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

Abstract

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.

Continuous enzyme aqueous two-phase extraction using a novel tubular mixer-settler in multi-step counter-current arrangement

“…Each stage of the bench-scale device contains: three-way inlet port, coalescence tubing and twoway outlet port [13,14]. As observed in Figure 1A, the counter-current arrangement of the system consists of three stacked coalescence tubing with peristaltic pumps (Watson Marlow The proposed platform as depicted in Figure 1B, requires a small bench space and it can be compacted based on the needed tubular coalescer length.…”

“…Vázquez-Villegas and collaborators [13,14] proposed a novel tubular mixer-settler for the continuous extraction of biomolecules with ATPS at bench scale. Such device allows the recovery of top and bottom phases in separated outlets and integrates typical ATPS operation 4 units (mixing, coalescence and separation) into one single process.…”

The increasing value of plant-made proteins

“…In this context, there have been several recent developments in bioprocess engineering [41,42], particularly in terms of medium optimization, standardization and streamlining of development and production, the increasing use of process analytical technologies (PAT) and online monitoring, automation, predictive scale-down models, disposable technologies, continuous production and purification processes and cost models to determine economic feasibility.…”

Novel aspects and future trends in the use of aqueous two‐phase systems as a bioengineering tool

“…Recently, continuous ATPS mixer‐settler units have been successfully used for the recovery of α‐amylase from soybean protein extract, with higher recovery efficiencies than their batch counterpart as well as dyes, Ig G and other antibodies in bench and pilot‐scale devices also with purities that can be compared with chromatographic operations . This said, the advantages of implementing continuous ATPS separation units present clear competitive advantages for the biotechnological market like reducing processing times, costs and increasing process yields that may substitute or reduce the need for other more complicated operations …”