Circular dichroism in biophysical characterization

In addition to providing scientists with a deeper understanding of biomolecular mechanisms and interactions in research and discovery, characterization of specific quality attributes for a potential therapeutic candidate is essential in the biopharmaceutical industry. These attributes are used to define a product’s identity, purity, potency and stability and, if critical, to correlate with safety and efficacy.

Typical characterization considerations of a biotherapeutic product


Extract from an FDA briefing by Sandoz for the biosimilar, filgrastim, given in 2015 showing the importance of quality attributes and complementary techniques to ensure safety and efficacy.

To ensure safety and efficacy, biophysical characteristics relating to structure and stability must be monitored for change throughout the drug development and manufacturing process.

Since the slightest change in higher order structure (HOS) can significantly impact efficacy and immunogenicity of a biotherapeutic, HOS is regarded as a ‘critical quality attribute’. As a result, by providing unique insights into the secondary and tertiary structure of proteins and their stability under native and stressed conditions, circular dichroism (CD) is an essential technique for characterizing a biotherapeutic.

Secondary and tertiary structure, stability

CD spectroscopy measures the difference between left-handed and right-handed circularly polarized light absorption of chiral samples as a function of wavelength. The difference in these absorbances is called “ellipticity” and is affected by peptide bond orientation in secondary structural elements and tertiary structural interactions of UV-active chromophore side chains such as aromatic amino acids.

Although suitable for studying chiral molecules of all types, Chirascan CD spectrometers are optimized for analysis in the far- and near-UV, giving insight into the tertiary and secondary structure of protein-based biomolecules. Thermal denaturation (temperature ramping) experiments provide information on stability, mechanisms of folding/unfolding including melting temperature (Tm).

In the biopharmaceutical industry, regulatory authorities are increasing their demands for results from characterization techniques to be supported by statistically-validated data. Results generated on a Chirascan Q100 system enable application of the weighted spectral difference method described in Dinh et al., anal. Biochem. 464, 2014 to yield data compatible with quality range testing (the Office of Biostatistics and Office of Biotechnology Products, CDER/FDA, recommends a quality range method of statistical analysis for quality attributes of intermediate criticality (Tier 2).). By making it possible to assess the significance of differences between two samples, the Chirascan Q100 facilitates objective HOS comparisons.

Complementary techniques used in biophysical characterization

Fourier transform infrared Spectroscopy (FTIR) analyzes proteins in the mid-spectral infrared region where the amide I band is most distinctive. This band contains information about the secondary protein structure as it is primarily dependent on the backbone structure.


Differential Scanning Calorimetry (DSC) is used to investigate the thermal stability of a protein solution by measuring the melting temperature (Tm) for any effective transition i.e. corresponding to the midpoint temperature during a transition, and it is given by the position of the corresponding local maximum in the heat capacity (Cp) profile. Cp represents the change in enthalpy of a system as temperature is increased during scanning. Cp profiles for a given protein depend on the conformational state and solution conditions, factors that directly affect the strength of intra- and intermolecular protein interactions.


Two dimensional Nuclear Magnetic Resonance Spectroscopy (2D NMR) correlates the spin states of two active nuclei in close physical and/or chemical proximity since the local electronic environment, such as solution conditions, temperature, and local chemical structure, of each NMR-active nucleus differentially shields it from an external magnetic field and gives it a specific frequency position. 2D NMR thereby provides a measure of atomic resolution higher order structure.


Sedimentation Velocity Analytical Ultra Centrifugation (SV-AUC) separates and characterizes biomolecules in a wide variety of solution conditions. Sedimenting boundaries of biomolecules have a characteristic speed (quantified by the sedimentation coefficient) that depends on the molecular weight and frictional coefficient of the species and the viscosity and density of the buffer. Curve fitting the speed and shape of sedimenting boundaries enables calculation of the molecular weight of each species to identify fragments, monomer, and aggregates.


Dynamic Light Scattering (DLS) estimates protein size and strength of the protein-protein interactions via analysis of the ‘intensity autocorrelation function’. Brownian motion causes constant fluctuation of molecules within the path of an incident light (i.e. a number of scatters). The size of fluctuations in scattering intensity correlates over short periods of time with the displacement of molecules, thus determining the average time that a molecule is located within the path of the incident light. This time relates to the diffusivity of the molecule, average hydrodynamic radius, and the net interactions experienced.

Static light scattering (SLS) collects time-averaged scattered intensities as a function of protein concentration at a given angle to obtain estimations of protein size and the strength of protein-protein interactions.


MS/MS Peptide Mapping provides primary sequence information. A peptide map results from signals generated as trypsin-digested peptides elute from a reverse-phase ultrahigh pressure liquid chromatography (UHPLC) column and pass through UV and MS detectors. Differing amino acid sequences give each peptide unique chromatographic properties and online MS/MS detection enables assignment of primary structure based on mass and fragmentation commensurate with the predicted amino acid sequence.

Peptide mapping under nonreducing conditions (excluding dithiothreitol as a reducing agent and using Lys C instead of trypsin) results in disulfide bonds that remain intact such that canonical disulfide linkages can be confirmed by mass in the mass spectrometer.  The theoretical molecular mass of canonical disulfide-containing peptides based on the expected amino acid composition is compared to measured molecular masses.


UHPLC-MS enables Intact Molecular Mass Analysis i.e. empirical determination of the molecular mass of a target protein as well as providing information on post-translational modifications. Measured molecular mass is compared to a theoretically calculated molecular mass based on the expected amino acid composition as an orthogonal confirmation of primary structure. Mass heterogeneity is determined by electrospray ionization mass spectrometry using LC-MS e.g. a UHPLC system coupled with a QTOF mass spectrometer.

Distribution of N-linked oligosaccharides is determined by enzymatic release of glycans from the biomolecule followed by labelling at their reducing terminus with the fluorescent dye 2-aminobenzamide. Hydrophilic liquid interaction UHPLC resolves the labelled glycans and quantifies them based on their relative fluorescence signal. Online MS/MS detection enables assignment of primary structure based on mass and fragmentation commensurate with a given glycan composition.


Capillary Zone Electrophoresis (CZE) is used to quantify charge purity and heterogeneity by separation of charge variants according to differential electrophoretic mobility in free solution within a uniform electric field applied across a buffer filled fused silica capillary.


Capillary Isoelectric Focusing (CIEF) separates charge variants based on differences in isoelectric point. Sample is mixed with an ampholyte solution to form a pH gradient when subjected to an applied electric field. charge variants migrate until they reach a pH where their net charge and electrophoretic mobility are zero i.e. pH is equal to the isoelectric point, pI.


Size Exclusion UltraHigh Performance Chromatography (SE-UPLC) reveals size heterogeneity by resolving species based on size and/or conformation. Samples in solution pass through a column packed with porous particles of a certain size. Larger molecules, such as aggregates, elute first having bypassed the pores. Smaller molecules, such as fragments, enter the pores, slowing down their rate of travel through the column, and elute later in order of decreasing size.


Capillary Electrophoresis Sodium Dodecyl Sulfate (CE-SDS) is analogous to size exclusion. Analytes complexed with SDS are loaded into a narrow bore glass capillary filled with an uncrosslinked polymer sieving matrix. A high voltage applied across the capillary drives analytes toward the outlet. Migration is retarded in a size-dependent manner by differential interaction with the sieving matrix.


Microflow Digital Imaging analyzes sub-visible particles (2-100 µm) in suspension by capturing bright-field images as sample passes through a flow cell positioned in the field of view of a microscopic system. Digital images are stored for subsequent analysis of counts, size, transparency and other morphological parameters.