|
|
Freeze-Drying – an Art or a Science? Freeze-drying (lyophilisation) is a widely used method for dehydrating a vast range of materials, including foodstuffs, pharmaceuticals, biotechnology products, vaccines, diagnostics and biological materials. It may be carried out on a range of scales, from bench top through pilot-scale to a full-scale manufacturing process and offers a number of advantages over conventional drying and many other processing methods. Despite its wide use, however, it is still apparent that many regard freeze-drying as somewhat of an art. This is perhaps not surprising, given the lack of available texts devoted to the process itself, with many published articles tending to describe specific applications of the process. Specialised training courses are now arguably the most effective means of learning about the various aspects of freeze-drying technology. This article seeks to provide a brief overview of the process. How does freeze-drying work? The process works by initially freezing a material, then removing the solvent (typically water, as we shall consider here) under vacuum. The main drying step (traditionally referred to as ‘primary drying’) involves the removal of bulk water from the frozen material, chiefly by sublimation (i.e. a direct phase change from ice to vapour without passing through the liquid state), but also typically through some evaporation and desorption. Additional (further) drying (traditionally termed ‘secondary drying’) involves the removal of what is termed ‘unfrozen’ water, which may be chemically associated with the bulk-dried material, by desorption. Achieving the frozen state Different materials freeze in different ways. This is true even for subtly different solutions or suspensions, which may respond in a dissimilar manner to each other even when subjected to the same thermal treatment. Few materials will crystallise in such a predictable manner as, for example, sodium chloride, which adopts what is termed ‘eutectic’ behaviour (i.e. it freezes at the same temperature and the same concentration irrespective of the initial concentration). Indeed, most solutions containing pharmaceuticals or biotech products will solidify to give an amorphous glass containing regions of unfrozen water - this is typically much more difficult to remove than the water in a frozen eutectic system, since it requires not only sublimation, but also evaporation and desorption. As well as considering the solutes, we also need to consider the ice crystal structure within the frozen material, since this will be affected by the cooling rate applied to the product and subsequent thermal treatment steps (such as annealing). One of the ultimate aims of the freezing programme in a lyophilisation cycle is to produce large, contiguous ice crystals in the product that are conducive to drying, rather than small, isolated crystals which are subsequently more difficult to remove from the frozen matrix. Main Drying As stated above, the main drying step of the process consists largely of sublimation. This is made possible by adopting appropriate temperature and pressure conditions in the drying chamber of the freeze-dryer. In this case, the liquid phase may be avoided by maintaining a sufficiently low temperature and low pressure (high vacuum) in the freeze-dryer, while providing enough energy (latent heat) to compensate for the phase change. The subliming vapour is then ‘trapped’ on the product condenser (sometimes referred to as the ‘vapour trap’), which is held at a lower temperature than the drying material. The rate of ice vapour removal from the product (and the subsequent deposition of this vapour onto the product condenser as solid ice) relies on the kinetics of heat and mass transfer, which change throughout the process as the product dries. In the early stages of sublimation, there is little impedance to the flow of vapour from the frozen material, while later in the process, the layer of the already-dry material creates a significant impedance to vapour flow. It has been put forward that the geometry of the freeze-dryer itself (i.e. non-linear path between the product and the vapour trap created by baffles, bends in pipework, etc.) is accountable for no more than 30% of the impedances within the system, yet the layer of dried product itself accounts for over 60%. Therefore, it is necessary to regulate heat input to the product during the various stages of the process. While in the past, freeze-dryers have not always allowed control of both temperature and pressure in the drying chamber, most modern production machines are equipped with both temperature and pressure control functions and many are programmable through a computer or PLC system. Characterising formulations for freeze-drying Every formulation will have a characteristic “critical temperature”, above which it will suffer processing defects during freeze-drying. Therefore, it is important to maintain the temperature below this until it is dried; however, maintaining the product too far below this temperature will lead to the drying process becoming unacceptably slow, since the kinetics of the drying process are temperature-dependent. Therefore, it is valuable, if not essential, to know the critical temperature of a formulation prior to freeze-drying, in order that suitable conditions can be adopted for its successful and safe processing in a reasonable timeframe. For a crystalline (eutectic) system, its critical temperature will typically be its ‘eutectic melting temperature’ (Teu). Exceeding this temperature will cause the material to melt during processing. For most formulations, however, which persist in an amorphous state, the critical temperature for freeze-drying will be their ‘collapse temperature’ (Tc). This is the temperature at which the material softens to the point of not being able to support its own structure, a phenomenon that may be closely linked with unacceptable product appearance, incomplete drying, poor stability and difficulty in reconstitution. We now know that collapse occurs at some point after a material has been warmed through its glass transition temperature (Tg’), although historically, the terms “glass transition”, “collapse” and “eutectic” have often been erroneously used interchangeably, in the mistaken belief that they were one and the same. Glass transitions in frozen materials may be determined using a number of thermoanalytical methods, such as a differential scanning calorimetry (DSC), or by instruments such as the BTL 'Lyotherm2' apparatus (see Figure 1), which provides an integrated differential thermal analyser (DTA) and impedance measurement capability within one instrument. The system is based upon the use of two different sample cells that can be cooled in a thermally insulated liquid nitrogen reservoir and heated in a controllable warming block. The data obtained from each technique is exported to Microsoft Excel, where it can be analysed in graphical format. Although techniques for the study of Tg’ are well established, there has been little commercially available equipment for the study of Tc itself until the launch of the BTL ‘Lyostat’ freeze-drying microscope a few years ago. The latest version, 'Lyostat 2' (see Figure 2), is a fully integrated freeze-drying microscope that enables collapse or eutectic melting temperatures to be observed in situ. Additional changes in the material may also be analysed using microscopy techniques such as polarised light analysis and differential interference contrast (DIC). Freeze-drying microscopy provides valuable information that can be fed back into both formulation and cycle development processes. Used in combination, instruments such as Lyostat and Lyotherm can provide a comprehensive means of formulation characterisation in terms of freeze-drying behaviour and critical temperatures. From the sublime… Additional (further) drying typically consists of the desorption of unfrozen water from the bulk dried product. This requires more energy than sublimation, since any physico-chemical interaction of the water with the bulk dried material needs to be broken before the water can be removed. This part of the process is governed by adsorption-desorption isotherms, and tends to be favoured by the use of higher temperature and lower pressure conditions than those typically employed in the main drying step. However, care often needs to be taken with heat-sensitive materials such as biologically active proteins, which may undergo damage (for example, denaturation or aggregation) if inappropriate conditions are employed even towards the end of secondary drying. The final product Generally, one looks for the final freeze-dried product to fulfil a number of set criteria, which for biotechnology products and pharmaceuticals might typically include maintenance of cosmetic acceptability, good reconstitution characteristics, appropriate moisture content and good shelf-life. Due to the complex interrelationship between the numerous product and process variables, some developmental work may be required to achieve these criteria. However, the advent of new technologies is enabling such work to be approached on a more rational basis. The science of freeze-drying The more we learn about freeze-drying, the more it reveals itself to be an exact (yet complex) science, rather than the ‘art’ (or even scientific curiosity) it was historically thought to be. However, there is clearly much more to be done in order for the process to be understood more fully and Biopharma Technology Limited strives to build upon its current position at the forefront of freeze-drying technology to rationalise the process further using a scientific approach. Figure 1: BTL 'Lyotherm 2' analyser Figure 2: BTL 'Lyostat 2' freeze-drying microscope system
|
|
|