A short introduction: the basic principle of freeze drying
Freeze drying is a complex operation where the solvent (usually water) is removed from the product by sublimation. Sublimation occurs when a frozen liquid goes directly to the gaseous state without passing through the liquid phase. This direct phase transfer “ice - water vapor” is a function of pressure and ice temperature which can be delineated by the phase diagram of water:
There are several advantages of the freeze drying process to stabilize delicate products (e.g. proteins, peptides, etc.): Properly freeze dried products (1) do not need refrigeration, (2) can be stored at ambient temperatures, (3) can be completely reconstituted with water for injection (WFI) within seconds and (4) are stable over a 2 year shelf life. The drawback, however, is that freeze drying is an expensive process (long process times, limited throughput) and requires specialized equipment.
The freeze drying process consists of three stages: freezing, primary (1°) drying and secondary (2°) drying. During the freezing step, the solution must be converted into a solid. The freezing procedure (e.g. freezing rate) has a great impact on the ice crystal size in the product which, in turn, may affect the drying rate during the 1° and 2° drying phase. In case of a formulation which contains excipients that tend to crystallize, it is important to assure that crystallization occurs quantitatively during the freezing procedure. Therefore, “annealing” steps are often included when crystalline materials are present. In addition, it is very important to perform the freezing step below a critical temperature. This temperature is the eutectic temperature (Teut) for crystalline materials or the glass transition temperature of the maximally freeze concentrated solute (Tg’) or collapse temperature (Tc) for amorphous products. If small “pockets” of unfrozen material remain in the product it may compromise the structural stability of the freeze dried product.
During 1° drying, conditions must be established in which ice can be removed from the frozen product via sublimation, resulting in a dry, structurally intact product. This requires very careful control of two parameters: shelf temperature (Ts) and chamber pressure (Pc). The rate of sublimation of ice from a frozen product depends (1) upon the difference in vapor pressure of the product compared to the vapor pressure of the ice at the condenser, (2) on the product resistance (Rp). In case of an existing pressure differential, molecules migrate from the higher pressure sample to a lower pressure area. Since vapor pressure is related to temperature, it is necessary that the product temperature is “warmer” than the condenser temperature. In turn, product resistance is a “formulation property” which is dependent on total solid content, type of excipient and even freezing rate.
As an important rule during 1° drying, the product temperature at the sublimation interface (Tp) must not exceed the “critical temperature” of the formulation. As mentioned above, the critical temperature is Teut for mainly crystalline and Tg’ or Tc for amorphous materials. Crystalline materials are easy to freeze dry since Teut is generally high (e.g. Teut of the very popular bulking agent mannitol is -3°C). However, note that crystalline materials do not stabilize a delicate structure like a protein or peptide. Here, amorphous sugars, polyols and other excipients must be used to stabilize the API during drying and storage. The drawback is that their collapse temperature is generally much lower than eutectic temperatures (e.g. sucrose: -32°C). When designing a freeze drying cycle, Tp should not increase significantly during primary drying. Therefore, a balance between the heat provided by the shelves and the heat removed by sublimation must be established. This balance is key to optimum drying.
After primary drying is complete, and all ice has sublimed, bound moisture is still present in the product. The product appears dry, but the residual moisture content may be as high as 7-8%. Continued drying at higher shelf temperature is necessary to reduce the residual moisture content to optimum values (for many formulations < 1%). This process is called isothermal desorption as the bound water is desorped from the product. 2° drying is normally continued at a product temperature higher than ambient but still below the glass transition (Tg) or melting temperature of the formulation. During this phase, chamber pressure and condenser temperature often remain the same.
During 1° and 2° drying additional equipment is necessary to measure and control important parameters such as pressure and temperature. Traditionally, thermocouples (TC) or resistance thermal detectors (RTD, PT-100) are used to determine the product temperature in a given vial. This invasive method also allows endpoint monitoring. For example, at the endpoint of primary drying the thermocouple reading increases up to a temperature close to Ts. This point indicates that the TC lost contact with the ice. Note that this does not guarantee that all ice is removed from the batch (even from this particular vial). More sensitive is the use of a Pirani and a capacitance gauge to determine the endpoint of primary (and secondary) drying. In general, the capacitance manometer is used to control the pressure in the chamber (absolute pressure measurement, independent of the type of gas). The reading of the Pirani gauge is dependent upon the type of gas which is present in the chamber: in the initial phase of primary drying the chamber is saturated with water molecules. Since the Pirani is calibrated for nitrogen, the reading is about 1.6 times higher that the actual pressure. However, close to the end of primary drying, the Pirani reading decreases due to a decrease of the total number of water molecules and an increasing nitrogen concentration (a controlled nitrogen purge is used to control the chamber pressure in many freeze dryers). The Pirani and the capacitance manometer will essentially read the same pressure at the end of 1° drying (see Figure). New process analytical technologies even allow the measurement of the temperature of the sublimation interface or instantaneous determination of the gas velocity and the mass flux.
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