Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco
Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco
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DOI：
10.1111/pbi.12132
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发表时间：
2014-02-01
影响因子：
13.8
通讯作者：
Fischer, Rainer
中科院分区：
文献类型：
--
作者：
Buyel, Johannes F.;Fischer, Rainer
絮凝是一种经济高效的方法，通过使分散的颗粒聚集在一起，允许通过沉淀、离心或过滤将其去除，从而提高澄清效率。任何给定过程的絮凝效果取决于进料流中颗粒的性质和浓度、絮凝剂聚合物的浓度、电荷密度和长度、剪切速率、进料流的性质(例如pH和离子强度)以及目标产品的性质。我们用实验设计的方法测试了一系列絮凝剂和工艺条件，以确定在生产HIV中和单抗(2G12)和在转基因烟草叶片中表达的荧光标记蛋白(DsRed)过程中最适合澄清步骤的聚合物。在我们测试的23种不同的絮凝剂中，聚合菌素P的降浊度效果最好，它是一种支化的阳离子聚乙烯亚胺，其电荷密度为13.0meq/g，在各种工艺条件下，该絮凝剂对浊度的降低率均在90%以上。我们开发了一个模型，预测其在不同工艺条件下的性能，这使我们能够根据工艺规模、深度过滤器类型和植物种类将深度过滤器的容量提高三倍。与不加絮凝剂的工艺相比，过滤耗材的成本降低了50%以上，并且2G12和DsRed的回收率都没有损失。
Flocculation is a cost-effective method that is used to improve the efficiency of clarification by causing dispersed particles to clump together, allowing their removal by sedimentation, centrifugation or filtration. The efficacy of flocculation for any given process depends on the nature and concentration of the particulates in the feed stream, the concentration, charge density and length of the flocculant polymer, the shear rate, the properties of the feed stream (e.g. pH and ionic strength) and the properties of the target products. We tested a range of flocculants and process conditions using a design of experiments approach to identify the most suitable polymers for the clarification step during the production of a HIV-neutralizing monoclonal antibody (2G12) and a fluorescent marker protein (DsRed) expressed in transgenic tobacco leaves. Among the 23 different flocculants we tested, the greatest reduction in turbidity was achieved with Polymin P, a branched, cationic polyethylenimine with a charge density of 13.0meq/g. This flocculant reduced turbidity by more than 90% under a wide range of process conditions. We developed a model that predicted its performance under different process conditions, and this enabled us to increase the depth filter capacity three-sevenfold depending on the process scale, depth filter type and plant species. The costs of filter consumables were reduced by more than 50% compared with a process without flocculant, and there was no loss of recovery for either 2G12 or DsRed.