Technically known as lyophilisation, lyophilization, or cryodesiccation is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. Freeze-drying works by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublime directly from the solid phase to the gas phase, skipping the liquid phase entirely. If you do not skip the liquid phase, you are not freeze drying, you are actually vacuum drying. This results in product degradation and loss of vital chemicals or nutrients.
The process of freeze-drying was invented in 1906 by Arsène d’Arsonval and his assistant Frédéric Bordas at the laboratory of biophysics of Collège de France in Paris. In 1911 Downey Harris and Shackle developed the freeze-drying method of preserving live rabies virus which eventually led to development of the first anti-rabies vaccine.
Modern freeze-drying was developed during World War II. Blood serum being sent to Europe from the US for medical treatment of the wounded required refrigeration, but because of the lack of simultaneous refrigeration and transport, many serum supplies were spoiling before reaching their intended recipients.
The freeze-drying process was developed as a commercial technique that enabled serum to be rendered chemically stable and viable without having to be refrigerated. Shortly thereafter, the freeze-dry process was applied to penicillin and bone, and lyophilization became recognized as an important technique for preservation of biologicals. Since that time, freeze-drying has been used as a preservation or processing technique for a wide variety of products.
These applications include the following but are not limited to: the processing of food, pharmaceuticals, and diagnostic kits; the restoration of water damaged documents; the preparation of river-bottom sludge for hydrocarbon analysis; the manufacturing of ceramics used in the semiconductor industry; the production of synthetic skin; the manufacture of sulfur-coated vials; and the restoration of historic/reclaimed boat hulls.
There are four stages in the complete drying process: pretreatment, freezing, primary drying, and secondary drying.
Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation revision (i.e., addition of components to increase stability, preserve appearance, and/or improve processing), decreasing a high-vapor-pressure solvent, or increasing the surface area. Food pieces are often IQF treated to make it free flowing prior to freeze drying. In many instances the decision to pretreat a product is based on theoretical knowledge of freeze-drying and its requirements, or is demanded by cycle time or product quality considerations.
In a lab, this is often done by placing the material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice in aqueous methanol, or liquid nitrogen. On a larger scale, freezing is usually done using a chest or walk-in freezer. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry. To produce larger crystals, the product should be frozen slowly or can be cycled up and down in temperature. This cycling process is called annealing. However, in the case of food, or objects with formerly-living cells, large ice crystals will break the cell walls (a problem discovered, and solved, by Clarence Birdseye), resulting in the destruction of more cells, which can result in increasingly poor texture and nutritive content. In this case, the freezing is done rapidly, in order to lower the material to below its eutectic point quickly, thus avoiding the formation of ice crystals. Usually, the freezing temperatures are between −50 °C and −80 °C (-58 °F and -112 °F). The freezing phase is the most critical in the whole freeze-drying process, because the product can be spoiled if improperly done. Amorphous materials do not have a eutectic point, but they do have a critical point, below which the product must be maintained to prevent melt-back or collapse during primary and secondary drying.
During the primary drying phase, the pressure is lowered (to the range of a few millibars), and enough heat is supplied to the material for the ice to sublime. The amount of heat necessary can be calculated using the sublimating molecules’ latent heat of sublimation. In this initial drying phase, about 95% of the water in the material is sublimated. This phase may be slow (can be several days in the industry), because, if too much heat is added, the material’s structure could be altered.
In this phase, pressure is controlled through the application of partial vacuum. The vacuum speeds up the sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser chamber and/or condenser plates provide a surface(s) for the water vapour to re-solidify on. This condenser plays no role in keeping the material frozen; rather, it prevents water vapor from reaching the vacuum pump, which could degrade the pump’s performance. Condenser temperatures are typically below −50 °C (−58 °F). It is important to note that, in this range of pressure, the heat is brought mainly by conduction or radiation; the convection effect is negligible, due to the low air density.
The secondary drying phase aims to remove unfrozen water molecules, since the ice was removed in the primary drying phase. This part of the freeze-drying process is governed by the material’s adsorption isotherms. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0 °C, to break any physico-chemical interactions that have formed between the water molecules and the frozen material. Usually the pressure is also lowered in this stage to encourage desorption (typically in the range of microbars, or fractions of a pascal). However, there are products that benefit from increased pressure as well. After the freeze-drying process is complete, the vacuum is usually broken with an inert gas, such as nitrogen, before the material is sealed.
At the end of the operation, the final residual water content in the product is extremely low, around 1% to 4%.
If a freeze-dried substance is sealed to prevent the reabsorption of moisture, the substance may be stored at room temperature without refrigeration, and be protected against spoilage for many years. Preservation is possible because the greatly reduced water content inhibits the action of microorganisms and enzymes that would normally spoil or degrade the substance.
Freeze-drying also causes less damage to the substance than other dehydration methods using higher temperatures. Freeze-drying does not usually cause shrinkage or toughening of the material being dried. In addition, flavors, smells and nutritional content generally remain unchanged, making the process popular for preserving food. However, water is not the only chemical capable of sublimation, and the loss of other volatile compounds such as acetic acid (vinegar) and alcohols can yield undesirable results.
Freeze-dried products can be rehydrated (reconstituted) much more quickly and easily because the process leaves microscopic pores. The pores are created by the ice crystals that sublimate, leaving gaps or pores in their place. This is especially important when it comes to pharmaceutical uses. Freeze-drying can also be used to increase the shelf life of some pharmaceuticals for many years.
Similar to cryoprotectants, some molecules protect freeze-dried material. Known as lyoprotectants, these molecules are typically polyhydroxy compounds such as sugars (mono-, di-, and polysaccharides), polyalcohols, and their derivatives. Trehalose and sucrose are natural lyoprotectants. Trehalose is produced by a variety of plant (for example selaginella and arabidopsis thaliana), fungi, and invertebrate animals that remain in a state of suspended animation during periods of drought (also known as anhydrobiosis).
Pharmaceutical and biotechnology
Pharmaceutical companies often use freeze-drying to increase the shelf life of the products, such as live virus vaccines, biologics and other injectables. By removing the water from the material and sealing the material in a glass vial, the material can be easily stored, shipped, and later reconstituted to its original form for injection. Another example from the pharmaceutical industry is the use of freeze drying to produce tablets or wafers, the advantage of which is less excipient as well as a rapidly absorbed and easily administered dosage form. Freeze-dried pharmaceutical products are produced as lyophilized powders for reconstitution in vials and more recently in prefilled syringes for self-administration by a patient. Examples of lyophilized biological products include many vaccines such as Measles Virus Vaccine Live, Typhoid Vaccine, Meningococcal Polysaccharide Vaccine Groups A and C Combined. Other freeze-dried biological products include Antihemophilic Factor VIII, Interferon alfa, anti-blood clot medicine Streptokinase and Wasp Venom Allergenic Extract. Many biopharmaceutical products based on therapeutic proteins such as monoclonal antibodies require lyophilization for stability. Examples of lyophilized biopharmaceuticals include blockbuster drugs such as Etanercept (Enbrel by Amgen), Infliximab (Remicade by Janssen Biotech), Rituximab and Trastuzumab (Herceptin by Genentech). Freeze-drying is also used in manufacturing of raw materials for pharmaceutical products. Active Pharmaceutical Product Ingredients (APIs) are lyophilized to achieve chemical stability under room temperature storage. Bulk lyophilization of APIs is typically conducted using trays instead of glass vials. Dry powders of probiotics are often produced by bulk freeze-drying of live microorganisms such as Lactic acid bacteria and Bifidobacteria.
Food and agriculture-based industries
Although freeze-drying is used to preserve food, its earliest use in agriculturally based industries was in processing of crops such as peanuts/groundnuts and tobacco in the early 1970s. Because heat, commonly used in crop and food processing, invariably alters the structure and chemistry of the product, the main objective of freeze-drying is to avoid heat and thus preserve the structural and chemical integrity/composition with little or no alteration. Therefore, freeze-dried crops and foods are closest to the natural composition with respect to structure and chemistry. The process came to wide public attention when it was used to create freeze-dried ice cream, an example of astronaut food. It is also widely used to produce essences or flavourings to add to food. Because of its light weight per volume of reconstituted food, freeze-dried products are popular and convenient for hikers. More dried food can be carried per the same weight of wet food, and remains in good condition for longer than wet food, which tends to spoil quickly. Hikers reconstitute the food with water available at point of use. Instant coffee is sometimes freeze-dried, despite the high costs of the freeze-driers used. The coffee is often dried by vaporization in a hot air flow, or by projection onto hot metallic plates. Freeze-dried fruits are used in some breakfast cereal or sold as a snack, and are a popular snack choice, especially among toddlers, preschoolers, hikers and dieters, as well as being used by some pet owners as a treat for pet birds. Most commercial freezing is done either in cold air kept in motion by fans (blast freezing) or by placing the foodstuffs in packages or metal trays on refrigerated surfaces (contact freezing). Culinary herbs, vegetables (such as vitamin-rich spinach and watercress), the temperature sensitive baker`s yeast suspension and the nutrient-rich pre-boiled rice can also be freeze-dried. During three hours of drying the spinach and watercress has lost over 98% of its water content, followed by the yeast suspension with 96% and the pre-boiled rice by 75%. The air-dried herbs are far more common and less expensive. Freeze dried tofu is a popular foodstuff in Japan (“Koya-dofu” or “shimi-dofu” in Japanese).
In chemical synthesis, products are often freeze-dried to make them more stable, or easier to dissolve in water for subsequent use. In bioseparations, freeze-drying can be used also as a late-stage purification procedure, because it can effectively remove solvents. Furthermore, it is capable of concentrating substances with low molecular weights that are too small to be removed by a filtration membrane. Freeze-drying is a relatively expensive process. The equipment is about three times as expensive as the equipment used for other separation processes, and the high energy demands lead to high energy costs. Furthermore, freeze-drying also has a long process time, because the addition of too much heat to the material can cause melting or structural deformations. Therefore, freeze-drying is often reserved for materials that are heat-sensitive, such as proteins, enzymes, microorganisms, and blood plasma. The low operating temperature of the process leads to minimal damage of these heat-sensitive products. In nanotechnology, freeze-drying is used for nanotube purification to avoid aggregation due to capillary forces during regular thermal vaporization drying.
Organizations such as the Document Conservation Laboratory at the United States National Archives and Records Administration (NARA) have done studies on freeze-drying as a recovery method of water-damaged books and documents. While recovery is possible, restoration quality depends on the material of the documents. If a document is made of a variety of materials, which have different absorption properties, expansion will occur at a non-uniform rate, which could lead to deformations. Water can also cause mold to grow or make inks bleed. In these cases, freeze-drying may not be an effective restoration method.
In bacteriology freeze-drying is used to conserve special strains. In high-altitude environments, the low temperatures and pressures can sometimes produce natural mummies by a process of freeze-drying. Advanced ceramics processes sometimes use freeze-drying to create a formable powder from a sprayed slurry mist. Freeze-drying creates softer particles with a more homogeneous chemical composition than traditional hot spray drying, but it is also more expensive. A new form of burial which previously freeze-dries the body with liquid nitrogen has been developed by the Swedish company Promessa Organic AB, which puts it forward as an environmentally friendly alternative to traditional casket and cremation burials.
Care for your freeze dryer
As is the case with all categories of laboratory equipment, the longevity of your freeze dryer depends on how you use it (application), how often you use it (frequency of use) and how well you care for it (maintenance). While it is true that some freeze dryers continue to lyophilize effectively for decades, the average life span of a freeze dryer in today’s laboratory environment is approximately 10 to 15 years.
Do you want to know the secrets to getting that much life out of your freeze dryer?
Let’s break down application, usage and maintenance—all those factors that determine how long and how well your freeze dryer works—and identify several Do’s and Dont’s to guide you.
DO . . .
Make sure your freeze dryer is compatible with the eutectic temperature of your sample(s).
First and foremost, ensure your sample is compatible with your freeze dry system before you use the unit. A good rule of thumb is to plan for your most challenging sample and choose a system based on that need. It’s not possible to modify a freeze dryer’s condenser temperature. The condenser temperature should be 10 to 15°C below the sample’s eutectic point. Low freezing point solvents, for instance, should not be used on -50°C units. Certain acids should not be used with bare stainless steel and should only be used with PTFE models. Systems that reach -84°C are ideal for lyophilizing samples with low eutectic temperatures (like those that contain acetonitrile). Systems that reach -105°C can handle samples containing small amounts of ethanol.
Perform regular vacuum pump maintenance.
Have you ever had problems with your freeze dryer not pulling sufficient vacuum? If so, you’re not alone—it’s one of the most common freeze dry troubleshooting issues. The good news is that performing regular pump maintenance can help solve the problem, both reducing immediate downtime and increasing unit life in the long term. After you’ve ensured you have the appropriate lyophilizer for your samples (see above), do the same for your pump: standard rotary vane pumps work well for aqueous based solutions, and hybrid or combination pumps are best suited for use with solvents or acids. A low maintenance option, the scroll pump, is a new offering, which does not use oil at all. In some cases, secondary acid or solvent traps can be used to extend the life of your vacuum pump by providing an additional barrier between the lyophilizer and the pump. Dry ice traps are also available if you don’t have the appropriate temperature differential between the eutectic temperature of your sample and the collector temperature.
Last but certainly not least, make sure to change the oil in your vacuum pump every 1,000 hours (or sooner if your application warrants). If in doubt, check the appearance of the oil. If it’s cloudy or darker than an iced tea color, it needs to be changed.
Note that some pumps, such as hydrocarbon free scroll pumps, can pull a deep vacuum for freeze drying without using oil. If you’ve chosen a scroll pump, make sure to change the scrolls after every 40,000 hours of use.
Clean the freeze dryer after each run.
It’s necessary to defrost and drain the condenser after each standard run. If using acids, note that you’ll also need to neutralize the chamber. Regardless of the sample used, you must rinse and wipe down any components that may have come into contact with chemicals to reduce the risk of damage. Don’t let water—especially chemically contaminated water—sit on the stainless, acrylic or rubber components of your freeze dryer.
DON’T . . .
Put samples in the freeze dryer that aren’t completely frozen.
A sample must be completely frozen to be included in a lyophilization run. If it’s not, a large volume of the sample will evaporate, thereby producing a high initial vapor load. The vapors can then pass through the condenser and into the vacuum pump where they can do damage. Or, in worst-case scenarios, liquid could be sucked directly into the pump, causing even more damage.
Note that if a sample starts to melt back in-process, simply make adjustments so that is remains frozen or remove it from the system entirely.
Overload your freeze dryer.
Overloading your freeze dryer can make sessions last longer or even cause them to be wholly unsuccessful. The vapor load that the condenser must accommodate is the greatest when a sample is first loaded.
The instantaneous load capacity rating is the quantity of vapor a freeze dryer can accommodate at one time. This is a different measurement than the ice holding capacity or the 24-hour collection rating. When freeze drying multiple, large volumes, make sure the condenser temperature does not rise shortly after the sample is loaded. If this does happen, you are close to exceeding the instantaneous load capacity. In these instances, consider staggering the loading so samples are started at varying intervals.
At the end of a run, it’s easy to remove your completed samples and forget to defrost and drain the condenser—an act that not only lengthens the process for the next user but can also cause damage to the unit if done frequently. Maintenance is important for vacuum pumps, too. Although it can be time consuming, changing the oil frequently is a must. Especially when there are multiple users of a single system, keeping a maintenance log of oil changes and compressor cleanings can help ensure none of these crucial tasks are neglected.
Source: Jessica Burdg, Science Journalist, Labconco May 10, 2016