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

Phillipp A B Braeuer,Lukas Gentner,Stefan Will,Julian F A Perlitz

doi:10.3390/s22031111

Phillipp A B Braeuer, Lukas Gentner + Show 2 more

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https://doi.org/10.3390/s22031111

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Abstract

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 β-sheet structure and a decrease in the other two structural elements, α-helix, and random coil, could be identified. In addition, it was observed that the degree of structural changes increased with increasing temperature.

Highlights

Protein-based drugs are often converted into their particulate form by special drying processes such as spray drying in order to improve the stability, shelf life, or application properties [1]
The third area marked in red between about 800 and 1800 cm−1 marks the so-called fingerprint region, which is of particular interest with regard to the structural changes of proteins
A self-assembled Raman sensor was attached to an optically accessible acoustic levitator, which allows for single droplets to be examined under defined experimental conditions

Summary

Introduction

Protein-based drugs are often converted into their particulate form by special drying processes such as spray drying in order to improve the stability, shelf life, or application properties [1]. The large number of droplets and the short timescale of these drying and particle formation processes—in the range of milliseconds to seconds—imply major challenges for the time-resolved in situ investigation of the structural changes of proteins [4] To overcome these challenges, single droplet evaporation and drying experiments have been established in recent years. The evaporation behavior and the particle formation process of a single droplet have been investigated by combining acoustic levitation with imaging techniques such as shadowgraphy [18,19] or with absorption techniques such as tunable diode laser absorption spectroscopy (TDLAS) [20]. The different secondary structural elements of the proteins, such as α-helix, β-sheet, random coil, and turns, can be characterized by appropriate evaluation methods [30]

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