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

(i)背景先前已在UCL Biochemical Engineering建立了离心、膜过滤、错流微滤和深层过滤的单个生物工艺单元操作的缩小模型[1-3]。超缩小（USD）工具通过仅使用毫升数量的材料进行高通量研究来产生工业相关数据，从而从根本上改变了早期制造开发的方法。最初，该方法是为离心而开发的，离心是生物制造中的一个主要步骤，早期工艺研究没有合适的缩小工具。在离心之前增加絮凝步骤用于产生更大的颗粒，这增强了离心机（4）中固液分离的效率。絮凝过程是复杂的，因为其有效性取决于絮凝材料的剂量、絮凝时间和絮凝剂添加速率（5）。此外，混合过程可能会增强絮凝作用或导致絮凝物破裂。涉及絮凝的工艺的放大需要基于这种时间依赖性子工艺（即“工艺动力学”）和工艺剪切的组合效应来表征。因此，在产生稳定的絮凝物（其增强离心分离）同时在初级回收期间保持高的总体产物产率之间存在权衡。使用目前涉及絮凝的实验室技术，难以确定在较大规模工艺中哪个参数将优化澄清回收率和产物产率之间的这种权衡。目前还没有建立规模缩小的絮凝-离心工艺技术，预测更大规模的操作。这突出表明，需要规模缩小的絮凝工具，以合理设计一次采油作业。(ii)宗旨和目标。拟议的项目建立在UCL开发的关于建立个人缩小模型的专有技术基础上。本研究的总体目标是获得更好的机械理解离心前的几个过程可能会受到时间依赖性的子过程（即“过程动力学”）和过程剪切的影响，以建立一个链接的规模缩小，预测在大规模的过程中经历的相互作用。具体目标如下：-创建生物制造中通常使用的细胞（例如酵母、CHO细胞或均质大肠杆菌）絮凝的缩小模型。大肠杆菌细胞），可以在受控条件下进行研究。为了研究这些细胞的絮凝机制作为絮凝剂类型和工艺条件的函数-为了建立絮凝-离心的缩小规模方法-为了应用缩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.