Lyophilization
Transcription
Lyophilization
[ Biotech Processes. David M. Fetterolf Lyophilization David M. Fetterolf “Biotech Processes” discusses fundamental information about biotechnology manufacturing useful to practitioners in validation and compliance. Reader comments, questions, and suggestions are needed to make this column a useful resource for daily work applications. The key objective for this column: Useful information. Contact column coordinator David Fetterolf at [email protected] or journal coordinating editor Susan Haigney at [email protected] with comments or suggestions for future discussion topics. KEY POINTS The following key points are discussed in this article: •Lyophilization, or freeze-drying, is used to remove moisture by sublimation •Products are lyophilized to increase shelf life •Freeze-dried products are reconstituted with water at time of use •Lyophilization processes are based on the physical properties of water, as described by the phase diagram •Sublimation is effected by control of product temperature and pressure within the lyophilization equipment •There are four major steps to the lyophilization process: formulation/filling, freezing, primary drying, and secondary drying •The major components of a lyophilizer are the chamber, condenser, and vacuum pump. •Freeze-drying is ancient technology, but lyophilizers have only been around for approximately 100 years •Lyophilizers are qualified by typical installation qualification (IQ), operational qualification (OQ), and performance qualification (PQ) protocols. Lyophilization processes are qualified by the For more Author information, go to gxpandjvt.com/bios 18 Journal three stages of process validation: process definition, process qualification, and continued process verification. INTRODUCTION Lyophilization, more commonly known as “freeze-drying,” is a means of dehydration (desiccation) used in the food, chemicals, pharmaceutical, and biotechnology industries. In all cases, lyophilization is used to improve the stability of a perishable product or make the product easier to store or transport. In the biotechnology industry, lyophilization is used as a final processing step for purified active pharmaceutical ingredients (APIs) or drug products to stabilize the protein for long-term storage. Freeze-drying is a process that removes water by first freezing the material within a lyophilizer. The ambient pressure is then reduced and the temperature is slowly increased within the lyophilization chamber to allow frozen water to sublimate (i.e., move from the solid phase directly to gas). Many food products (e.g., coffee, fruits, vegetables, meats, and ice cream) can be freeze-dried and subsequently stored at room temperature. The resulting product generally retains its original shape and is much lighter and easier to carry. For example, hikers frequently pack freeze-dried food to reduce weight in their packs. The freeze-dried products are easily reconstituted with water. Freeze-drying is also used to preserve museum artifacts, remove moisture, and prevent degradation and mold growth. Similarly, in the biotechnology industry, protein products, antibodies, oligonucleotides, and vaccines are lyophilized to increase the shelf life by reducing the risk of degradation during storage. Again, these products are much lighter and take up much less space, which make them easier to store and ship. The end user (i.e., doctor, patient, downstream manufacturer, etc.) simply reconstitutes the freeze-dried powder prior to [ ABOUT THE AUTHOR David M. Fetterolf is a consultant with BioTechLogic, Inc. He provides manufacturing and CMC support for clients with biopharmaceutical products from development through commercial launch. David can be reached by e-mail at [email protected]. of Validation T echnology [Winter 2010] iv thome.com David M. Fetterolf, Coordinator. injection or other use. This article discusses the fundamental principles behind lyophilization and the specific stages of the lyophilization cycle. It also briefly describes the types of equipment used in lyophilization and the types of validation studies that are typically performed for this unit operation. Figure 1: Phase diagram for water. PHASE DIAGRAMS The principles of lyophilization are based on the physical properties of water that are illustrated by the phase diagram for water. A phase diagram for a substance describes the solid, liquid, and gaseous states of a substance as a function of temperature and pressure. In the lyophilization process, the temperature and pressure conditions within the lyophilizer are controlled to enable the sublimation of water and its removal from the dosage form. Water is removed from the dosage form as a gas. Figure 1 provides the phase diagram for water. As previously stated, the phase diagram for a substance provides information on its state as a function of temperature and pressure. In Figure 1, temperature is on the x-axis, with values ranging from below 0°C to above 100°C. Pressure is on the y-axis, with values from an absolute vacuum (0 mm Hg or 0 microns) to beyond 760 mm Hg, or atmospheric pressure (760,000 microns). The three states of water are indicated: solid (ice), liquid (water), and gas (water vapor). The lines between each phase represent equilibrium conditions. The following are phases of water at specific pressures as temperature is increased, as described in Figure 1: •Pressure 760 mm Hg or atmospheric pressure (1 atm). We know water freezes, and ice thaws, at 0°C. Between 0°C and 100°C, water is liquid. At 100 °C, water boils and water vapor condenses. •Pressure 380 mm Hg, or midway down the pressure scale. As temperature increases, ice melts at slightly above 0°C. As temperature increases further, water boils at approximately 82°C. •Pressure 4.58 mm Hg. As temperature increases to 0.0098°C, ice, water, and water vapor exist in equilibrium. This is known as the triple point of water. •Pressure below 4.58 mm Hg. As temperature increases, solid ice converts directly to water vapor gas. Liquid water does not exist at these pressure and temperature conditions. Water Phase Diagram And The Lyophilization Process The various steps in lyophilization can be plotted on the water phase diagram to understand how temperature gxpandjv t.com and pressure enable sublimation. In sublimation, frozen water is converted directly to water vapor gas, avoiding the water liquid state. The following provides an example of a lyophilization process for a product dissolved in water. The process begins at ambient temperature and pressure and proceeds with changes to each paramater, as follows: •Atmospheric pressure and room temperature. Product in solution is aseptically filled into vials. Water is in the liquid state. •Atmospheric pressure and temperature lowered to -10°C. Product freezes to ice. •Pressure is reduced to approximately 4 mm Hg and temperature remains below 0°C. Product remains as solid ice. •Pressure maintained at 4 mm Hg and temperature increased to 20°C or higher. Water begins to sublime directly into the gaseous state. Transition to the liquid state does not occur at this pressure and temperature. Water continues to sublime until all ice has sublimated. This is termed “primary drying.” •Temperature is continually increased until all adsorbed moisture is eliminated. Pressure may or may not be increased. This is termed “secondary drying.” These conditions enable the dosage form to maintain its integrity without losses due to boiling. There is no liquid state in the sublimation process. Figure 2 shows the stepwise description of the example lyophilization process described previously. As you can see, the steps form a curve around the triple point, thus avoiding the liquid state of water. Journal of Validation T echnology [Winter 2010] 19 Biotech Processes. Figure 2: Example lyophilization process. LYOPHILIZATION PROCESS FOR BIOPHARMACEUTICAL PRODUCTS There are four major stages in the lyophilization process for biopharmaceutical products, as follows: •Formulation/filling •Freezing •Primary drying •Secondary drying. Stage 1. Formulation And Filling As briefly discussed in a previous article in this series (1), formulation is exchanging the product matrix (buffer) into the final buffer or adding water or other raw materials (e.g., excipients) to create the final prelyophilized product in solution. For drug products, formulation prior to lyophilization usually includes the addition of water (i.e., water for injection) and excipients to the drug substance to obtain the desired product concentration. If the lyophilization is to occur in the vials that will be used for administration to the patient, the intent of the formulation process is to create the actual final drug formulation, which is a suitable matrix for stabilization of the protein. The lyophilization process will then remove the water, which is then re-added just prior to use (i.e., reconstitution). Many times, chemicals that do not increase or decrease product efficacy are added to the formulation matrix to protect the product during each stage of the lyophilization process and/or during long-term storage. These types of chemicals, also called stabilizers (2), can prevent unwanted changes in the drug, such 20 Journal of Validation T echnology [Winter 2010] as unfolding, during lyophilization. Commonly used stabilizers for biologics are sugars and glycols. At the end of the lyophilization process, biotech drug products resemble a fluffy white powder, or “cake.” Because the aesthetics of the cake sometimes play an important role in product marketability, bulking agents are often added to make the cake appear fluffier. Bulking agents can also help to prevent “collapse” of the drug product, which can occur if the product is heated too rapidly during the drying stages. These bulking agents are not intended to change the chemical properties of the product. Some examples of bulking agents typically used in the biopharmaceutical industry are mannitol, dextran, and polyethylene glycol. Once the product is in the proper form and all excipients are added, the last step prior to placing the material into the lyophilizer is filling the product into the proper container. For biotechnology products, the containers are usually glass vials, which come in a variety of shapes, sizes, and colors. Although not always, vials are typically used if no further processing is needed. Once the product is filled into the vials, each vial is partially stoppered (i.e., not fully pushed into place) such that the vial is vented so water vapor can escape during lyophilization. Other types of containers such as trays can be used to lyophilize large quantities of product. Trays are typically used when lyophilization is an intermediate processing step. Regardless of the container choice, aseptic technique is used during filling and lyophilization processes if the drug product is a parenteral. Stage 2. Freezing Once the product is placed into the lyophilizer chamber, the product (inside the vials or trays) is frozen. This is done by cooling the lyophilizer shelves, which are in contact with the product container, to freeze the contents. This freezing process separates the water from the product, and also decreases chemical activity of the product. What results is an amorphous (without any clear shape) solid product and water crystals. Typical shelf temperatures for lyophilization of protein products are around -40°C or lower. From the simple phase diagram shown in Figure 3, lowering the temperature of a liquid at constant pressure results in a phase change from liquid to solid (point 1 to point 2). It is clear that the temperature of the shelves, type of container, amount of product in each vial or tray, height of liquid, etc. can impact the rate of freezing, which, in turn, impacts the cake form and structure (i.e., morphology), drying rate, and (in some cases) product stability. In general, fast rates of freeze are harder to control, and iv thome.com David M. Fetterolf, Coordinator. Figure 3: Phase diagram for freezing. Figure 4: Phase diagram for drying. therefore, are more variable. They also tend to produce a finer structure, which results in a slower rate of water transfer during the subsequent drying step. There is also some evidence that the higher surface area resulting from smaller crystals can lead to increased product degradation. These types of consequences (i.e., increased vs. decreased cycle time, potential degradation, etc.) are kept in mind when designing and optimizing the overall lyophilization cycle. from the cake, the temperature will slowly increase to the temperature of the shelf. An equivalent temperature of the product and shelves is a signal that primary drying has ended. As mentioned previously, the drying and heating rate must be carefully controlled. The heating of the product must be kept below the glass transition temperature (Tg) of the solution, which is the point in the freezing process at which the physical state changes from an elastic liquid to a brittle but amorphous solid glass and the point at which ice formation ceases (3). If heat is applied too quickly or to temperatures above Tg, the cake can melt or collapse, which could lead to degradation and aesthetic issues mentioned previously (4). The cake could be difficult to reconstitute at a later point, as well. Although rare, drying the product too fast at this stage could result in the product being carried off with the exiting water vapor. Stage 3. Primary Drying After freezing, two types of water exist within the product; these include the following: •Mobile water free from the amorphous solid •Bound/trapped water within the amorphous solid. The intent of primary drying is to remove the mobile water from the product, which is accomplished by lowering the lyophilizer chamber pressure (i.e., pulling a vacuum). By the phase diagram in Figure 4, one can see that lowering the pressure at constant temperature results in a phase change from solid to gas (i.e., sublimation–point 2 to point 3). Sublimation at atmospheric conditions is commonly seen when frozen carbon dioxide (dry ice) is left at room temperature. The solid turns to a gas without first changing into the liquid form. Because the product temperature decreases during the sublimation process, heat is added via the lyophilizer shelves to keep the cake at a relatively constant temperature—that is, the shelves are providing the heat of sublimation. However, as water is removed gxpandjv t.com Stage 4. Secondary Drying At the end of primary drying, there is no mobile water left in the product. However, the water trapped within the amorphous solid is more difficult to remove. To do this, temperature is increased at the low pressures used for primary drying. Again, as in primary drying, it is important that the temperature is not increased too quickly, and that it stays below the Tg, which (coincidentally) increases as water is removed (3). This results in a porous, fluffy cake with little residual moisture. Increasing the temperature too quickly, or above Tg, could result in collapse and make reconstitution difficult (5). Journal of Validation T echnology [Winter 2010] 21 Biotech Processes. Secondary drying can be a lengthy process, lasting up to several days. Typical residual moisture levels after secondary drying are less than 1%, but are dependent on the needs of each individual product. Karl Fisher Titration (ASTM E203-08) (6) is the most common test used to determine residual moisture levels. Because the dry product will act as a sponge and pull water from ambient conditions, the product containers are closed/capped as soon after secondary drying as possible. Most lyophilizers have the capability of pushing stoppers into place while the product is still under vacuum. If using trays, they are sealed immediately upon release of the vacuum and product removal from the lyophilizer chamber. To fully define the lyophilization process, development studies are performed to characterize the freeze-drying parameters. A risk-assessment is then performed to determine potentially critical parameters, which are then carried into a DOE framework to fully define the design and control spaces. Typical product quality attributes that are monitored during these types of studies include, but are not limited to, residual moisture, potency, purity, etc. at all places within the lyophilization chamber (i.e., product uniformity). Then, the lyophilization process is qualified and further monitored and evaluated during continued process verification; both under prospective protocols. Lyophilization Cycle Optimization CONCLUSION The four stages of lyophilization described previously are intended to provide a basic understanding of the principles behind lyophilization. The discussion of the addition of annealing steps (e.g., to modify water crystal structure), solvents, inert gases, temperature optimization, pressure optimization, and other parameters during lyophilization is beyond the scope of this article; however, these are common ways to aid in the optimization of moisture removal. See the articles listed in the references and recommended resources sections for more information. All four stages of the lyophilization process (i.e., formulation, freezing, primary drying, and secondary drying) are equally important to the successful performance of any lyophilization process and are instrumental in producing a stable product for longterm storage. Any change in one step has the potential to greatly impact the subsequent steps, overall product quality, or final moisture level. It is important to understand the basic principles of lyophilization and then apply them to each individual product and lyophilization process. Proper process qualification and continuous process monitoring can then be performed to ensure proper validation. VALIDATION OF LYOPHILIZATION EQUIPMENT AND PROCESSES A freeze-dryer, or lyophilizer, is made up of a chamber, condenser, and vacuum pump. The basics of freezedrying food were used by ancient Peruvian Incas; however, laboratory versions of lyophilizers have only been around for approximately 100 years. As you can imagine, designs of laboratory and manufacturing-scale lyophilizers have greatly evolved over the last century. Equipment has increased in complexity, which makes validation of the lyophilization equipment and process a time-consuming activity. In addition to the cooling (freezing), heating, and vacuum control functions described in the previous sections, many freeze-dryers now incorporate computerized control and monitoring systems, clean-in-place (CIP), and sterilize-in-place (SIP) functionality. The reliability and reproducibility of these functions must be validated (through IQ, OQ, and PQ protocols) to ensure consistent moisture removal and overall product quality; therefore, typical validation of a lyophilizer involves multiple protocols (or multiple sections) focusing on verifying the performance of each function. 22 Journal of Validation T echnology [Winter 2010] REFERENCES 1.Houp, Rachel C., “Biotech Processes: Ultrafiltration/ Diafiltration,” Journal of Validation Technology, Autumn 2009. 2.Carpenter, J.F., Pikal, M.J., Chang, B.S., and Randoloph, T.W., “Rational Design of Stable Lyophilized Protein Formulations: Some Practical Advice,” Pharmaceutical Research, Vol. 14, No. 8, 1997. 3.BioPharm International, “Guide to Formulation, Fill, and Finish,” The BioPharm International Guide, August 2004. 4.FTS Systems, Inc., “Basic Theory of Freeze Drying,” Dura-Dry MP Instruction Manual, February 1991. 5.Virtis, “Freeze Drying 101,” http://www.virtis.com/literature/freeze101.jsp. 6.ASTM International, “ASTM E203-08, Standard Test Method for Water Using Volumetric Karl Fischer Titration,” http://www.astm.org/Standards/E203.htm. iv thome.com David M. Fetterolf, Coordinator. RECOMMENDED RESOURCES Jennings, T.A., Lyophilization—Introduction and Basic Principles, CRC Press LLC, Boca Raton, Florida, 1999. Carpenter, J.F. and Chang, B.S., “Lyophilization of Protein Pharmaceuticals, Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation,” Interpharm Press, Buffalo Grove, IL, pp. 199 – 264, 1996. Carpenter, J.F. and Manning, M.C. (editors), Rational Design of Stable Protein Formulations: Theory and Practice (Pharmaceutical Technology), Springer, 1st Edition, April 30, 2002. Tang, X. and Pikal, M.J., “Design of Freeze Drying Processes for Pharmaceuticals: Practical Advice,” Pharmaceutical Research, Vol. 21, No. 2, February 2004. “Product Technologies for Lyophilization,” Genetic Engineering and Biotechnology News, November 15, 2006. JVT gxpandjv t.com GLOSSARY Glass transition temperature (Tg). The point in the freezing process at which the physical state changes from an elastic liquid to a brittle but amorphous solid glass. This is the point at which ice formation ceases. Karl Fischer Titration. Most common method by which residual moisture is determined in a product sample. Phase diagram. Information about the solid, liquid, and gaseous states of a substance as a function of temperature and pressure. ARTICLE ACRONYM LISTING CIP DOE IQ OQ PQ SIP Tg Clean in Place Design of Experiments Installation Qualification Operational Qualification Performance Qualification Sterilize in Place Glass Transition Temperature Journal of Validation T echnology [Winter 2010] 23
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Lyophilization
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