(T1530-08-49) Capillary Mediated Vitrification (CMV) is a User-Friendly Alternative to Lyophilization for the Production of Thermostable Bio-molecules

Purpose: Lyophilization is the most widely used method for desiccation of biological molecules across the biopharma industry. By freezing liquid samples and subjecting them to a deep vacuum, the biological molecules are dried through sublimation, theoretically resulting in thermostable products. However, in practice, the stability of lyophilized products varies based on several factors, including the degree of process optimization and the complexity of the biomolecule, often leaving them vulnerable to temperature-induced damage. While other techniques for drying biomolecules such as spray drying and thin film freezing exist, they are limited in application due to significant yield losses and product-specific process optimization requirements. Capillary Mediated Vitrification (CMV) is a novel method for biomolecule preservation that leverages the process of capillary evaporation to remove solvents from samples without freezing or boiling, producing amorphous thermostable products in less than 1 hour. In this work, we compared the thermostability of CMV stabilized thermolabile luciferase against catalog equivalent lyophilized products at elevated temperature.
Methods: The procured commercial lyophilized vials were stored in their original packaging (glass bottles with rubber stoppers in a heat sealed polyethylene secondary packaging containing a single desiccant) in a 37 °C incubator for up to 4 weeks. The procured liquid luciferase was mixed with an excipient buffer and loaded onto porous scaffolds and placed in a vitrification chamber to perform the CMV process. After a 30 minute cycle, the vitrified scaffolds were packaged in a zip sealable Mylar Foil pouch with a single desiccant in sets of 3 and stored in the same incubator as the lyophilized vials at 37 °C for up to 4 weeks. At the end of each week of thermal stress, one lyophilized vial and one set of 3 CMV samples were withdrawn from the incubator. The lyophilized samples were reconstituted as per manufacturer instructions. The CMV samples were reconstituted by depositing each scaffold into one conical tube and eluting with 1 ml of PBS. All the samples were diluted 1.3-fold and run in triplicate in a 96 well plate using an in-house luciferase assay. The luminescence values were obtained using a Synergy H1 microplate reader. In the absence of a matched reference for the lyophilized samples, week 1 data from either set was used as a reference standard to analyze the other 3 weeks of data for the respective sample type. The standard curves were analyzed by linear regression and the relative activity for the CMV and lyophilized sample dilutions was determined by direct interpolation. The relative activity reported was calculated as a percentage of the week 1 data. The entire process was repeated a second time and the data reported is an average of the two independent trials.

Results: Recombinant luciferase was chosen as the model system for this study due to its high sensitivity to thermal stress, and the availability of structurally identical liquid and lyophilized forms from the same vendor (Millipore Sigma). The data indicates that lyophilized luciferase degrades at a faster rate than CMV luciferase when stored at elevated temperature. After 4 weeks of storage, CMV luciferase was within 84% of the activity shown after 1 week of storage, while the lyophilized luciferase had dropped to 52% of its week 1 activity. The slope of the lyophilized luciferase activity over time was 2.5 times greater than that of the CMV luciferase samples. The quality of the data was impacted, to an extent, by the variability in activity between the different lyophilized vials from the same lot.

Conclusion: The results from this study show that Capillary Mediated Vitrification can be a viable alternative to lyophilization in production of thermostable biological materials. In addition to producing products that have a higher degree of stability, the shorter cycle time (30 minutes compared to 24-72 hours), ease of use (minimal process optimization), and low capital expense requirements could make CMV an attractive process to replace lyophilization in the biopharma industry.

Acknowledgements: Upkara, Inc. holds the license for the Capillary Mediated Vitrification patent with the purpose of commercializing the technology. All authors are employees of the company.

Key differences between lyophilization and capillary mediated vitrification

Relative recovery of luciferase enzyme activity at 37 C over time in CMV and lyophilized samples