Molecular Dynamics Simulations Reveal How Competing Protein-Surface Interactions for Glycine, Citrate, and Water...

Area of Science:

• Biochemistry and Biophysics
• Protein Formulation Science
• Computational Chemistry

Background:

• Designing stable protein therapeutics requires minimizing aggregation, a challenge often addressed by experimental screens based on thermal transition midpoints (Tm) and forced degradation.
• Current methods offer limited prediction of long-term storage stability, especially at low temperatures, highlighting the need for a deeper mechanistic understanding of excipient effects.
• Understanding how excipients like glycine interact with proteins can lead to improved formulation design strategies for enhanced therapeutic stability.

Purpose of the Study:

• To investigate the complex impact of glycine concentration on the stability of an antibody Fab fragment.
• To elucidate the underlying mechanisms of glycine's stabilizing effects using molecular dynamics (MD) simulations.
• To correlate formulation behavior with changes in protein dynamics and excipient-protein interactions.

Main Methods:

• Experimental determination of antibody Fab fragment stability, including thermal transition midpoints (Tm) and entropy changes (ΔSvh).
• Forced degradation studies at elevated temperatures to assess aggregation kinetics at various glycine concentrations.
• All-atom molecular dynamics (MD) simulations to analyze protein dynamics (RMSF) and molecular interactions at different glycine concentrations.

Main Results:

• Glycine concentration showed a complex effect on Fab stability: Tm increased monotonically, but native ensemble dynamics (ΔSvh) peaked at 30 mg/mL.
• Aggregation kinetics at 65 °C were similar at 0 and 20 mg/mL glycine but significantly slower at 50 mg/mL.
• MD simulations revealed concentration-dependent glycine-protein interactions, shifting from preferential hydration at low concentrations to preferential exclusion at higher concentrations, impacting protein dynamics and stabilizing mechanisms.

Conclusions:

• Glycine's stabilizing effect is concentration-dependent, involving a complex interplay of self-interaction, water displacement, and buffer component interaction.
• At low concentrations, glycine preferentially interacts, reducing protein dynamics on short timescales, while higher concentrations lead to preferential exclusion and altered flexibility.
• The observed slowing of aggregation at high glycine concentrations correlates with reduced Fab ensemble flexibility, suggesting a macromolecular crowding effect that enhances stability.