(T1130-11-71) Predicting the Stability of Lyophilised Protein Mixtures with Terahertz Spectroscopy

Purpose: The use of protein drugs is increasing as they can provide specific therapeutic effects that small-molecule drugs cannot. The challenge with protein drugs is to design the protein drugs to have long-term storage stability and shelf life. One method to increase the stability and shelf life is by removing all residual water through lyophilisation. It is well studied how the role of water affects protein dynamics that can lead to misfolding propensity and protein aggregation. However, the protein dynamics in lyophilised protein mixtures have not been studied equally thoroughly.

Methods: The use of terahertz time-domain spectroscopy (THz-TDS) allows the study of protein dynamics which can be used to predict the room temperature stability within a day. THz-TDS is a technique which probes molecular mobility, and mobility is related to the stability of the formulation. This study used THz-TDS to investigate the stability of lyophilised protein mixtures with different protein sizes (bacitracin, lysozyme, HSA) and different protein concentrations. The samples were measured with TeraPulse 4000 spectrometer over temperatures between 80 to 420 K.

Results: Samples with low protein concentrations (up to 33%) showed that the molecular mobility increases with temperature. As the protein concentration becomes more dominant (more than 66%), the molecular mobility no longer increases with temperature around 300 K, possibly due to protein jamming or protein confinement. The largest protein (HSA) showed a more pronounced effect of protein confinement as there was a decrease in molecular mobility after reaching a local maximum. Proteins of smaller size (bacitracin, lysozyme) become trapped in a confined state around 300 K but can exit the confinement with more energy supplied by increasing the temperature.

Conclusion: THz-TDS allows the investigation of the molecular mobility of different systems over a range of temperatures. The change in molecular structure can be inferred from the changes in molecular mobility. Larger protein size shows a higher degree of jamming. The addition of sucrose helps stabilise the mixture. Samples with lower concentrations of protein did not exhibit protein jamming. Increasing protein concentration increases the degree of protein jamming and suggests that the lyophilised mixture is less stable at higher protein concentrations.

References: T. Shmool et al., “Observation of high-temperature macromolecular confinement in lyophilised protein formulations using terahertz spectroscopy,” Int. J. of Pharmaceutics: X., vol. 1, 2019.

Mobility of bacitracin (smallest protein), lysozyme and HSA (largest protein) at 1 THz.

Mobility of different concentrations of HSA in sucrose mixtures at 1 THz.