The concept of cosolvent exclusion was developed by a group of Timasheff’s laboratory in 1970-1990 and is currently used widely to explain the effects of a variety of cosolvents on the stability and solubility of macromolecules. Not surprisingly, these concepts have had substantial influence in the fields of formulation, protein folding and unfolding, but they have perhaps more surprisingly found their way into the field of chromatography. A variety of excluded cosolvents have been used to enhance binding and resolution of proteins and other macromolecules in ion exchange, hydroxyapatite, affinity, and hydrophobic interaction chromatography. These cosolvents include salting-out salts, amino acids and polymers, and frequently polyethylene glycol (PEG). A new mode of chromatography, termed “steric exclusion chromatography,” was recently introduced. It employs hydroxylated solid phase surfaces. Steric exclusion of the PEG stabilizes the association of macromolecules with the solid phase. Elution is achieved by reducing the PEG concentration. Magnetic particles are also used in this chromatography. This review summarizes the concepts of preferential cosolvent exclusion and its applications in column chromatography.

Introduction

The theoretical foundation and supporting data on protein solvent interactions were developed primarily by pioneering work conducted in Timasheff’s laboratory. Early studies focused on the mechanisms of protein denaturants, such as organic solvents and guanidine hydrochloride or urea, as they were found to play a critical role in understanding the unfolding and refolding pathways of proteins.1-6 These additives (cosolvents) exerted their effects at very high concentrations, where conventional stoichiometric binding measurements were neither practical nor meaningful. Timasheff’s group employed equilibrium experiments, for example, dialysis, which measured differences in cosolvent concentration between the vicinity (inside the dialysis bag) of protein molecule and the bulk phase (dialyzing solvent).7 At high cosolvent concentrations, many different interactions between the protein surface and cosolvent were found to occur.8-11 Water molecules, which were at high concentration in aqueous solution, were also bound to the protein or other macromolecules.12,13 All these interactions could cause difference in cosolvent concentration around the protein surface. These experiments provided a wealth of information about the effects of cosolvents on protein denaturation and stability in aqueous solution. Protein denaturants, for example, organic solvents and urea, were observed to bind to the proteins in the denatured, unfolded state at high cosolvent concentration, leading to stabilization of the denatured structure.1-6 Protein stabilizers, for example, sugars and polyols, were found to show lower cosolvent concentration inside the dialysis membrane than the dialyzing solvent.14-22 This showed that they were excluded from protein surfaces. Carpenter and Crowe and others23-27 demonstrated that these excluded cosolvents also stabilized proteins against freezethaw stresses, and they have since been shown to prevent proteolysis.28 Other important applications of preferentially excluded cosolvents are found in the field of chromatography. Chromatography plays an essential role in purification of biopharmaceuticals such as proteins, peptides, nucleic acids, and viruses. Affinity and hydrophobic interaction chromatography (HIC) permit the application of various excluded salts to modulate binding and elution. Anion and cation exchange chromatography are less tolerant of salts but invite application of nonionic and zwitterionic excluded cosolvents such as polyethylene glycol (PEG) and glycine. Many of these applications have been found particularly to enhance fractionation of native proteins from fragments and aggregates.