Factorial and Economic Evaluation of an Aqueous Two-Phase Partitioning Pilot Plant for Invertase Recovery From Spent Brewery Yeast.

Aqueous two-phase systems (ATPS) have been reported as an attractive biocompatible extraction system for recovery and purification of biological products. In this work, the implementation, characterization, and optimization (operational and economic) of invertase extraction from spent brewery yeast in a semi-automatized pilot plant using ATPS is reported. Gentian violet was used as tracer for the selection of phase composition through phase entrainment minimization. Yeast suspension was chosen as a complex cell matrix model for the recovery of the industrial relevant enzyme invertase. Flow rates of phases did not have an effect, given that a bottom continuous phase is given, while load of sample and number of agitators improved the recovery of the enzyme. The best combination of factors reached a recovery of 129.35 ± 2.76% and a purification factor of 4.98 ± 1.10 in the bottom phase of a PEG-Phosphate system, also resulting in the removal of inhibitor molecules increasing invertase activity as reported by several other authors. Then, an economic analysis was performed to study the production cost of invertase analyzing only the significant parameters for production. Results indicate that the parameters being analyzed only affect the production cost per enzymatic unit, while variations in the cost per batch are not significant. Moreover, only the sample load is significant, which, combined with operational optimization results, gives the same optimal result for operation, maximizing recovery yield (15% of sample load and 1 static mixer). Overall res ults of these case studies show continuous pilot-scale ATPS as a viable and reproducible extraction/purification system for high added-value biological compounds.

An integrated practical implementation of continuous aqueous two-phase systems for the recovery of human IgG: From the microdevice to a multistage bench-scale mixer-settler device.

Aqueous two-phase systems (ATPS) are a liquid-liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer-settler device in one stage, multistage and multistage with recirculation configuration. Single-stage pure IgG extraction with a polyethylene glycol (PEG) 3350-phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation.

Low-abundant protein extraction from complex protein sample using a novel continuous aqueous two-phase systems device.

The present work describes the application of a novel continuous aqueous two-phase system prototype for the recovery of biomolecules. The prototype is an alternative platform for protein recovery and α-amylase from soybean extracts was used as a model system. The system was selected as an example of low-abundant protein present in complex mixtures. Compared with batch systems, continuous operation in this prototype seems to increase partition coefficient with higher recovery efficiencies. Processing time is reduced at least three times in the continuous system when compared to batch mode, while hold up (volumetric quantity of the opposing phase in a determined phase sample) decreases with decreasing phases flow. Furthermore, similar partition coefficient (Kp > 4) with a higher top phase enzyme recovery (81%) is also obtained in this system probably due to better contact surface between phases, compared with that obtained in batch (79%). A continuous aqueous two-phase system process with purification factor 40-fold higher than batch experiments was achieved. These preliminary results exhibit the potential of continuous systems for the recovery of low-abundant proteins from complex mixtures. The promising performance of this prototype can raise the attention of the industry for the adoption of aqueous two-phase system processes.