Structural changes and inactivation of enzymes during drying and particle formation from levitated microdroplets
Pharmacy
Final Report Abstract
The main objective of the research project was to investigate structural changes of enzymes during drying and particle formation of single droplets in an acoustic levitator. Changes relevant to pharmaceutical industrial processes, including spray drying, can be seen in situ and under controlled circumstances in such a levitator. The activity of samples removed from the levitator during the drying process, which was determined ex situ, was correlated with the data gathered by optical measurement techniques. The experimental investigations were accompanied by numerical investigations in which a model was set up to describe the coupled time-dependent problem consisting of fluid mechanics, heat transport/diffusion, drying and particle formation from a single droplet of a protein solution. A commercial acoustic levitator was equipped with a temperature-controlled levitator chamber for conducting drying experiments under defined conditions and with measuring equipment (Raman, fluorescence, absorption spectroscopy, shadow recording) for investigating structural changes and the drying process. By removing the droplets or particles from the levitator, it was also possible to determine the inactivation by bioassay and the aggregation state as a function of time. Furthermore, the influence of excipients (carbohydrates, amino acids) on the activity of proteins was investigated. The investigations of the aggregation behavior during single drop drying showed that the critical point of drying ("crust formation") correlates with the formation of insoluble aggregates, presumably due to the rapid temperature increase at the critical point without cooling effect of the evaporating solvent. The activity determinations showed a correlation with the drying air temperature. The higher the drying air temperature, the shorter the retention of the initial activity and the higher the degree of subsequent inactivation, with the maximum rate of inactivation occurring shortly after the critical point. During investigations of the aggregation and inactivation behavior, comparative measurements in cuvette experiments showed that in addition to the purely thermally induced agglomeration and inactivation, further agglomerating and inactivating effects act on the protein structure. The addition of trehalose and arginine as excipients during drying showed a stabilizing effect. The spectroscopic investigations showed that the secondary structural changes occur in all structural parts and are faster and more pronounced with increasing drying temperature. The correlation of the structural changes with the results of the absorption spectroscopy also showed that the changes were mainly observed around the critical point of drying. The numerical approach showed that the levitation process has an influence on the drying and particle formation and thus slows down the aggregation of the dissolved molecules on its surface, which significantly delays the critical time of crust formation. In addition, a high degree of agreement between the simulation results and experimental measurement data was achieved through the appropriate choice of levitation parameters. In conclusion, it can be stated that all essential project goals were reached and the combination of experimental and numerical observation of the single-drop drying of protein solutions can be regarded as suitable for a better understanding of the aggregation and inactivation processes that take place as well as structural changes in the protein and to classify them in the temporal progress of the drying. The developed methodology is therefore very suitable for the investigation of such processes and offers an excellent basis for further work in the field of pharmaceutical technology.
Publications
- “Optical Investigation of the Drying Behaviour of Protein Microdroplets.”, Sensors Day 2017 Cambridge, Großbritannien, 20. Oktober 2017
Perlitz, J.F.A., Koch, H., Prihoda, F., Lee, G., Will, S.
- "Correlation of the kinetics of aggregation and inactivation of L-glutamate dehydrogenase during drying and particle formation of a levitated microdroplet", Drying Technology 37.2 (2018)
Keil, N., Will, S., Lee. G.
(See online at https://doi.org/10.1080/07373937.2018.1445097) - "Numerical study of droplet evaporation of two-phase flows with heat and mass transfer." Physics of Fluids 30 (2018)
Bänsch, E., Götz, M.
(See online at https://doi.org/10.1063/1.5017936) - “In situ investigation of the protein particle formation process during drying in an acoustic levitator.” PARTEC 2019 International Congress on Particle Technology Nürnberg, Deutschland, 09.-11. April 2019
Perlitz, J.F.A., Koch, H., Prihoda, F., Lee, G., Will, S.
- “Temperature-induced Enzyme Denaturation Characterized by Light Scattering, Intrinsic Fluorescence, SEC and Activity Measurements.”, 3rd European Conference on Pharmaceutics, Bologna, Italien, 25.-27. März 2019
Prihoda, F., Perlitz, J.F.A., Will, S., and Lee, G.
- “Untersuchung der Proteinein-zeltropfentrocknung in einem akustischen Levitator mit Hilfe laseroptischer Messmethoden”, 10. ProcessNet-Jahrestagung und 34. DECHEMA-Jahrestagung der Biotechnologien 2020 Aachen, Deutschland, 21.-24. September 2020
Perlitz, J.F.A., Prihoda, F., Bänsch, E., Lee, G., Will, S.
(See online at https://doi.org/10.1002/cite.202055027) - "Measurement of Water Mole Fraction from Acoustically Levitated Pure Water and Protein Water Solution Droplets via Tunable Diode Laser Absorption Spectroscopy (TDLAS) at 1.37 µm", Applied Sciences 11.11 (2021)
Perlitz, J.F.A., Broß, H., Will, S.
(See online at https://doi.org/10.3390/app11115036) - "Measurement of Secondary Structure Changes in Poly-L-lysine and Lysozyme during Acoustically Levitated Single Droplet Drying Experiments by In Situ Raman Spectroscopy." Sensors 22.3 (2022)
Perlitz, J.F.A., Gentner, L., Braeuer, P.A.B., Will, S.
(See online at https://doi.org/10.3390/s22031111) - "Numerical study of single droplet drying in an acoustic levitator before the critical point of time." Chemical Engineering Science 248 (2022)
Doß, M., Bänsch, E.
(See online at https://doi.org/10.1016/j.ces.2021.117149) - “Numerical study of single droplet drying in an acoustic levitator,” 14th Annual Meeting, InterPore 2022, Abu Dhabi, United Arab Emirates, 30. Mai - 02. Juni 2022
Doß, M., Ray, N., Bänsch, E.
