Circular Dichroism (CD) Spectroscopy Technology

Circular dichroism (CD) spectroscopy, an important tool for characterizing protein higher-order structure (HOS) and thermal stability, has been widely used in biosimilar comparability studies to demonstrate similarity in protein HOS (secondary structure and/or tertiary structure). With decades of rich experience in developing and performing CD analytical methods, CD Formulation uses CD technology to provide reliable analytical support for the development and characterization of your protein/peptide drugs, from early development to late product release testing.

What is Circular Dichroism (CD) Spectroscopy?

CD is an absorption spectroscopy method based on the difference in absorption of left- and right-circularly polarized light. It is used to measure the difference in the absorbance of a substance to right- and left-circularly polarized light. In the regular secondary structure of a protein or polypeptide, the peptide bonds are arranged in a highly regular manner. Proteins or polypeptides with different secondary structures will produce CD bands with different positions and different absorption intensities. Therefore, the secondary structure information of a protein or polypeptide chain can be obtained based on the CD spectrum, thereby revealing the structure of the protein or polypeptide.

Fig. 1 Schematic representation of the Circular Dichroism instrument configuration. (Pignataro MF, et al., 2020)

Circular Dichroism (CD) Spectroscopy Principle

Circular dichroism is an absorption technique based on Beer's law:

A=εlc

Where A is absorbance, ε is the molar absorption constant, l is the cell path length, and c is the chromophore concentration.

This technique uses the circular dichroism characteristics of proteins and the different absorption of left and right circularly polarized light by asymmetric molecules to analyze the structure of proteins. The main photoactive groups in proteins or peptides are peptide bonds, aromatic amino acid residues, and disulfide bonds. When they absorb plane circularly polarized light differently, absorption differences will occur. The existence of this absorption difference leads to different amplitudes of the polarized light vector, and circularly polarized light becomes elliptically polarized light, which is the circular dichroism of proteins. CD scanners can be used to analyze the secondary and higher-level structures of protein and peptide samples to a certain extent.

Our Services Related to Circular Dichroism (CD) Spectroscopy

Thanks to decades of experience in supporting protein/peptide biopharmaceutical development and manufacturing using CD technology, our team of highly qualified experts offers a range of CD-related services to accelerate the implementation and success of your projects.

Our experienced team of experts has completed hundreds of protein and peptide CD analysis projects, allowing support for all stages of your protein/peptide drug development and manufacturing - from early studies to downstream process monitoring and GMP batch release testing.

Utilizing cutting-edge CD technology, we support the following protein/peptide characterization plans, including but not limited to:

Protein and Peptide HOS (Secondary, Tertiary) Characterization

For HOS characterization of proteins and peptides.

1) Far-UV CD measurements are performed using circularly polarized light in the wavelength range of approximately ≤ 200-250 nm. Since each type of secondary structural element (e.g. α-helix, β-sheet, etc.) has a unique spectral CD profile, the far-UV CD is sensitive to changes in secondary structure.

Our scientists will perform far-UV CD measurements at different solution conditions (pH, excipients, etc.) to determine the secondary structure of your protein of interest, and we will also compare with library/database information to determine the secondary structure.

2) Near-UV CD measurements are performed in the wavelength range of approximately 250-300 nm, where tyrosine, tryptophan and phenylalanine residues as well as disulfide bonds absorb light and are sensitive to CD.

Our scientists perform near-UV CD measurements to provide information on changes in the tertiary structure of your protein of interest due to formulation conditions, high temperature, storage, etc.

Fig. 2 CD spectra of polypeptides and proteins with representative secondary structures. (Yao H, et al., 2018)

Protein Thermal Conformation Stability Studies

Temperature changes can affect the molecular structure of proteins and peptides and lead to aggregation, which in turn affects the stability and efficacy of protein drugs during storage. Our scientists conduct thorough protein thermal conformational stability studies by using a CD spectrophotometer equipped with a temperature control system to monitor the relationship between the single wavelength of the target peak and temperature.

Throughout the CD analysis process, our scientists obtain thermodynamic parameters such as Gibbs free energy, enthalpy ( DH ) and entropy ( DS ) changes, and melting temperature ( T m ) by fitting the thermal melting curve to study whether the protein undergoes structural changes when heated.

Protein-Protein Interaction Analysis

CD can also be used to study protein interactions. Our scientists determine the interactions between proteins or between proteins and nucleic acids, including binding sites, binding modes, and binding strengths, by monitoring changes in protein conformation, thereby revealing the mechanism and kinetics of protein interactions.

Available Types of Circular Dichroism (CD) Spectroscopy Analysis

UV-CD Spectroscopy

This technique provides detailed information about the content and arrangement of secondary structural elements such as alpha helices, beta sheets, and random coils and is primarily used to analyze the secondary and tertiary structure of proteins.

UV/Vis-CD Spectroscopy

UV/Vis Circular Dichroism extends the analysis range to the near-UV and visible regions of the spectrum. This type of circular dichroism spectroscopy is used to study charge transfer transitions and electronic properties of molecules.

Vibrational CD Spectroscopy

Vibrational CD combines the advantages of CD and infrared spectroscopy (IR), providing information on molecular vibrational modes by measuring differences in the absorption of light in chiral molecules. This type of circular dichroism spectroscopy is primarily used to understand protein molecular structure and interactions.

Advantages of Our Circular Dichroism (CD) Spectroscopy Technology

• A non-invasive technique that does not require extensive sample preparation or modification.
• No crystallization required: Unlike X-ray crystallography, CD doesn't require crystallization of proteins or other molecules, making it suitable for studying systems that are difficult to crystallize.
• Temperature and pressure effects: Allows for performing under different temperature and pressure conditions to support formulation development and stability studies of proteins and peptides.
• Study dynamic processes and interactions such as protein folding, conformational changes, and protein-ligand binding in real-time.
• Simple, fast, and non-destructive.
• Far-UV circular dichroism can predict secondary structure.
• Near-UV CD can reflect changes in tertiary structure.

Custom Circular Dichroism (CD) Spectroscopy Services

Why Choose Our Circular Dichroism (CD) Spectroscopy Technology?

• Our team consists of highly skilled professionals with extensive experience in CD spectroscopy, supporting the analysis of nuances in protein characterization and molecular behavior.
• Our analytical laboratories are equipped with state-of-the-art CD instruments capable of providing high-resolution data, ensuring accurate and reliable readouts for a wide range of samples, including recombinant proteins, monoclonal antibodies, bispecific antibodies, enzymes, and peptides.
• Our services include detailed analysis and interpretation of CD data, providing insights into secondary structure, conformational changes, and interactions with ligands or other biomolecules.
• We provide flexible experimental design and customized solutions to meet your specific research needs, whether you require routine analysis or specialized projects involving complex samples.
• We value our partnership with our clients and are committed to providing ongoing support and guidance during the project to ensure the smooth implementation of your plans.
• Fast turnaround time: comprehensive reports are provided in 1 to 4 weeks, depending on sample size and selected services.

Publication

CD Formulation aims to provide a powerful analytical tool for protein and peptide HOS (secondary, tertiary) characterization. Please feel free to contact us if you are interested in our services. Learn how our CD spectroscopy technology can support the smooth implementation of your protein/peptide biopharmaceutical program.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.