(802e) Interfacing Aqueous Two-Phase Systems With Countercurrent Chromatography: Track Record and Future Potential for Bioseparations

Countercurrent Chromatography (CCC) is a continuous form of liquid-liquid extraction, which the Brunel Institute for Bioengineering (BIB) team has developed from academic know-how to industrial competitive  process scale. CCC has been used extensively for the separation and isolation of molecules across a wide range of polarity, typically natural products using aqueous-organic two-phase systems.

To extend CCC to bioseparations, biocompatible solvent systems, such as Aqueous Two-Phase Systems (ATPS) containing polyethylene glycol (PEG) and salt, are required. These differ from the conventional aqueous-organic systems as they are more viscous, have lower density difference and lower interfacial tension resulting in slower phase settling times.

Successful separations of biomolecules have been achieved using both hydrostatic  and hydrodynamic  CCC technologies at various scales.

Further protein separation studies to the purification of monoclonal antibody (mAb) products from various cell cultures, using PEG-inorganic salts ATPS opened up a new area of research. Clear separation of the mAb from impurities has been achieved with retention of biological activity  using new invention allowing independent variation of mixing and settling. The need to optimise the CCC operational mode to preserve bioactivity will be discussed.

Additionally, CCC instruments have the potential to perform as a reactor/separator. In this mode a catalyst is immobilised by partitioning to the liquid stationary phase and substrate passed through the column dissolved in the liquid mobile phase. Reaction occurs in the zones of mixing, together with separation of product from substrate by the chromatographic action of the CCC.  This approach has been successfully applied to enzymatic biotransformation of a chiral mixture of amino acids when the high value product of the bioreactor was continuously separated from the product and biocatalyst as it is formed, using a PEG-phosphate ATPS.

Results of our current research with industry will be presented in more details.