Observation of High-Temperature Macromolecular Confinement in Lyophilised Protein Formulations Using Terahertz Spectroscopy

09 May 2019, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Characterising the structural dynamics of proteins and the effects of excipients are critical for optimising the design of formulations. In this work we investigated four lyophilised formulations containing bovine serum albumin (BSA) and three formulations containing a monoclonal antibody (mAb, here mAb1), and explore the role of the excipients polysorbate 80, sucrose, trehalose, and arginine on stabilising proteins. By performing temperature variable terahertz time-domain spectroscopy (THz-TDS) experiments it is possible to study the vibrational dynamics of these formulations. The THz-TDS measurements reveal two distinct glass transition processes in all tested formulations. The lower temperature transition, T, is associated with the onset of local motion due to the secondary relaxation whilst the higher temperature transition, T, marks the onset of the α relaxation. For some of the formulations, the globular BSA as well as mAb1, the absorption at terahertz frequencies does not increase further at temperatures above T. Such behaviour is in contrast to our previous observations for small organic molecules as well as linear polymers where absorption is always observed to steadily increase with temperature due to the stronger absorption of terahertz radiation by more mobile dipoles. The absence of such further increase in absorption with higher temperatures therefore suggests a localised confinement of the protein/excipient matrix at high temperatures that hinders any further increase in mobility. Additionally, we used Fourier-transform infrared spectroscopy (FTIR), circular dichroism (CD), and solid-state nuclear magnetic resonance (ssNMR) experimental data to further investigate the structural changes associated with our terahertz spectroscopy measurements. None of these techniques were able to resolve the subtle changes associated with vibrational confinement observed by terahertz spectroscopy. No evidence was found for any conformational changes in the protein structure upon reconstitution. We found that subtle changes in excipient composition had an effect on the transition temperatures Tand T as well as the vibrational confinement in the solid state. Further work is required to establish the potential significance of the vibrational confinement in the solid state on formulation stability and chemical degradation as well as what role the excipients play in achieving such confinement.


Terahertz spectroscopy
Structural Relaxation Dynamics
protein stabilities
lyophilization conditions
Glass Transition

Supplementary materials

Supplementary info


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.