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Circular Dichroism (CD) Assay Services
Welcome! We use exclusively Chirascan spectrophotometers from Applied Photophysics for our CD measurements.
Circular Dichroism (CD) is a biophysical technique used to characterize the higher order structure and conformational stability of biological samples, including proteins & peptides, nucleic acids, and small molecules.
CD is the difference in absorbance between left and right circularly polarized light by a chiral molecule that contains a chromophore. The technique lends itself to many applications, including quantifying stability, elucidating the secondary and tertiary structures of proteins, and conducting robust statistical comparisons of higher order structural changes in biotherapeutics.
Secondary Structure
Far-UV CD (260-180 nm) reports on secondary structure motifs, such as α-helices and β-sheets. Structural elements are readily quantifiable with commercially available software such as ProtaCAL.
Tertiary Structure
Near-UV CD (350-250 nm) reports on elements of tertiary structure, such as the orientation, solvent exposure, and overall fold of Trp, Tyr, & Phe residues, as well as disulfide bonds.
Reliably measure protein Tm values
CD measures protein conformational stability via thermal denaturation temperature ramps, yielding Tonset, Tm, van’t Hoff enthalpy, & ΔG.
HOS & Quality Control
Thanks to the robust automation in our Chirascan Q100 system, we can conduct statistically meaningful higher order structure comparisons on different batches of proteins or biotherapeutics.
Critical Data for Biotherapeutic Development
CD spectroscopy is now recognized as a crucial part of the biophysical characterization process where HOS comparisons are essential for defining critical quality attributes of biotherapeutics and strengthening the totality of evidence for regulatory submission.
Label-Free
CD utilizes the intrinsic chromophores present in most biomolecules, meaning there’s no need for labeling or adding dyes.
High-Throughput
We can run full CD characterization and referencing on up to 96 samples in a single automated experiment with extremely high precision.
Low & Nondestructive Sample Requirements
Depending on the experiment, CD can be run with protein concentrations as low as 0.1 mg/mL. Sample can be recovered and returned to you.
Formulation Screening
CD is a powerful screening tool to quickly determine the appropriate buffer, pH, ionic strength, and excipients needed to keep your protein happy and properly folded.
Proteins & Peptides
Proteins and peptides are the most well-studied CD substrates. Distinguish not only α-helix & β-sheet, but also 310-helix, π-helix, random coils, turns, and so much more.
Nucleic Acids
Nucleic acid structural motifs, including different G-quadruplex topologies, can be easily distinguished.
Small Molecules
CD is an indispensable technique for characterizing chiral small molecules, including natural products with unknown stereochemistry.
What sets Ichor’s CD services apart?
Ichor’s CD services include sample handling, standard assay execution, data analysis & reporting, and development of custom workflows for unique projects. No project is too big or small.
Ichor exclusively uses Chirascan CD spectrophotometers from Applied Photophysics. Custom samples requiring specialized accessories can be run on the Chirascan V100, whereas up to 96 samples can be run in a single experiment on the Chirascan Q100. We pass the savings and efficiency on to you.
Ichor provides full workflow support, including protein production, sample preparation and processing, CD assay execution, and statistical analysis complete with a full report deliverable.
At the conclusion of your study, our scientists will sit down and walk you through your data deliverables.
Chirascan spectrophotometers can record simultaneous multidimensional datasets comprising CD, absorbance, fluorescence, and temperature data throughout the entire UV, visible, and near-IR wavelength range from 163 nm to 1150 nm. Raw data can be exported in CSV or Excel formats, among others, and publication quality spectra can be prepared upon request. Further data analysis is also possible with software from Applied Photophysics:
- Pro-Data Viewer – for basic spectral analysis and fitting
- Global3 – for global fitting of temperature ramp data
- qBiC – for HOS statistical comparisons
We offer a range of orthogonal biophysical techniques, including nanoDSF (for high-throughput protein conformation stability analyses), in addition to Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) – both widely heralded for binding interaction studies.
Higher Order Structure Analysis (HOS)
(Three images – 1) spectral overlays of multiple sample lots with error in gray, 2) WSD equation, and 3) WSD plot showing sample distribution about a mean +/- 2 SD.
Ichor’s CD services go beyond routine formulation screening experiments and into the realm of statistically powerful secondary and tertiary structure comparisons (often referred to as Higher Order Structure, or HOS). Comparison of spectra is conducted via the Weighted Spectral Difference (WSD) method. The resulting values and their standard deviations can be used as an objective measure of their similarity, which allows the data to be subjected to a quality range test and be included as part of the product’s Critical Quality Attributes.
CD is indispensable in biosimilar development:
“Circular dichroism" has been used/proposed in 96% of biosimilar applications involving mAbs and other biotherapeutics.”
–Regulatory consideration for characterization of HOS in biotechnology products, M. T. Gutierrez Lugo, Ph. D., OBP/CDER/FDA. 5th International Symposium on HOS of Protein Therapeutics 2016.
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 HOS and evaluation of their statistical significance early in the development process.
Examples include comparisons of innovator versus biosimilar lots (above) and forced degradation studies, which are used to investigate stability, storage conditions, and expected shelf life.
Overlay of CD vs. wavelength temperature isotherms.
Overlay of fluorescence emission vs. wavelength temperature isotherms.
Thermal Denaturation – Temperature Ramp Experiments
All Chirascan systems can perform continuous, multi-wavelength temperature ramp (T-ramp) experiments. Chirascan systems allow for the simultaneous acquisition of multiparameter data, including CD, absorbance, fluorescence, and temperature. Both CD and fluorescence spectra as a function of temperature can be used to determine the melting temperature (Tm) of a protein via global fitting with Applied Photophysics’ Global3 software.
Image of Chirascan Q100 instrument with integrated autosampler.
Closeup image of plate in autosampler.
High-Throughput Sample Analysis for HOS and T-Ramp Studies
The Chirascan Q100 is designed to run up to 96 samples per experiment in complete, unattended operation. This includes highly precise and reproducible sample handling and robust cleaning protocols, both prerequisites for statistically powerful HOS comparisons and time-consuming temperature ramp experiments. All samples and reference buffers are stored in temperature-controlled plates prior to analysis to ensure sample integrity throughout the experiment.
Chirascan platform accessories provide dedicated tools for specialized applications—for orthogonal data beyond circular dichroism, less common sample types, and additional measurement modes. Accessories are available for virtually every sample type and experiment, including:
- 6-Cell Turret – for increased sample throughput
- Titrator – for automated titrations
- pH Probe – for monitoring in-sample pH
- Solid Sample Holder – for measurements of solid (disc) samples
- Integrating Sphere – for measurements of powders and other opaque samples
- Stopped-Flow – for following kinetics by CD and other signal modes
- Circularly Polarized Luminescence (CPL) – for measuring the CD of luminescent compounds and calculating glum
- CCD Fluorometer – for fast, full emission spectrum fluorescence data
- Total Fluorescence – for detection of the total fluorescence emission as a function of excitation wavelength
- Scanning Emission Monochromator – for scanning the fluorescence emission wavelengths at a fixed excitation wavelength
- Couette Cell – for investigations of relative molecular orientation (Linear Dichroism)
- Optical Rotary Dispersion (ORD) – for measuring optical rotation/activity as a function of wavelength (Polarimetry)
- Magnetic Circular Dichroism (MCD) – for measuring the absorption of circularly polarized light by a sample in a magnetic field
Featured Capabilities / Case Studies – Courtesy of Applied Photophysics
A primer on what CD can do
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.
- HOS comparisons can be made between different sources or lots of biotherapeutics, as well as in forced degradation experiments to monitor change under stressed conditions and assess stability and shelf life.
- 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.
Protein Therapeutics
Chirascan systems provide invaluable information for the characterization of protein biotherapeutics.
Whether these are recombinant enzymes or regulatory proteins, or monoclonal antibodies (mAbs) and constructs derived thereof, including bispecifics and antibody-drug conjugates—Circular Dichroism spectroscopy is an essential orthogonal technique and indispensable in any toolset for biophysical characterization.
To assist with the complex task of characterizing protein-based biotherapeutics such as mAbs, the National Institute of Standards and Technology (NIST) undertook a holistic study using a humanized mAb, the NISTmAb, in 2016.
The study utilized the biophysical techniques most commonly employed during biotherapeutic development, and the NISTmAb is now available to biopharmaceutical companies as a representative reference molecule.
Applied Photophysics had been approached by the NIST to contribute CD data to this project. A primary standard of the NISTmAb, PS 8670, was compared with the NISTmAb reference material, RM 8671 using the automated Chirascan Q100 system, equipped with 21 CFR, Part 11 compatible software, to investigate both the secondary and tertiary HOS.
Zhai et al. 2021, Struct. Dyn. 8, 024102. DOI: 10.1063/4.0000086
Peptide Therapeutics
Inhibiting the fibrilization of a GLP-1-like peptide
Despite having lower complexity compared to protein therapeutics, peptide therapeutics have their own developability challenges. Peptide drugs often suffer from low stability by having a strong propensity for self-aggregation. CD spectroscopy is well-suited for assessing the structural integrity of peptides, and Chirascan systems provide the required sensitivity.
The study outlined here aimed at finding a suitable formulation for the GLP-1-like peptide G48.
- By monitoring peptide structure with a Chirascan system and evaluation at a range of conditions, including different concentrations, pH values, and excipients, it was found that undesired peptide fibrillation was successfully inhibited in presence of polysorbate 80.
- Moreover, CD data reveals that peptide aggregation depends on net charge, as different magnitudes of structural changes are observed at neutral pH as compared to acidic or basic pH.
- See the original paper for this study here: https://pubmed.ncbi.nlm.nih.gov/31812799/
RNA Therapeutics
Multi-Method Benchtop Analyses
In this white paper, the thermal stability of lipid nanoparticles (LNPs) developed for mRNA drug delivery was investigated in a three-way collaboration between Applied Photophysics, SINTEF (Norway), and Malvern Panalytical (UK).
CD data for mRNA-LNPs showed a transition corresponding to mRNA melting and a high-temperature transition attributed to LNP disintegration. Wavelength-dependent differences suggested that the latter is not the same one as observed for empty LNPs.
Together with insight from DSC data, these preliminary findings suggest that mRNA-LNPs are indeed more than just the sum of their constituent components and there is much remaining to investigate with these promising new therapeutic modalities.
CD spectroscopy with Chirascan systems can provide a better understanding of your RNA drug product.
https://www.photophysics.com/use-cases-collection/rna-therapeutics/
Quality Control
Identify the Presence of Contaminants
Whenever there is risk of your product containing proteins that are structurally different, HOS comparisons using a Chirascan Q100 can confirm structural purity.
- In this example, an antibody-based therapeutic was spiked with different amounts of bovine serum albumin (BSA), representative for contaminants that are structurally different, and with different amounts of an aggregated fraction of the same product (Agg.), representative for contaminants that are structurally highly similar.
- The highest contents of BSA and aggregated product resulted in far-UV CD spectra that are easily visually distinguished from the spectrum of the pure product.
- Smaller amounts of contaminants were only distinguishable by means of statistical analysis. Similarity between samples was assessed by calculating Weighted Spectral Difference values.
Do you know how pure your drug product is?
Frequently Asked Questions
Learn more about Ichor’s CD services below:
CD stands for Circular Dichroism. CD is an absorbance-based biophysical technique in which the difference in absorbance between left and right circularly polarized light by a chiral molecule is measured. CD is a conceptually simple technique that follows Beer’s Law, however it is capable of providing extremely detailed and sensitive measurements on the minute structural elements and motifs of biomolecules. Nearly all biomolecules have chromophores that absorb in the UV or visible light regions and are therefore naturally amenable to CD analysis.
CD provides invaluable information on the secondary and tertiary structure of proteins and nucleic acids, it allows for the measurement of chemical & thermal stability, and it is used in the statistical quantification of changes to higher order structure. There are few other techniques (nanoDSF is another) that can provide comparable structural information, particularly with the high-throughput, low-cost, low sample consumption profile of a benchtop instrument. If you are producing proteins or therapeutic antibodies in-house, CD is a must-have technique to characterize proper folding and to quality control your product.
Our end-to-end CD services include novel CD assay development. Assays can be developed around your specific biomolecule of interest and characterization needs. Once a method is developed, samples can be run at scale using our automated Chirascan Q100 instrument to obtain exceptionally reproducible and statistically comparable results.
Because CD is a technique based on the preferential absorbance of one handedness (left or right) of circularly polarized light by a chiral molecule, any optically active biomolecules (e.g., proteins, peptides, nucleic acids, natural products, etc.) are amenable to the technique. The primary prerequisite is that the molecule absorbs in the range of ~170 nm to 1150 nm (UV, visible, or near-IR). Concentration requirements are generally sample dependent, but typically span the range from approximately 0.1 mg/mL to >10 mg/mL.
The most common CD applications are 1) secondary and tertiary structural determination, 2) thermal stability (temperature ramp experiments), and 3) higher order structure comparisons. Other common applications include small molecule ligand binding studies and even protein-protein interaction studies. CD is a comparative technique and is most powerful when used as such.
You will receive all raw and processed data produced by the instrument. This includes the plate map and sample preparation procedure, raw data in Excel or CSV format (CD, absorbance, and fluorescence, if applicable, as a function of wavelength & temperature), and a PowerPoint or PDF report containing all processed and reportable data. Experiment-specific deliverables:
- Secondary structure data (far-UV): Absolute structural composition can be calculated via the ProtaCAL Software Suite.
- Temperature ramp data: Globally fitted CD and/or fluorescence melt curve data, which provides the onset and melting temperatures (Tonset & Tm), in addition to thermodynamic parameters such as the van’t Hoff enthalpy and ΔG.
- Higher Order Structure (HOS) comparison: Weighted Spectral Difference (WSD) statistical calculations of near- or far-UV spectral similarity.
Additionally, publication quality custom figures can be generated upon request.
The actual cost will depend on the scope (i.e., number of samples) and complexity of the project, as well as whether there is any synergy with our other service offerings. Analyses can be run manually for small numbers of samples or in automation for large numbers of samples, such as for statistical comparisons. We are thus able to offer substantial discounts for larger sample volumes.
Turnaround times are typically on the order of 2-3 weeks from receipt of all materials.
