What is Lyophilization ?
Lyophilization is a process which extracts the water from foods and other products so that the foods or products remain stable and are easier to store at room temperature (ambiant air temperature).
Lyophilization is carried out using a simple principle of physics called sublimation. Sublimation is the transition of a substance from the solid to the vapour state, without first passing through an intermediate liquid phase. To extract water from foods, the process of lyophilization consists of:
- Freezing the food so that the water in the food become ice;
- Under a vacuum, sublimating the ice directly into water vapour;
- Drawing off the water vapour;
- Once the ice is sublimated, the foods are freeze-dried and can be removed from the machine.
Traditional Lyophilization Technology:
Traditional lyophilization is a complex process that requires a careful balancing of product, equipment, and processing techniques.
For nearly 30 years, lyophilization has been used to stabilize many types of chemical components. In their liquid form, many such biochemicals and chemical reagents are unstable, biologically and chemically active, temperature sensitive, and chemically reactive with one another.
Because of these characteristics, the chemicals may have a very short shelf life, may need to be refrigerated, or may degrade unless stabilized. When performed properly, the process of lyophilization solves these problems by putting reagents into a state of suspended activity.
Lyophilization gives unstable chemical solutions a long shelf life when they are stored at room temperature. The process gives a product excellent solubility characteristics, allowing for rapid reconstitution. Heat- and moisture-sensitive compounds retain their viability.
Most proteins do not denature during the process, and bacterial growth and enzyme action, which normally occur in aqueous preparations, can be eliminated. Thus, lyophilization ensures maximum retention of biological and chemical purity.
The fundamental process steps are:
- Freezing: The product is frozen. This provides a necessary condition for low temperature drying.
- Vacuum: After freezing, the product is placed under vacuum. This enables the frozen solvent in the product to vaporize without passing through the liquid phase, a process known as sublimation.
- Heat: Heat is applied to the frozen product to accelerate sublimation.
- Condensation: Low-temperature condenser plates remove the vaporized solvent from the vacuum chamber by converting it back to a solid. This completes the seperation process.
The first step in the lyophilization process is to freeze a product to solidify all of its water molecules. Once frozen, the product is placed in a vacuum and gradually heated without melting the product.
This process, called sublimation, transforms the ice directly into water vapor, without first passing through the liquid state. The water vapor given off by the product in the sublimation phase condenses as ice on a collection trap, known as a condenser, within the lyophilizer’s vacuum chamber.
To be considered stable, a lyophilized product should contain 3% or less of its original moisture content and be properly sealed.
Lyophilization Equipment :
A lyophilizer consists of a vacuum chamber that contains product shelves capable of cooling and heating containers and their contents. A vacuum pump, a refrigeration unit, and associated controls are connected to the vacuum chamber.
Chemicals are generally placed in containers such as glass vials that are placed on the shelves within the vacuum chamber.
Cooling elements within the shelves freeze the product. Once the product is frozen, the vacuum pump evacuates the chamber and the product is heated. Heat is transferred by thermal conduction from the shelf, through the vial, and ultimately into the product.
Lyophilization Container Requirements:
The container in which a substance is lyophilized must permit thermal conductivity, be capable of being tightly sealed at the end of the lyophilization cycle, and minimize the amount of moisture to permeate its walls and seal.
The enclosed reagents can only remain properly lyophilized if the container in which they are processed meets these requirements.
Lyophilization Heat Transfer:
Successful lyophilization is heavily dependent on good thermal conductivity. For this reason, containers used in the lyophilization process must be capable of meeting a number of heat-transfer requirements.
Such containers should be made of a material that offers good thermal conductivity; should provide good thermal contact with the lyophilizer shelf, which is the source of heat during processing; and should have a minimum of insulation separating the source of heat from the product requiring heating.
Poor thermal conductivity often results from the use of containers made of materials with low coefficients of heat transfer. It can also be caused by the shape, size, or quality of the container.
It may come from thermal barriers, such as excessive amounts of material, which can act as insulation, preventing energy from being transferred to the point at which the frozen ice and dried product interface.
Poor thermal conductivity often results in a product that is not successfully lyophilized. In a serum vial, the surface of the frozen cake must sublime first to allow the ice vapor to escape.
Thereafter, the sublimation front moves as the drying proceeds. Generally, the sublimation front simultaneously moves downward toward the bottom of the serum vial and inward toward the center of the frozen cake (the core).
If sublimation is not controlled—and it cannot be controlled when significant thermal barriers exist—then portions of the chemicals may actually be vacuum-dried rather than freeze-dried.
The processed product will then not possess the defined and reproducible characteristics of a properly lyophilized material, such as maximized retention of activity, optimized shelf life, rapid reconstitution, and a consistent finished cake.
Sealing Lyophilized Products:
A properly lyophilized product must be sealed within its container prior to removal from the ultradry atmosphere that exists at the end of the lyophilization cycle.
A product that has been dried to less than 3% residual moisture will, when exposed to an environment with greater than its own moisture level, absorb as much moisture as it can. The product’s quality will immediately be degraded.
All of the desirable characteristics of a lyophilized product—such as increased shelf life, enhanced chemical performance, and rapid reconstitution—will be compromised. This reintroduction of moisture can lead to loss of product, product failures in the field, false results, and even product recalls.
The most common mistake that companies make is to use packages that cannot be sealed inside the lyophilizer prior to repressurization. For example, the manufacturing process for some diagnostic products may require lyophilizing the product inside a large number of screw-top tubes.
There is no practical way to seal these tubes inside of a lyophilizer prior to terminating the batch, so the company will assemble a large production crew to apply the tops manually—often in a room incompatible with lyophilization.
The recently stabilized chemistry will be jeopardized by exposure to unacceptably high and variable moisture levels during the manual sealing process. Exposing lyophilized material to atmospheric moisture in this way may result in an unstable product.
Lyophilization has many advantages compared to other drying and preserving techniques.
- Lyophilization 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.
s one or more of the following criteria: unstable; heat liable; minimum particulates required; accurate dosing needed; quick; complete rehydration needed; high value.
Some other less common applications of lyophilization are recovery of water-damaged books and manuscripts and preservation of archaeological specimens, tissue for spare-parts surgery, museum specimens for display such as plants and animals, and vegetable matter for research programs.
Principles of Lyophilization Equipment
Wet samples can be frozen by placing them in a vacuum. The more energetic molecules escape, and the temperature of the sample falls by evaporative cooling. Eventually it freezes. About 15% of the water in the wet material is lost.
The simplest form of lyophilizer would consist of a vacuum chamber into which wet sample material could be placed, together with a means of removing water vapor so as to freeze the sample by evaporative cooling and freezing and then maintain the water-vapor pressure below the triple-point pressure. The temperature of the sample would then continue to fall below the freezing point and sublimation would slow down until the rate of heat gain in the sample by conduction, convection, and radiation was equal to the rate of heat loss as the more energetic molecules sublimed away were removed.
This simple approach creates numerous difficulties. When a material is frozen by evaporative cooling it froths as it boils. This frothing can be suppressed by low-speed centrifugation. Centrifugation also helps to dry faster by reducing material thickness and exposing a greater surface area.
An alternative is to freeze the material before it is placed under vacuum. This is commonly done with small laboratory lyophilizers where material is frozen inside a flask. The flask is then attached to a manifold connected to the ice condenser. To speed the process the material can be shell-frozen by rotating the flask in a low-temperature bath, giving a large surface area and small thickness of material.
For larger-scale equipment it is usual to place the material on product-support shelves inside the drying chamber, which can be cooled so that the material is frozen at atmospheric pressure before the vacuum is created. Without a controlled heat in[put to the sample its temperature would fall until drying was virtually at a standstill. For this reason it is usual to arrange a heat supply to the product-support shelves so that, after their initial use for freezing the product, they can be used to provide heat to replace the energy lost with the subliming water vapor and maintain the product at a constant low temperature.
One milliliter of ice produces more than 1,000,000 ml. of water vapor at typical lyophilization cycle pressures. The more energy-efficient vacuum pumps cannot handle large quantities of water vapor. For this reason it is usual to fit a refrigerated trap (called the ice condenser) between the lyophilization chamber and the vacuum pump. Modern lyophilizers incorporate refinements.
The most important are listed below:
- Separated drying chamber and ice condenser to reduce cross-contamination
- Provision of an isolation valve between chamber and ice condenser to allow for end-point determination and simultaneous loading and defrosting
- Construction of the chamber and ice condenser as pressure valves to allow for steam sterilization at 121°C or higher
- Cooling and heating of the product -support shelves by a circulating intermediate heat-exchange fluid to give even and accurate temperature
- Additional instruments to control, monitor, and record process variables
- Movable product-support shelves to close the slotted bungs used in vials and to facilitate cleaning and loading
- Automatic control system with safety interlocks and alarms, duplicated vacuum pumps, refrigeration systems, and other moving parts to enable drying to proceed without endangering the product in the event of mechanical breakdown.