Institute for Molecular Biotechnology, Worringer Weg 1, RWTH Aachen University, 52074 Aachen, Germany.
Biotechnol J. 2014 Mar;9(3):415-25. doi: 10.1002/biot.201300369. Epub 2013 Dec 10.
The extraction of biopharmaceutical proteins from intact leaves involves the release of abundant particulate contaminants that must be removed economically from the process stream before chromatography, for example, using disposable filters that comply with good manufacturing practice. We therefore scaled down an existing 200-kg process for the purification of two target proteins from tobacco leaves (the monoclonal antibody 2G12 and the fluorescent protein DsRed, as monitored by surface plasmon resonance spectroscopy and fluorescence imaging, respectively) and screened different materials on the 2-kg scale to reduce the number of depth filtration steps from three to one. We assessed filter cost and capacity, filtrate turbidity, and protein recovery when the filter materials were challenged with extracts from different tobacco varieties and related species grown in soil or rockwool. PDF4 was consistently the most suitable depth filter because it was the least expensive, it did not interact significantly with the target proteins, and it had the greatest overall capacity. The filter capacity was generally reduced when plants were grown in rockwool, but this substrate has a low bioburden, thus improving process safety. Our data concerning the clarification of plant extracts will help in the design of more cost-effective downstream processes and accelerate their development.
从完整叶片中提取生物制药蛋白时，会释放出大量的颗粒状污染物，在进行色谱分析之前，必须从工艺流中经济地去除这些污染物，例如使用符合良好生产规范的一次性过滤器。因此，我们对现有的 200 公斤从烟草叶片中纯化两种目标蛋白（单克隆抗体 2G12 和荧光蛋白 DsRed，分别通过表面等离子体共振光谱和荧光成像监测）的工艺进行了缩小规模，并在 2 公斤规模上筛选了不同的材料，以将深度过滤步骤从三个减少到一个。我们评估了过滤成本和容量、滤液浊度以及当过滤材料受到来自不同烟草品种和在土壤或岩棉中生长的相关物种的提取物挑战时的蛋白质回收率。PDF4 始终是最合适的深度过滤器，因为它最便宜、与目标蛋白的相互作用不显著，并且具有最大的总容量。当植物在岩棉中生长时，过滤器的容量通常会降低，但这种基质的生物负荷低，从而提高了工艺安全性。我们关于植物提取物澄清的数据将有助于设计更具成本效益的下游工艺，并加速其开发。
