Lyophilization: A Primer

Freeze drying, or lyophilization, is a stabilization method that is widely used in the pharmaceutical industry for drugs, vaccines, antibodies, and other biological material. Because the product is dried without excessive heating, proteins and other products that would be thermally denatured can be successfully preserved without loss of activity.

Freeze-dried products have a very high surface area, which enables them to be reconstituted quickly. This quick reconstitution is particularly important in the case of emergency vaccines and antibodies, which need to be administered as soon as possible.

TAKESHI NISHIJIMA/MARIA TOUTOUDAKI/GETTY IMAGES; DAN WARD

Every formulation has different freeze-drying characteristics and, therefore, different processing requirements. To ensure cycles are both robust and efficient, they should be tailor-made for each formulation. Failure to do so can lead to inconsistent dryness across samples, reduced stability during storage, and reduced activity on rehydration.

There are three main business advantages of optimizing a product's lyophilization cycle:

• Financial gain: optimal lyophilization cycles use only the energy and time required, shortening process time and increasing product throughput.

• Product excellence: a well-dried product exhibits a long shelf life and maximum activity on rehydration.

• Quality and regulatory assurance: consistency throughout batches is assured and regulatory submissions are completed with the inclusion of lyophilization cycle data.

The freeze-drying cycle

Lyophilization is a complex drying process that involves removing the solvent from a material by sublimation. Sublimation is achieved through varying the temperature and pressure of the material so that the solvent does not pass through the liquid stage, but moves directly from the solid phase to the gas phase (see Figure 1). Lyophilization takes place in three main stages: freezing, primary drying, and secondary drying. Each stage has its own challenges.

Figure 1: During freeze drying the temperature and pressure are controlled so that the frozen solvent moves directly from the solid to the gas phase without passing through the liquid phase.

Freezing. The material is frozen. The rate of freezing, and the final temperature to which the material is lowered, both have a significant impact on the quality of the final product. The rate at which the temperature is lowered affects the structure of the ice matrix, which has an impact on the ease of flow of the sublimated vapor out of the sample. Annealing, a technique of raising and then lowering the temperature of a frozen material, can be used to encourage crystallization or to provoke a more favorable ice structure.

In delicate materials such as proteins, there is a risk of damage from ice crystal growth. In general, the faster the rate of freezing, the larger the ice crystals formed and the greater the risk of damage. A slower freezing cycle will result in smaller crystals that cause less damage, but the resulting structure will cause a greater impediment to the flow of vapor and therefore slow the drying process.

During the freezing stage, it is vital that the material is cooled below its critical temperature (T_crit) to ensure it is fully frozen. Every formulation has a different T_crit that is affected by the combination and proportions of the elements within it, such as the solvent, excipients, and the active ingredient. It is vital that the critical temperature is determined for every different formulation. Knowing the T_crit not only makes it easy to ensure that the T_crit is achieved during freezing, but also means that energy is not wasted by taking the temperature lower than required. Methods for determining T_crit are discussed below.

Primary drying. The frozen material is initially dried by sublimation. During primary drying the pressure of the drying chamber is reduced to a very low level, while the temperature is raised slightly to allow the solvents to sublime. Throughout this stage the temperature must be kept below the critical temperature (T_crit) so that the material does not melt or its structure collapse.

One of the effects of sublimation is cooling of the product, which slows the process of drying. The rate of sublimation can decrease by as much as 13% for each unnecessary 1°C decrease in temperature (1). To counter this cooling and provide energy to drive the sublimation process, heat is added through the freeze-dryer shelf. The energy transfer during primary drying must be balanced so that sufficient heat is used to encourage sublimation without risking collapse.

Collapse is the most serious processing defect in freeze drying, resulting in reduced shelf life, reduced stability, decreased product activity, and poor reconstitution (see Figure 2).

Figure 2: A selection of vials containing the same freeze-dried material. The fill depth of all four vials was identical before processing. The three vials to the right have all undergone serious process defects.

Secondary drying. Secondary drying is a desorption process that removes any solvent that is left chemically bound in the material after primary drying. The moisture level at the beginning of this stage may be around 5–10%, with a final moisture content of typically less than 5%.

To facilitate the desorption process, the temperature is raised and the pressure reduced to a minimum (see Figure 3). This is the slowest phase of the lyophilization process. Depending on the final moisture level required, it could last several days. Therefore, any increases in efficiency can have a significant impact on manufacturing throughput.

Figure 3: A simplified freeze-drying chart, showing the variations in temperature and pressure throughout the lyophilization cycle.

PRODUCT CHARACTERIZATION FOR CYCLE DEVELOPMENT

Fully characterizing each formulation provides the data necessary to ensure that the cycle designed is optimal for the product and the equipment. Without this information, there is no way to determine the basic process parameters or to scientifically verify the success of the resulting cycle.

Process conditions that are too aggressive will damage the product, decreasing stability and activity, and risking complete batch failure. Process conditions that are too conservative will add unnecessary energy costs, increase batch duration, and reduce turnaround time. A poorly designed cycle can experience some or all of these problems.

Collapse temperature. The most important characteristic of a material for freeze drying is its critical temperature. In simple crystalline materials this is the eutectic temperature (T_eu),although more commonly the collapse temperature (T_c) is relevant. T_c is applicable to products which will form amorphous solids, such as pharmaceutical formulations.

T_c and T_eu are typically ascertained using freeze-drying micro-scopy (FDM), a quick and well-understood process in which a small amount of product is frozen under a microscope. FDM can be carried out on quantities as small as 70 µL (2). Such quick feedback makes it feasible to check the freeze-drying characteristics of each new product formulation, helping the formulation technologist understand the product's response to freeze drying. In the interests of achieving optimum efficiency, FDM can also be used to determine the relative rates of drying for different formulations, or for the same formulation at different temperatures.

In addition to the identification of critical temperature, FDM can also provide a visual indication of the potential for skin formation and the effects of annealing on the ice structure, solute crystallization, and critical temperature.

Frozen state mobility. It is common to think of freezing as a simple, discrete process whereby something is either a solid or a liquid. However, in complex formulations comprising many separate elements, solidification cannot be relied on as an indication of complete freezing and changes may still be taking place within the frozen structure.

A solid that has a non-crystalline (amorphous) structure is referred to as a glass and the point at which the product changes from a liquid to solid is known as the glass transition temperature (T_g). However, due to the complex nature of most pharmaceutical and biotechnological products, glass transition occurs over a range of temperatures. Changes in molecular mobility can occur even in product frozen below its collapse temperature, and these changes can have significant impact on the product's shelf life and long-term activity.

In the event that changes are taking place in the frozen state, it may be necessary to adjust the cycle or to adjust the formulation. However, in most cases the possibility of frozen state flexibility is ignored until problems with the dry product occur. To avoid late-stage redevelopment work, it is advisable to conduct the analysis early on in cycle development, ideally at the same time as FDM.

Typical frozen state analyses include differential scanning calorimetry (DSC) and joint differential thermal analysis (DTA)/impedance analysis.

DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. DSC is used to detect physical transformations such as phase transitions, endo- or exo-thermic events such as crystallization events, and glass transitions.

DTA is a technique similar to DSC. When used in conjunction with impedance analysis (ZSinΦ), a fixed frequency dielectric analysis, the molecular mobility of a frozen sample can be explored to a very high degree of accuracy.

Refining the cycle

Once initial cycle parameters have been defined, the next step is to run a test batch on a research freeze dryer with product monitoring capabilities. Monitoring process conditions such as chamber pressure and product temperature enable the endpoints of primary drying and secondary drying to be determined.

Where primary drying should end and secondary drying begin is dependent on the individual properties of the product and the stated process requirements. But as the two stages are so different in processing terms, when and how the change should occur is of vital importance to the success of the process and minimizing cycle time.

The end of secondary drying, and the freeze-drying process overall, is difficult to define and pinpoint. A range of tolerance for final moisture content must be decided upon, weighing the desired stability and activity of the product against the cost of continuing the process for further hours or days.

A conservative freeze-drying cycle that has been arrived at by trial and error might produce satisfactory product reliably and repeatably. However, there will be no scientific evidence of the suitability of the process other than exhaustive quality assurance testing. By providing evidence of the analysis, cycle feedback and overall process of cycle development, the suitability of the cycle can be easily verified by internal and external auditors.

In the instance that previously robust batches lose consistency or product stability slips, the original data can be used for troubleshooting.

Freeze-drying cycles are optimized not only with regards the formulation, but also the freeze drying equipment and batch parameters such as fill depth, batch size, and container type. For optimum efficiency in manufacturing scale-up, the cycle should be designed for the specific process equipment used.

The following real example of how this technology has been used to improve efficiency speaks volumes about how much of a difference characterizing a freeze-drying cycle makes.

A vaccines manufacturer had a 70-hour freeze-drying cycle for a product, which was limiting manufacturing capability. Freeze-drying company Biopharma Technology Ltd was asked to analyze the product's thermal characteristics. The cycle had been designed to freeze the product below -45 °C and maintain the product below -40 °C throughout primary drying. FDM analysis showed a collapse temperature at -18.2 °C; DTA/impedance analysis showed a significant softening event at -23 °C. Raising the designated freezing temperature to a still-conservative -28 °C enabled the freezing step to be significantly shortened, as well as saving the cost in energy of cooling the chamber and product through unnecessary extra degrees. The temperature setpoint of primary drying could also be raised to increase the rate of sublimation. Process monitoring subsequently indicated that the product was being left in primary drying conditions for much longer than necessary and the duration of this stage was cut by 40%.

Analysis of the product dried using the new cycle demonstrated that while the total process time was reduced by 15 hours, the product was just as good as before.

References

1. Tang, X.; Pikal, M., J. Pharm. Res. 21(2), 191–200, (2004).

2. Formulation Characterisation 2: Thermal and Other Methods, Biopharma Technology Ltd.

Katriona Scoffin is a science writer and Laura Ciccolini, PhD, is commercial director of Biopharma Technology Ltd, Winchester, England.

All figures courtesy of the authors.