||Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase Al. Three osmolytes, sucrose, glycerol, and L-arginine, significantly increased the stability of protein disulfide isomerase Al at -20 degrees C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP beta-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20 degrees C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20 degrees C under isochoric cooling lost 22\% of its function after 15 days and 0.6M sucrose prevented insulin deactivation. (C) 2020 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.