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Protein stabilization in spray drying and solid-state storage by using a 'molecular lock' - exploiting bacterial
Wiktoria Brytan1, Tewfik Soulimane1, Luis Padrela1
1SSPC - The Science Foundation Ireland Research Centre for Pharmaceuticals, Department of Chemical Sciences, Bernal Institute, University of Limerick Limerick Ireland Luis.Padrela@ul.ie.
RSC Chemical Biology
|January 6, 2025
Summary
Large enzyme stabilization for spray drying was achieved using a C-terminal extension, acting as a molecular lock. This excipient-free method enhances protein stability and activity during formulation and storage.
Area of Science:
- Biochemistry
- Protein Engineering
- Formulation Science
Background:
- Spray drying of large biomolecules like therapeutic enzymes faces stability challenges due to stresses during solid-state formation.
- Current formulations often rely on excipients, but alternative stabilization strategies are needed.
- Thermophilic proteins sometimes utilize C-terminal extensions to enhance molecular rigidity and stability.
Purpose of the Study:
- To investigate the stabilizing effect of a unique C-terminal extension on a large enzyme, aldehyde dehydrogenase from *Thermus thermophilus* (ALDH*Tt*), during spray drying.
- To explore an excipient-free approach for stabilizing large biotherapeutics against drying stresses.
- To elucidate the mechanism by which the C-terminal extension protects protein structure and function.
Main Methods:
- Engineering a thermostable aldehyde dehydrogenase (ALDH*Tt*) with a C-terminal extension.
- Comparing the stability and activity of the engineered ALDH*Tt* with a mutant lacking the extension after spray drying.
- Utilizing a design of experiments approach for optimizing the spray drying process of the excipient-free enzyme.
Main Results:
- The C-terminal extension acted as a 'molecular lock,' preserving the oligomeric state of the ALDH tetramer during spray drying.
- Removal of the extension led to aggregation and dissociation, reducing enzyme stability.
- The ALDH*Tt* with the C-terminal extension retained approximately 24% more activity post-spray drying and up to 16% more activity during storage compared to the mutant.
- An excipient-free spray drying process for ALDH was successfully developed.
Conclusions:
- A C-terminal extension can effectively stabilize large enzymes like ALDH*Tt* against spray drying stresses by maintaining oligomeric integrity.
- This strategy offers a promising excipient-free approach for formulating large biotherapeutics as stable dry powders.
- The findings provide insights into protecting oligomeric proteins during solid-state formation and broaden the application of spray-dried enzymes in biocatalysis.
