Advanced Process Designs and Operating Modes of the TAPPIR®-Technology for Continuous Aqueous Two-Phase Extraction

Gentle and cost-efficient separation techniques for biochemical products such as enzymes and proteins are of major interest for the biotech-industry. Aqueous Two-Phase Extraction (ATPE) has been shown to offer a promising approach in this regard [1]. This is mainly based on the high biocompatibility of the Aqueous Two-Phase System (ATPS) utilized, resulting from a water content of up to 70-90 wt.-% and the absence of organic solvents. However, ATPE has not yet made it into industrial application due to the high experimental effort for screening suitable ATPS and poor space-time yields due to long phase separation times resulting from low density differences and small interfacial tensions.

While the first challenge is currently addressed by predictive modeling of ATPS using ePC-SAFT, a state-of-the-art equation of state [2], the long phase separation times in “conventional” ATPE design (Mixer-Settler-Approach) require additional equipment and energy to enable faster phase separation [3]. To avoid this bottleneck, the Tunable Aqueous Polymer Phase Impregnated Resins (TAPPIR^®)-Technology is applied. As one phase of the ATPS is immobilized in porous particles enabling extraction through dispersion of these particles in the other aqueous phase, a subsequent liquid-liquid separation becomes no longer necessary. In order to implement this technique in an industrial process, in this work the TAPPIR^®-Technology is transferred to a column, where one aqueous phase is used as the mobile phase, whereas the other aqueous phase is stationarily impregnated in the particles enabling a semi continuous flow-through separation of biomolecules.

Within this work, advanced process designs and operating modes using the TAPPIR^®-Technology in packed bed will be presented. A polyethylene glycol 4000 – tri sodium citrate ATPS in a packed bed of Stuttgarter Masse particles was used for the purification of lysozyme from ovalbumin. It was shown, that lysozyme can be fully extracted into the polymer-rich phase in flow through mode when sufficient residence time is provided (Figure 1) whilst the impurity is separated in the raffinate. The recovery of the purified lysozyme was demonstrated by back extraction using a second salt rich phase as mobile phase. A sharp segregation between the raffinate and the product phase was achieved by extending the operation mode with a short flush stage. To achieve higher yields, design parameters such as the column length and process parameters like the flow rate were investigated. In particular, further improved process designs (e.g. interconnection of multiple columns, recycle concepts of the raffinate) can lead to improved yields and moreover shift the semi continuous process to a fully continuous separation of biomolecules. Subsequently, this technique is transferred to an advanced system utilizing thermodynamically optimized ATPS and enhanced particles for the purification of an industrially relevant enzyme. It will be shown that a sophisticated process design of the TAPPIR^®-Technology in a column offers an improved approach for the industrial implementation of ATPE as a separation technique for biomolecules.

References

[1] J. A. Asenjo, B. A. Andrews, J. Chem. Technol. Biotechnol. 83 (2) (2008) 117-120.

[2] M. Wessner, G. Schembecker, C. Brandenbusch, AIChE Annual Meeting (2020)

[3] U. Gündüz, A. Tolga, J. Chromatogr. B 807 (1) (2008) 13-16.