Biophysical Analytical Techniques for Antibody Drug Characterization

The discovery and successful development of antibody drugs depends on the comprehensive characterization of the molecule before and after formulation. The rapid development of sensitive biophysical methods is facilitating the successful development of antibody drugs by providing early analysis of drug properties and insights into mechanisms of action. Efficient biophysical characterization techniques are indispensable analytical tools in the development of antibody drugs. Creative Proteomics offers a powerful suite of biophysical techniques to characterize and evaluate the conformational integrity, aggregation propensity, binding affinity and pharmacokinetic properties of antibody drugs. All of our biophysical characterization assays are rigorously validated and regulatory compliant and can be used for routine lot release or stability studies of your antibody drug.

Introduction to Biophysical Characterization of Antibody Drugs

Biophysical methods are often applied to the analysis of higher order protein structures, which are essential to ensure the safety, efficacy, stability and ultimate quality of antibody drugs. Below are some of the biophysical techniques we use in characterizing different quality attributes of antibody drugs:

Biophysical Characterization Platform at Creative Proteomics

Creative Proteomics' biophysical characterization platform is equipped with a comprehensive range of technologies and equipment for the structural and active characterization of antibody drugs. Our platform is staffed by a dedicated characterization team with extensive experience and knowledge in providing customized solutions for customer characterization needs based on the functional features of the instrumentation. Below is a description of the advanced biophysical technologies available on our platform:

Circular Dichroism (CD)

Circular dichroism (CD) is a light absorption phenomenon based on the principle that antibody molecules are analyzed based on the differences in the way they affect the left and right polarization of light in the far UV region (190-250 nm) or near UV region (230-250 nm). Far-UV CD is more sensitive to secondary structures, while near-UV CD is more sensitive to tertiary structures. Different molecular structures produce different light intensity signals and can therefore be used to analyze molecular structure.

Nuclear Magnetic Resonance (NMR)

Nuclear Magnetic Resonance (NMR) technology is ideal for structural analysis of antibody drugs and plays a uniquely important role in the higher-order structural elucidation of proteins. Our advanced inspection technologies and data analysis systems provide our customers with accurate information on critical quality attributes.

Fourier Transform Infrared (FT-IR) Spectroscopy

Fourier transform infrared (FT-IR) spectroscopy is an analytical method that uses the near-infrared region (800-2500 nm) of the electromagnetic spectrum. It measures the absorbance of a sample at different wavelengths in the NIR region, providing information related to the secondary structural features of proteins.

Sedimentation Velocity Analytical Ultra Centrifugation (SV-AUC)

Sedimentation rate experiments report the relative percentages of monomer, multimer and aggregate species by recording the values of the generated sedimentation coefficients. Thus, the SV-AUC is often used as an orthogonal method to SEC to characterize aggregates of antibody drugs.

Sedimentation Equilibrium Analytical Ultra Centrifugation (SE-AUC)

SE-AUC involves the analysis of the sedimentation behavior of macromolecules in a centrifugal field. It is a method based on the principles of centrifugation and sedimentation, which allows for the determination of the molecular weight, homogeneity, and association properties of macromolecules in solution. This information can be used to determine the molecular weight and level of aggregation of an antibody drug, and can also be used to study antibody-antigen binding affinity.

Dynamic Light Scattering (DLS)

The main application of DLS is the detection of protein aggregation at the micrometer scale. In addition, DLS can be used to detect protein-protein interaction forces under electrostatic, van der Waals, hydrogen bonding and hydrophobic forces.

Fluorescence Polarization Anisotropy (FPA)

Fluorescence polarization anisotropy (FPA) determines binding constants from the interaction of relatively small fluorescently emitting ligands with larger receptor molecules., and is commonly used in the determination of binding activity of antibody drugs.

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) technique is a novel analytical technique based on optical principles. We used the SPR technique to characterize the functional activities related to antibody drugs, including antigen-antibody affinity and the binding activities of Fc with FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcRn and C1q.

Bio-Layer Interferometry (BLI)

BLI technology uses fiber-optic biosensors to detect changes in the thickness of the sensor's optical layer in real time as molecules bind and dissociate. Creative Proteomics scientists use BLI technology to determine the affinity and kinetics of antibody drugs to characterize their functional activity.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) is conducted by heating the molecule at a constant rate and the detectable changes in heat capacity associated with thermal denaturation are recorded. We analyze the high-level structure and stability of antibody drugs through DSC thermograms of proteins.

Differential Scanning Fluorimetry (DSF)

Differential scanning fluorimetry (DSF) detects the amount of fluorescent dye binding to structurally altered proteins during heating. We commonly use it to evaluate the thermal stability of antibody drugs.

Isothermal Titration Calorimetry (ITC)

ITC is an important method for studying bio-thermodynamics and kinetics. Our highly sensitive ITC technology allows for the rapid determination of a variety of parameters such as antibody affinity (Ka), antigen binding heat (H), and antigen-antibody binding ratio (n), which can also be used to indicate antigen-antibody interactions in vivo and in vitro.

Size-Exclusion Chromatography with Multi-Angle Laser Light Scattering (SEC-MALLS)

Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) detection is used to separate proteins based on size and to determine the molar mass of the separated proteins. It is often used in our laboratory for impurity and aggregate characterization of antibody drugs.

Native size-exclusion chromatography-mass spectrometry (nSEC-MS)

nSEC-MS combines the principles of size exclusion chromatography (SEC) and mass spectrometry (MS) to provide detailed information about the molecular weight, structure, and heterogeneity of antibody drugs.

Creative Proteomics is committed to using our biophysical technologies to provide you with comprehensive and reliable characterization services. Contact us today to learn more about how our biophysical technologies can help support your antibody drug development program.