(732e) Tunable Aqueous Polymer Phase Impregnated Resins: A New Approach for Protein Recovery

Tunable Aqueous Polymer Phase Impregnated Resins:

A new approach for protein recovery

Nowadays, a variety of specialized biotechnological products is needed in food, cosmetic and pharmaceutical industry and the demand increases steadily. As a consequence, the Downstream Processing has to face complex and diverse challenges like high product sensitivities towards temperatures, organic solvents, or pH values. Additionally, comparatively low product concentrations, a high number of unspecified impurities, and strict demands for final product quality complicate the Downstream Processing.

Current Downstream Processing techniques suffer from several drawbacks such as high costs, irreversible product deactivation, and possible product contamination due to leaching of ligands, for example. Thus, Downstream Processing often represents the bottleneck of the whole production process and its costs can make up to 80% of the whole production costs.

Hence, there is a constant need for efficient alternatives to conventional Downstream Processing methods for the purification of biotechnological products. One of these alternative techniques is Aqueous Two-Phase Extraction (ATPE). For ATPE, two partially immiscible aqueous phases are used as extraction system. Aqueous two phase systems (ATPS) can consist of two aqueous polymer or salt phases, or the combination of one aqueous polymer and one aqueous salt phase. The main advantage of ATPE as a Downstream Processing technique for biotechnological molecules is the biocompatibility due to the high water content of approximately 80 wt.-%.The low interfacial tension between the two aqueous phases allows fast mass transfer rates and emulsification is reached at lower agitation speed. Thus, proteins that are sensitive to mechanical stresses are less damaged. Beside the advantages, an industrial application of ATPE is still limited because of some major drawbacks: The low interfacial tension and low density difference between the two aqueous phases lead to intensive and stable emulsifications and therefore, long phase separation times. Thus, the footprint for the required mixer/ settler equipment is high, or additional equipment like centrifuges are needed. Various attempts have been made to decrease the demixing time of ATPS, like differently designed extraction columns or acoustic and temperature driven phase coalescers. Another approach to overcome the long settling times of classical ATPE is the so called ‘Tunable Aqueous Polymer-Phase Impregnated Resins’ (TAPPIR^®)-Technology (Schembecker et al., 1 April 2011, AZ 10 2011 001 743.). This technology eliminates the need of phase emulsification and separation by immobilizing one aqueous phase inside porous solids prior to extraction. The phase contact is achieved by the suspension of the impregnated solids in the second aqueous phase. Thus, emulsification and phase separation is realized in one single step. The separation principle of the TAPPIR^®-Technology is based on the technique of solvent impregnated resins (SIR) introduced by Warshawsky. In contrast to SIR, the TAPPIR^®-Technology is meant to be used as novel approach for Downstream Processing of biotechnological products. The challenges in downstream processing of this product division are different from those present in the SIR applications where no special attention has to be paid on product sensitivities. For example, the use of organic solvents should be avoided because of possible denaturation effects of the proteins. Thus, for the TAPPIR^®-Technology ATPS are used to create a more biocompatible environment and gentle extraction conditions.

Regarding the expensive phase forming polymers like PEG to form ATPS, the TAPPIR^®-Technology offers the opportunity to reuse the impregnated particles for multiple separation runs. Thus, the recycle of the impregnated particles might lead to an overall cost reduction and could increase the industrial acceptance to ATPE. In terms of process integration, a potential advantage might be the combination of ATPE in the TAPPIR^®-Technology with size exclusion separation effects due to different pore sizes of the used particles.

However, the TAPPIR^®-Technology is new and thus, many factors need to be investigated in regard to their impact on the separation system. The selection of the ATPS determines the separation efficiency and thus, the partitioning of product and contaminants between immobilized and bulk aqueous phase. The type of the ATPS in combination with the concentration of the phase forming components and other properties, such as the molecular weight of the polymer, determine its physicochemical properties, such as the density difference between the phases, the phases’ viscosities and the interfacial tension. Additionally, neutral additives such as sodium chloride offer the possibility to individually influence the partitioning behaviour of product and contaminants without having significant impact on the miscibility. The form, size and especially porosity and pore size of the solid particles influence transport processes, hydrodynamics, and impregnation behaviour. Regarding the number of influencing factors, the set-up or optimization of the TAPPIR^®-Technology can be complex. On the other hand it offers the possibility to react flexible and to tailor a selective and customized purification strategy for biotechnological products.

To show the applicability of the TAPPIR^®-Technology the partitioning of proteins in an aqueous polymer-salt system is investigated. The biodegradable and sustainable aqueous polyethylene glycol (PEG) /sodiumcitrate system and macroporous particles, which had no adsorptive capacities towards the proteins investigated, are used. The effects of particle material and size and the addition of the neutral additive sodium chloride are determined in regard to separation efficiency. The whole process of target protein extraction into the immobilized aqueous PEG phase and the back-extraction into a second aqueous sodiumcitrate phase is investigated. To evaluate the performance of the TAPPIR^®-Technology, the results are compared to classical ATPE experiments. It could be shown, that the TAPPIR^®-Technology can efficiently separate the target protein out of the mixture. Additionally, it could be demonstrated, that the target protein is extracted back into a second aqueous sodiumcitrate phase. Comparing the overall recovery of target protein the results achieved via TAPPIR^®-Technology are comparable to the one yielded by classical ATPE. Thus, these new technology can be an efficient alternative to classical purification strategies and can offer new alternatives for the use of ATPE.