Trehalose and mannitol based lyoprotection of Taq DNA polymerase for cold-chain-free long-term storage

Polymerase chain reactions (PCR) are most reliable and precise means for nucleic acid analysis of biological samples. A cold-chain system with temperature at around -20 °C is generally necessary for storage and transportation of PCR-related reagents. In order to facilitate ambient temperature storage and transportation, this study prepared Taq DNA polymerase and 5 × HS-Taq Mix (as low as 0.5 U/sample) into stable solid formulations using an optimized freeze-drying process and lyoprotectant formulations comprising trehalose dihydrate (3.3∼5%, w/v) and mannitol (10%, w/v). The lyocakes were characterized by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). In the optimized freeze-drying process, trehalose dihydrate mainly formed an amorphous structure and acted as both cryoprotectant and lyoprotectant, while mannitol crystallized to serve as a bulking agent. The enzyme activities of Taq and 5 × HS-Taq Mix samples were measured via real-time quantitative PCR (qPCR). Long-term storage stability test demonstrated that freeze-dried samples with optimized formulations showed no remarkable reduction in amplification efficiencies for target sequence compared to freshly prepared corresponding samples after being stored at 37 °C and 55% relative humidity (RH) for 0, 1, 4, 8 and 12 weeks.