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Lyophilization, or freeze-drying, is a deceptively simple process used in the manufacture of a variety of pharmaceutical products. Some examples include parenteral drugs (those administered intravenously), in vitro diagnostic products, blood serum and plasma, antibiotics, and vaccines. Generally, lyophilization is desirable when one wishes to remove excess water from a product without handling the product excessively or to increase the stability of a product prior to packaging. The process was first developed during the Second World War as a method of preserving whole blood and blood plasma for use at front-line hospitals; today, a wide variety of products are produced using this technique and many products which would have no effect at all in solution have turned out to be very powerful antibiotics through the use of lyophilization.
II. BASIC PRINCIPLES
Lyophilizers consist of four basic components: a chamber for vacuum drying, a vacuum source, a heat source, and a vapor removal system. An industrial system is designed for batch operation; some systems are designed for continuous drying operation. At its heart, lyophilization consists of three distinct steps: freezing the product, primary drying, and terminal drying. All three steps are interdependent: a failure in one step means complete failure of the process.
Lyophilization is dependent upon the process of sublimation, wherein water passes directly from the solid state to vapor by lowering the temperature of the solution below its triple point (sometimes referred to as its eutectic point). For an aqueous solution, this is typically found at 4.579 mm Hg (1 atm = 760 mm Hg) and 0.0099 degrees Celsius. However, for most pharmaceuticals, the temperature is frequently lowered as far as -40°C and the pressure as low as 100 microns (0.1 mm Hg) to prevent the frozen water from melting, which would result in frothing as the liquid water and the ice vaporize simultaneously. In addition, the rate of change in temperature and pressure, determined by pre-production validation, has a great effect on the product stability. The key process parameters, then, are the shelf temperature (the temperature of the shelf holding the vials of product), chamber pressure, and time. These three parameters directly effect other variables such as the condenser temperature and the product temperature.
III. FREEZING
The freezing step is extremely critical in that this step establishes many of the product’s final characteristics. The physical structure (amorphous or crystalline) of the product is often dependent upon the method and rate of freezing. The surface area and porosity of the final product will also be established during this step. These have a great bearing on the reconstitution of the product. Additionally, the survival of biological materials (blood cells or other tissues) is dependent upon the care taken during freezing. Rapid freezing results in smaller ice crystals which will not disrupt cell membranes, maintaining the integrity and efficacy of the product after the terminal drying phase. A slower freezing cycle, however, allows a crystalline matrix to form within the product itself, making it possible for some drugs to be reconstituted more quickly. The product is typically dissolved in a suitable solvent, generally water for injection (WFI) which has been specially filtered by a 0.22 micron bacteria-retentive filter. A vessel (usually a vial or ampule) is then filled with the solution and partially stoppered under aseptic conditions. Freezing is accomplished either by placing the vessel in a refrigerated tray within the lyophilizer or pre-freezing in another chamber. During freezing, large volumes of material are generally agitated to produce thin layers of liquid which may be frozen and dried quickly. The vessel may be spun independently on its axis (shell freezing) or placed in a centrifuge with a number of other vessels (vertical-spin freezing). If a small amount of material is required (as in a laboratory operation), the material may be held static, resulting in a plug at the bottom of the vessel. In laboratory-scale processing, liquid carbon dioxide, dry ice, or an acetone/dry ice combination is used as the refrigerant; for industrial scale processing, standard ammonia or Freon refrigeration units are used because they are more economical and can be regulated more readily. The phase change is monitored electronically (by determining the difference in resistance between the fluid and the solid as a function of temperature), by thermal analysis (measuring the difference in temperature between the sample and a reference), or by differential scanning calorimetry (the difference in heat input between the sample and a reference isothermal, given that the change in temperature is a linear function). Alternatively, one could also use a microscope to observe the formation of ice crystals.
IV. PRIMARY DRYING
During primary drying, about 95% of the water in the solution is removed. As mentioned above, the pressure is lowered below 4 mm Hg by pumps, steam ejectors, or a combination thereof, at which point the solid ice sublimes into water vapor and is carried off by a vacuum-pump driven condenser. Alternatively, liquid or solid dessicants may be used (particularly in large-volume biological products such as blood serum or penicillin). Generally, the product is held under these conditions for about 24 hours or longer, depending on the nature of the product. The drying time must be validated for each individual product through the use of trial runs. For certain products (antibiotics and parenterals), stainless steel blades may be used to agitate the product and break up any large concentrations of ice which may have formed.
V. TERMINAL DRYING
The terminal drying phase removes the remaining 5% of the moisture and accounts for 20% of the drying time. The remaining 5% consists of water which is bound or adsorbed into the product. The temperature of the drying chamber is raised above 25°C and the pressure kept below 0.100 mm Hg. Convection or radiation is used to achieve the drying temperature. The final percentage of moisture necessary in the product depends upon the results of the stability studies performed on the product in the development stage. In some cases, it is not desirable to remove all moisture (many protein complexes are dependent upon water to maintain the secondary and tertiary structure of the proteins and, accordingly, their chemical activity). After terminal drying is completed, the vessels are then completely stoppered using a hydraulic or screw-rod mechanism built into the machine. In some cases, particularly when the vessels lie on trays, the vessels lying below each tray are stoppered by the tray directly above, using that tray as a platen.
VI. PROCESS VALIDATION
While the process may look simple on its face, in reality, lyophilization presents many difficulties to a pharmaceutical manufacturer. Not the least of these is scaling up the manufacturing process from the trial run to production lots. In this case, the eutectic point of the product must be precisely known in order to facilitate the establishment of the cycle parameters for primary drying. Additionally, differing product strengths, vial sizes, and batch sizes will often each have their own cycle parameters.
Another problem, particularly in sterile drug manufacturing, is that of ensuring that the lyophilizer is properly sterilized. While the drying chamber itself may be sterilized through the use of a chemical sterilant, the associated plumbing is somewhat more difficult to handle. Many manufacturers use ethylene oxide, commonly used to sterilize surgical equipment, but this can leave behind undesirable residue. The most satisfactory solution by far is that of autoclaving, or the use of steam under pressure. The autoclaving cycle is also useful as a positive pressure check to ensure that all seals and gaskets are intact and properly functioning. Condensate remaining after the steam sterilization cycle is removed by a drain line in the floor of the chamber, with sterile nitrogen gas injected into the chamber at a pressure above atmospheric pressure to ensure that non-sterile air does not enter the chamber.
During the sterilization cycle, biological indicators are often used to validate the process. If the indicators are positive after the sterilization cycle, it is most likely indicative of poor steam penetration in some part of the system (frequently under the trays which hold the vials, which are designed in many cases to collapse for cleaning. Many manufacturers have resorted to a "two-phase" sterilization cycle: one in which the trays are collapsed, the other in which they are configured as they would be for the manufacturing cycle.
VII. FINISHED PRODUCT TESTING
Finished product testing is very important in any pharmaceutical manufacturing operation and especially with concern to lyophilized dosage forms. Among the tests which are performed are dose uniformity testing, moisture and stability testing, and sterility testing.
Dose uniformity testing as recognized by the United States Pharmacopoeia (USP) may be accomplished through either of two methods: content uniformity and weight variation. Weight variation is applied to solids with or without added substances which have been prepared from solution and freeze-dried in final containers. In this case, the weights of the samples will fall along a standard distribution and should (ideally) fall within 1 standard deviation (s) of the mean desired weight. Potency may be tested by reconstituting the sample and assaying the contents; however, these results must be correlated with the sample weights in order to have any meaning.
Sample weight is also important in stability testing. Generally, the most concentrated reconstituted solution will degrade faster than a less concentrated solution. Therefore, the amount of moisture remaining in the product is critical in determining the stability of the product over the long term. In general, expiration dates and stability are based upon those batches which have the higher moisture content (the worst-case scenario). Products are generally produced much stronger than the USP standard to account for decay during processing and storage, and to assure potency throughout the product’s shelf life.
Sterility testing is dependent upon the solution used to reconstitute the product. Generally, sterile WFI should be used to reconstitute products, as bacteriostatic WFI can kill cells present as contaminants and mask the true level of contamination in the dosage form.
VIII. ADVANTAGES AND DISADVANTAGES
While this may seem like a lot of trouble to go to just to dry out a solution, lyophilization does present some very strong advantages to the pharmaceutical manufacturer.
Generally, liquids are much easier to process under aseptic conditions than powders or solids, as liquids have no interstices in which microbes can escape sterilization processes. Also, dry powders resulting from lyophilization are exceptionally stable and thus amenable to further processing without damaging the efficacy of the product. Also, since pressure and not temperature is at the heart of the drying process, most of the water (95%, as mentioned earlier) can be removed without heating the product excessively, which would result in the degradation of chemical bonds and ultimately the effectiveness of the product. Finally, the process allows for rapid and easy reconstitution for use by a medical practitioner.
Chief among the disadvantages of lyophilization is the cost and complexity of the equipment involved. Both in terms of the initial investment and in terms of maintenance costs, lyophilizers are extremely expensive pieces of equipment. While much of the process can be monitored through automation, a great deal of training is still required to operate a lyophilizer. Also, handling and processing times are increased dramatically. From start to finish, the complete lyophilization cycle can take as much as 48 hours. Also, as mentioned previously, the entire process must be revalidated not only for new products, but also for differing batch sizes, potency levels, and even container types. The revalidation process is itself extremely costly and must be monitored with precision in order to assure reproducibility on a production batch scale.
An additional problem is posed in that much of the system is hydraulically controlled, and the risk of contamination by hydraulic fluids is ever-present. This can be mitigated through the use of filters and convoluted paths between the injection and evacuation ports and the chamber itself. However, this approach also makes sterilization more difficult. As the equipment gets older, sterilization becomes more difficult: seals and gaskets may need to be replaced more frequently, and the chamber itself suffers fatigue from constant pressurization and depressurization cycles. While a lyophilization system offers many advantages from a pharmaceutical technology perspective, from a quality perspective it should be seen as a very high maintenance system.
IX. CONCLUSION
While lyophilization has been in use for over 50 years, and the process itself has changed only superficially in that time, it is nonetheless an extremely complex method of pharmaceutical manufacture requiring meticulous attention to detail at every level in order to produce a viable product. Without constant validation and monitoring, a production batch worth millions of dollars can easily be turned into useless slush. Quality systems, obviously, are of great import in the lyophilization process and should not be taken lightly.
Rippie, Edward G. Theory and Practice of Industrial Pharmacy, 2d. edition. Philadelphia: Lea and Febinger, 1976.
Trappler, Edward H, president of Lyophilization Technology, Inc. Lyophilization - Basic Concepts and Design. Lecture given August 10, 1993.
U.S. Food and Drug Administration. "Guideline for the Manufacture of In Vitro Diagnostic Products." Rockville, MD: U.S. Government Printing Office, 1994.
U.S. Food and Drug Administration. "Guidance for Industry for
the Submission Documentation for Sterilization Process Validation in Applications
for Human and Veterinary Drug Products." Rockville, MD: U.S. Government
Printing Office, 1994.
