When protein technology aficionados meet in San Diego for “Pep Talk” in January 2008, the topic of lyophilization will be covered for the first time at this meeting. This critical approach to macromolecular processing is essential for the preservation of perishable material.
Lyophilization, also known as freeze-drying, is a venerable warhorse, having been invented over half a century ago. Long of tooth, its basic technology changed little for many years.
“Pep Talk” gurus, however, will find that the field has undergone an extreme makeover, driven largely by advances in computer technologies. The outcome is a great improvement in the survival of fragile biological structures when the samples pass back and forth through the dehydration process.
Ordinarily, lyophilization targets the preservation of proteins. Radical redesign of these strategies now allows the recovery of viable virus particles and even bacteria after a cycle of freezing and thawing.
Foam Cake Structures
Much of the improvement in lyophilization technology has come through a thorough understanding of the basic material science of pharmaceutical solids and the “thermal history” or processing conditions that the product is subjected to during the drying process.
“We seek to improve the survivability of proteins through the process that typically may include loss of secondary, tertiary, and quartenary protein structures,” according to Vu Truong, Ph.D., vp, R&D, Aridis Pharmaceuticals (www.aridispharma.com). “Complex, large proteins such as the antihemophilic Factor VIII are a particular challenge.”
Because of the poor performance of traditional lyophilization procedures, Dr. Truong looked for other drying processes, settling on foam drying. “Foamy cake structures, or foam drying, is a half-century-old drying process dismissed as an unconventional and poorly designed freeze-drying strategy because the resulting material looked like a collapsed cake,” Dr. Truong comments.
“By altering the freeze-drying parameters slightly we are able to achieve the collapsed cake structures, which give the material a white-transparent look,” he explains. Given the right formulation, the foam-dried material adopts an amorphous, glassy structure that is found to be lower in stored energy as compared to the same formulation when freeze-dried or spray-dried. In this condition, both the global glassy-state motions and local molecular motions of the dried structure, as measured by neutron scattering, are lowered. This means longer glass relaxation time.
In scanning electron microscope pictures, the foam-dried material takes on a thick vitreous appearance that exhibits less specific surface area, so that the encased protein is less exposed at the air-solid interface as compared to the thinner, honeycomb structures of a typical freeze-dried cake.
These findings translate into longer shelf life, especially at room temperature. But in cases that still require refrigeration (at 2–8ºC or below), like with vaccines, there is great interest in improving storage stability to simplify product distribution.
Dr. Truong’s approach of foam-drying along with the addition of sugars as a cryo- and dessicoprotectant greatly improves product stability. His team was guided in this respect by Mother Nature. Bacterial species such as thermophilic bacteria produce thermoprotectants, unusual polysaccharides that enable them to resist extremely warm conditions and also help avoid formation of ice crystals inside the membrane of the virus or bacterium cell, which is the prime source of lowered viability.
Aridis has introduced other processing improvements, since the cake structures formed by foam drying are extremely nonuniform and inconsistent. For this reason, steps are taken to control the foaming action, and the material is thoroughly milled and mixed to generate product consistency.
The application of these approaches allowed Aridis to realize room temperature preservation of live viruses, effective powderization of live viral vaccines, and greatly improved overall stability.
Protecting Potent Compounds
Another concern in lyophilization techniques is contamination. Normally, freeze-drying must be done in open systems to allow the transport of water vapor from the product to the ice condenser. Because of the exposed configuration, the substance faces a risk of contaminating the work space as well as enduring contamination through microbial infestation and particulates.
Single-use technology is widely available to provide containment of freeze-dried materials. This option allows the transport of solvent vapor from the frozen product and provides a balance between aseptic processing needs and product containment needs.
W. L. Gore & Associates (www.gore.com) has released a new freeze-drying container for research and stability studies, scale-up evaluations, and multiproduct lyophilization operations. Gore™ Lyoguard® Freeze-Drying Containers hold up to 60 mL of product and constitute single-use, autoclavable packages with removable, screw-on lids for access to the pharmaceutical cake. The devices can treat molecules in a closed system, while allowing sublimation of solvent.
This design avoids the risk of cross-contamination and uncontrolled release of materials as well as greatly circumscribes the cleaning and cleaning validation of the freeze-drying equipment.
Gore uses a proprietary polytetrafluoroethylene membrane incorporated into the container’s removable lid, which allows vapor to flow freely in and out of the container. The bottom part of the container is made of polypropylene and a medical-grade film, the flexibility of which enables the containers to conform to dryer shelves, thereby transferring heat efficiently and uniformly.
Circumventing Stabilizing Excipients
“Molecules hate to be freeze-dried, so we have to come up with a formulation that will optimize their performance,” says J. Jeff Schwegman, Ph.D., founder and CSO at BioConvergence (www.bioc.us). Critical areas under investigation by the company include the characterization of the physical structure of the target biomolecules during lyophilization and the determination of the optimal pH and minimal toxicity involved in the structural stabilization procedure.
The role of excipients in formulation development is a particular problem, since many of these substances have poor freezing properties, according to Dr. Schwegman. “For this reason we have to monitor the agents and their concentrations closely, so there is a balance between the thermal properties of the solution and the added stabilizing excipients.”
Dr. Schwegman and colleagues thus employ a variety of approaches including infrared spectrometry and use of accelerated stability studies in formulation development. He has also adopted infrared microscopy as a means of following changes in protein structure during the lyophilization process. This method yields more information than IR spectroscopy, which can be used only at the end of the freeze-drying process.
If not for IR microscopy, structural perturbation data and other changes would not be revealed, confounding the formulation of the excipient. Freeze-drying in the presence of mannitol and sucrose provides a considerable reduction in the level of perturbation.
Diversity of Applications
Lyophilization methods continue to grow with new types of products spilling into the market as methods of purification become more diversified. “Cancer therapies, recombinant antibodies, bacterial therapeutics, and vaccine technology have generated new pressures on the field of lyophilization,” remarks Edward Trappler, Ph.D., president of Lyophilization Technology (LTI; www.lyotechnology.com). Additionally, delivery systems utilizing liposomes and new chemical entities conjugated to polymers require novel approaches in lyophilization.
LTI’s use of mixed solvents for lyophilization has given rise to a subspecialty within this field. The company uses organic compounds for preserving polypeptides and proteins purified by chromatography. Mixed solvents including combinations of ethanol or t-butanol have also been investigated for processing finished drug products. This provides the ability to dissolve and process compounds that are poorly soluble or insoluble in water. Other features of this method include crystallization of the amorphous solute while the morphology of the crystalline solute may be unaltered.
Improvements constantly taking place in analytical and automation technology have enhanced lyophilization development and manufacturing. It is now possible to monitor the process in real time using mass spectrometry so as to optimize performance. “We tried to automate loading of lyophilizers 15 years ago but it proved to be difficult,” Susan Behrens, Ph.D., senior director, biological sciences & strategy, Merck & Co. (www.merck.com) notes. “Now using sensors, computers, and robotics we can monitor, control and automate every step of the process, protecting sterility at the same time.”
“Since the early days of the technology, viruses reflected the most demanding side of the equation, as they are large and complex structures,” points out Dr. Behrens. “We still have some distance to go in developing a really robust technology that will handle their lyophilization without loss of viability.”
She adds, though, that “vaccines and biologicals are on common ground when it comes to the lyophilization process.” This commonality in production includes the range of upstream processes such as fermentation and cell culture and downstream purification with lyophilization as the final step for some products.
As the demand for vaccine products is expected to double in the next five years, the ability to transfer knowledge gained from large-scale protein production will be vital. Merck’s investigations include studies on the excipients used for stabilization during freezing, including sugars, albumin, and gelatin.
Given the demand for vaccine preparations in underdeveloped countries, it is not surprising that Merck is considering the prospect of formulations so robust they could survive at tropical room temperatures of around 40ºC. “Although the science is not there yet, it is a theoretically achievable goal,” she states, “given that many viruses survive in the body of their host at close to that temperature.”
Into the Void
Looking into the future, the improvements in lyophilization technology suggest that perhaps the freeze-dried preservation of living material could be expanded from viruses and certain bacteria to larger structures, including mammalian cells. This concept has been pursued by Xuchu Ma, Ph.D., and coworkers at UC Davis who have shown that certain proteins and sugars found in animals capable of surviving desiccation might aid this process. They transfected mammalian cells with the gene for the stress protein p26 from the brine shrimp Artemia into mammalian cells. These were then loaded with trehalose, achieving a sharp increase in survival during air-drying.
Bizarre little creatures known as tardigrades, multicellular invertebrates with phenomenal resistance to desiccation, have been the subjects of additional studies. Poorly understood at present, the organism appears to mobilize sugars and heat shock proteins in a complex symphony to survive desiccation, in which these molecules complex with the cellular proteins, protecting them from destructive rounds of denaturation and loss of function.
Complex and involving many genetic systems, engineering desiccation into mammalian cell lines would represent a formidable challenge, but also an exciting possibility for long-term storage under ambient conditions of complex biological entities. HSP70, an important heat shock protein, is ubiquitous among living creatures and seems to be involved in the protection from desiccation in tardigrades and perhaps also a wide range of species including mammalian cells, according to Ingemar Jönsson at Kristianstad University in Sweden.
So the ultimate challenge to lyophilizers, mammalian cells to which one simply adds water to restore their viability, may not be quite as divorced from reality as it appears at first glance.