Teresa Cunha and Raquel Aires Barros 1 Introduction

Purification of proteins using aqueous two-phase systems (ATPS) has been successfully carried out on large scale for more than a decade (1-17).

One of the major advantages of ATPS is that the scaling up is possible through the use of conventional extraction equipment used for organic-aqueous extraction in chemical industry. The large-scale application of ATPS is mainly limited by the theoretical understanding of phase equilibrium and protein partitioning, the selectivity of these systems and cost of the polymers (1821); the possibility to recycle the polymers must also be considered (22-24).

Large-scale protein recovery using ATPS requires two operations: (i) mixing of the phase components followed by phase dispersal, and (ii) phase separation.

In batch processes, equilibration is usually done in agitated vessels, and in mixer-settler devices. A few minutes of gentle stirring are normally enough to obtain phase and partition equilibrium (6). The fast approach to equilibrium is owing to the low interfacial tension between the two phases, which enables the formation of very small droplets and thus a large interface for mass transfer with low energy input (6). Phase separation is performed either by settling under gravitational force for fast-settling systems like PEG-salt systems, or by continuously operating common centrifugal separators (6).

An alternate scheme is the use of a column-type extractor to improve extraction efficiency using ATPS. The selection of a particular continuous contacting column, depends on the needs of the operation, the properties of the biomolecules, and the type of ATPS involved (13). Columns can be operated with countercurrent flow of both phases or by keeping one phase stationary. When operating an extraction column with ATPS, the problem is not the generation of sufficient exchange surface between phases as in the case of organic

From: Methods in Biotechnology, Vol. 11: Aqueous Two-Phase Systems: Methods and Protocols Edited by: R. Hatti-Kaul © Humana Press Inc., Totowa, NJ

solvents, but the avoidance of very small droplet formation, which will decrease the performance and lead to flooding of the column (2).

For the design of commercial-scale columns, data on the following parameters are needed: protein mass-transfer coefficients, dispersed phase holdup, and the extent of mixing in both phases. Usually, these parameters are dependent on the phase velocities, and physical properties, and also on the column geometry (25).

In this chapter we present the equipment and the methodology for aqueous two-phase extrasction of proteins on large scale using mixer-settlers and column contactors. The reader is referred to the literature refs. given in the chapter for details of the specific equipment/procedure.

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