Separation of Antibodies by Liquid-Liquid Aqueous Partition and by Liquid-Liquid Partition Chromatography

Abstract

In this review, we describe the use of liquid-liquid aqueous partition as a method for the separation of antibodies. Water-based two-phase systems made up of polyethylene glycol and dextran have, by far, been the most frequently used systems. The distribution of a molecule in these systems depends on its exposed surface properties and is described by its partition coefficient. The separation may be performed in a single step in a batch experiment or in several steps using various forms of automated counter-current extraction methods, referred to in this review as liquid-liquid partition (LLP). The sensitivity and selectivity of the two-phase technique can be considerably improved by employing a column chromatographic approach, liquid-liquid partition chromatography (LLPC). In LLPC, the bottom-phase of the two-phase system is adsorbed onto a support and packed into a column which is eluted with the corresponding top-phase. In the first part of this review, the methodology behind UP and UPC is described and outlined in broader terms, before the properties and prestanda of the two approaches are compared. In the second part, the results obtained by LLP and LLPC on antibodies are described in more detail. This review shows, that liquid-liquid aqueous partition is a powerful tool for antibody analysis, that is for purification and fractionation, detection and separation of conformational isomeric forms, examination of surface properties related to antigen specificities and for providing new interesting information about the events upon antigen-antibody complexation and about possible ligand-induced conformational changes.