Combining Unfolding Reversibility Studies and Molecular Dynamics Simulations to Select Aggregation-Resistant Antibodies

Berner, Carolin; Menzen, Tim; Winter, Gerhard und Svilenov, Hristo L. (2021): Combining Unfolding Reversibility Studies and Molecular Dynamics Simulations to Select Aggregation-Resistant Antibodies. In: Molecular Pharmaceutics, Vol. 18, No. 6: pp. 2242-2253

Abstract

The efficient development of new therapeutic antibodies relies on developability assessment with biophysical and computational methods to find molecules with drug-like properties such as resistance to aggregation. Despite the many novel approaches to select well-behaved proteins, antibody aggregation during storage is still challenging to predict. For this reason, there is a high demand for methods that can identify aggregation-resistant antibodies. Here, we show that three straightforward techniques can select the aggregation-resistant antibodies from a dataset with 13 molecules. The ReFOLD assay provided information about the ability of the antibodies to refold to monomers after unfolding with chemical denaturants. Modulated scanning fluorimetry (MSF) yielded the temperatures that start causing irreversible unfolding of the proteins. Aggregation was the main reason for poor unfolding reversibility in both ReFOLD and MSF experiments. We therefore performed temperature ramps in molecular dynamics (MD) simulations to obtain partially unfolded antibody domains in silico and used CamSol to assess their aggregation potential. We compared the information from ReFOLD, MSF, and MD to size-exclusion chromatography (SEC) data that shows whether the antibodies aggregated during storage at 4, 25, and 40 degrees C. Contrary to the aggregation-prone molecules, the antibodies that were resistant to aggregation during storage at 40 degrees C shared three common features: (i) higher tendency to refold to monomers after unfolding with chemical denaturants, (ii) higher onset temperature of nonreversible unfolding, and (iii) unfolding of regions containing aggregation-prone sequences at higher temperatures in MD simulations.

• Item Type: Journal article
• Faculties: Chemistry and Pharmacy > Department of Pharmacy
• Subjects: 500 Science > 540 Chemistry
• ISSN: 1543-8384
• Language: English