Protein stability and critical stabilizers in frozen solutions

Freezing is a common unit operation during the processing and storage of therapeutic proteins. Upon freezing, protein molecules and other solutes are excluded from the ice crystals, resulting in the formation of a freeze concentrated solution (FCS). Protein destabilization can be prevented due to the low temperature and the formation of a viscous FCS. However, the changes in temperature, pH, salt concentration, and the formation of ice-solution interfaces can induce protein destabilization. The current paper reviews the stresses associated with freezing and thawing and discusses the stabilization strategies. At subambient conditions, protein unfolding is thermodynamically favored when the solution is cooled below the cold denaturation temperature. The formation of a viscous FCS, specifically at a temperature below the glass transition temperature (T_g'), immobilizes and stabilizes the proteins. Protein can adsorb at the ice/solution interface, followed by unfolding and aggregation. Therefore, freezing and thawing rates need to be carefully controlled to minimize the ice surface area (due to the formation of small ice crystals) and to avoid ice recrystallization. Besides, stabilizers, including sugars, surfactants, and amino acids, are added as stabilizers (cryoprotectants) to prevent protein destabilization upon freezing and frozen storage. In situ spectroscopic (infrared, Raman, and solid-state NMR) and scattering (X-ray and neutron) techniques provide useful insights into protein stability and phase transitions of excipients in frozen solutions. Finally, we discuss the importance of the freezing process and excipient selection in the successful lyophilization of protein formulations. The review paper comprehensively discusses the challenges during the cryopreservation of therapeutic proteins and provides valuable insights into the critical stabilizers in frozen protein solutions.