Biotech Processes. [David M. Fetterolf

Lyophilization

David M. Fetterolf

“Biotech Processes” discusses fundamental information three stages of process validation: process defini- about biotechnology manufacturing useful to practitio- tion, process qualification, and continued process ners in validation and compliance. Reader comments, verification. questions, and suggestions are needed to make this column a useful resource for daily work applications. INTRODUCTION The key objective for this column: Useful information. Lyophilization, more commonly known as “freeze-dry- Contact column coordinator David Fetterolf at dfet- ing,” is a means of dehydration (desiccation) used in the [email protected] or journal coordinating edi- food, chemicals, pharmaceutical, and biotechnology tor Susan Haigney at [email protected] with industries. In all cases, lyophilization is used to improve comments or suggestions for future discussion topics. the stability of a perishable product or make the product easier to store or transport. In the biotechnology indus- KEY POINTS try, lyophilization is used as a final processing step for The following key points are discussed in this article: purified active pharmaceutical ingredients (APIs) or drug • Lyophilization, or freeze-drying, is used to remove products to stabilize the protein for long-term storage. moisture by sublimation Freeze-drying is a process that removes water by first • Products are lyophilized to increase shelf life freezing the material within a lyophilizer. The ambient • Freeze-dried products are reconstituted with water pressure is then reduced and the temperature is slowly at time of use increased within the lyophilization chamber to allow • Lyophilization processes are based on the physi- frozen water to sublimate (i.e., move from the solid phase cal properties of water, as described by the phase directly to gas). Many food products (e.g., coffee, fruits, diagram vegetables, meats, and ice cream) can be freeze-dried and • Sublimation is effected by control of product tem- subsequently stored at room temperature. The resulting perature and pressure within the lyophilization product generally retains its original shape and is much equipment lighter and easier to carry. For example, hikers frequently • There are four major steps to the lyophilization pack freeze-dried food to reduce weight in their packs. process: formulation/filling, freezing, primary The freeze-dried products are easily reconstituted with drying, and secondary drying water. Freeze-drying is also used to preserve museum • The major components of a lyophilizer are the artifacts, remove moisture, and prevent degradation and chamber, condenser, and vacuum pump. mold growth. Similarly, in the biotechnology industry, • Freeze-drying is ancient technology, but lyophi- protein products, antibodies, oligonucleotides, and vac- lizers have only been around for approximately cines are lyophilized to increase the shelf life by reduc- 100 years ing the risk of degradation during storage. Again, these • Lyophilizers are qualified by typical installation products are much lighter and take up much less space, qualification (IQ), operational qualification (OQ), which make them easier to store and ship. The end user and performance qualification (PQ) protocols. (i.e., doctor, patient, downstream manufacturer, etc.) Lyophilization processes are qualified by the simply reconstitutes the freeze-dried powder prior to

For more Author ABOUT THE AUTHOR information, David M. Fetterolf is a consultant with BioTechLogic, Inc. He provides manufacturing and CMC go to support for clients with biopharmaceutical products from development through commercial launch. gxpandjvt.com/bios [David can be reached by e-mail at [email protected].

18 Journal of Validation Technology [Winter 2010] ivthome.com David M. Fetterolf, Coordinator.

Figure 1: Phase diagram for water. injection or other use. This article discusses the funda- mental 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.

PHASE DIAGRAMS The principles of lyophilization are based on the physical properties of water that are illustrated by the phase dia- gram 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 lyophiliza- tion process, the temperature and pressure conditions within the lyophilizer are controlled to enable the sub- limation 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 sub- stance provides information on its state as a function and pressure enable sublimation. In sublimation, frozen of temperature and pressure. In Figure 1, temperature water is converted directly to water vapor gas, avoiding the is on the x-axis, with values ranging from below 0°C to water liquid state. The following provides an example of a above 100°C. Pressure is on the y-axis, with values from lyophilization process for a product dissolved in water. The an absolute vacuum (0 mm Hg or 0 microns) to beyond process begins at ambient temperature and pressure and 760 mm Hg, or atmospheric pressure (760,000 microns). proceeds with changes to each paramater, as follows: The three states of water are indicated: solid (ice), liquid • Atmospheric pressure and room temperature. Prod- (water), and gas (water vapor). The lines between each uct in solution is aseptically filled into vials. Water phase represent equilibrium conditions. The following is in the liquid state. are phases of water at specific pressures as temperature • Atmospheric pressure and temperature lowered to is increased, as described in Figure 1: -10°C. Product freezes to ice. • Pressure 760 mm Hg or atmospheric pressure (1 • Pressure is reduced to approximately 4 mm Hg and atm). We know water freezes, and ice thaws, at temperature remains below 0°C. Product remains 0°C. Between 0°C and 100°C, water is liquid. At as solid ice. 100 °C, water boils and water vapor condenses. • Pressure maintained at 4 mm Hg and temperature • Pressure 380 mm Hg, or midway down the pres- increased to 20°C or higher. Water begins to sublime sure scale. As temperature increases, ice melts directly into the gaseous state. Transition to the liquid at slightly above 0°C. As temperature increases state does not occur at this pressure and temperature. further, water boils at approximately 82°C. Water continues to sublime until all ice has subli- • Pressure 4.58 mm Hg. As temperature increases mated. This is termed “primary drying.” to 0.0098°C, ice, water, and water vapor exist in • Temperature is continually increased until all adsorbed equilibrium. This is known as the triple point moisture is eliminated. Pressure may or may not be of water. increased. This is termed “secondary drying.” • Pressure below 4.58 mm Hg. As temperature increases, solid ice converts directly to water vapor These conditions enable the dosage form to maintain gas. Liquid water does not exist at these pressure its integrity without losses due to boiling. There is no and temperature conditions. liquid state in the sublimation process. Figure 2 shows the stepwise description of the example lyophilization Water Phase Diagram And The process described previously. As you can see, the steps Lyophilization Process form a curve around the triple point, thus avoiding the The various steps in lyophilization can be plotted on the liquid state of water. water phase diagram to understand how temperature gxpandjvt.com Journal of Validation Technology [Winter 2010] 19 Biotech Processes.

Figure 2: Example lyophilization process. 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. Bulk- ing 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 proper- ties 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 excipi- ents 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, LYOPHILIZATION PROCESS FOR sizes, and colors. Although not always, vials are typically BIOPHARMACEUTICAL PRODUCTS used if no further processing is needed. Once the product There are four major stages in the lyophilization process is filled into the vials, each vial is partially stoppered (i.e., for biopharmaceutical products, as follows: not fully pushed into place) such that the vial is vented • Formulation/filling so water vapor can escape during lyophilization. Other • Freezing types of containers such as trays can be used to lyophi- • Primary drying lize large quantities of product. Trays are typically used • Secondary drying. when lyophilization is an intermediate processing step. Regardless of the container choice, aseptic technique is Stage 1. Formulation And Filling used during filling and lyophilization processes if the As briefly discussed in a previous article in this series drug product is a parenteral. (1), formulation is exchanging the product matrix (buffer) into the final buffer or adding water or other Stage 2. Freezing raw materials (e.g., excipients) to create the final pre- Once the product is placed into the lyophilizer chamber, lyophilized product in solution. For drug products, the product (inside the vials or trays) is frozen. This is done formulation prior to lyophilization usually includes by cooling the lyophilizer shelves, which are in contact with the addition of water (i.e., water for injection) and the product container, to freeze the contents. This freez- excipients to the drug substance to obtain the desired ing process separates the water from the product, and also product concentration. If the lyophilization is to decreases chemical activity of the product. What results is occur in the vials that will be used for administration an amorphous (without any clear shape) solid product and to the patient, the intent of the formulation process water crystals. Typical shelf temperatures for lyophilization is to create the actual final drug formulation, which of protein products are around -40°C or lower. is a suitable matrix for stabilization of the protein. From the simple phase diagram shown in Figure The lyophilization process will then remove the 3, lowering the temperature of a liquid at constant water, which is then re-added just prior to use (i.e., pressure results in a phase change from liquid to solid reconstitution). (point 1 to point 2). Many times, chemicals that do not increase or It is clear that the temperature of the shelves, type of decrease product efficacy are added to the formulation container, amount of product in each vial or tray, height matrix to protect the product during each stage of the of liquid, etc. can impact the rate of freezing, which, in lyophilization process and/or during long-term stor- turn, impacts the cake form and structure (i.e., morphol- age. These types of chemicals, also called stabilizers ogy), drying rate, and (in some cases) product stability. (2), can prevent unwanted changes in the drug, such In general, fast rates of freeze are harder to control, and

20 Journal of Validation Technology [Winter 2010] ivthome.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 pro- from the cake, the temperature will slowly increase duce a finer structure, which results in a slower rate to the temperature of the shelf. An equivalent tem- of water transfer during the subsequent drying step. perature of the product and shelves is a signal that There is also some evidence that the higher surface area primary drying has ended. resulting from smaller crystals can lead to increased As mentioned previously, the drying and heating product degradation. These types of consequences rate must be carefully controlled. The heating of the (i.e., increased vs. decreased cycle time, potential deg- product must be kept below the glass transition tem- radation, etc.) are kept in mind when designing and perature (Tg) of the solution, which is the point in the optimizing the overall lyophilization cycle. freezing process at which the physical state changes from an elastic liquid to a brittle but amorphous solid Stage 3. Primary Drying glass and the point at which ice formation ceases (3). After freezing, two types of water exist within the If heat is applied too quickly or to temperatures above product; these include the following: Tg, the cake can melt or collapse, which could lead to • Mobile water free from the amorphous solid degradation and aesthetic issues mentioned previously • Bound/trapped water within the amorphous solid. (4). The cake could be difficult to reconstitute at a later point, as well. Although rare, drying the product The intent of primary drying is to remove the too fast at this stage could result in the product being mobile water from the product, which is accomplished carried off with the exiting water vapor. by lowering the lyophilizer chamber pressure (i.e., pulling a vacuum). By the phase diagram in Figure Stage 4. Secondary Drying 4, one can see that lowering the pressure at constant At the end of primary drying, there is no mobile water temperature results in a phase change from solid to left in the product. However, the water trapped within gas (i.e., sublimation–point 2 to point 3). Sublima- the amorphous solid is more difficult to remove. To tion at atmospheric conditions is commonly seen do this, temperature is increased at the low pressures when frozen carbon dioxide (dry ice) is left at room used for primary drying. Again, as in primary drying, temperature. The solid turns to a gas without first it is important that the temperature is not increased changing into the liquid form. too quickly, and that it stays below the Tg, which Because the product temperature decreases during (coincidentally) increases as water is removed (3). the sublimation process, heat is added via the lyophi- This results in a porous, fluffy cake with little residual lizer shelves to keep the cake at a relatively constant moisture. Increasing the temperature too quickly, or temperature—that is, the shelves are providing the above Tg, could result in collapse and make reconsti- heat of sublimation. However, as water is removed tution difficult (5). gxpandjvt.com Journal of Validation Technology [Winter 2010] 21 Biotech Processes.

Secondary drying can be a lengthy process, lasting To fully define the lyophilization process, devel- up to several days. Typical residual moisture lev- opment studies are performed to characterize the els after secondary drying are less than 1%, but are freeze-drying parameters. A risk-assessment is then dependent on the needs of each individual product. performed to determine potentially critical param- Karl Fisher Titration (ASTM E203-08) (6) is the most eters, which are then carried into a DOE framework common test used to determine residual moisture to fully define the design and control spaces. Typical levels. Because the dry product will act as a sponge product quality attributes that are monitored during and pull water from ambient conditions, the product these types of studies include, but are not limited to, containers are closed/capped as soon after secondary residual moisture, potency, purity, etc. at all places drying as possible. Most lyophilizers have the capabil- within the lyophilization chamber (i.e., product uni- ity of pushing stoppers into place while the product formity). Then, the lyophilization process is qualified is still under vacuum. If using trays, they are sealed and further monitored and evaluated during con- immediately upon release of the vacuum and product tinued process verification; both under prospective removal from the lyophilizer chamber. protocols.

Lyophilization Cycle Optimization CONCLUSION The four stages of lyophilization described previously All four stages of the lyophilization process (i.e., are intended to provide a basic understanding of the formulation, freezing, primary drying, and second- principles behind lyophilization. The discussion ary drying) are equally important to the successful of the addition of annealing steps (e.g., to modify performance of any lyophilization process and are water crystal structure), solvents, inert gases, tem- instrumental in producing a stable product for long- perature optimization, pressure optimization, and term storage. Any change in one step has the potential other parameters during lyophilization is beyond the to greatly impact the subsequent steps, overall prod- scope of this article; however, these are common ways uct quality, or final moisture level. It is important to aid in the optimization of moisture removal. See to understand the basic principles of lyophilization the articles listed in the references and recommended and then apply them to each individual product and resources sections for more information. lyophilization process. Proper process qualification and continuous process monitoring can then be per- VALIDATION OF LYOPHILIZATION formed to ensure proper validation. EQUIPMENT AND PROCESSES A freeze-dryer, or lyophilizer, is made up of a chamber, condenser, and vacuum pump. The basics of freeze- REFERENCES drying food were used by ancient Peruvian Incas; 1. Houp, Rachel C., “Biotech Processes: Ultrafiltration/ however, laboratory versions of lyophilizers have only Diafiltration,” Journal of Validation Technology, Autumn been around for approximately 100 years. As you can 2009. imagine, designs of laboratory and manufacturing-scale 2. Carpenter, J.F., Pikal, M.J., Chang, B.S., and Randoloph, lyophilizers have greatly evolved over the last century. T.W., “Rational Design of Stable Lyophilized Protein Equipment has increased in complexity, which makes Formulations: Some Practical Advice,” Pharmaceutical validation of the lyophilization equipment and process Research, Vol. 14, No. 8, 1997. a time-consuming activity. In addition to the cool- 3. BioPharm International, “Guide to Formulation, Fill, ing (freezing), heating, and vacuum control functions and Finish,” The BioPharm International Guide, August described in the previous sections, many freeze-dryers 2004. now incorporate computerized control and monitoring 4. FTS Systems, Inc., “Basic Theory of Freeze Drying,” systems, clean-in-place (CIP), and sterilize-in-place Dura-Dry MP Instruction Manual, February 1991. (SIP) functionality. The reliability and reproducibil- 5. Virtis, “Freeze Drying 101,” http://www.virtis.com/lit- ity of these functions must be validated (through IQ, erature/freeze101.jsp. OQ, and PQ protocols) to ensure consistent moisture 6. ASTM International, “ASTM E203-08, Standard Test removal and overall product quality; therefore, typical Method for Water Using Volumetric Karl Fischer Titra- validation of a lyophilizer involves multiple protocols tion,” http://www.astm.org/Standards/E203.htm. (or multiple sections) focusing on verifying the per- formance of each function.

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RECOMMENDED RESOURCES GLOSSARY Jennings, T.A., Lyophilization—Introduction and Basic Prin- Glass transition temperature (Tg). The point ciples, CRC Press LLC, Boca Raton, Florida, 1999. in the freezing process at which the physical state Carpenter, J.F. and Chang, B.S., “Lyophilization of Protein changes from an elastic liquid to a brittle but amor- Pharmaceuticals, Biotechnology and Biopharmaceuti- phous solid glass. This is the point at which ice for- cal Manufacturing, Processing and Preservation,” Inter- mation ceases. pharm Press, Buffalo Grove, IL, pp. 199 – 264, 1996. Karl Fischer Titration. Most common method by Carpenter, J.F. and Manning, M.C. (editors), Rational Design of which residual moisture is determined in a product Stable Protein Formulations: Theory and Practice (Pharmaceuti- sample. cal Technology), Springer, 1st Edition, April 30, 2002. Phase diagram. Information about the solid, liquid, Tang, X. and Pikal, M.J., “Design of Freeze Drying Processes and gaseous states of a substance as a function of for Pharmaceuticals: Practical Advice,” Pharmaceutical Re- temperature and pressure. search, Vol. 21, No. 2, February 2004. “Product Technologies for Lyophilization,” Genetic Engineer- ARTICLE ACRONYM LISTING ing and Biotechnology News, November 15, 2006. JVT CIP Clean in Place DOE Design of Experiments IQ Installation Qualification OQ Operational Qualification PQ Performance Qualification SIP Sterilize in Place Tg Glass Transition Temperature

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