Timothy E. Pearcy
For nearly 30 years, lyophilization has been used to stabilize many types of chemical components used in IVDs. 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.
Three incompatible components of a clinical chemistry assay processed as LyoSpheres and ready for reconstitution by sample to measure analyte level spectrophotometrically.
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 (see sidebar, below).
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.
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 Capability. 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.
Vapor Transmission. Water-vapor transmission rates are related to the volume of water vapor and air that can travel through the walls and seal of a package. As a general rule, the more porous a packaging material and the weaker the seal on the package, the greater the potential for compromising protected lyophilized components. As soon as a lyophilized product is exposed to moisture levels higher than approximately 3%, its stability has been compromised.
The problems associated with high vapor-transmission rates are compromised shelf stability and jeopardized product performance.
Glass Serum Vials
The glass serum vial with a slotted stopper is an ideal container for lyophilizing IVD reagents because it meets all three requirements for successful freeze-drying. To begin with, such containers are made of a material, glass, that conducts heat well. In fact, the heat conductivity property of glass is one of the main reasons it is used for serum vials, which were designed specifically to meet the heat and mass transfer requirements of a lyophilizer.
|A single tube containing eight different-sized LyoSpheres. From left to right: (a) 250 µl is 0.907 cm diam; (b) 200 µl is 0.845 cm diam; (c) 150 µl is 0.723 cm diam; (d) 120 µl is 0.685 cm diam; (e) 100 µl is 0.635 cm diam; (f) 6. 60 µl is 0.526 cm diam;(g) 40 µl is 0.444 cm diam; (h) 25 µl is 0.376 cm diam.|
Another key advantage of the conventional serum vial and lyophilization stopper is that it allows the dried chemistries within it to be sealed inside a freeze dryer. This capability enables the manufacturer to attain and maintain the requisite residual moisture level of 3% or less, thereby protecting sensitive biologicals from exposure to excessive moisture levels.
Glass serum vials are also capable of limiting the amount of vapor that may enter the vial, thereby protecting lyophilized products from the transmission of water vapor or other forms of moisture or further contaminants. A glass serum vial sealed at a vacuum level of approximately 20 atm with a slotted stopper held in a place with an aluminum compression crimp seal has a leak rate low enough to provide years of shelf stability. Few other containers have a leak rate as low as a properly sealed serum vial.
For all of these reasons, the glass serum vial has become the traditional container of choice for IVD manufacturers that produce lyophilized products. This type of process-driven container continues to serve IVD manufacturers well for many products.
However, the glass serum vial with slotted stopper cannot meet all of the many needs that companies have to provide diverse and unique delivery formats. Lyophilizing inside of a serum vial means that the vial also serves as the product's delivery device, since there is no practical means of extracting a lyophilized cake from a vial and then repackaging it into an another delivery device. The result is something akin to serving pasta in the pot in which it was boiled; effective, but not especially elegant.
Nonstandard Lyophilization Containers
Many diagnostic tests are required to be simple, easy to use, self-contained, and mistakeproof. But using a serum vial is a multistep process in itself. It is no wonder that IVD manufacturers have therefore explored alternative types of containers for the processing and delivery of lyophilized reagents. From the manufacturers' point of view, the package in which a reagent is delivered should not be restricted to the container in which it was processed. Rather, such packaging should be designed to enhance the usability of the product.
Virtually any container other than a serum vial with a slotted stopper can be defined as a nonstandard lyophilization container. Some IVD companies are attempting to use such nonstandard containers for lyophilizing products, but the alternatives that have so far been explored have generally proven inadequate to lyophilize, seal, and house lyophilized chemistries. Many have poor heat-transfer characteristics, cannot be sealed within a lyophilizer, or offer unacceptably high moisture-vapor transmission rates. As a result of such inadequacies among nonstandard containers, some products may inadvertently be produced, distributed, and sold as lyophilized when in fact they do not possess the stability and quality of a properly lyophilized product.
Like the serum vial, any other container used in the lyophilization process must provide good heat transfer, allow good vapor transfer during lyophilization, have the capability to be sealed inside the freeze dryer, and provide a high-quality vapor barrier for its lyophilized contents. A number of IVD companies are already processing their chemistries in containers not specifically designed for lyophilization, such as chromatography vials, 96-well microtiter plates, microwell tubes, plastic reservoirs, tissue-culture tubes, sample cups, foil-sealed pouches, blood-collection tubes, squeeze-dropper tubes, custom-designed plastic-formed devices, screw- and snap-cap containers, and glass and plastic ampules.
There is substantial motivation for companies to develop or use such nonstandard lyophilization containers. Companies develop new products, need to reduce the costs of established products, need to interface with new diagnostic instruments, or differentiate themselves from their competitors. However, manufacturers must also account for the dry, clean, and protective environment that must be maintained to preserve the integrity and stability of a lyophilized component. Many companies using nonstandard delivery devices believe they are handling their lyophilized components acceptably. Unfortunately, if a nonstandard lyophilization container is used, it is likely that overall quality will be compromised.
One format that has proven attractive to manufacturers over the years is that of a self-contained lyophilized bead. When lyophilized as beads, immunochemical and clinical chemistry reagents requiring two or three components that are incompatible as liquids because of their pH level or reaction to one another can coexist compatibly. Because such lyophilized beads can be stable and nonreactive, chemicals can be packaged together in the same delivery device. This allows for a great deal more flexibility in designing products than when there is a need to incorporate disparate chemistries within a single glass serum vial. However, the goal of producing such lyophilized beads has proven an elusive one, and manufacturers have encountered considerable difficulties in developing a freeze-drying process suitable for the task.
A sample of the many different delivery devices that can be used for packaging IVD reagents and other chemicals processed as LyoSpheres.
One of the problems that manufacturers have faced is how to control the variable sizes and volumes of the beads, which can translate into inconsistent aliquots of lyophilized chemicals. In addition, manufacturers have had difficulty developing adequate procedures and equipment to maintain the integrity of lyophilized products outside of the lyophilizer's protective environment.
As a result of such problems, early attempts to produce consistent, accurate, and stable beads of lyophilized chemicals on a commercial scale proved unsuccessful. Some companies attempted to commercialize the technology for producing lyophilized beads before a complete, comprehensive, and tested set of procedures and equipment had been developed. Their efforts resulted in product failures in the field because of inconsistent final moisture levels in the batches of beads, and created skepticism about the potential of the technology.
Refined Bead Technology
The technology that allows lyophilized beads to be processed and packaged inside a variety of containers has since been refined. Biolyph (Minneapolis) has developed the processing equipment and procedures for converting solutions of unstable liquid suspensions into stable beads, trade-named LyoSpheres, and for packaging them in almost any type of delivery device. The company has demonstrated the viability of its technology by processing and packaging more than 45 million diagnostic tests to detect human disease, environmental contaminants, and agricultural pathogens in a wide variety of delivery devices.
Biolyph's specialized equipment enables the processing and packaging of lyophilized beads without jeopardizing the stable environment in which they are processed. This includes their dispensing, lyophilization, postlyophilization handling, and packaging.
The company's refined process results in small-diameter beads, each containing a precise amount of concentrated liquid chemical dispensed and lyophilized as a sphere. The chemicals are formulated exactly as if they were being lyophilized conventionally in a serum vial, except for their concentrations. Ideally, each bead contains no more than 250 µl of liquid volume. When converting products that are currently processed in serum vials, a concentration step is required to reduce the total volume of liquid in order to accommodate the bead's size requirements.
A 25-µl LyoSphere has a diameter of approximately 0.376 cm, while a 250-µl LyoSphere has a diameter of approximately 0.907 cm. The beads do not contain any polymers or other additives—they are the suspension of the diagnostic solution by itself. Their structure comes from the solids left behind following the removal of ice through sublimation. The beads are shelf stable, consistent, and capable of being packaged under controlled conditions inside an array of delivery devices. The beads are water soluble and can be rehydrated within a few seconds.
Because lyophilized beads do not require process-driven packaging, manufacturers can select their product delivery devices according to whatever package works best for their product and market. Possibilities among existing types of packaging include crushable glass onion-skin tubes, plastic tissue-culture tubes, laminated foil pouches, standard glass or plastic serum vials, microtiter plates, squeeze-droppers, and blood-collection tubes. Manufacturers may also design their own delivery devices.
Refined technologies are enabling manufacturers to process liquid components that are incompatible or competitive with one another into individual beads, and then to package them together in a single delivery device. Containers that have previously been deemed inadequate can be used successfully for chemicals that have been lyophilized. The lyophilized beads create opportunities for manufacturers to simplify test-activation procedures and to reduce packaging costs and material waste. Using this process, manufacturers are no longer restricted to containers whose primary virtues are related to the processing requirements for the product.
The key benefits of this technology include reduced manufacturing costs, elimination of traditional packaging restrictions, opportunities for creating new product-delivery formats, and brand differentiation. Cost savings can be achieved by eliminating separate packaging for incompatible chemistries. Meanwhile, product development and marketing personnel can design optimized delivery devices for a particular test and its end-users.
Traditional lyophilization is a complex process that requires a careful balancing of product, equipment, and processing techniques. Below are the basic equipment and processing elements of traditional 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 used in IVD products 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.
Processing. 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.
The key components of a lyophilizer and each component's function, along with the five significant stages of processing that a standard serum vial goes through.
Timothy E. Pearcy is the founder and managing director of Biolyph (Minneapolis), a contract service company specializing in product lyophilization and packaging.