Towards a Unified Biophysical Characterization Platform: Combining Dynamic Light Scattering and Raman Spectroscopy to Determine Protein Structure and Stability
The combination of dynamic light scattering (DLS) with Raman spectroscopy has the capability to characterize a wealth of chemical, structural, and physical parameters of therapeutic proteins. Raman spectroscopy simultaneously derives protein secondary structure markers (amide I and III) and tertiary structure markers (aromatic side chains, disulfide bond, hydrogen bonding, local hydrophobicity). These markers can be monitored under controlled conditions by the determination of spectral peak position, shape, and/or intensity. Raman is able to make these structural determinations at formulation concentrations, 50 mg/mL or greater, rather than at the diluted concentrations required by conventional methods, i.e. typically less than a few mg/mL for circular dichroism (CD).
DLS using backscatter detection is capable of measuring the hydrodynamic radius of proteins at high concentrations (over 50 mg/mL). As the technique is based on light scattering, and scattering intensity scales with r6, it is exquisitely sensitive to the formation of aggregates. Therefore, changes in size distribution and polydispersity as a function of a variety of perturbations (temperature, pH, salt concentration, etc.) can be monitored, enabling the derivation of protein interaction and kinetic information, i.e. kD, relaxation times.
Combining these two techniques into a single system allows for the measurement of size and structure from a single small volume sample under identical conditions. This combined approach is unique in that it can indicate, for example, if a protein has changed its structure and then aggregated, aggregated without structural change, or changed structure and remained un-aggregated. It is therefore able to provide useful insights into the mechanism of aggregate formation.