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Thermal Stability of Proteins

January 26, 2021

Protein formulations exist in a thermodynamic metastable state. One consequence of protein formulation metastability is their sensitivity to temperature changes (Frokjaer and Otzen, 2005). Proteins are incredibly heat sensitive. At temperatures not much higher than body temperature (greater than 40°C) proteins can denature. Protein denaturation is the complete loss of higher order (quaternary, tertiary, and secondary) structure in the protein leaving only the primary structure, the amino acid sequence, intact. This fragility is not limited to high temperatures, but also low temperatures (Bhatnagar et al, 2007). Excipients like surfactants, sugars, and salts can be incorporated into formulations to improve protein stability under thermal stress.

surfacant, salt, sugar

Other excipients can be added to improve their stability and functionality at moderate temperatures. Free amino acids like histidine and arginine are commonly added to protein formulations to reduce viscosity by screening protein-protein interactions (Dear et al, 2019). However, what is not known is how these excipients perform under high temperature. Viscosity measurements are a useful tool that can guide formulation development. Investigating viscosity changes of protein formulations under stress can provide insight on the underlying structure within the fluid and how different fluid components interact. Inducing thermal stresses can reveal that excipients known to improve formulation performance under ideal conditions can behave inversely when exposed to a high stress environment.thermal stresses

To observe how excipients perform under high temperature, the team at RheoSense examined viscosity changes of two model proteins, bovine serum albumin (BSA) and bovine gamma globulin (BgG), as they are heated and cooled. Viscosity data of six protein formulations was collected on the VROC® initium. We observed that proteins in formulations enriched in salt are more resistant to thermal denaturation than proteins in formulations containing amino acid excipients, demonstrating that excipients have strengths and weaknesses depending on their environment. Understanding these differences between excipients and the knowing the environment your fluids will experience, will help guide and improve formulation development.

To learn more about the parameters of the test performed and see the full data sets collected, download our application note “Thermal Stability of Proteins”.

Download CTA Thermal Stability-1

Written by: Zachary Imam, PhD, RheoSense Research Scientist

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