Aqueous two-phase systems for protein separation: Phase separation and applications

Aqueous two-phase systems (ATPS) that are formed by mixing a polymer (usually polyethylene glycol, PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers, and water can be effectively used for the separation and purification of proteins. The partitioning between both phases is dependent on the surface properties of the proteins and on the composition of the two phase system as has been recently reviewed by Asenjo and Andrews [1]. This paper analyses and reviews some elements that are important for implementation of these processes which are related to phase separation and continuous processing of ATPS. Phase separation for ATPS formed by PEG and salts has been studied and has been found to depend on which of the phases is continuous. Profiles of dispersion heights can be represented as a fraction of the initial height and are independent of the dimensions of the separator. This is important for the design of large scale aqueous two-phase separations. The kinetics of phase separation has been investigated as a function of the physical properties of the system. The settling rate is a crucial parameter for equipment design and it has been studied as a function of viscosity and density of the phases as well as the interfacial tension between them. Correlations that describe the rate of phase separation have been developed. Working in a continuous bottom-phase region is advantageous to ensure fast separation. A mathematical model to describe the continuous, study state operation of these systems has been investigated. Two simulations to show the effect of phase ratio on purification have been carried out which clearly show the effectivity of using such models. The practical application of ATPS has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. Examples include the purification of α-amylase and the large scale "in situ" purification of IGF-1 carried out by Genentech. The production scale purification of chymosin from recombinant Aspergillus supernatant is the most successful industrial application of this technology. Other applications include the separation and purification of human α-antitrypsin from transgenic sheep milk, the purification of monoclonal antibodies, tPA from CHO supernatant and recombinant VLPs (virus like particles) from yeast cells.