Lyophilization, or freeze drying, is often used to increase the stability and shelf life of pharmaceutical products. While liquid formulations are usually preferred for injectable protein therapeutics (in terms of convenience for the end user and ease of preparation for the manufacturer), this form is not always feasible given the susceptibility of proteins to denaturation and aggregation under stresses such as heating, freezing, pH changes, and agitation, all of which could result in the loss of biological activity (1). Moreover, water mediates hydrolysis and deamidation; these reactions as well as other chemical degradation pathways, such as oxidation, are accelerated under aqueous conditions, making the formulation unsuitable for long-term storage (1).
Lyophilization generally results in improved stability profiles. “Protein products that have been freeze dried are more stable than liquid formulations,” observes James Searles, director of pharmaceutical development at Hospira One 2 One, a CMO with expertise in parenteral manufacturing. Another advantage is that distribution is less complicated because cold-chain issues are mitigated and the vibrational stress during shipment is minimized (2). In addition, contamination due to extractables and leachables from the container closure is also less of an issue for lyophilized products because the formulation is only in contact with the closure for short periods instead of the entire shelf life. The disadvantages of lyophilization, however, include the lengthy and complex manufacturing process, the higher overall production cost, and the larger capital investment needed for manufacturing (2).
The fundamental goal of lyophilization is to impart desirable characteristics into the product, such as long-term stability, short reconstitution time, maintenance of the characteristics of the original liquid formulation upon reconstitution (e.g., solution properties, isotonicity, structures or conformation of proteins, and particle-size distribution for suspensions), and elegant cake appearance (3). Nonetheless, developing a freeze-drying cycle for protein formulations has its challenges.
“Given their larger size and the importance of higher-order structures to their function, proteins require intense analytical focus,” explains Searles. “In addition, while small molecule degradation reaction rates can often be gauged using higher incubation temperatures over shorter times, many mechanisms of protein damage cannot be simulated in such a way, and would, therefore, require long stability studies before one has confidence in the a given formulation.”
The importance of stability
The main consideration in lyophilization of protein formulations is long-term stability, which is related to water content, says Enric Jo, director and plant manager of Reig Jofre, a European development and manufacturing group serving the pharmaceutical market. “For small-molecule drugs, it is possible in most cases to formulate them without excipients, or just by adding a bulking agent or pH modifier, to obtain a liquid formulation that is stable enough to endure the necessary duration spent before it is freeze-dried. The moisture content in the products is usually sufficiently low to ensure that the formulation remains stable for long periods.”
For proteins, however, the situation is more complicated because proteins are labile molecules. Their stability is related to the water content of the formulation, but at the same time, the active form of a protein is related to the conformational structure that needs some water content to avoid denaturation processes. According to Jo, these stability issues can be avoided through formulation optimization and adequate process control.
“A new stability concept can be described as thermodynamic stability. This stability refers to the position of equilibrium between native and unfolded conformations,” says Jo. “The problem is further complicated because while a protein may exhibit thermodynamic instability during freeze drying and unfold, if no irreversible reactions (e.g., aggregation) occur during storage or during reconstitution, the reconstituted protein may refold completely within seconds and display perfect pharmaceutical stability.”
There is a complex and poorly understood web of interactions between a given protein, the formulation, and the lyophilization cycle used, observes Searles. “For example, a recent study describes how freezing and annealing protocols had dramatic effects on subsequent rates of aggregation, oxidation, and deamidation over long-term stability, and that the fraction of protein at the solid-gas interface throughout the highly porous lyophilized cake was by far the most important factor (4). Interestingly, the formulation characteristics affected the protein damage only by affecting the fraction of protein at the interface.”
The first step in the development of freeze-drying cycles for protein therapeutics is to understand the specificity of the protein molecule in the given formulation. As Yves Mayeresse, director of GlaxoSmithKline’s vaccines leadership team, points out, “Knowing the way the protein has been produced and purified can increase the understanding of its degradation pathway.” Mayeresse adds that it is also important to envisage what the commercial product will look like after a few years. “Having the final goal in mind will greatly help define the path towards it,” he says. “The first milestone is to produce a quality product, and to achieve this, a robust formulation and a robust freeze-drying process are needed.”
Jeff Schwegman, founder and CEO of AB Bio Technologies notes that one of the biggest challenges is to develop a freeze-drying cycle for a formulation that has not been properly designed. “A properly designed formulation takes into account any issues with associated freezing and drying stress and corrects these issues before cycle design begins,” he says.
Schwegman goes on to explain that the freezing and drying behavior of proteins is usually not an issue. “These molecules have high glass-transition temperatures making them easy to dry. Freezing is not an issue as these molecules form a glass at relatively high temperatures.” Schwegman believes that formulation variables are the critical issues here. “These variables have to be balanced correctly for stabilization without overdoing such that they adversely affect the thermal properties of the protein formulation and how it freeze-dries,” he explains.
“The worst-case scenario is trying to design a lyophilization cycle around a formulation that someone without any knowledge of lyophilization has developed. For example, most proteins are developed in phosphate buffered saline, which is one of the worst things to freeze-dry with proteins.”
The best freeze-drying cycles are those that have been optimized to be as short as possible but with enough of a safety factor to avoid a process deviation when challenged with fluctuations in equipment performance, observes Searles. “The quality-by-design (QbD) approach is perfect for achieving this” he says.
According to Mayeresse, the criteria for successful development of freeze-dried protein formulations can be easily defined using the process validation approach and the QbD principle. “A good starting point is to know the target product profile and the critical quality attribute (CQA) of the product. The definition of the formulation composition is critical for the whole process,” he remarks. Mayeresse suggests that the number of stabilizers used should be kept at the minimum and one should choose stabilizers that are generally recognized as safe. Schwegman adds that if the protein shows sensitivity to freezing stress that cannot be corrected through stabilizers, then a freezing-rate or annealing study should be initiated to work out the optimal freezing protocol.
Freezing is the most critical step when developing a lyophilization cycle for protein formulations. The product must be frozen at a temperature that is low enough to be completely solidified. The microstructure established by the freezing process usually represents the microstructure of the dried product (5). The solute can crystallize or remain substantially amorphous with freezing; understanding the physical form of the solute (i.e., whether crystalline or amorphous) after freezing is important from the standpoint of drying characteristics, appearance of the final product, and product stability during storage (5).
As Jo points out, the complex physical changes occurring during the freezing process can contribute to denaturation of proteins. “During this phase, the structure in which the proteins will be embedded is created,” Mayeresse explains. “If the molecule is not locked in the right conformation, it can end up with an important loss of potency.” Mayeresse further adds that the difficulty is linked to the fact that freezing is mainly governed by a kinetic process.
“Considering that it is possible to freeze in different ways (e.g., slow rate, quick rate, quench freezing with liquid nitrogen, or annealing), it is important to assess the impact on protein integrity,” says Jo. “An extremely quick quench freezing with liquid nitrogen can yield a better process in terms of time necessary for desorption because the ice crystals present a very large ice surface area, thereby improving the secondary drying. Curiously, it also has the capacity to improve the output of primary drying.”
Jo, however, cautions that this increased ice surface areas may affect the stability of the proteins. “Studies of unfolding during freezing provide evidence that proteins can unfold as a result of adsorption to the ice surface, although the available experimental data cannot be generalized.” He explains that there is an optimum freezing rate range for a given formulation based on protein degradation considerations and ice crystal size. During development, it is important to investigate the scope of annealing for each formulation considering that the main process to dry the product occurs during the freezing step.
For primary drying, as long as the glass-transition temperature is known, the shelf temperature, chamber pressure, and phase duration can be easily defined using available tools or based on scientific knowledge, notes Mayeresse. According to Schwegman, the primary drying protocol is developed using information from the thermal characterization study while the secondary drying protocol is developed using information on the thermal stability of the molecule and through the use of a sample thief to pull samples at various moisture levels to determine the optimal amount of residual moisture. “For secondary drying, the temperature of the shelf and duration may have an impact on product potency, but keeping these parameters at a reasonable value will avoid this hurdle,” Mayeresse adds. “This phase also determines the residual moisture level. The remaining humidity can increase degradation reactions during the product shelf life and should, therefore, be kept at a low level, typically below 3%.”
Quality by design
The entire development process of a protein formulation should be a QbD approach, says Schwegman. “Preformulation studies determine where the product is in its happiest environment, and the formulation and lyophilization studies are designed to achieve those conditions through the addition of excipients and how the formulation is freeze-dried.”
Mayeresse agrees that the QbD approach is an advancement towards a more systematic and efficient way of developing new protein formulations and processes. “In the past, development was mainly based on trial and error until a good product was obtained. The QbD approach is by far a better concept that is well-recognized by regulatory authorities compared with the traditional method of having three good batches.”
According to Schwegman, many of the critical process parameters (CPPs) can be determined before cycle development starts including the optimal freezing protocol, the critical temperatures, and acceptable moisture levels, among others. Jo adds that the CPPs for the freeze drying process are the temperature and pressure, both during freezing and drying, as well as the time (i.e., the speed at which each step is performed and the related ramps). “These CPPs should be established in accordance with the critical material attributes (CMAs) of the formulation, such as the total solidification temperature (Tts), the melting temperature (Tm), the glass-transition temperature of the freeze concentrate (Tg’), and the collapse temperature (Tco). All these CMAs provide us with the thermal fingerprint of the formulation.”
The CQAs for freeze-drying protein formulations are numerous, according to Mayeresse, their relative importance is a function of the product type that will be developed. The specific CQAs to take into account with proteins are those related with aggregation, pH, water content, native structure, and related substances that appear as a result of degradation reactions, such as deamidation, oxidation, and so on, observes Jo. The major CQAs include potency, residual moisture content, long-term stability, cake elegance, appearance, reconstitution time, clarity after reconstitution, and product-related impurities (i.e., aggregates, fragmentation, and oxidation), and the list is not exhaustive.
As Mayeresse explains, an issue during freezing could impact the potency of the product given that this phase is crucial for product integrity. “An issue during primary drying will first impact the cake elegance creating collapse, which could potentially lead to higher moisture content. This level of residual humidity may impact potency during storage, but should be detected at release if the tests are adequately defined,” he adds. “An issue during secondary drying can impact not only moisture content but also product integrity if a high temperature over an extended period is encountered. In this case, the process boundary defined during the development process should highlight the outcome of such a deviation.”
Jo recommends that an analysis of the behavior of the protein (in relation to aggregation, the equilibrium of the folded and unfolded structure, the effects of pH shifts, and the degradation reactions) should be carried out, depending on the type and quantity of proposed added excipients, to reach both thermodynamic and kinetic stabilities. “Once the appropriate design of experiment (DoE) for the formulation is defined, the consequences, applying a conservative cycle based on the thermal fingerprint of the formulation, should be studied to determine if the CQAs are within the expected ranges,” explains Jo. “The approach yields a formulation that will be optimized by a second DoE.” Such a strategy allows for checks at different CPP levels to improve the cycle and establish the design space, according to Jo. “Both the first and second DoE are the result of an appropriate risk assessment, and when applied over the cycles, we can define the control strategy by means of a principal component analysis over the outputs of the process,” says Jo.
Process analytical technology
The implementation of QbD requires using appropriate process analytical technologies (PAT) to monitor CPPs during lyophhilization to ensure that the product meets the desired quality attributes. “During the past 10 years, different PAT tools have been investigated by different teams from different companies,” notes Mayeresse. “Today there is no global recommendation on which tool should be used.”
“Important PAT advances for lyophilization include wireless temperature probes, drying-rate measurement systems, and endpoint-detection instruments,” Searles comments. Wireless temperature probes used in non-GMP engineering batches can give product temperatures in automatically loaded lyophilizers, allowing comparison of this critical parameter to laboratory development runs. Drying-rate measurement systems and instruments for endpoint detection used in the laboratory as well as manufacturing dryers allow successful bridging between equipment. “These parameters are all cornerstones of the QbD approach in lyophilization,” says Searles.
“Useful PAT tools exist at a pilot plant level that are not different from those used to analyze the process for small molecules,” observes Jo. “The application of dew-point probes, the combination of capacitance and Pirani vacuum gauges, and others can be some of the simplest PAT tools,” he continues. “More sophisticated tools can be applied during the process, such as near infrared spectroscopy (NIR), to determine the evolution and the quality of the species that disappear (water) and appear (dry product) during the process.”
Mayeresse points out that PAT tools for freeze-drying can increase the knowledge of the process. “The best approach is to use a tool that provides global monitoring of the load,” he says. “In this regard, the microbalance system gives useful information but is focalized on one vial. A pressure rise test gives a global picture of the load inside the freeze-dryer. These PAT tools can be equally useful for a scale-down model, giving more comparison information between large-scale and small-scale freeze-dryers and allowing a smooth transition between them.”
The choice of a PAT tool should be thought in terms of implementation, Mayeresse emphasizes. “If the decision has been taken to use a specific tool as part of the release data for the product, it should be available on all the industrial equipment at the different sites, or it will be difficult to justify its presence or absence during audit.”
The application of some PAT tools at an industrial scale can be a challenge, as Jo notes. “For instance, dew-point probes cannot be steam sterilized. On the other hand, NIR can be more or less easy to introduce in a pilot plant, but it is difficult to introduce it in large-scale equipment,” says Jo. “The difficulties increase when you want to establish a validated NIR model that explains the process,” he goes on to explain. “A conformational stability of the protein obtained by means of NIR (to understand the mechanistic criteria of the process) is difficult to establish during cooling, freezing, and sublimation. Nevertheless, the use of the relationship between the data provided by the capacitance and Pirani gauges and other combined variables using the output signals of the equipment, enables you to obtain the relevant information to guarantee the control strategy for the process and its suitability in commercial batches.”
While a QbD approach, together with the application of PAT tools in the lyophilization of protein formulations, will indeed increase the knowledge of the product and process starting at the development scale, Mayeresse points out that adjustments have to be made for its implementation. “You will need to devote more time towards development, and you will need a good capacity to interpret results coming from a multivariate environment in contrast to the past, where development mainly involved a univariable analysis,” says Mayeresse.
“The complexity in the development is due to the necessity to define the protein formulation and the freeze-drying cycle at the same time,” explains Jo. “Obviously, if the formulation is not optimal, it will be very difficult to obtain the best cycle in the freeze dryer,” he concludes.
1. J.F. Carpenter et al., “Rational Design of Stable Lyophilized Protein Formulations: Theory and Practice” in Rational Design of Stable Protein Formulations: Theory and Practice, J.F. Carpenter and M.C. Manning, Eds. (Kluwer Academic/Plenum, New York, 2002), pp. 109-133.
2. J.T. Blue, “Key Considerations for the Successful Lyophilization of Proteins,” SP Scientific webcast Aug. 26, 2010, accessed Apr. 3, 2014.
3. F.K. Bedu-Addo, Pharm Technol (2004).
4. Y. Xu et al., J Pharm Sci, doi 10.1002/jps.23926, accessed Apr. 3, 2014.
5. L.A. Gatlin and S.L. Nail, Bioprocess Technol 18: 317-367 (1994).
About the Author
Adeline Siew is the scientific editor for Pharmaceutical Technology.