Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying

We performed molecular dynamics simulations of Reteplase in the presence different excipients to study stabilizing mechanisms and identify role during freeze drying. To simulate freeze-drying process, we divided process into five distinct steps: (i) protein–excipient formulations at room temperature, (ii) ice-growth (iii)–(iv) partially solvated fully dried formulations, (v) reconstitution. Furthermore, coarse-grained (CG) were employed explore protein-aggregation arginine. By using a representation, could observe collective behavior interactions between protein molecules aggregation process. The CG revealed that arginine prevented intermolecular catalytic domain Reteplase, thus reducing propensity. This suggests played by interacting with protein-specific regions. From simulations, several events: collapse structure, recovery drying-induced damages reconstitution, (iii) stabilization local aggregation-prone region via direct excipients. Complementary nanoDSF, size-exclusion chromatography, CD spectroscopy investigate effect on structure stability.