An alternating flow-direction method for increasing productivity in the purification of large biotherapeutic modalities using size exclusion chromatography

The purification of large biotherapeutic modalities such as viral coat proteins, plasmid DNA, mRNA, therapeutic viruses and vesicles is more challenging than the purification of smaller and more established products such as monoclonal antibodies. This is because these entities, due to their large size, have limited access to binding sites present in the pores of conventional resin-based chromatographic media. However, this transport limitation could potentially be exploited for their purification using size exclusion chromatography (SEC). Here, the strategy is to isolate these in the void fraction of an appropriate SEC column. However, challenges such as low capacity, low productivity and poor scalability typically associated with SEC would first need to be addressed. In this study, we propose an alternating flow-direction-based SEC technique as an approach for increasing the productivity of preparative SEC. The feed is introduced into the SEC device from opposite directions in an alternating manner. By doing so, the separation time could be significantly reduced. Proof of concept for this technique was obtained using a z² cuboid SEC device, having a volume of 24 mL, and packed with Sephacryl S 200 resin. The effect of alternating flow direction on the separation time was examined based on a case study for the purification SARS-CoV-2 delta spike protein from small molecular weight impurities present in cell-free supernatant. Compared to conventional unidirectional SEC, the time (or volume of mobile phase) required for purifying the spike protein could be reduced by about 42 %.