Lyophilization is a process by which water is removed from biological material for long term stability. More specifically, Lyophilization occurs when a biological material is frozen and subsequently the pressure around the material is lowered below the triple point of the substance, so the frozen water in the material sublimes. This process causes a shift in phases directly from the solid phase to the gas phase as seen in a typical phase diagram.
THE LYOPHILIZATION PROCESS
In the initial step of the lyophilization process the biological material is cooled down below its triple point (refer to above graph). As the temperature and pressure are reduced below the triple point, sublimation occurs. The triple point is defined as the lowest temperature at which the solid and liquid phases of the material can coexist. By running the freeze-dry process below the triple point the sublimation phase transition occurs instead of the transition into liquid phase in the subsequent steps.
For inert material the product is frozen slowly or cycled up and down in temperature for annealing. The final desired state is for large crystals to form.
In the case of biological substances, the strategy is different since the formation of large crystals results in degradation of the cell wall for cellular organisms, or loss of enzymatic activity, aggregation, or denaturation in enzymes and proteins. In the case of living organisms flash freezing is the method used to lower the in vivo product to below its eutectic point, thus avoiding the formation of ice crystals in the organism. Usually, the freezing temperatures are between 55 C and 80 C.
The freezing phase is the most critical part of the freeze-drying process since the stability of the sample is essential for optimal recovery and performance of the freeze dried product.
The terms freeze-drying and lyophilization are used interchangeably in many industries.
Lyophilization also has a tendency to result in the loss of activity or aggregation of proteins, therefore stabilizers are added to provide a micro infrastructure for stabilization of the biomolecule.
Furthermore, the proper control of pressure and temperature conditions will increase the effectiveness of the lyophilized biomolecule.
Protectants can alleviate the stresses by several mechanisms including the formation of an amorphous glassy state, replacing water, hydrogen bonding to proteins, physical dilution and separation of protein molecules, etc. Common protectants/stabilizers include sugars, polyols, polymers, and surfactants, as well as some proteins and amino acids. We use trehalose and mannitol (normally 8% w/v) as protectants for lyophilization.
In order to freeze-dry biomolecules effectively stabilizers can also be included with the biomolecule of interest in the Lyophilization process. These stabilizers help in the formation of a glassy state that supports the transition of the biomolecule in phase transition. LSAT uses many different stabilizers to support the integrity of the biomolecules. These stabilizers include a mixture of proteins, amino acid, gels substances, polyols, polymers, surfactants, salts, and sugars. Depending on the application we advise on the most effective method to freeze-dry your product.
Trehalose is a non-reducing disaccharide, well-known for its ability to stabilize biomolecules and microorganisms during prolonged periods of desiccation.
Mannitol is also commonly used as a stabilizer as well as a bulking agent in the process of lyophilization. It was reported to reduce aggregation for some proteins during lyophilization.
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