Statistically Validating HOS Analysis with Circular Dichroism

The purpose of this article is to provide information to researchers on how to create objective, statistically-validated HOS decisions in an informed manner while maintaining project momentum.

From QTPP Onwards: Run Effective Comparability Programs During Biosimilar Development

Determine Innovator HOS Characteristics

Circular dichroism (CD) analysis of the tertiary structure of chiral molecules in a highly absorbing chiral formulation buffer indicates differences within the tryptophan region of Fab fragments from innovator lots. See Figure 1.

Figure 1. The analysis of a tertiary structure. Spectra is normalized for protein concentration by simultaneous absorbance measurements using the Chirascan™ Q100 at n=3.

Objective, Quantifiable Evaluation of Differences and Similarities

The detected differences within the tertiary structure of innovator lots as a result of dissimilar geographical areas were found to be statistically significant. Following this evaluation, a comparison between the innovator and biosimilar established the statistical significance of minor differences observed in the tertiary structure (data not shown). Figure 2 represents the Tier 2 quality range test that was applied.

Figure 2. The results of a Tier 2 quality range test, in which a +/-2SD acceptance criteria by the Office of Biostatistics and the Office of Biotechnology Products, CDER/FDA was applied. Weighted spectral difference, provided by Dinh, N., et al. (2014). Analytical Biochemistry. 464:60-62.

The highlighted components of this technique include:

  • The quantification of any differences and similarities under normal or stressed conditions
  • Monitoring changes present throughout biotherapeutic development and scale-up phases
  • Strengthen the total available evidence for any required regulatory submissions
  • Define an acceptable range for HOS variability within a control strategy for ensuring quality, safety and efficacy in the manufacturing process

From Lead Characterization Onwards: Define Characteristics, Measure Significance of Change

Monitor Change Under Stressed Conditions (Forced Degradation)

The high sensitivity of the aforementioned CD analysis of IgG1 samples were subjected to a range of degradation conditions, in which slight differences in the tertiary structure were shown when compared to the control sample. These results are further shown in Figure 3.

Figure 3. The analysis of the tertiary structure, in which the spectra was normalized for protein concentration by simultaneous absorbance measurements using a Chirascan™ Q100 at n=6.

Quantifiable Assessment of Changes in Tertiary Structure

The statistical analysis performed here provided an objective confirmation of the spectral results. All degradation conditions have been shown to influence the local environment of the aromatic side changes, however no changes were detected in the secondary structure (results not shown). The effects of the sample condition and their significance is further shown in Table 1.

Table 1. Weighted spectral difference, provided by Dinh, N., et al. (2014). Analytical Biochemistry. 464:60-62.

Sample Condition Expected affect p-value
T1 0.3% H2O2, 20 °C, 3 hours Oxidation 0.001
T2 2 M glucose, 40 °C, 1 week Glycation 0.001
T3 pH 8.5, 40 °C, 1 week Asn deamidation/Asp isomerization ≤ 0.001
C Sample preparation only(dialysis) No effect (Control) 0.807

p-value > 0.05; differences not significant at 2σ confidence interval
p-value < 0.05; differences significant at 2σ confidence interval

More than Just Circular Dichroism – Dedicated Chirascan Accessories

CCD Fluorometer

The CCD fluorometer, which is controlled by the Chirascan software, generates an emission spectra in seconds to provide and changes in the CD, fluorescence and absorbances in a single experiment.

6-Cell Turret

The 6-cell turret, which is controlled by the Chirascan software, allows for users to analyze up to 6 samples or thermal denaturation data sets each run. The magnetic stirring and Precision Peltier temperature control components of this accessory allow for optimal conditions for thermal denaturation experiments.

Tirator and pH Probe

The tirator and pH probe are used to constantly monitor any concentration and pH dependent changes in the CD, fluorescence or absorbance data during an experiment. Both of these accessories are completely automated, as they are controlled by the Chirascan software. Upon detecting a change in either the concentration of pH of the sample, this dual syringe titration system will change the concentration of the solution while ensuring that the volume is constant.

LD Couette Cell

The LD Couette Cell provides users with information on the conformation and relative orientation of molecules by conducting an examination of the macromolecular structures and their interactions. The structures of any molecule used here can either be intrinsically oriented or oriented prior to the start of the experiment.


The Stopped-Flow accessory allows users to characterize fast reactions, while complementing CD spectra with useful kinetic information. Any reactions that occur in solution can therefore be studied closely as the rate constants, changes in absorbance, CD, fluorescence or fluorescence polarization are characterized by the stopped-flow accessory.

Integrating Sphere and Solid Sample Holder

The integrating sphere allows users to detect and changes in the chirality of solid state samples while simultaneously guaranteeing a high signal to noise ratio during the collection of CD data from highly scattering samples, such as powders and solids. Transparent samples, such as KBr discs, can be rotated on a wheel in the sample holder to eliminate any potential birefringence effects.

Also Available

  • Optically Rotary Dispersion Accessory: Characterizes chiral molecules at a high concentration where absorption bands are obscured by buffers alt or solvent absorption. Additionally, this accessory can also be used when measuring at wavelengths where chiral molecules are unable to absorb light.
  • Fluorescence Anisotropy Detector: Characterizes ligand binding events.
  • Near IR Extension Kit: Expands the scanning range of a Chirascan V100 to 1700 nm.
  • Magnetic CD Accessory: Used to study specific molecules, such as metalloproteins.

This information has been sourced, reviewed and adapted from materials provided by Applied Photophysics.

For more information on this source, please visit Applied Photophysics.

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