ETHANOL SODIUM CHLORIDE PHOSPHATE MOBILE PHASE FOR SIZE-EXCLUSION CHROMATOGRAPHY OF POLY(ETHYLENE GLYCOL) MODIFIED PROTEINS

摘要

The use of an aqueous sodium chloride-phosphate mobile phase for size-exclusion chromatography of poly(ethylene glycol) modified (PEGylated) proteins resulted in premature degradation of silica-based gel filtration columns. This column degradation was manifested as peak tailing and loss of resolution. An aqueous ethanol-sodium chloride-phosphate mobile phase was identified which extended column lifetimes without loss of resolution or change in peak shape. We describe the effects of sodium chloride, phosphate, and ethanol concentrations on column performance. Peak shape and column lifetimes were better when an aqueous ethanol-sodium chloride-phosphate mobile phase was used than with an aqueous salt mobile phase. Also, circular dichroism was utilized to investigate possible solvent effects on the conformation of each protein.