Thermal Gradient Mid- and Far-Infrared Spectroscopy as Tools for Characterization of Protein Carbohydrate Lyophilizates

Abstract

Protein drugs play an important role in modern day medicine. Typically, these proteins are formulated as liquids requiring cold chain processing. To circumvent the cold chain and achieve better storage stability, these proteins can be dried in the presence of carbohydrates. We demonstrate that thermal gradient mid- and far-infrared spectroscopy (FTIR and THz-TDS, respectively) can provide useful information about solid-state protein carbohydrate formulations regarding mobility and intermolecular interactions. A model protein (BSA) was lyophilized in the presence of three carbohydrates with different size and protein stabilizing capacity. A gradual increase in mobility was observed with increasing temperature in formulations containing protein and/or larger carbohydrates (oligo- or polysaccharides), lacking a clear onset of fast mobility as was observed for smaller molecules. Furthermore, both techniques are able to identify the glass transition temperatures (Tg) of the samples. FTIR provides additional information as it can independently monitor changes in protein and carbohydrate bands at the Tg. Lastly, THz-TDS confirms previous findings that protein–carbohydrate interactions decrease with increasing molecular weight of the carbohydrate, which results in decreased protein stabilization.

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

“…and Zeitler, 2019). We also showed that this appears to be universally so for all organic molecular materials that we have investigated to date including polyalcohols (Sibik et al, 2013(Sibik et al, , 2014Ruggiero et al, 2017), pharmaceutical drugs, Sibik and Zeitler, 2016;Kissi et al, 2018) proteins (Sibik and Zeitler, 2016;Mensink et al, 2017b) or polymers (Shmool and Zeitler, 2019). Ngai et al recently highlighted the same phenomenon for a further set of protein samples by carefully re-analysing data previously published in the literature using a range of techniques.…”

Observation of high-temperature macromolecular confinement in lyophilised protein formulations using terahertz spectroscopy

“…We have previously shown in our own work that local dipole mobility is associated with the onset of Tg,β, regardless of whether these motions originate from side groups or larger domains purely depending on the height of the respective energy barriers (Sibik and Zeitler, 2016, Shmool and Zeitler, 2019). We also showed that this appears to be universal for all organic molecular materials that we have investigated to date including polyalcohols (Sibik et al, 2013, Sibik et al, 2014, Sibik and Zeitler, 2015, Ruggiero et al, 2017), pharmaceutical drugs (Sibik et al, 2015, Sibik and Zeitler, 2016, Kissi et al, 2018), proteins (Sibik and Zeitler, 2016, Mensink et al, 2017) and polymers (Shmool and Zeitler, 2019). Ngai et al recently highlighted the same phenomenon for a further set of protein samples by carefully re-analysing data previously published in the literature using a range of techniques (Ngai et al, 2019).…”

“…2. In line with previous experiments for a range of organic molecular materials, three distinct temperature regions can be identified for each material and Tg,β was defined as the intersection point of the two best-fit linear fits at low temperatures, and Tg,α was defined as the intersection point of the two best-fit linear lines at high temperatures (Sibik et al, 2014, Mensink et al, 2017, Shmool and Zeitler, 2019). It is worth noting that the values of Tg,α, as determined from the THz-TDS experiments, are in good agreement with our own calorimetric measurements, Tg,DSC, as well as the values reported in the literature for these materials (Table 2, see ESI) (Duddu and Dal Monte, 1997, Srirangsan et al, 2010, Wang et al, 2009).…”

“…The terahertz data further confirmed the information from the ssNMR measurements regarding the miscibility of sugar and protein phases as discussed above. There was no indication from the terahertz data that any phase separation had taken place (Mensink et al, 2017).…”

Freezing of Aqueous Solutions and Chemical Stability of Amorphous Pharmaceuticals: Water Clusters Hypothesis

“…Wsat (iii) What are the relationships between water clustering patterns and proton transfer in pharmaceutically relevant amorphous solids, for example, proteins embedded into sugar glasses? (iv) Are there any fundamental chemical mechanisms which could support the hypothesis on relationships between a particular relaxation mode (e.g., as measured by the neutron scattering 7 or THz spectroscopy 125 ) and a particular reaction step, for example, proton transfer in water-mediated deprotonation of amide in deamidation reaction?…”