Measurement of Secondary Structure Changes in Poly-L-lysine and Lysozyme during Acoustically Levitated Single Droplet Drying Experiments by In Situ Raman Spectroscopy

Drying processes such as spray drying, as commonly used in the pharmaceutical industry to convert protein-based drugs into their particulate form, can lead to an irreversible loss of protein activity caused by protein secondary structure changes. Due to the nature of these processes (high droplet number, short drying time), an in situ investigation of the structural changes occurring during a real drying process is hardly possible. Therefore, an approach for the in situ investigation of the expected secondary structural changes during single droplet protein drying in an acoustic levitator by time-resolved Raman spectroscopy was developed and is demonstrated in this paper. For that purpose, a self-developed NIR-Raman sensor generates and detects the Raman signal from the levitated solution droplet. A mathematical spectral reconstruction by multiple Voigt functions is used to quantify the relative secondary structure changes occurring during the drying process. With the developed setup, it was possible to detect and quantify the relative secondary structure changes occurring during single droplet drying experiments for the two chosen model substances: poly-L-lysine, a homopolypeptide widely used as a protein mimic, and lysozyme. Throughout drying, an increase in the beta-sheet structure and a decrease in the other two structural elements, alpha-helix, and random coil, could be identified. In addition, it was observed that the degree of structural changes increased with increasing temperature.

Automated summary

This paper presents an in situ investigation method for studying the secondary structure changes during protein drying using time-resolved Raman spectroscopy in an acoustic levitator. The developed setup successfully quantifies the relative secondary structure changes for two model substances, showing an increase in beta-sheet structure and a decrease in alpha-helix and random coil structures during drying.