Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying
Abstract
:1. Introduction
2. Materials and Methods
2.1. Structure
2.2. Coarse-Grained Simulations
2.3. Freeze-Drying Simulations
2.4. Materials
2.5. Sample Dialysis and Preparation
2.6. Freeze-Thaw Cycles and Lyophilization
2.7. Differential Scanning Fluorimetry (nanoDSF)
2.8. Circular Dichroism (CD) Spectroscopy
2.9. High-Performance Size Exclusion Chromatography (HP-SEC)
3. Results and Discussion
3.1. Protein–Protein Interaction Study of Reteplase
3.2. Molecular Dynamics Simulations of the Freeze-Drying Process
3.3. Local Structural Changes and Effects of Excipients
3.4. Experimental Validation
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Excipient | Concentration | Number of Excipient Molecules | Number of Ions |
---|---|---|---|
No Excipient | No Excipient | No Excipient | 1 Na+ |
ARG | 10% w/w | 284 | 283 Cl− |
ARG | 100 mM | 51 | 50 Cl− |
ARG | 10 mM | 5 | 4 Cl− |
TXA | 10% w/w | 314 | 1 Na+ |
TXA | 100 mM | 51 | 1 Na+ |
TXA | 10 mM | 5 | 1 Na+ |
SUC | 10% w/w | 144 | 1 Na+ |
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Ko, S.K.; Björkengren, G.; Berner, C.; Winter, G.; Harris, P.; Peters, G.H.J. Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying. Pharmaceutics 2023, 15, 1854. https://doi.org/10.3390/pharmaceutics15071854
Ko SK, Björkengren G, Berner C, Winter G, Harris P, Peters GHJ. Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying. Pharmaceutics. 2023; 15(7):1854. https://doi.org/10.3390/pharmaceutics15071854
Chicago/Turabian StyleKo, Suk Kyu, Gabriella Björkengren, Carolin Berner, Gerhard Winter, Pernille Harris, and Günther H. J. Peters. 2023. "Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying" Pharmaceutics 15, no. 7: 1854. https://doi.org/10.3390/pharmaceutics15071854
APA StyleKo, S. K., Björkengren, G., Berner, C., Winter, G., Harris, P., & Peters, G. H. J. (2023). Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying. Pharmaceutics, 15(7), 1854. https://doi.org/10.3390/pharmaceutics15071854