Countercurrent chromatography of proteins using aqueous two-phase systems

Abstract

The biotechnology industry requires high - capacity protein manufacturing processes that retain protein functionality. Large - scale countercurrent J- type centrifuges have been developed by the Brunel Institute for Bioengineering that successfully purify small organic molecules using aqueous - organic phase systems. Aqueous two - phase systems (ATPS) have been used historically to purify bio-molecules whilst retaining their biological function. This thesis focuses on extending CCC to the separation of proteins, using a model ATPS of PEG-1000 and potassium dihydrogen phosphate to separate a mixture of lysozyme and myoglobin. Initial studies were on the behaviour of this phase system in aJ- type CCC centrifuge fitted with a multilayer column; the variable parameters examined were centrifuge rotational speed, mobile phase flow rate and direction, and type of mobile phase. A set of optimum conditions were identified that gave good retention and stability of the phases in the column. These conditions were applied to separate a mixture of the proteins lysozyme and myoglobin in the same centrifuge. However, the proteins did not elute as predicted from their equilibrium distribution ratios. It appears that the wave - like mixing and settling behaviour of the phases in the centrifuge was insufficient for the proteins to achieve equilibrium partitioning. A centrifugal partition chromatography (CPC) centrifuge was introduced to the study. This provided full protein separation, credited to the cascade phase mixing created by this design of centrifuge. The experimental parameters were used in an experiment on a pilot - scale CPC instrument. The CPC study was extended to the isolation of a target protein (phosphomannose isomerase) from a fermentation supernatant. CPC gave partial purification of the protein with retention of enzyme activity. This thesis demonstrates the importance of phase mixing in CCC, which has led to a new column design by BIB with the potential of industrial - scale protein purifications.