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Using mass spectrometry to investiga...

Jones, Tara Tibbs.

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Using mass spectrometry to investigate protein-surface interactions during hydrophobic interaction chromatography.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Using mass spectrometry to investigate protein-surface interactions during hydrophobic interaction chromatography./
作者:	Jones, Tara Tibbs.
面頁冊數:	155 p.
附註:	Source: Dissertation Abstracts International, Volume: 64-10, Section: B, page: 5079.
Contained By:	Dissertation Abstracts International64-10B.
標題:	Engineering, Chemical. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3108771

Using mass spectrometry to investigate protein-surface interactions during hydrophobic interaction chromatography.
Jones, Tara Tibbs.

Using mass spectrometry to investigate protein-surface interactions during hydrophobic interaction chromatography. - 155 p.

Source: Dissertation Abstracts International, Volume: 64-10, Section: B, page: 5079.

Thesis (Ph.D.)--University of Virginia, 2003.

Hydrophobic Interaction Chromatography (HIC) is a purification tool used at the analytical scale for the separation of therapeutic biomolecules. The many variables that affect retention and selectivity of proteins during HIC as well as the complexity of the hydrophobic effect have prevented the complete understanding and advancement of a robust, modeling approach to guide process development. In previous work, conformational changes for unstable proteins were observed to greatly affect retention (Wu et al., 1986), (Jones and Fernandez, 2003). However, we have shown with hydrogen-deuterium isotope exchange (HX) and mass spectrometry (MS) that conformational changes do not contribute to selectivity variations for three stable proteins on popular commercial HIC media, suggesting that conformational changes are not a factor for HIC selectivity changes observed for stable proteins. A closer investigation of unstable proteins showed a robust correlation between conformational changes and increased adsorption affinity caused either by increased mobile phase modulator or increased hydrophobicity of the surface despite the different selectivity patterns of the resins, indicating that conformation is not solely responsible for selectivity for unstable proteins. One possible explanation of the unexpected patterns is preferred binding orientations of the proteins that may be different for each resin. As a test of feasibility, chemical modification techniques and MS were used as an attempt to detect preferential binding. Finally, HX and MS were used to investigate the effect of a stabilizing metal additive on conformation and chromatography. The results showed a dramatic, chromatographic peak sharpening with increasing calcium concentrations, as well as increased protein stability on the surface. Despite the reported improvements to chromatography yield and purity, additives are not typically explored as a complement to HIC due to the added number of experiments, expensive, and specialized handling procedures (Gagnon and Grund, 1996). This study further supports the need for a robust HIC model approach to help guide purification process development and suggests relatively simple models of conformational change may be useful.Subjects--Topical Terms:

1018531
Engineering, Chemical.

Using mass spectrometry to investigate protein-surface interactions during hydrophobic interaction chromatography.
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520 $a Hydrophobic Interaction Chromatography (HIC) is a purification tool used at the analytical scale for the separation of therapeutic biomolecules. The many variables that affect retention and selectivity of proteins during HIC as well as the complexity of the hydrophobic effect have prevented the complete understanding and advancement of a robust, modeling approach to guide process development. In previous work, conformational changes for unstable proteins were observed to greatly affect retention (Wu et al., 1986), (Jones and Fernandez, 2003). However, we have shown with hydrogen-deuterium isotope exchange (HX) and mass spectrometry (MS) that conformational changes do not contribute to selectivity variations for three stable proteins on popular commercial HIC media, suggesting that conformational changes are not a factor for HIC selectivity changes observed for stable proteins. A closer investigation of unstable proteins showed a robust correlation between conformational changes and increased adsorption affinity caused either by increased mobile phase modulator or increased hydrophobicity of the surface despite the different selectivity patterns of the resins, indicating that conformation is not solely responsible for selectivity for unstable proteins. One possible explanation of the unexpected patterns is preferred binding orientations of the proteins that may be different for each resin. As a test of feasibility, chemical modification techniques and MS were used as an attempt to detect preferential binding. Finally, HX and MS were used to investigate the effect of a stabilizing metal additive on conformation and chromatography. The results showed a dramatic, chromatographic peak sharpening with increasing calcium concentrations, as well as increased protein stability on the surface. Despite the reported improvements to chromatography yield and purity, additives are not typically explored as a complement to HIC due to the added number of experiments, expensive, and specialized handling procedures (Gagnon and Grund, 1996). This study further supports the need for a robust HIC model approach to help guide purification process development and suggests relatively simple models of conformational change may be useful.
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