Investigation of the reversibility of freeze/thaw stress-induced protein instability using heat cycling as a function of different cryoprotectants

Abstract

Formulation conditions have a significant influence on the degree of freeze/thaw (FT) stress-induced protein instabilities. Adding cryoprotectants might stabilize the induced FT stress instabilities. However, a simple preservation of protein stability might be insufficient and further methods are necessary. This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein. Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG200 and Tween20. In terms of heat-induced reversibility of aggregates, glycerol showed the best performance followed by sucrose, NaCl and Tween20 systems. The analysis was performed using a novel approach to visualize complex interplays by a clustering and data reduction scheme. In addition, solubility and structural integrity were measured and confirmed the obtained results. Graphic abstract

HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions

“…The formulation design process has improved in recent years through the availability of Design of Experiment (DoE) approaches [6,7], and high-throughput screens that measure the impact of formulations on thermal-unfolding transition mid-points (Tm) and aggregation onset temperatures (Tagg) [8][9][10]. Formulations are also assessed through intensive degradation studies with aggregation kinetics accelerated by elevated temperatures, shear flow, agitation, or through repeated freezing and thawing [11][12][13].…”

Enhancing the Productivity and Stability of Superoxide Dismutase from Saccharomyces cerevisiae TBRC657 and Its Application as a Free Radical Scavenger

“…Thus, water molecules will be removed from the target protein, forming a more compact structure. Some additives can form a hydrophobic interaction with the target enzyme, leading to reduced movement and also reduced water activity of the enzyme, which causes the formulated enzymes to be more stable in the environment [41][42][43][44]. Thus, these top 3 formulated SODs were selected and further analyzed for their SOD activities and storage stability at 4 • C for 6 months.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…Dimethyl sulfoxide (DMSO) acts as a stabilizer because it is preferentially excluded from the protein surface [22,40,41]. Addition of DMSO but also of glycerol or ethylene glycol serve as cryoprotectants preventing protein solutions from freezing at −20 • C and allowing multiple use of a unique sample without freeze-thaw cycles [38,42]. Adding polymers in solution such as poly(ethylene glycol) (PEG), alginate, or chitosan maintains a hydration shell around the protein according to the exclusion mechanism [38,[43][44][45].…”