Solid state chemistry of proteins IV. What is the meaning of thermal denaturation in freeze dried proteins?
This research addresses the thermodynamic significance of the denaturation endotherm observed during differential scanning calorimetry (DSC) scans of proteins in dry formulations, such as freeze dried solids. Human growth hormone formulations are the chosen representative examples. We employ observations of denaturation temperature, glass transition temperature, and the differences between estimated molecular mobilities to argue that unfolding is under partial thermodynamic control. Further, unfolding during a DSC scan is simulated using a three state kinetic model, which is a two state unfolding model followed by aggregation. Kramers-type rate constants are used, where the preexponential term is dominated by viscous forces. Simulation results are in qualitative agreement with experiment, and clearly show that while the denaturation endotherm is impacted by irreversibility, caused by nonzero scan rate and aggregation, the position of the endotherm peak is changed only slightly. Thus, the denaturation peak is a good approximation for the thermodynamic denaturation temperature. Using data for denaturation temperature, heat of denaturation, and heat capacity of denaturation, free energy versus temperature curves were calculated. We find that even formulations with added saccharides are thermodynamically unstable near ambient temperature; significant denaturation in the solid state is prevented by low mobility.
Pikal
Michael J
MJ
School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269-3092, USA. michael.pikal@uconn.edu
Rigsbee
Daniel
D
Akers
Michael J
MJ
D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical; D005612:Freeze Drying; D019382:Human Growth Hormone / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D044367:Phase Transition; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D013816:Thermodynamics; D014783:Viscosity
D011506:Proteins; D019382:Human Growth Hormone