Assure Batch Uniformity for Freeze-Dried Products

March 16, 2005
Success requires precise control of process conditions, extensive equipment testing, and analysis of bulk solution, dry and reconstituted product; surrogates can be used for some tests.
The need to assure batchuniformity in freeze-driedproducts is evident in thevariation in the extent ofshrinkage between the contents of these two vials.By Edward H. Trappler, President, Lyophilization TechnologyBringing any new pharmaceutical to market requires coordinated efforts in product design, formulation development and process engineering throughout the development phase. Moving that new product to commercial-scale manufacturing requires careful validation, and demonstrating batch uniformity is an essential part of this critical process.Lyophilization, or freeze-drying, is becoming more important in developing and manufacturing unstable, sensitive pharmaceuticals. However, lyophilization poses special challenges to achieving and ensuring batch uniformity. First, the product itself is sensitive to the presence of water and to process conditions. Then, the process involves manipulating subambient temperature and subatmospheric pressure conditions. Success requires close control of process parameters such as temperature and time, and equipment operating performance. It also depends on understanding factors unique to each lyophilizer — even a vial’s position on the freeze dryer tray can have a major impact on product quality and batch uniformity.Controlling critical processing parameters is imperative to ensuring that batches are uniform and the process reproducible from batch to batch. Completing a comprehensive Installation Qualification (IQ), Operational Qualification (OQ) and Peformance Qualification (PQ) assures that the equipment can produce material of sufficient quality.USP has issued compendial requirements for content uniformity [1] to assure that the dosage in each container is within an acceptable variation. However, to date, only limited information is available on assessing finished product attributes for lyophilized preparations [2, 3]. Characteristics and quality of the lyophilized product, both dried material and the reconstituted solution, can verify the level of batch uniformity.This article will discuss some of the issues involved in achieving batch uniformity for lyophilized pharmaceuticals, summarize important research, and suggest strategies, at every step of the process, for ensuring the batch uniformity of lyophilized products.Pinpoint the sources of variabilityFortunately, research has provided a scientific basis for improving control of the freeze-drying process, by correlating specific variables to product quality. The most important factors, by far, are:
  • Sublimation rates and residual moisture. Both of these critical process variables may be influenced by a vial’s position on the lyophilizer shelf, as well as by variations in process conditions, time, temperature control, heat and mass transfer [4]. Sublimation rates appear to be lowest at the vial tray’s center, and highest at its corners [5,6], and empirical evidence is so strong that one researcher has even developed a three-dimensional mathematical model to correlate tray placement and sublimation rate [7]. Further studies have examined the influence of annealing during freezing, and quantified primary drying rate coefficients of samples at various positions in the shelf [8].

  • Quality of API, excipient, and bulk solution. The behavior of the material during the processes depends on the characteristics of the starting bulk solution, and characteristics of starting ingredients must be analyzed and correlated with final product attributes to verify batch uniformity. This analysis should include any variation in preparation and dispensing prior to placing the product into the lyophilizer.

  • Quality of packaging components. Container construction can have an impact on product during processing, particularly during sublimation. Studies have shown that container construction can influence heat transfer [9], temperature profiles [10] and product behavior [11].

  • Relative capacity and performance of the lyophilizer equipment.

  • Concentration changes, thermal stress, and dehydration that occur during freezing.

  • Stability of the bulk solution throughout the fill day, as well as that of the solution in the final containers. Storage affects the stability of bulk solutions [12]. Nevertheless, a number of manufacturers formulate bulk solution the day before filling occurs, which assumes that the solution will be stable for over 24 hours. Since filling a batch may take anywhere from a few to over eight hours, each container of solution holds product that has been in the presence of water for a different length of time. As a result, the quality of product in the first filled container may differ from that of the last.
Take actionOnce the root causes of variation are understood, an action plan can be developed. The following steps will help in this process:
  • Compare liquid and solid state product at comparable stages of the cycle.

    Because the first and last containers filled may vary in quality, product of different age in the liquid state should be compared with dried product removed at comparable stages in the filling cycle. This analysis will ensure that process conditions during lyophilization have not exacerbated any changes in product quality that may already have occurred.

  • Location, location, location: Consider product’s position in the lyophilizer.

    It is also important to correlate dried product quality to the vial’s position in the lyophilizer. One should expect the quality of the first and last containers filled, both unprocessed liquid and dried product, to be comparable. However, location may influence the product’s residual moisture, an important factor in promoting adequate stability, as well as its physical appearance, reconstitution time, and the quality of the reconstituted solution.

  • Define and monitor critical parameters, and, where possible, monitor product temperature.
    Lyophilizers such as this one from Hull are becoming more important in developing and manufacturings unstable, sensitive pharmaceuticals.

    Critical parameters such as shelf temperature, chamber pressure, and time should be accurately and precisely controlled. Even less critical factors such as product temperatures should be monitored, since temperature data can help assess processing conditions and pinpoint any environmental influences within the lyophilizer.Unfortunately, as with product sampling, product temperature information is only available for a few containers in each batch. In addition, automated loading systems prevent operators from inserting temperature sensors into product. Nevertheless, whenever possible, product temperature should be monitored at selected locations in the lyophilizer. These values should then be compared with attributes of finished product from these locations, to correlate processing variations to dried product quality. The aim is to quantify variations in temperature profile during processing that may be attributed to location on the shelf, and identify problematic locations in the lyophilizer, where product sampling might be appropriate during initial batch processing.
  • Verify equipment uniformity.

    The performance capabilities of each individual freeze dryer will influence process reproducibility, batch uniformity, and consistency of the finished product. Qualifying equipment performance is, thus, an integral part of assuring reproducibility, consistency and uniformity. Complete and comprehensive Equipment Qualification studies are necessary, including Installation and Operational Qualification, which ensures that the equipment has been properly installed, adequate utilities are available, and the lyophilizer is functioning properly.

    Focusing on function and performance capabilities, the OQ demonstrates the processing capability of the lyophilizer. Demonstrating adequate shelf temperature uniformity is a critical part of both these studies.

  • The OQ tests.

    OQ tests determine how well the equipment can perform critical functions during lyophilization, most notably, the heat transfer required to freeze and then sublimate ice in the product. Temperature must be closely controlled from the time that product is loaded onto the lyophilizer shelves until it is removed after stoppering. A comprehensive OQ study must therefore test:

    • cooling and heating rates,
    • control at set point,
    • temperature uniformity.

    Each lyophilizer’s specific design and capabilities should also be tested. Shelf temperature uniformity should be verified across any one shelf and all shelves of the lyophilizer, and any variations must be within an acceptable range to assure product batch uniformity. Temperature uniformity, in turn, should involve testing at various locations across each shelf, such as at the center and four corners, and comparing the temperature of those five locations for all of the shelves.

    At any location on the shelf, the temperature should be compared to either the mean of the measured values or the temperature indicated on the controlling instrument. The allowable range may be described relative to the mean of the measurements.

    The sublimation/condensation test: Equipment vendors aspire to provide lyophilizers with uniform shelf temperature. Appropriately tested under “no load” conditions during the Factory and Site Acceptance Tests (FAT, SAT), uniform shelf temperatures may again be demonstrated under load conditions during the sublimation/condensation test as part of a comprehensive OQ.

    The sublimation/condensation test challenges the shelves’ ability to provide sufficient heat to achieve acceptable sublimation rates. The test also demonstrates the condensation rate and ice-load capacity of the condenser. Rates of sublimation for each shelf may be expressed as kilograms of ice per hour and reflect the relative heat provided. Comparing the rates achieved for each shelf of the lyophilizer demonstrates each shelf’s relative heat-transfer capacity, as well as any potential influence of relative shelf position within the lyophilizer.

    Pressure-control capability tests: An OQ study should also verify the equipment’s pressure-control capability. Pressure control tests demonstrate the accuracy and precision of maintaining the chamber pressure across the anticipated pressure range. Test results should then be compared to target values at low, intermediate and high pressures.

    There is a growing interest in applying Process Analytical Technology to measure the gas composition within the lyophilizer [12]. Initially, it is assumed that the composition of the atmosphere is uniform, independent of lyophilizer size and equipment configuration, which may include the size and number of shelves, and the shelves’ position relative to the condenser. However, little has been published to date showing the composition of the atmosphere relative to location, and therefore uniformity, within the lyophilizer.

  • Quantify product uniformity within a batch.

    To justify the time limits set for bulk storage, assay methods may be used to monitor active ingredients and the presence of any degradation products between the time that product is formulated through the end of filling. Aging may result in differences in formulation purity, influencing the phase transition of the product formulation. Storage of bulk solution or filled final containers over an extended period of time may:

    • result in a pH shift

    • consume one component of a buffering system

    • induce chemical degradation of the API

    • alter the desired secondary, tertiary or quaternary structure of a peptide or protein product

    • allow polymerization, aggregation or denaturation of active ingredients that have both hydrophilic and hydrophobic character.

    For biopharmaceuticals, conformational changes should be monitored, to justify allowable bulk-storage conditions such as temperature or atmospheric conditions, including an allowable time for bulk solution storage and filling. For any pharmaceutical product, batches should be monitored during initial scaleup to determine any differences in purity, presence of degradation products, and changes to the active ingredient content from the beginning to the end of the filling operation. Any differences can also be monitored by routinely sampling the first and last vials placed in the lyophilizer.
  • Figure 1. Product lyophilized with retention of structure established during freezing. Note uniform structure, dense fine texture, and white color associated with pharmaceutical elegance.

    Quantify product uniformity within the lyophilizerAfter completing OQ studies, and before processing the first batch of actual product, one should demonstrate the equipment's performance capabilities under load conditions, using a predefined set of processing parameters: shelf temperature, chamber pressure, and time. These values should then be compared to the intended target values.At this point, one should assess product response in areas within the equipment that are expected to impart the greatest degree of variation, such as the shelf perimeter. In addition, one can compare the range of product temperatures at the completion of each critical step in the process: loading, freezing, primary and secondary drying. The distribution of times at which monitored vials reach the drying end point and the product temperature begins to rise when sublimation is completed may also be evaluated. As will be discussed later, surrogate materials can be used in these tests.It is important to note that the type of container and placement of the thermocouple within the container [9] strongly influences when these sudden increases, or “breaks,” in product temperature take place. These variations are generally caused by differences in heat transfer to the container as well as mass transfer of the water vapor through the dried product [13]. The graph illustrates the variation in product temperature for formulations containing mannitol, a sugar alcohol, and sucrose, a polysaccharide, as the main constituents in various formulations. The significance placed upon, and conclusions drawn from, any variation in product temperature and the time when the temperature “breaks” must also account for any influences due to the formulation.Mapping the lyophilization chamber provides enough data to quantify any effects of location and surrounding environmental influences, such as differences in the heat transfer capacity of a particular shelf relative to others within the shelf bundle. The result of such a study can be used to identify appropriate locations for monitoring and product sampling during actual product validation studies, as well as to demonstrate sufficient batch uniformity.These trial runs also verify adequate process parameter control of shelf temperature and chamber pressure under load conditions. The batch size and process parameters needn’t duplicate those for any actual product. Instead, the testing should provide the opportunity to design an appropriate model to challenge the equipment capabilities. Several models have been studied and well characterized [14].Quantify finished product attributesIn successful lyophilization, product should retain the physico-chemical attributes of the starting solution and the structure established during freezing [3]. The dried cake should be uniform in structure, color and texture — ideally, a dense white cake with fine, uniform structure, showing good physical strength and friability, as shown in Figure 1 (above).With some formulations, drying may cause the cake to shrink as shown in Figures 2 and 3. When shrinkage is due to the solute nature or its concentration, it tends to be uniform throughout the batch. However, structural alteration, whether localized or complete, can be attributed to “collapse” [16], which can occur when the frozen or partially dried material exceeds the phase transition and the material becomes fluid. Figure 4 shows samples exhibiting various degrees of collapse that can sometimes occur, even within a single batch.
    Figure 2. Example of lyophilized preparation exhibiting a slight amount of shrinkage.

    Figure 3. Variation in extent of shrinkage due to a frail structure resulting from a formulation consisting of low solute concentration.

    Figure 4. Range in physical appearance of random samples from different locations within a lyophilizer.

    In some instances, collapse may be considered a cosmetic concern. However, the presence of collapsed material is suspect, particularly when collapsed dried material is randomly dispersed throughout a batch. Collapse may also lead to some entrapment of water, yielding product with higher residual moisture. Product degradation may occur because of hydrolysis. In some cases, the assay will fail to meet compendial limits. When this occurs, the presence of collapse becomes a greater concern and is characterized as a product defect.It is therefore imperative to establish an acceptable range of moisture content by establishing “residual moisture” as a critical finished product attribute. Suitable techniques have been developed [17] while new methods involving NIR could allow each container within a batch to be tested nondestructively [18,19].Reconstitution time and the constituted solution appearance are also critical finished product attributes. Lyophilized material is generally highly hygroscopic and reconstitution should take less than two minutes — longer with collapsed material, but resulting solution should be clear and free of any insoluble materials [1], and its expected appearance should be a quality attribute established and supported by development data.Quality of the constituted product of a lyophilized preparation would be the same as for any ready-to-use liquid preparation. However, once diluent is added, shelf life is limited. Reconstituted product may need to be used immediately or stored for a limited time at selected conditions, typically 2-8° C. The reconstituted solution’s stability should be established during development and measured as part of the stability testing.Use surrogates to demonstrate uniformity All this testing can be prohibitive when using actual product. For sterile product, there is also the extraordinary burden of maintaining a high level of sterility assurance because of the extra manipulations required when placing samples and temperature probing. A well-designed model can be used as a surrogate for product, allowing physical appearance, residual moisture, and reconstitution time to be tested at different locations within the lyophilizer.Surrogate testing will help identify suitable locations for monitoring and sampling during product-specific performance qualification studies. Select excipients that are influenced by the rate of freezing, so that they can reveal subtle differences in measurable in-process characteristics such as temperature, and attributes of dried product such as residual moisture.The excipient, concentration, and fill volume all influence the variation in physical structure and density, and therefore affect the rate of mass transfer of water vapor during sublimation [14]. For example, dilute solutions of excipients such as mannitol, polyvinylpyrrolidone and simple ionic salts in the range of 5% to 12% w/v solidify with a dense, uniform structure, regardless of the rate at which the material is cooled during freezing. Such preparations would not provide sufficient visual cues to allow environmentally-influenced variations to be seen. However, excipients such as dextran, sucrose, and lactose allow structural differences to be observed clearly.These differences can then indicate when product is solidified at different rates of cooling during freezing—an effect that may be influenced by position within the lyophilizer. Lyophilization Technology is currently developing data on the use of these materials. These differences may provide indications of any variation due to position because of variations in both product temperature and residual moisture.References1. USP 28 / NF 23, Rockville, Md.: United States Pharmacopeial Convention, Inc. (2005).2. Avis, K., Bashir, J. Evaluation of Excipients in Freeze-Dried Products for Injection, Bulk Parent. Drug Assoc., 27 (2): 68-83 (1973).3. Daukas, L., Trappler, E. Assessing the Quality of Lyophilized Parenterals, Pharmaceutical and Cosmetic Quality, 2 (5): 21-24 (1998).4. Greiff, D. Factors Affecting the Statistical Parameters and Patterns of Distribution of Residual Moisture in Arrays of Samples Following Lyophilization, J. Parenteral Science Technology, 44(3): 119-128 (1990).5. Kobayashi, M., Harashima, K., Sunama, R., and Yao, A., Inter-vial Variance of the Sublimation Rate in Shelf Freeze-Dryer. Proceeding of the 18th International Congress of Refrigeration. 1991, Montreal, Canada, International Institute of Refrigeration, p. 1711.6. Placek, J., Kamei, D., Lee, G., et al. In-homogeneity Phenomena in Lyophilization, poster presentation at the AAPS Annual Meeting, New Orleans, La. Nov. 16, 1999.7. Rajniak, P., Placek, J., Reynolds, S., and Hunke, W. Mathematical Modeling of Primary Drying, poster presentation at the AAPS Annual Meeting, New Orleans, La. Nov. 16, 1999.8. Searles, J., Carpenter, J., and Randolph, T. Primary Drying Rate Heterogeneity During Pharmaceutical Lyophilization, American Pharmaceutical Review, 3 (3): 6-24, (2000). 9. Pikal, M. J., Roy, M. L., and Shah, S. Mass and Heat Transfer in Vial Freeze-Drying of Pharmaceuticals: Role of the Vial, J. Pharmaceutical Science, 77 (9): 1224-1237 (1984).10. Day, L. Influence of Vial Construction and Material on Uniformity of Product Temperature During Freezing and Freeze Drying of Model Product Formulations, presented at the Annual Meeting of the PDA, Boston, 1995.11. Cannon, A., Shemeley, K. Statistical Evaluation of Vial Design Features That Influence Sublimation Rtes During Primary Drying, Pharmeutical Research, 21 (3): 536-542 (2004).12. Guide to Inspection of Lyophilization of Parenterals, USFDA, Shanley, A. Process Models and Gas Analysis Help Optimize Lyophilizer Operations, Pharmaceutical Manufacturing, 3 (4): 41-44 (2004).14. Pikal, M., Shah, S., Senior, D., and Lang, A. Physical Chemistry of Freeze Drying: Measurement of Sublimation Rates by a Microbalance Technique, J. Pharmeutical Science, 72 (6): 635-650 (1983).15. Scheaffer, G., Sum, L., and Trappler, E. Techniques in Demonstrating Batch Uniformity for Lyophilized Products, presented at the Annual Meeting of the PDA, Boston, November, 1995.16. MacKenzie, A. P. Collapse During Freeze Drying — Qualitative and Quantative Aspects, In: Freeze Drying and Advanced Food Technology (W. A. Goldblith, L. Rey and W. W. Rothmayer, eds.), Academic Press, New York, 278-307 (1975).17. May, J.C., Grimm, E, Wheeler, R..M., and West, J. Determination of Residual Moisture in Freeze-Dried Vial Vaccines: Karl Fischer, Gravometric and Thermogravimetric Methodologies, J. Biological Stand. 10: 249-259 (1982).18. Last, I.R., and Prebble, K.A. Suitability of Near-Infrared Methods for the Determination of Moisture in a Freeze-Dried Injection Product Containing Different Amounts of the Active Ingredient, J. Pharmaceutical & Biomedical Analysis, 11 (11/12): 1071-1076 (1993).19. Brulls, M., Folestad, S., Sparen, A., and Rusmuson, A. In-Situ Near-Infrared Spectroscopy Monitoring of the Lyophilization Process, Pharmeutical Research, 20 (3): 494-499 (2003).About the AuthorEdward Trappler is president and founder of Lyophilization Technology, Inc. (Ivyland, Pa.), a contract research and technical services firm dedicated to freeze-drying. He has over 25 years experience in lyophilization, ranging from product development to equipment application engineering. Ed has worked for a number of pharmaceutical firms including Elkin-Sinn, Inc. and McNeil Pharmaceuticals, and also for the equipment vendor Hull Corp. He has written and presented numerous papers and courses in freeze-drying in the United States, Europe, Japan and China. You can email him at [email protected].