Towards continuous aqueous two-phase extraction (CATPE)

Aqueous Two-Phase Extraction (ATPE) in mixer-settlers offers a gentle and biocompatible environment to separate proteins from complex mixtures. We have developed an aqueous two-phase system with inexpensive and biocompatible PEG 1500 or 4000 and ammonium citrate. We have purified several dehydrogenases [1] to near homogeneity after forward extraction into a PEG-heavy top phase at pH \u3e 9 and back extraction into a bottom phase at pH 4-6; in selected cases, we were able to obtain pure protein in the bottom phase without forward extraction into the top phase. We have scaled up the PEG 1500/4000-ammonium citrate to a 5-10 L scale, with phase separation times of less than five minutes.[2] We currently extend the system to the separation of Qα virus-like particles. However, ATPE technology is characterized by complex phase separation and very limited number of separation stages not offering enough separation efficiency. These limitations can be overcome by the novel Tunable Aqueous Polymer Phase Impregnated Resins (TAPPIR) technology which immobilizes one phase out of a biphasic aqueous extraction system in porous material (Figure 1) [3]. By immobilizing these impregnated resins in columns continuous operation similar to Simulated Moving Bed systems become possible. TAPPIR provides high separation efficiency along with high capacity, avoids long phase separation times (especially for highly viscous polymer phases) and offers an answer to the non-ecological image of ATPE through immobilizing and re-using phase forming material. The application of the TAPPIR technology has been shown for the separation of lysozyme and myoglobin using a polyethylene glycol 4000/citrate aqueous two-phase system in batch experiments [4]. In addition, the influence on protein partitioning of the porous solids\u27 properties like solid material, particle and pore size has been investigated. It could be demonstrated that the same partitioning levels can be reached for the TAPPIR as for classical ATPE mixer/settler experiments and that the leaching of the immobilized phase is negligible [5]. The presentation will introduce the TAPPIR technology, describe the advantages over chromatography and present a process concept for continuous operation with zero waste

Progress towards continuous aqueous two-phase extraction via TAPPIR

At ICB II, we presented Aqueous Two-Phase Extraction (ATPE) as a non-chromatographic alternative for protein purification. We had developed an aqueous two-phase system with inexpensive and biocompatible PEG 1500 or 4000 and ammonium citrate. We purified several enzymes, more specifically a series of dehydrogenases [1], to near homogeneity after forward extraction into a PEG-heavy top phase at pH \u3e 9 and back extraction into a bottom phase at pH 4-6; in selected cases, we were able to obtain pure protein in the bottom phase without forward extraction into the top phase. Scale-up of the PEG 1500/4000-ammonium citrate to 5-10 L scale still often gave phase separation times of less than five minutes.[2] However, ATPE technology is characterized by complex phase separation and very limited number of separation stages not offering enough separation efficiency. Furthermore, conventional ATPE does not lend itself to continuous operation. Please click Additional Files below to see the full abstract

Tailor-made aqueous two-phase systems for application in continuous separation of potent biomolecules

Aqueous Two-Phase Extraction (ATPE) using Aqueous Two-Phase Systems (ATPS) has long been shown to be a viable and promising alternative in the work-up of potent biomolecules (e.g. enzymes, proteins, therapeutics) from fermentation broth. Although ATPE has significant advantages over common separation strategies, such as a high biocompatibility, gentle separation profile due to low interfacial tension, good scalability and high efficiencies, industrial applications have not yet been realized. Reasons typically given are based on the ATPS “physiochemical” properties such as viscosities and low density differences between the phases, which lead to long phase separation times. However, these challenges can be addressed using advanced technology such as the “Tunable Aqueous Polymer-Phase Impregnated Resins” (TAPPIR)-Technology immobilizing one phase of an ATPS inside porous solids, which are then transferred into a chromatography column. The second aqueous phase serves as mobile phase. The main advantage of this technique is the simple and efficient emulsification and liquid–liquid phase separation through the packed-bed column design. In addition, the extraction phases, i.e. both the back extraction phase and the immobilized phase, can be reused enabling a low-waste production process. The remaining bottleneck for an industrial application is the identification of the “base” ATPS, which enables the desired extraction of the biomolecule with the required yield and purity to be competitive to existing processes. State-of-the-art ATPS design so far is based on a “trial-and-error” based approach identifying ATPS that work for a given task but often perform in suboptimal fashion. In the present work, we will present a novel thermodynamics-based strategy for the identification and characterization of tailor-made ATPS for the continuous separation of highly potent industrial enzymes by ATPE. By consideration of the molecular interactions in solution, we are able to define potentially suitable ATPS based on a predictive modeling approach using ePC-SAFT, a state-of-the-art equation of state. The objective of this step is to supply a thermodynamically optimized combination of ATPS-phase formers that lead to optimal water condition (low concentration of phase formers, large process window), in principal enabling optimal separation. This initial selection is refined by taking into account molecular interactions of the biomolecule (enzyme), by measuring and modeling biomolecule-biomolecule and biomolecule-phase former interactions. These interactions are experimentally captured using advanced light scattering techniques that are both time and cost efficient. It will be shown that, based on the description of molecular interactions through osmotic virial coefficients (B22 and B23) as well as the diffusion interaction parameter (kD) between the molecules in solution, the phase behavior of the biomolecule in an ATPS can be made accessible, but was previously inaccessible with other phase diagram estimation strategies One major advantage of our predictive modeling approach is the estimation of the partition coefficient of the biomolecule between the two aqueous phases based on a minimal set of experimental data, i.e. B22, B23, kD, and phase composition data. Furthermore, the influence of the ATPS phase-formers on protein solubility and stability can be judged qualitatively, an ideal complement in the development of ATPS. Lastly, we applied the thermodynamics-based strategy to the separation of an industrially relevant dehydrogenase from fermentation broth. The design-driven process development led to the identification of a tailor made ATPS that outperformed the reference ATPS from previous works in terms of solubility and stability of the biomolecule enabling a cost-efficient use of the TAPPIR technology

Investigation of Novel Amine Dehydrogenases for the Asymmetric Synthesis of Amines

This presentation was given at SERMACS

Energising the E-factor: The E+-factor

[EN] The E-factor has become an important measure for the environmental impact of (bio)chemical reactions. However, summing up the obvious wastes generated in the laboratory neglects energy-related wastes (mostly greenhouse gases) which are generated elsewhere. To estimate these wastes, we propose to extend the E-factor by an energy-term (E-factor). At the example of a lab-scale enzyme fermentation, we demonstrate that the E-factor can constitute a multiple of the classical E-factor and therefore must not be neglected striving for a holistic estimation of the environmental impact.This workwas supported by the European Union Project H2020-BBI-PPP-2015-2-720297-ENZOX2 and F.H. gratefully acknowledges funding by European Research Council (ERC Consolidator Grant No. 648026) and the for financial support through a Netherlands Organisation for Scientific Research VICI grant (no. 724.014.003). J.M.R, B.R and A.S.B. gratefully acknowledge support from the United States National Science Foundation grant IIP-1540017

Substrate Investigations for Amine Dehydrogenase

This presentation was given at the Center of Pharmaceutical Development

Optimization of Reaction Conditions for an Amine Dehydrogenase Reaction

This presentation was given at SERMACS

Optimization of Reaction Conditions of Amination by Amine Dehydrogenase Enzymes

This presentation was given at the Armstrong Student Scholarship Symposium