White Paper

White Paper:
Beyond α-Helix and β-Sheet: the Expanding Role of Circular Dichroism

Perceived for many years as a low-resolution biophysical technique, circular dichroism (CD) spectroscopy has advanced well beyond its conventional textbook description. This white paper highlights the expanding role of CD as it undergoes a fundamental transition from its origins in basic academic research to becoming an indispensable tool for the biopharmaceutical industry—a biophysical characterization technique that offers far more than the proportion of α-helix and β-sheet in a protein.

  • Near-UV circular dichroism spectra reveal minor changes in tertiary structure, complementing the traditional use of far-UV scans to detect change in secondary structure
  • Objective, statistically-validated higher order structure (HOS) comparisons of circular dichroism spectra confirm similarities or differences between samples, replacing subjective, visual comparisons
  • Comprehensive information about structure and stability is revealed in a single continuous, multi-wavelength thermal denaturation experiment

In short, state-of-the-art CD analysis provides unique insights into changes in secondary and tertiary structure, as well as kinetic and thermodynamic information, enabling researchers to publish with confidence and contributing to informed decision-making during biotherapeutic development.

Section 1: The Changing Role of CD Analysis

A brief overview of how circular dichroism has moved beyond α-helix and β-sheet to become a critical tool for biomolecular characterization.

Section 2: Protein Secondary and Tertiary

Structure in Focus

Using NISTmAb, a monoclonal antibody reference material, we show how modern circular dichroism spectrometers provide detailed insight into the secondary and tertiary structure of proteins. Offered by the National Institute of Standards and Technology, U.S. Department of Commerce, the NISTmAb standard enables scientists to evaluate the performance of methods used to determine physicochemical and biophysical attributes of monoclonal antibodies.

Real Life Examples: Case studies illustrate how state-of-the-art circular dichroism enables researchers to publish with confidence and contributes to informed decision-making during biotherapeutic development.

Section 3: Assessing Protein Stability Through Thermal Denaturation

Including an introduction to the theory and practice of thermal denaturation experiments, this section features a case study demonstrating characterization of the thermal stability of a biomolecule using CD and fluorescence measurements.

Section 4: Evaluating Statistical Significance of Changes Detected in Higher Order Structure

As the biopharmaceutical industry faces increasing demands for objective, statistically-validated data in regulatory submissions, state-of-the-art CD spectrometry enables detection of minor changes in higher order structure (HOS) and evaluation of their statistical significance - exemplified in this section by a forced degradation study of a monoclonal antibody.

Section 5: The Physical Principles Behind Circular Dichroism

For those new to CD spectroscopy, or as a refresher for experienced users, this section covers the basic physical principles behind the technique.

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