Establishing Ultra Scale-down Tools for the Rational Design of Linked Bioprocesses: Flocculation and Centrifugation

(我)的背景。在此之前，UCL生化工程已经建立了单个生物过程单元操作的缩小模型，包括离心、膜过滤、横流微过滤和深度过滤[1-3]。超缩小（USD）工具从根本上改变了早期制造开发的方法，只需使用毫升量的材料就可以进行高通量研究，从而产生与工业相关的数据。最初，该方法是为离心开发的，这是生物制造的一个重要步骤，没有合适的缩小比例的工具可用于早期工艺研究。在离心之前加入絮凝步骤可以产生更大的颗粒，从而提高离心机中固液分离的效率(4)。絮凝过程是复杂的，其效果取决于絮凝材料的用量、絮凝时间和絮凝剂的添加速度(5)。此外，混合过程可能会增强絮凝或导致絮凝体破碎。涉及絮凝的过程的放大需要根据这些依赖时间的子过程的综合效应来表征。“过程动力学”)和过程剪切。因此，在产生稳定的絮凝体以增强离心作用的同时，在初级回收期间保持高的总体产品收率之间存在权衡。在更大规模的过程中，确定哪个参数可以优化澄清回收率和产品收率之间的权衡，这很难用目前的实验室技术来确定。目前还没有确定的絮凝-离心工艺的规模化技术，可以预测更大规模的操作。这凸显了为合理设计初次采油作业而需要缩小絮凝工具的必要性。宗旨和目标。拟议的项目建立在伦敦大学学院建立个人缩小模型的技术基础上。本研究的总体目标是对离心前的几个过程有更好的机制理解，这些过程可能受到时间相关子过程(即。“过程动力学”)和过程剪切，以建立一个关联的缩小比例，预测在大范围内经历的过程相互作用。具体目标如下：-创建生物制造中通常使用的细胞絮凝的缩小模型（如酵母，CHO细胞或均质大肠杆菌细胞），可以在受控条件下进行研究。-研究这些细胞的絮凝机制，作为絮凝剂类型和工艺条件的功能-建立絮凝-离心的小规模方法-应用小规模方法来理解和解决大规模遇到的挑战。

(i) Background. Scale-down models of individual bioprocess unit operations have previously been established at UCL Biochemical Engineering on centrifugation, membrane filtration, crossflow microfiltration and depth filtration [1-3]. The ultra scale-down (USD) tools radically transform the approach to early manufacturing development by enabling high throughput studies using only millilitre quantities of material to yield industrially-relevant data. Initially, the approached was developed for centrifugation, a major step in biomanufacturing for which no suitable scale-down tool is available for early process studies. The addition of a flocculation step prior to centrifugation is employed to create larger particles which enhances the efficacy of the solid-liquid separation in the centrifuge (4). The flocculation process is complex as its effectiveness is dependent on dosage of flocculating material, flocculation time, and rate of flocculant addition (5). Additionally, mixing processes could either enhance flocculation or cause flocs to break-up. The scale-up of processes involving flocculation need to be characterised based on the combined effect of such time-dependent sub-processes (i.e. 'process kinetics') and process shear. Thus, there is a trade-off between creating stable flocs which enhances centrifugation while maintaining a high over-all product yield during primary recovery. Identifying which parameter in the larger scale process would optimize this trade-off between clarification recovery and product yield is difficult to determine using current laboratory techniques involving flocculation. There is currently no established scale-down technique for flocculation-centrifugation processes which predicts larger scale operations. This highlights the need for scale-down tool for flocculation for the rational design of primary recovery operations.(ii) Aim and objectives. The proposed project builds on the know-how developed at UCL on establishing individual scale-down models. The overall aim of this research is to gain a better mechanistic understanding of several processes before centrifugation which may be influenced by both time-dependent sub-processes (i.e. 'process kinetics') and process shear to establish a linked scale-down that predicts the process interaction experienced at large scale. Specific objectives are as follows:- To create a scale-down model of flocculation of cells typically used in biomanufacturing (such as yeast, CHO cells or homogenized E. coli cells) which can be studied under controlled conditions.- To investigate the mechanism of flocculation of these cells as a function of flocculant type and process conditions- To establish a scale-down methodology for flocculation-centrifugation - To apply the scale-down methodology in understanding and addressing challenges encountered at large scale.