Optimization of Freeze Drying Cycle Protocol Using Real Time Microscopy and Integrated Differential Thermal Analysis-Electrical Impedance

The objective of the research work was to use freeze dry microscopy (FDM) and differential thermal analysis (DTA)/electrical impedance (EI) for human serum albumin (HSA) freezing solution to determine the freeze drying cycle (FDC) to ensure acceptable cake integrity, reconstitution time and long-term stability. The HSA solutions with stabilizers were freeze dried at −40°C and 0.06hPa for 72h. HSA content was determined by reverse phase high performance liquid chromatography (RP-HPLC). Optimized composition was used to determine critical temperature by FDM and DTA/EI. Based on observations FDC was designed and the optimized composition was processed. Freeze dried products were visually inspected for morphology. Reconstitution time was determined by Thiermann method and residual moisture analysis by Karl Fischer titration. Freeze dried products were investigated for glass transition temperature using differential scanning calorimetry (DSC) and for amorphization using x-ray diffractometry (XRD), respectively. FDM and integrated DTA/EI studies provided specific information required for primary drying to process HSA compositions. Highest HSA recovery was observed in composition containing 1%w/v HSA, 25%w/v sucrose, 1.0%w/v sodium dihydrogen phosphate and 0.3%w/v polyvinylpyrrolidone K30. Reconstitution times of products were 15–20 sec. Residual moisture content was below 3%w/w retaining 99.56%w/v of HSA. XRD pattern indicated amorphous nature of material. DSC thermogram showed Tg well above storage temperature. Results revealed need of thorough understanding of freezing and annealing temperatures, shelf temperature, vacuum level and its duration during processing for freeze drying. Development and validation of efficient and economical FDC for HSA can be designed using FDM and DTA/EI.