Freeze drying is a process in which ice (or other frozen solvents) is removed from a material through a process known as sublimation and bound water molecules are removed via desorption.
Freeze drying and lyophilization are names that are used interchangeably, based on the location and industry where the drying is occurring. Controlled freeze drying ensures that the temperature of the product is sufficiently low during the process to prevent any changes in the characteristics and appearance of the dried product. It is an excellent technique for maintaining a wide range of heat-sensitive materials, for example, tissues, plasma, proteins, pharmaceuticals, and microbes.
When a solid (ice) directly changes to a vapor without first going through a liquid (water) phase, it is referred to as sublimation. Having a complete understanding of the concept of sublimation is essential to gain insights into the freeze-drying process. As demonstrated below on the phase diagram for water, low pressures are needed for sublimation to occur.
Sublimation is a phase change, and can occur by adding heat energy to the frozen product. Within the freeze-drying process, sublimation can be simply described as:
- Freeze—The product is fully frozen, often in a tray, flask, or vial.
- Vacuum—The product is subsequently placed under a deep vacuum, well below the water’s triple point.
- Dry—Heat energy is subsequently added to the product, which causes the ice to sublime.
The following is a summary of the steps needed to lyophilize a product in a batch process:
- Loading/container (bulk, vials, flask)
- Freezing (thermal treatment) at atmospheric pressure
- Primary drying (sublimation) under vacuum
- Secondary drying (desorption) under vacuum
- Backfill and stoppering (for product in vials) under partial vacuum
- Removal of dried product from freeze dryer
Apart from offering a prolonged shelf life, effective freeze drying should produce a product that has a short reconstitution time with tolerable potency levels. The process must be repeatable, with well-defined time, pressure, and temperature parameters for each step. For many applications, functional and visual characteristics of the dried product are also vital.
The following are the key components of freeze-drying equipment:
- Refrigeration system
- Vacuum system
- Control system
- Product manifold or chamber
The (ice) condenser, which is positioned within the freeze dryer, is cooled by the refrigeration system. This refrigeration system can also be used for cooling shelves in the product chamber for product freezing.
The vacuum system includes a separate vacuum pump that is linked to an attached product chamber and an airtight condenser.
Differing greatly in their complexity, control systems often include pressure and temperature sensing abilities. Sophisticated controllers will facilitate the programming of a complete “recipe” for freeze drying, and will also include options to track how the freeze-drying process is advancing. The application and use (i.e. lab versus production) decide the selection of a control system for the freeze dryer.
Usually, product chambers are larger chambers with a system of shelves on which the product is placed, or a manifold with attached flasks.
The function of the condenser is to attract the vapors being sublimed off of the product. Since the condenser is preserved at a lower energy level in relation to the product ice, the vapors will condense and return to the solid form (ice) in the condenser.
Next, the sublimated ice collects in the condenser and is removed manually toward the end of the freeze-drying cycle (defrost step). The freezing point and collapse temperature of the product govern the condenser temperature needed. The refrigeration system should be able to keep the condenser’s temperature much below the product’s temperature.
In shelf freeze dryers, the condenser can be placed either in a separate chamber (external condenser) connected to the product chamber (internal condenser) by a vapor port or inside the product chamber.
Manifold freeze dryers depend on ambient conditions to give the sublimation heat to the product. Since an equal amount of heat is removed by vaporization of the solvent, this heat input no longer melts the product. State-of-the-art shelf freeze dryers can provide a heat source to speed up or control the drying process and they can apply the refrigeration system to facilitate product freezing inside the unit.
Freeze dryers can be easily divided by the type of product chamber: (1) shelf dryers where the product is located in a tray or directly on a shelf, (2) manifold dryers where the product is usually pre-frozen and in flasks, and (3) combination units that come with both drying options.
Freeze dryers can also be divided by use and size: (1) pilot units for process development and scale-up; (2) laboratory bench-top units for research and development; and (3) larger production-sized units. It is worth noting that pilot-sized freeze dryers are also used for product R&D and for small volume production applications, as well as in process scale-up work.
The selection of a freeze dryer relies on the characteristics of a product amongst many other application-based variables, including the container in which the product will be dried in, the number of ports or shelf area needed to accommodate the amount to be dried in each batch, the overall volume of ice to be condensed, and whether there are any organic solvents present. Another factor that needs to be considered is the type and shape of the product being dried and its end application.
Product Containers and Containment Systems
It is important that a suitable container is selected for the product. Trays, flasks, and vials are the most common product containers. If possible, a container should be selected that maintains the product’s maximum thickness to less than ¾” (2 cm). For certain applications where product contamination is a major concern, exclusive containers made of Tyvek® and Gore-Tex® are also available.
In addition, product trays with removable-bottoms can be used when working with vials. After the tray is loaded with vials, it is placed on a shelf in the freeze dryer, which is then followed by sliding the bottom part of the tray. This technique enables the vials to rest directly on the shelf and boosts the transfer of heat to the product. Glove boxes are examples of special containment systems that can be used for freeze drying specific products, particularly when harmful materials are present.
Physical Properties of Materials and Formulation
In order to develop an effective lyophilization process, the physical properties of freeze dried materials should be properly understood. Even though some products are simple crystalline materials, most of the products that are lyophilized are amorphous, meaning that they form glassy states when they are are frozen.
Formulation and processing development are key steps that are usually performed to render a product ready for freeze drying and usable for its particular application. The type of excipients added to a formulation can considerably impact the thermal properties of the product and its potential to be freeze dried in a reasonable amount of time.
Recipe for Freeze Drying
Lyophilization in a shelf freeze dryer demands the design of a working cycle or process, which is sometimes referred to as a “recipe”. Multiple steps are usually involved in both drying and freezing of the product. It is important to determine individual time, pressure, and temperature settings for each step.
For each particular product or formulation that is lyophilized, it is essential to develop a freeze drying process that is based on the amount of product, the special properties of the product, and the container used. There is no universal "standard" recipe that will work with every product.
The sample must be fully and completely frozen before pulling a vacuum and initiating the drying process. If an unfrozen product is placed under a vacuum, it may expand outside of the container.
In the case of simple manifold freeze dryers, the product is placed in a flask or vial based on quantity, and subsequently frozen in a separate piece of equipment. Shell baths, standard laboratory freezers, and direct immersion in liquid nitrogen are other options.
In shell (bath) freezing, a flask containing the sample is rotated in a freezing bath, thus allowing the sample to freeze on the flask walls. This method of freezing increases the surface area of the product and decreases its thickness. It is not recommended to freeze a large block of sample in the base of a flask, as the sample will be too thick to enable efficient removal of water - the flask might also break due to uneven stress.
More sophisticated shelf freeze dryers have freezing capabilities that are integrated into the product shelf, making it possible to freeze the product within the freeze dryer. A product is either loaded in bulk form directly onto a product tray, or pre-loaded into vials that are subsequently transferred to the shelf.
With shelf freeze dryers, the cooling rates affecting the crystal size and product freezing rates can be precisely controlled. The speed of the freeze-drying process can be improved by larger ice crystals, thanks to the larger vapor pathways that are left behind in the dried part of the product as the ice crystals are sublimated.
Due to the effects of super-cooling, shelf cooling rates do not necessarily produce larger ice crystals. When the super-cooled liquid finally freezes, it occurs very readily, leading to smaller ice crystals. In a clean room environment (where very few particulates for ice nucleation exist), there is a considerably greater amount of super-cooling. There are some biological products that cannot endure large ice crystals - these products should be freeze dried with smaller ice crystal sizes.
A key step in establishing and improving a freeze-drying process is the determination of a product’s critical collapse temperature. It is this critical temperature that determines the maximum temperature that can be endured by the product at the time of primary drying without collapsing or melting. Dielectric resistance analysis and thermal analysis (freeze-dry microscopy and differential scanning calorimetry) are common techniques used to determine a product’s critical temperature.
Frozen products can be categorized as either amorphous or crystalline glass in structure. Crystalline products have a well-defined “eutectic” freezing/melting point, which is its collapse temperature. It is much more difficult to freeze dry amorphous products, which have a corresponding “glass transition” temperature.
In amorphous products, the collapse temperature is usually a few degrees warmer than that of its glass transition temperature. While most freeze dried materials are in fact amorphous, the word “eutectic” is usually applied (incorrectly) to illustrate the melting/freezing of any product.
According to the US FDA Guide to Inspections of Lyophilization of Parenterals (http://www.fda.gov/ora/inspect_ref/igs/lyophi.html), the manufacturer should be aware of the product’s eutectic point (critical collapse temperature). It is good practice to define the collapse temperature for all new ingestible or injectable drug formulations that need to be freeze dried.
Without knowing the product’s critical temperature, a trial-and-error method is needed to establish suitable primary drying temperatures. Initially, it is possible to use a slow conservative cycle with low pressures and temperatures. The pressure and temperature can then be increased on subsequent cycles until proof of melt-back or collapse is observed—which indicates that the product was excessively warm.
There are some amorphous products (for example, glycine or mannitol) that form a metastable glass with incomplete crystallization when initially frozen. Such products can gain from a thermal treatment process, also known as annealing. In order to get a more complete crystallization, the temperature of the product is cycled during annealing (for instance, from −40 °C to −20 °C for a few hours and then back to −40 °C). Additional advantages of annealing are larger crystal growth and corresponding shorter drying times.
More attention is required in the freeze-drying process when using organic solvents. Lower temperatures are needed to freeze and condense the organic solvents, as these can easily bypass the condenser and ultimately damage the vacuum pump. There are freeze-dryer refrigeration designs available that offer the lower shelf and condenser temperatures required to freeze and subsequently condense certain organic solvents.
Liquid nitrogen (LN2) traps or unique filter cartridges may be needed to catch or condense specific solvents with extremely low freezing temperatures. When handling volatile and/or potentially dangerous materials, safety considerations should be made.
The drying portion of the freeze drying process is comprised of two parts: primary drying and secondary drying. Most of the water removed from the product during the freeze-drying process is done so via sublimating all the free ice crystals at the time of the primary drying step. During primary drying, organic solvents are also removed.
Primary drying, or sublimation, is a slow process that is performed at cooler temperatures (safely below the critical collapse temperature of the product). Sublimation needs heat energy to push the phase change process from solid to gas. All the three heat transfer methods, such as convection, conduction, and radiation, should be taken into account when freeze drying a product.
Within a simple manifold dryer, heat is mainly transferred to the product/flask through radiation and convection from the surrounding environment. With little control over heat flow into the product, it becomes more of a challenge to regulate the process. When working with products that have low collapse temperatures, the flask may need to be wrapped or insulated to decelerate the rate of heat transfer and prevent collapse.
Within a shelf freeze dryer, the bulk of the heat is transferred into the product via conduction and the surface contact of the container/tray/ product has to be maximized with the shelf. Yet, convection and radiation effects also need to be taken into consideration for the purpose of product uniformity and process control.
Radiant heat coming from the interior walls of the product chamber will cause vials/product placed on the perimeter of the shelf to dry more rapidly when compared to drying with the product positioned in the middle of the shelf (known in freeze drying as the “edge effect”).
Radiation that comes through the acrylic doors, which are often used on R&D and pilot freeze dryers, has an even major impact and the product positioned in the front of these dryers will usually dry the fastest of all. For this reason, production freeze dryers are developed with small viewports and metal doors. For protective purposes, a piece of aluminum foil can be hung before the product on the inside of a pilot freeze dryer—this will obviously block the view of the product and not enable observation at the time of the process.
Since shelf contact is usually unreliable, convective heat transfer can help in promoting even product drying. System pressures in the range of 100 to 300 mTorr will typically promote a sufficient amount of convection. At ultra-low system pressures below 50 mTorr, fewer gas molecules are present to offer convection, and slower/uneven drying is possible.
Primary drying is generally a top-down procedure with a well-defined sublimation front shifting through the product as it dries.
Below the interface is a product with ice crystals that still needed to be sublimed, and above the ice surface interface is a dried product, or “cake”. Toward the end of primary drying, when all the free ice crystals have been completely sublimed, the product will seem to be dried. The moisture content, however, can still be in the 5%–10% range owing to the presence of “sorbed” water molecules adhered to the product.
Pressure and Temperature During Primary Drying
As stated before, each frozen product possesses a unique critical temperature. The product temperature should be maintained safely below this critical temperature at the time of primary drying to prevent collapse. The temperature of the product depends on the vapor pressure at the ice interface, and in turn, this vapor pressure depends on the system vacuum level set point as well as the rate of heat transfer into the product (which is regulated by altering the shelf temperature).
After identifying a target product temperature (which is usually several degrees colder than the critical temperature), the system vacuum level and the shelf temperature are the only two variables that have to be determined/controlled. At the time of primary drying, the shelf temperature and the system pressure are set and are regulated in combination to produce a suitable product temperature.
One recommended method is to use the vapor pressure of ice table to set the system pressure. Using thermocouples, the temperature of the product is tracked and the shelf temperature setpoint is gradually increased until the product achieves its target temperature.
After obtaining the target product temperature, the shelf temperature is maintained constant for the balance of primary drying. Some products that have high resistance to vapor flow in the dried part of the cake may require the shelf temperature to be reduced at the end of primary drying to maintain the target product temperature and prevent collapse.
It is not advisable to randomly and repetitively raise the shelf temperature at the time of primary drying, as is common in some older legacy cycles.
The use of the vapor pressure of ice table is a scientific method to establish a suitable pressure for freeze drying. A standard guideline is to select a system pressure that is 20%–30% of the ice vapor pressure at the target product temperature. Sublimation will occur when the vacuum level setpoint is deeper than the ice vapor pressure at the present product temperature. The vacuum levels for freeze drying usually range from 50 to 300 mTorr, with 100 to 200 mTorr being the most typical range.
With the pressure and temperature parameters set, primary drying is subsequently continued for a length of time that is adequate for all of the ice crystals to be sublimed.
Since the majority of commercial freeze dryers cannot reliably control vacuum much below 30 mTorr, at extreme cold product temperatures (below −40 °C), a system pressure setpoint that is 20%–30% of the ice vapor pressure cannot be realized. At these cold product temperatures, freeze drying takes place very slowly.
With manifold freeze drying, the process is driven by the room’s ambient temperature and the system pressure set point. Due to the lack of control over the rate of heat transfer into the product, the majority of manifold dryers are operated conservatively at lower pressures so as to keep the temperature of the product lower.
Determination of the End of Primary Drying
There are a number of analytical techniques available for establishing whether primary drying is complete. Monitoring the product temperature with a thermocouple probe is the most basic technique.
During active primary drying, the quantified product temperature will be colder than the shelf temperature set point because the shelf’s heat is being used for the sublimation phase change. As soon as the sublimation of ice crystals is over, the temperature of the product will increase and approach the temperature of the shelf. When the temperature of the product equals the temperature of the shelf, it can be concluded that primary drying is complete.
Note: the specific vial containing the thermocouple wire will usually dry faster when compared to other vials on the shelf because the wire will conduct more amounts of heat into that specific vial. Likewise, if bulk drying, the area around the thermocouple wire will dry more rapidly when compared to other areas in the product tray.
A modest amount of additional drying time (30 minutes to 2 hours, depending on the product characteristics) should be allowed after the product thermocouple temperature increases, to guarantee that all of the ice in the entire batch of product has been fully sublimated.
Since a product will dry from the top down, the thermocouple’s tip must always be located at the extreme bottom and center of the container. It is acceptable if the thermocouple touches the container’s bottom. When drying in vials it is good practice to insert the thermocouple in a vial in the middle of the shelf. Effects of radiant heating will cause product/vials located on the perimeter of the shelf to dry more rapidly.
More primary drying endpoint determination tools are also available on larger freeze dryers fitted with sophisticated process control systems. One such technique involves a comparison of parallel pressure readings between a capacitance manometer and a Pirani gauge.
A capacitance manometer invariably provides an actual pressure reading in the product chamber, but the Pirani gauge will provide a false high reading in the presence of water vapor. Little or no water vapor is present when the Pirani pressure reading decreases and reaches the true pressure reading of the capacitance manometer.
One more tool is available with freeze-dryer designs that come with external condensers. It is possible to add an isolation valve to the vapor port that joins the product chamber to the condenser. For a brief period of time, this isolation valve can be closed and the resultant rise in pressure in the product chamber can be determined. When this pressure rise nears zero, no more water vapor is produced through sublimation.
Besides the free ice that is sublimed at the time of primary drying, a considerable amount of water molecules remain attached to the product. It is this water that is removed (desorbed) at the time of secondary drying. As all the free ice has been removed in primary drying, the temperature of the product can now be raised substantially, without fear of collapse or melting.
Secondary drying essentially begins at the time of the primary phase; however, at increased temperatures (usually in the 30 °C to 50 °C range), desorption continues much more rapidly. Secondary drying rates depend on the temperature of the product. System vacuum could be continued at the same level applied during primary drying; however, lower vacuum levels will not improve secondary drying times.
In amorphous products, the temperature increase from primary to secondary drying may need to be regulated at a slow ramp rate to prevent collapse.
Secondary drying is progressed until the product has satisfactory moisture content for long-term storage. Based on the application, the moisture content present in fully dried products is usually between 0.5% and 3%. In the majority of cases, the drier the product, the longer is its shelf life. However, there are some complex biological products that may actually become too dry for optimum storage results, and accordingly, the secondary drying process must be controlled.
At the time of secondary drying, a “sample thief” mechanism could be applied to occasionally remove the vials from the freeze dryer for the determination of residual moisture content.
Apart from designing a recipe that effectively dries a product, it is also quite useful to optimi (shorten) the cycle length, particularly if there is potential for process scale-up or repetition for production. Freeze drying can also be a multi-day process. The cycle time can be considerably lowered by analyzing a number of factors:
- Freezing and annealing—maximize crystallization and crystal size to boost drying rates.
- Product thickness—molecules of water vapor undergo resistance as they come out from the dried part of the product. Thinner samples show less resistance to vapor flow and result in faster drying. When drying bulk product in flasks, shell freezing can be useful.
- Critical collapse temperature—this is the most vital piece of information for cycle optimization. The capacity to run primary drying at higher product temperatures considerably decreases the drying time by producing a larger pressure differential between the pressure at the condenser and the vapor pressure over ice in the product. Each 1 °C increase in the temperature of the product can reduce the primary drying time by as much as 13%.
Cycle optimization utilizing eutectic/collapse temperature information needs an iterative method of taking real-time measurements of the temperature of the product at the time of primary drying and subsequently making related modifications to the shelf temperature settings. This can be done manually with the help of product thermocouples or, if drying in vials, an automated SMART system can be employed.
Process Scale-Up Considerations
Usually, laboratory pilot-sized shelf freeze dryers are employed to develop a cycle to be used for process scale-up to a larger production-sized unit. Moreover, a similarity in shelf temperature uniformity and heat transfer characteristics is essential to make sure that a lyophilization process developed in the laboratory can be effectively transferred to a production freeze dryer.
One of the most vital factors that need to be considered is the variation between the clean room environment, which is typical of a production freeze dryer, and the laboratory environment that most pilot units are operated in. The variation in particulates can significantly impact product freezing and the size of the ice crystal.
Usually, production freeze dryers are configured for operation in a clean room environment and can be used for steam sterilization (SIP) and clean-in-place (CIP). If required, process compliance to US FDA regulation 21 CFR 11 is another production consideration. However, this regulation calls for certain standards of process security and control.
Storage of Dried Product
Lyophilized products are highly hydroscopic and hence it is important to seal them in airtight containers after freeze drying to avoid rehydration from atmospheric exposure. It is possible to configure freeze dryers with a “stoppering” capability so as to seal the product while it is still under partial vacuum within the unit. Stoppering is normally done on vials with partly inserted stoppers.
The shelves are collapsed such that each shelf drives down the stoppers/vials placed on the neighboring shelf. Backfilling with an inert gas (for example, dry nitrogen) before stoppering/sealing the product is also a common practice.
Freeze-Dryer Care and Maintenance
As well as cleaning the system and defrosting the condenser after each cycle, routine freeze-dryer maintenance usually involves visualyl checking all gaskets and seals and periodically changing the vacuum pump oil. Sophisticated controllers provide the ability to run a periodic system test and/or leak test to make sure that the unit is working to original factory specifications.
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