title: Preparative purification of a recombinant protein by hydrophobic interaction chromatography: modulation of selectivity by the use of chaotropic additives.
abstract: Development and implementation of a chaotropic wash step following protein loading on a hydrophobic interaction chromatographic (HIC) column is described for the purification of a recombinant protein. Various agents that reduce protein affinity in hydrophobic interaction chromatographic systems were screened for their utility in a wash step following protein loading on a Phenyl Fast Flow Sepharose HIC column. A combination of sodium thiocyanate, glycerol, and urea was selected as a suitable additive for the wash buffer that selectively eluted most of the major impurities present in the feed stream. Eluate purity, as monitored by reversed-phase chromatography and SDS-PAGE, was significantly increased by incorporation of this wash step in the purification process. Incorporation of this wash step on HIC enabled a reduction in the overall number of chromatographic steps in the downstream purification process for this recombinant protein, resulting in improved process yields and significant economic advantages. The effect of varying concentrations of each of the three wash additives on yield was studied. While the step yield decreased with an increase in concentration for urea and sodium thiocyanate, an optimum was observed with respect to glycerol concentration. The preferential interaction theory is employed to explain this effect.
authors.affiliation: ICOS Corporation, 22021, 20th Avenue SE, Bothell, Washington 98021, USA. shuklaa@immunex.com
authors.forename: Abhinav A
authors.initials: AA
authors.lastname: Shukla
authors.forename: Joshua
authors.initials: J
authors.lastname: Peterson
authors.forename: Laura
authors.initials: L
authors.lastname: Sorge
authors.forename: Patsy
authors.initials: P
authors.lastname: Lewis
authors.forename: Shannon
authors.initials: S
authors.lastname: Thomas
authors.forename: Steve
authors.initials: S
authors.lastname: Waugh
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D011994:Recombinant Proteins