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

Abstract

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.

Developability Assessment During the Selection of Novel Therapeutic Antibodies

“…In contrast to DSF, studying temperature dependence of intrinsic tryptophan fluorescence does not require extrinsic dyes. If additional information on unfolding pathways are of interest, DLS can be combined with Raman spectroscopy . Concomitant DLS and Raman spectroscopy provides insights into stress‐induced changes in hydrodynamic size as well as secondary and tertiary structure.…”

DLS and zeta potential – What they are and what they are not?

“…Hence, broadened peaks emerge while confidence in the data falls. Agglomeration is enhanced by increasing concentrations and thus, DLS is reliable only at dilute concentrations (typically 50-100 µg/ml) and unsuitable for many therapeutic formulations where much higher (up to few mg/ml) concentrations are used [79].…”

Recent applications of light scattering measurement in the biological and biopharmaceutical sciences

“…Let us first consider the recently published intensity distribution of the diameter of hydration of purified human serum albumin (HSA) at 20°C [50]. This distribution, scaled to a maximum value of unity, is reproduced as a solid curve in Figure 10A.…”

“…Relative distribution of scattering intensity plotted against log d h for HSA at 20°C, replotted from [50], solid curve. Relative distribution of w/v concentration calculated as described in text, dashed curve.…”