这是一种具有重要免疫调节功能且在制药领域颇具前景的蛋白质。新的rhAFP工艺仅采用易于放大和验证的单元操作，并降低了现有包涵体处理方法的复杂性。建立该工艺的一个关键要求是在蛋白质复性之前获得高纯度的rhAFP，原因如下：（i）rhAFP一旦复性就容易与疏水性污染物结合；（ii）rhAFP在复性过程中会以依赖污染物的方式聚集。在这项工作中，在使用两个简单的色谱步骤进行纯化之前，将从细胞悬液中直接提取蛋白质与DNA沉淀 - 离心步骤相结合。复性采用单步、氧化还原优化的稀释复性方案，通过反相高效液相色谱分析、酶联免疫吸附测定和圆二色光谱法确定复性是否成功。对每个单元操作后的DNA和蛋白质污染物负载量进行定量分析表明，污染物水平降低到了与传统工艺流程相当的水平。在这个基于尿素的工艺中，识别并最小化了由于氨甲酰化导致的蛋白质微化学修饰，从而得到了一种最终复性和纯化的产品，该产品从氨甲酰化变体中得到了显著纯化。重要的是，这项工作首次确凿地证明，化学提取工艺可以替代更复杂的传统包涵体处理工艺流程，而不会影响产品纯度和产量。这种高度强化和简化的工艺有望普遍应用于制备其他以包涵体形式表达的治疗候选药物。
