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 of different excipients to study the stabilizing mechanisms and to identify the role of excipients during freeze drying. To simulate the freeze-drying process, we divided the process into five distinct steps: (i) protein-excipi...

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Bibliographic Details
Published in:	Pharmaceutics Vol. 15; no. 7; p. 1854
Main Authors:	Ko, Suk Kyu, Björkengren, Gabriella, Berner, Carolin, Winter, Gerhard, Harris, Pernille, Peters, Günther H J
Format:	Journal Article
Language:	English
Published:	Switzerland MDPI AG 30-06-2023 MDPI
Subjects:	coarse-grained simulations formulation development freeze drying Investigations Molecular dynamics protein stability Proteins Reteplase Simulation Simulation methods Spectrum analysis Tranexamic acid Germany arginine freeze drying coarse-grained simulations Reteplase molecular dynamics formulation development protein stability
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Summary:	We performed molecular dynamics simulations of Reteplase in the presence of different excipients to study the stabilizing mechanisms and to identify the role of excipients during freeze drying. To simulate the freeze-drying process, we divided the process into five distinct steps: (i) protein-excipient formulations at room temperature, (ii) the ice-growth process, (iii)-(iv) the partially solvated and fully dried formulations, and (v) the reconstitution. Furthermore, coarse-grained (CG) simulations were employed to explore the protein-aggregation process in the presence of arginine. By using a coarse-grained representation, we could observe the collective behavior and interactions between protein molecules during the aggregation process. The CG simulations revealed that the presence of arginine prevented intermolecular interactions of the catalytic domain of Reteplase, thus reducing the aggregation propensity. This suggests that arginine played a stabilizing role by interacting with protein-specific regions. From the freeze-drying simulations, we could identify several protein-specific events: (i) collapse of the domain structure, (ii) recovery of the drying-induced damages during reconstitution, and (iii) stabilization of the local aggregation-prone region via direct interactions with excipients. Complementary to the simulations, we employed nanoDSF, size-exclusion chromatography, and CD spectroscopy to investigate the effect of the freeze-drying process on the protein structure and stability.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1999-4923 1999-4923
DOI:	10.3390/pharmaceutics15071854