Isothermal chemical denaturation of large proteins: Path-dependence and irreversibility

Wafer, Lucas N.; Kłoczewiak, M; Polleck, Sharon; Luo, Yin

doi:10.1016/j.ab.2017.10.001

Cited by 10 publications

(17 citation statements)

References 33 publications

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“…Apparent free energy of unfolding (ΔG), Cm and mvalues were calculated for the different transitions. 40,41 Different unfolding models (e.g. two-state, three-state) were tested for each protein to find the best fit.…”

Section: Isothermal Chemical Denaturation (Icd)mentioning

confidence: 99%

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization

et al. 2020

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Section: Isothermal Chemical Denaturation (Icd)mentioning

confidence: 99%

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization

et al. 2020

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“…The unfolding of Bis-mAb in urea is not completely reversible, and the degree of reversibility depends strongly on the formulation conditions (see below). Therefore, the thermodynamic analysis and extraction of Gibbs free energy of unfolding from the ICD curves would be inaccurate 31 . A further complication for the analysis is that Bis-mAb does not exhibit clearly defined transitions that can be well described by a two-state or a three-state unfolding model (data not shown).…”

Section: The Resistance To Urea-induced Unfolding Of Bis-mab Depends Mostly On Phmentioning

confidence: 99%

Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

Svilenov

Winter

2020

Journal of Pharmaceutical Sciences

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“…were also investigated but showed worse fit quality than the three-state model after an f-test comparison. The three-state model was also used by other groups to evaluate isothermal chemical denaturation data with mAbs 11,21 . Since all samples were evaluated with the three-state model, two Cm values were determined -Cm1 for the transition taking place at lower denaturant concentration and Cm2 for the other transition.…”

Section: Isothermal Chemical Denaturation (Icd)mentioning

confidence: 99%

“…two-state, three-state unfolding, etc.) 11,12 . A recent paper shows that neither reversibility nor equilibration times in an ICD experiment with mAbs are trivial 11 .…”

Section: Introductionmentioning

confidence: 99%

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A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

Svilenov

Gentiluomo

Frieß

et al. 2018

Journal of Pharmaceutical Sciences

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The early-stage assessment of the physical stability of new monoclonal antibodies in different formulations is often based on high-throughput techniques that suffer from various drawbacks. Accordingly, new approaches that facilitate the protein formulation development can be of high value to the industry. In this study, a dynamic light scattering plate reader is used to measure the aggregation (by means of the increase in the hydrodynamic radius [R]) of monoclonal antibody samples that were subject to incubation and subsequent dilution from different concentrations of a denaturing agent, that is, guanidine hydrochloride. The increase in the R of the protein samples is dependent not only on the denaturant concentration used but also on the buffer in which the incubation/dilution was performed. We also compare the aggregation after dilution from a denaturant with other high-throughput stability-indicating methods and find good agreement between the techniques. The proposed approach to probe the physical stability of monoclonal antibodies in different formulation conditions offers a unique combination of features-it is isothermal, probes both the resistance to denaturant-induced unfolding and the colloidal protein stability, it is entirely label-free, does not rely on complex data evaluation, and requires very short instrument measurement time on standard equipment.

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Isothermal chemical denaturation of large proteins: Path-dependence and irreversibility

Cited by 10 publications

References 33 publications

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization

Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

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