Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr 71-76, D-13355 Berlin, Germany.
Microb Cell Fact. 2010 May 20;9:35. doi: 10.1186/1475-2859-9-35.
Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.
Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.
Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditions from the first screening phase not only shortens the developmental line by granting that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.
由于许多因素会影响产物以可溶性和活性形式的积累，因此重组蛋白的生物工艺开发既耗时又费力。目前，在大多数情况下，开发路线的特点是在分批条件下进行筛选阶段，然后再开发补料分批过程。在补料分批条件下进行筛选不仅可以减少工作量，还可以保证所选有利条件在生产规模上也适用。
在这里，首次将高通量多因素克隆文库筛选与 96 孔板中的补料分批技术相结合，并直接衍生出一种策略，可直接扩展到生物反应器规模。以一种难以表达的蛋白质（核糖核酸酶抑制剂）为例，证明了通过（i）应用具有不同强度启动子和核糖体结合位点的载体文库以及各种融合伙伴，以及（ii）早期使用补料分批技术，可以在一条连贯的线路上进行各种载体构建和生长条件的筛选。结果表明，EnBase 技术为微井规模的受控培养条件提供了简单的解决方案。此外，从较小的培养体积中获得的高细胞密度为各种分析提供了材料。在实际情况下，目标蛋白高产的关键因素是（i）融合伙伴，（ii）使用矿物盐培养基和补料分批技术，以及（iii）预诱导生长速率。最后，结果表明，在微井板和摇瓶规模上选择的有利条件也适用于生物反应器。
培养介质和培养条件对筛选程序的成功有重大影响。因此，应用受控培养条件至关重要。从第一个筛选阶段开始连续使用补料分批条件不仅可以缩短开发路线，因为可以保证所选条件与规模扩大相关，而且在我们的情况下，标准批培养根本无法选择正确的克隆或条件。
