Hydrophobic interaction chromatography of proteins IV

Haimer, Emmerich; Tscheliessnig, Anne; Hahn, Rainer; Jungbauer, Alois

doi:10.1016/j.chroma.2006.11.003

Cited by 63 publications

(31 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Under such conditions, it is expected that adsorption/desorption and folding/unfolding rates will be slow relative to the experiments in this work, requiring kinetic modeling that explicitly accounts for the loss of unlabeled protein from the resin by both the desorption and unfolding pathways. The presence of unlabeled, adsorbed protein after an initial equilibration period that was much longer than k À1 unf ;surf also confirms that unfolding on the surface can be reversible, in contrast to recent work that models surface unfolding as an irreversible process (Haimer et al, 2007). An irreversible unfolding process is inconsistent with the data in this study, in which most of the adsorbed protein is observed to be in the native conformation after a 30-min equilibration period, but undergoes unfolding at a sufficiently high rate that essentially all of the protein is fully labeled within 1 min.…”

Section: Resultssupporting

confidence: 68%

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Deitcher

Xiao

O’Connell

et al. 2009

Biotech & Bioengineering

View full text Add to dashboard Cite

A two-conformation, four-state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four-state model to the effect of salt concentration on the adsorption and unfolding of the model protein alpha-lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild-type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of alpha-lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild-type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (approximately 6-8 kJ/mol) due to increased instability of the protein. Finally, the four-state model is used to accurately predict the apparent adsorption strength of a disulfide bond-reduced variant.

show abstract

Section: Resultssupporting

confidence: 68%

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Deitcher

Xiao

O’Connell

et al. 2009

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…In the model formulation, the protein could undergo an irreversible unfolding process or ''spreading'' following the initial adsorption to the surface from the liquid phase. Jungbauer et al [36] and Haimer et al [37] evaluated the kinetics of the conformational change of various model proteins using different HIC stationary phases. In the studies, the authors showed the rate of the conformational change depended on a variety of parameters, including the protein type, adsorbent ligand type, and solution salt concentration.…”

Section: Introductionmentioning

confidence: 99%

“…Several authors have applied the spreading model to predict the adsorption properties of membrane adsorbers [38] and HIC adsorbents [37]. Haimer et al coupled the spreading model with the continuity equation to describe the mass transport process within a HIC column.…”

Section: Introductionmentioning

confidence: 99%

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

McCue

Engel

et al. 2008

Bioprocess Biosyst Eng

View full text Add to dashboard Cite

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.

show abstract

“…HIC shows similar capacity to ion exchange chromatography (IEC) and a high level of resolution. Since it exploits a different principle than IEC and other separation techniques it can be used as an orthogonal method to achieve the purification of complex protein mixtures (Haimer et al, 2007). In this chapter, the theoretical principles underlying macromolecule retention in HIC are reviewed and discussed in sight of their application for predicting macromolecule behavior in HIC.…”

Section: Introductionmentioning

confidence: 99%

Hydrophobic Interaction Chromatography: Fundamentals and Applications in Biomedical Engineering

Mahn¹

2012

Biomedical Science, Engineering and Technology

View full text Add to dashboard Cite

Hydrophobic interaction chromatography of proteins IV

Cited by 63 publications

References 40 publications

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

Hydrophobic Interaction Chromatography: Fundamentals and Applications in Biomedical Engineering

Contact Info

Product

Resources

About