Stability of protein formulations at sub-zero temperatures by isochoric cooling

Conference Dates

July 14-17, 2019


Optimization of protein formulations at sub-zero temperatures is required for many applications such as storage, transport and lyophilization, particularly for high-value proteins used for clinical and diagnostic applications. Using isochoric cooling (constant volume) is possible to reach sub-zero temperatures without freezing aqueous solutions. This accelerates protein damage at sub-zero temperatures as protein unfolds by cold denaturation and diffusion in the liquid phase still exists, therefore speeding up optimization of protein formulations (Rosa et al., 2013). The proof of concept for the use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase (PDIA1). Three typical osmolytes, sucrose, glycerol and L-arginine, increased very significantly the long-term stability of PDIA1 at -20°C with all of them tested under isochoric cooling within the short time frame of around 700 h. The extremely stable redox green fluorescent protein 2 (roGFP2) was selected to evaluate the applicability of isochoric cooling on the optimization of highly stable proteins. This derivative of GFP is 2.6 fold more stable than the already very stable GFP b-barrel. Nevertheless, it was possible to denature to some extent roGFP2 at -20°C under isochoric cooling and to assign a stabilizing effect to sucrose. The isochoric method was also applied to insulin, a widely used therapeutic protein. Insulin damage was evaluated through the magnitude of a signalling event elicited by insulin on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented this deactivation.

Rosa, M., C. Lopes, E. P. Melo, S. K. Singh, V. Geraldes, M. A. Rodrigues (2013) Measuring and modelling hemoglobin aggregation below freezing temperature. J. Phys. Chem. B 117, 8939-8946.

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