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Related Concept Videos

Protein Denaturation01:28

Protein Denaturation

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The function of proteins depends on their native three-dimensional structure, which is dictated by the amino acid sequence of the specific protein. Folding of the polypeptide chain takes place under specific conditions that energetically favor the folded conformation. In contrast, protein denaturation occurs spontaneously under unfavorable conditions that disrupt the integrity of the folded conformation. Thus, the chemical and physical environment of a protein, such as significant changes in pH...
8.1K

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Isothermal Chemical Denaturation: Data Analysis, Error Detection, and Correction by PARAFAC2.

Dillen Augustijn1, Alina Kulakova2, Sujata Mahapatra2,3

  • 1Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark.

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Summary

This study characterizes protein conformational stability in biotherapeutics using isothermal chemical denaturation. PARAFAC2 modeling accurately analyzes protein fluorescence data, enabling outlier correction for improved stability assessments.

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Area of Science:

  • Biochemistry
  • Biopharmaceutical Development
  • Protein Science

Background:

  • Protein conformational stability is crucial for biotherapeutic efficacy and preventing aggregation.
  • Isothermal chemical denaturation (ICD) is a method to assess chemical stability and optimize formulation conditions (pH, salt, excipients).
  • Intrinsic protein fluorescence, particularly tryptophan fluorescence, serves as a reporter for protein unfolding.

Purpose of the Study:

  • To investigate protein fluorescence emission spectra during isothermal chemical denaturation for seven monoclonal antibodies (mAbs).
  • To evaluate the impact of salt and excipients on protein fluorescence peak shapes and tailing across a physiological pH range.
  • To apply and validate the PARALLEC2 method for modeling denaturation data and detecting/correcting outliers.

Main Methods:

  • Isothermal chemical denaturation (ICD) was performed on seven monoclonal antibodies (mAbs) using GuHCl as a denaturant.
  • Intrinsic protein fluorescence emission spectra were recorded as a function of denaturant concentration.
  • Data were analyzed using the PARAFAC2 tensor decomposition method to model fluorescence changes and identify outliers.

Main Results:

  • Protein fluorescence peak shapes remained largely preserved under typical biotherapeutic salt (0-140 mM NaCl) and excipient concentrations.
  • Tryptophan fluorescence showed only minor peak tailing variations across a broad pH range (5.5-9.0).
  • PARAFAC2 modeling provided robust, automated analysis, revealing correlations between experimental setup and measurement errors, with a method for outlier correction.

Conclusions:

  • Salt and excipients commonly used in biotherapeutics have minimal impact on the characteristic fluorescence profiles of folded and unfolded proteins.
  • The PARAFAC2 method is effective for analyzing complex fluorescence data from protein denaturation studies, offering automated outlier detection.
  • A novel correction method based on PARAFAC2 scores allows for the retrieval of complete transition curves, enhancing the accuracy of biotherapeutic stability assessments.