Preparative Purification of a Recombinant Protein by Hydrophobic Interaction Chromatography: Modulation of Selectivity by the Use of Chaotropic Additives
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...
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| Veröffentlicht in: | Biotechnology progress 2002, Vol.18 (3), p.556-564 |
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| creator | Shukla, Abhinav A. Peterson, Joshua Sorge, Laura Lewis, Patsy Thomas, Shannon Waugh, Steve |
| description | 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. |
| doi_str_mv | 10.1021/bp020038a |
| format | Article |
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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. 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| subjects | Biological and medical sciences Biotechnology Chromatography, Liquid - methods Electrophoresis, Polyacrylamide Gel Fundamental and applied biological sciences. Psychology Methods. Procedures. Technologies Others Recombinant Proteins - isolation & purification Sensitivity and Specificity Spectrophotometry, Ultraviolet Various methods and equipments |
| title | Preparative Purification of a Recombinant Protein by Hydrophobic Interaction Chromatography: Modulation of Selectivity by the Use of Chaotropic Additives |
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