What is an Lyophilization?
Lyophilization, also known as freeze-drying, is a process used for preserving biological material by removing the water from the sample, which involves first freezing the sample and then drying it, under a vacuum, at very low temperatures. Lyophilized samples may be stored much longer than untreated samples.
Why Lyophilization Is Used?
The lyophilization process
Lyophilization involves the removal of water or other solvents from a given product by a process called sublimation. This occurs when the ice of a frozen product converts directly to the gaseous state without passing through the liquid phase. This enables the preparation of a stable product that is easy to use and store at ambient temperatures.
A low pressure environment is pre-requisite to allow this process to take place. In order to start the removal of water, the pressure inside the freeze dryer must be below the “triple point value” for the product, whilst also maintaining the temperature of the sample below its freeze point in the lyophilization process.
Pre-freezing – first stage of the lyophilization process
The sample is frozen, which means the water in the product is converted to ice, thereby the phase has changed from liquid to solid.
Slow pre-freezing will produce lager ice crystals, which are easier to lyophilize, whilst fast pre-freezing results in smaller crystals.
Primary drying – Second stage of the lyophilization process
In the second stage of lyophilization the sublimation process starts. The ice formed in the pre-freeze step is removed from the sample by the direct transition of the “solid” ice to a vapour without passing through a liquid phase. The resultant vapour is collected by the condenser, which has a lower temperature and pressure than the product. The vapour is thus converted back to ice on the condenser surface.
The “energy” required for this process to occur is provided by a gentle heating of the sample, which will start the sublimation process and eventually the sample will dry.
If too much energy (heat) is applied to the sample during this stage the condenser of the lyophilizer may not be able to condense the volume of vapours fast enough, the ice condenser temperature will subsequently rise along with its vapor pressure, thus increasing the risk of the sample melting.
Secondary drying – Third stage of the lyophilization process
Finally, any residual water present, which is strongly bound to the molecules of the sample, is converted to vapour and removed from the sample.
This water has invariably a vapour pressure lower than that of water in its “free” form.
Removal of the water in this final stage of lyophilization will be performed at higher product temperatures, consequently, any biological activity of the sample will not be impaired or affected. This usually involves increasing the temperature and lowering the pressure to provide enough energy to break down the molecular bonding. A process called desorption.
Advantages of Lyophilization
Lyophilization has many advantages over the other drying and preserving techniques.
- It maintains food/ biochemical and chemical reagent quality because they remains at a temperature that is below the freezing-point during the process of sublimation.The use of lyophilization is particularly important when processing lactic bacteria, because these products are easily affected by heat.
- Food/biochemicals and chemical reagents which are lyophilized can usually be stored without refrigeration, which results in a significant reduction of storage and transportation costs.
- Lyophilization greatly reduces weight, and this makes the products easier to transport. For example, many foods contain as much as 90% water. These foods are 10 times lighter after lyophilization.
- Because they are porous, most freeze-dried products can be easily rehydrated. Lyophilization does not significantly reduce volume, therefore water quickly regains its place in the molecular structure of the food/ biochemicals and chemical reagents.
Disadvantages of Lyophilization
The principle disadvantages are:
- High capital cost of equipment
- High energy cost
- Lengthy process time (typically 4-10 hrs. per drying cycle)
- Possible damage to products due to change in pH and tonicity.
Is lyophilization the same as freeze drying?
Lyophilization and freeze drying are terms that are used interchangeably depending on the industry and location where the drying is taking place. Controlled freeze drying keeps the product temperature low enough during the process to avoid changes in the dried product appearance and characteristics. It is an excellent method for preserving a wide variety of heat-sensitive materials such as proteins, microbes, pharmaceuticals, tissues & plasma.
Applications of Lyophilization
The most important application of industrial freezing drying is explained below:
Pharmaceutical Industry and Biotechnology
Pharmaceutical companies often use freeze-drying to increase the shelf life of products, such as vaccines and other injectable. By removing the water from the material and sealing the material in a vial, the material can be easily stored, shipped and later reconstituted to its original form for injection.
Freeze-drying is used to preserve food and make it very light weight. The process has been popularized in the forms of freeze- dried ice cream; an example of astronaut food. The coffee is often dried by vaporization in a hot air flow, or by projection on hot metallic plates. Freeze-dried fruit is used in some breakfast cereal. Culinary herbs are also freeze-dried, although air-dried herbs are far more common and less expensive. However, the freeze-drying process is used more commonly in the pharmaceutical industry.
In chemical synthesis, products are often lyophilized to make them more stable, or easier to dissolve in water for subsequent use. In bio separations, freeze-drying can be used also as a late- stage purification procedure, because it can effectively remove solvents. Also it is capable of concentrating substances with low molecular weights that are too small to be removed by a filtration membrane.
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. In bacteriology freeze- drying is used to conserve special strain. 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. Freeze drying is also used for floral preservation and wedding bouquet preservation has become very popular with brides who want to preserve their wedding day flowers.
Types of Lyophilizers
Lyophilizers come in all shapes and sizes. Note that freeze dryers are often classified by the type of chamber: manifold benchtop units, shelf consoles, and combination models. Another classification offers four categories according to the specific research goals:
1) lab freeze dryers: with a shelf area of 0.1 sq.m. to 1 sq.m.; used for simple removal, Phase 1 clinical trials, and scale-up production;
2) production units: with a shelf area of 1 sq.m. to more than 40 sq.m.; employed for high-volume productions, as well as Phase 2 and Phase 3 trials;
3) sterilizable lyophilizers: units that are used when sterilization between cycles is needed, which is done through pressurized steam or hydrogen peroxide;
4) non-sterile freeze-dryers: units that are usually more affordable than sterilizable units. Interestingly, the presence of additional features, such as shelf style (bulk or stoppering), condenser location (internal or external), and material used (316L or 304L), are also utilized to classify lyophilizers.
Factors to Consider When Buying a Lyophilizer
Lyophilizers are invaluable tools in lab settings used to increase the shelf life of vaccines and other pharmaceutical products; freeze dryers can also facilitate the transportation of biopharmaceuticals. Additionally, some medicines can be freeze-dried in order to be easily absorbed, while some proteins and antibodies are freeze-dried for stability. Given the wild applications of lyophilization, it’s no surprise there’s a variety of lyophilizers experts can choose from. Whether it’s to set up a new laboratory or replace old equipment, there are three major factors to consider before purchasing a new unit:
Before buying a freeze dryer, experts should refine their research and production goals. Both the sample size and type are crucial factors to consider. While benchtop models are ideal for small volumes, floor standing models are beneficial for large scale applications. When it comes to sample types, a difference of 15-20°C is needed between the eutectic temperature of the sample and the collector chamber. While the majority of biological samples can be frozen by a standard system reaching -50°C, some samples, such as High-Performance Liquid Chromatography/HPLC preparations in acetonitrile/CH3CN, will need a cascade-type collector reaching -84°C to accommodate lower freezing points. An ultra-low temperature lyophilizer (of up to -105°C), on the other hand, might be required to accommodate preparations with methanol (with a eutectic point of -97.6°C). For acidic samples that can harm the stainless steel coils of a system, Polytetrafluoroethylene/PTFE coating is recommended, as well as a hybrid-type pump. Last but not least, the volume of liquid should be considered. While lyophilizers with a small capacity can accommodate tissue samples with small amounts of liquid, food products may hold higher amounts and require units with a bigger capacity. Note that collector capacities can vary from 2.5 to 18 liters, with different shelving and holders.
As explained above, different research goals require different equipment. Capacity parameters, materials used, and design types are all factors to consider. Yet, we should note that all lyophilizers follow three vital processes to execute water removal: 1) freezing to preserve the sample’s physical form; 2) sublimation drying that removes 95% of the water; 3) secondary drying or desorption drying used to remove any remaining water molecules. Interestingly, after lyophilization, products contain 1-4% moisture and are nitrogen sealed for further use. Note that lyophilization differs from evaporation (the use of heat to eliminate moisture), and sublimation refers to the direct transition from a solid to a gaseous state.
Lyophilization is an expensive process due to equipment costs, long processing times, and energy costs. Prices can vary according to the unit’s parameters (e.g., temperature range, capacity, type, and brand). Note that full-sized models can process multiple samples in a single cycle; some units may even have more than one condenser for almost continuous use. Control systems can also vary in complexity, with some allowing the programming of a whole freeze-drying recipe, which can also affect costs. Furthermore, drying accessories, which can be customized according to the research goals, should be considered. To test tubes and serum vials, for instance, flasks can be used, while for bulk samples, a tray dryer will be preferable. Additional equipment, such as glove boxes for toxic materials and accessories to stopper under vacuum, can increase the final price of a unit.
Safety Guidelines for Lyophilizer
- Mammalian cells can not be preserved by lyophilization because it can desteoy mammalian cells. Many microorganisms and proteins survive lyophilization well,because they rehydrate easily and quickly because of the porous structure left after the ice sublimes.
- Wear appropriate eye protection at all times when working with or near a lyophilizer.
- Specimens shell-frozen in ampoules are dried on a vacuum manifold or in a chamber-type drier at low negative pressure. If the glass neck of the ampoule is sealed off while the ampoule is still under vacuum, it may cause implosion, either during the sealing or later when the evacuated ampoule is being opened. To avoid this, after drying is completed, and before sealing is done, bring the pressure within the ampoule back to normal by gradually introducing dry nitrogen, avoiding turbulent disturbance of the dry product.
- The narrow or constricted neck of the ampoule is contaminated if the specimen is allowed to run down the wall of the neck during filling. Subsequently, when the ampoule is sealed with a torch, the dried material on the wall becomes charred or partially decomposed; residues of this material may adversely affect the dried material when it is reconstituted. To avoid this, a syringe with a long cannula or a Pasteur-type pipette should be used to fill the vial. Do not allow the delivery end of the cannula or pipette to touch the neck of the vial.
- All ampoules used for freeze-drying of cultures, toxins, or other biohazardous material should be fabricated of Pyrex-type glass. This type of glass requires a high-temperature torch using an air-gas or oxygen-gas mixture for sealing. These hard glass ampoules are much less apt to form gas bubbles that burst inwardly during sealing under vacuum than the soft glass ampoules and are more resistant to breakage during handling and storage.
- The filling of ampoules and vials with infectious specimens, the subsequent freeze-drying, and sealing or closing of ampoules and vials in the preparation of dry infectious specimens should be performed in a biological safety cabinet. The same is true for the preparation of ampoules and vials containing liquid specimens not subject to freeze-drying.
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