Protein instability during HIC: Describing the effects of mobile phase conditions on instability and chromatographic retention

Xiao, Yunzhi; Freed, Alexander S.; Jones, Tara Tibbs; Makrodimitris, Kostantinos; O’Connell, John P.; Fernandez, Erik J.

doi:10.1002/bit.20826

Cited by 24 publications

(23 citation statements)

References 39 publications

(46 reference statements)

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“…Both the solvophobic (Horvath et al, 1976) and preferential interaction (Perkins et al, 1997) theories include an additional term to describe ''salting-in'' effects at low salt concentrations; however, our previously published data are well described by a linear salt dependence (Xiao et al, 2006), indicating that ''salting-out'' behavior is dominant. The free energy of protein unfolding in solution has been generally shown to increase linearly with salt concentration for non-denaturing salts:…”

Section: Theoretical Aspects the Four-state Model Of Protein Adsorptimentioning

confidence: 88%

“…In previous publications (Xiao et al, 2006(Xiao et al, , 2007a, we have presented a thorough description of a two-conformation, four-state model to describe the equilibrium thermodynamics of protein adsorption and unfolding in HIC. For brevity, we present here only the relationships directly relevant to the analysis of data in this study.…”

Section: Theoretical Aspects the Four-state Model Of Protein Adsorptimentioning

confidence: 99%

“…We have previously proposed a two-conformation adsorption model that includes the effects of salt concentration and temperature on both stability and adsorption to describe the effects of secondary protein unfolding on HIC behavior, specifically wild-type bovine a-lactalbumin adsorbed to Phenyl Sepharose TM 6FF low substitution resin (Xiao et al, 2006(Xiao et al, , 2007b. The choice of a-lactalbumin as a model protein was motivated by the ability to modify the solution phase protein stability by changing the available calcium for binding (Ikeguchi et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

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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

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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.

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Section: Theoretical Aspects the Four-state Model Of Protein Adsorptimentioning

confidence: 88%

Section: Theoretical Aspects the Four-state Model Of Protein Adsorptimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

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“…2), added ammonium sulfate decreases the reversibility of the adsorption process relative to low salt conditions. Other experiments and a thermodynamic framework developed in our group focusing in the linear portion of the isotherm offers a resolution of this difference, suggesting that salt affects native and unfolded protein adsorption differently (Xiao et al, 2006). Further studies of proteins adsorbed to hydrophobic interaction chromatography surfaces (Fogle et al, 2006) apply this hypothesis to high loading conditions as studied here and again show that even a salt that is stabilizing to proteins in solution, ammonium sulfate, can be destabilizing upon adsorption.…”

Section: Effect Of Protein Loading Adsorption Time and Mobile Phasementioning

confidence: 89%

QCM‐D sensitivity to protein adsorption reversibility

Jordan

Fernandez

2008

Biotech & Bioengineering

Self Cite

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Using a quartz crystal microbalance with dissipative monitoring (QCM-D) we have determined the adsorption reversibility and viscoelastic properties of ribonuclease A adsorbed to hydrophobic self-assembled monolayers. Consistent with previous work with proteins unfolding on hydrophobic surfaces, high protein solution concentrations, reduced adsorption times, and low ammonium sulfate concentrations lead to increased adsorption reversibility. Measured rigidity of the protein layers normalized for adsorbed protein amounts, a quantity we term specific dissipation, correlated with adsorption reversibility of ribonuclease A. These results suggest that specific dissipation may be correlated with changes in structure of adsorbed proteins.

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“…Several models have also been proposed to describe protein adsorption and unfolding on HIC resins. For instance, Xiao et al [12] proposed a four state model where native and unfolded species can adsorb to and desorb from the surface. Muca et al [13] and Marek et al [14] used a three-state model to describe a band splitting behavior of model proteins on HIC resin Butyl Sepharose 4FF.…”

Section: Motivations and Backgroundmentioning

confidence: 99%

Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

Guo

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This work elucidates the molecular interactions that are responsible for protein unfolding and aggregation on the surface of certain cation exchanger resins. The chromatographic behavior of a monoclonal antibody (mAb) that exhibits a two-peak elution behavior is studied for a range of strong cation exchange resins and with varying load buffer pH and composition. The two-peak elution behavior is very pronounced for the tentacle and polymer-grafted resins, but is essentially absent for hydrophilic macroporous resins. Confocal Laser Scanning Microscopy (CLSM) shows that this behavior is related to the unique kinetics of protein binding in the tentacle-type resin Fractogel. Hydrogen-Deuterium Exchange Mass Spectrometry (HXMS) proves that the two-peak elution behavior is tied to conformational changes that occur when the mAb binds. Circular dichroism suggests that the propensity of different mAbs to form stabilizing intermolecular structures can be related to their chromatographic behaviors. Another mAb exhibiting a threepeak elution behavior on the CEX resin POROS XS was also investigated. Dynamic Light Scattering (DLS) shows that the third peak contains significant levels of aggregates formed in the column that only slowly revert to monomeric species after elution. Circular dichroism and HXMS analyses of the eluted fraction, in-line fluorescence detection, and bound-state HXMS analysis indicate that the aggregates are generated by a destabilized, unfolded intermediate that is slowly formed on the resin. The two early eluting peaks observed regardless of hold time are shown to comprise exclusively monomeric species and form as a result of the presence of weak and strong binding sites on the resin having, respectively, fast and slow binding kinetics. This work has important practical implications in downstream processing for the industrial production of biopharmaceuticals as well as broad scientific value from a biomolecular perspective.ii Acknowledgements I would like to give my deepest gratitude to my academic advisor, Professor Giorgio Carta, for his guidance and support during my PhD study. I will never forget the many hours we spent together discussing my research results, drawing sketches of protein aggregation mechanisms, and fixing all sorts of experimental equipment.

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Protein instability during HIC: Describing the effects of mobile phase conditions on instability and chromatographic retention

Cited by 24 publications

References 39 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

QCM‐D sensitivity to protein adsorption reversibility

Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

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