Analytical biochemistry

Concomitant Raman spectroscopy and dynamic light scattering for characterization of therapeutic proteins at high concentrations.

PMID 25475399


A Raman spectrometer and dynamic light scattering system were combined in a single platform (Raman-DLS) to provide concomitant higher order structural and hydrodynamic size data for therapeutic proteins at high concentration. As model therapeutic proteins, we studied human serum albumin (HSA) and intravenous immunoglobulin (IVIG). HSA concentration and temperature interval during heating did not affect the onset temperatures for conformation perturbation or aggregation. The impact of pH on thermal stability of HSA was tested at pHs 3, 5, and 8. Stability was the greatest at pH 8, but distinct unfolding and aggregation behaviors were observed at the different pHs. HSA structural transitions and aggregation kinetics were also studied in real time during isothermal incubations at pH 7. In a forced oxidation study, it was found that hydrogen peroxide (H2O2) treatment reduced the thermal stability of HSA. Finally, the structure and thermal stability of IVIG were studied, and a comprehensive characterization of heating-induced structural perturbations and aggregation was obtained. In conclusion, by providing comprehensive data on protein tertiary and secondary structures and hydrodynamic size during real-time heating or isothermal incubation experiments, the Raman-DLS system offers unique physical insights into the properties of high-concentration protein samples.