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Breakthroughs in analytics and alternatives to traditional freeze drying promise to reshape biological development and the cold chain.
The past year has been a busy one for vendors and contract development and manufacturing organizations (CDMOs) that provide lyophilization equipment and services. On the bulk lyophilization side, demand for lyophilization for antibody-drug conjugates (ADCs), monoclonal antibody therapies (mAbs) for COVID-19, and some of the raw materials used in COVID-19 vaccines has been growing, says Scott Ross, global product specialist for bulk lyophilization with W.L. Gore and Associates.The company’s Lyoguard trays are being used in the production of mAbs; diagnostics tests for COVID-19 virus or antibodies; and some of the key raw materials used in messenger RNA (mRNA) vaccines, including synthetic oligonucleotides, adjuvants, and lipid nanoparticles, he says.
“For ADCs, lyophilization is likely to become a de facto standard,” Ross says, noting that operator safety concerns and the need to reduce turnaround times at CDMOs are driving use of contained tray systems. In addition, real-time monitoring of temperature and other parameters is becoming routine, he says.
As it has lifted demand for bulk lyophilization, the COVID-19 pandemic has also highlighted deficiencies with vial-based lyophilization, a process that has seen few changes in the past 50 years. By offering a powdered form of vaccine that could be shipped at ambient temperatures and reconstituted at the point of administration, lyophilization could have simplified the distribution of new vaccines, including those based on mRNA, which must be transported and stored as frozen liquids at extremely low temperatures, then mixed and manipulated before patients can be injected. Of the five COVID-19 vaccines that are now being used globally to prevent COVID-19, only Russia’s Sputnik V—which also comes in frozen liquid form—actually uses traditional vial freeze drying.
In the end, it’s a question of capacity. It would take three years for the industry to have enough capacity to lyophilize 300 million vials of product, says Alina Alexeenko, professor of chemical engineering, aeronautics, and astronautics at Purdue University and cochair of LyoHUB, a consortium of users and equipment and services vendors which was established four years ago to promote best practices and establish a roadmap for the future of this technology.
Fellow co-chair and Purdue professor Elizabeth Topp, who is also chief science officer at the Dublin, Ireland-based National Institute of Biopharmaceutical Research and Training (NIBRT), agrees.“Global lyophilization capacity wouldn’t allow us to process a billion vaccines a year, and stability testing is another hurdle. If you’re pushing a vaccine out the door in nine months for emergency use authorization, you clearly won’t have time to do a shelf stability study on a lyophilized formulation and guarantee 18 months stability,” she says. However, new analytical methods and process development strategies promise to improve processing and simplify distribution, administration, and the cold chain. “We can expect to see more lyophilized vaccines in the future,” says Steve Nail, senior LyoHUB scientific advisor who was formerly a Purdue professor and is now principal scientist at Baxter Biopharmaceutical Solutions, a CDMO in Bloomington, Ind.However, he says, the equipment used to process the materials will look quite different from the traditional stainless steel lyophilizers, with heat-transfer fluid circulating through shelves.
“The technology is up for disruption,” says Alexeenko. She notes that innovative drying processes (e.g., spray-freeze drying [both continuous and non-continuous]; spin freezing; thin- film freezing; and microwave drying), show promise to simplify the processing, transportation, and distribution of biopharmaceuticals and vaccines.
Lyophilization poses challenges, particularly for some proteins. A number of stresses are associated with freezing and freeze drying, as well as with agitation and phase interfaces, says Nail.Another problem is the fact that lyophilization involves very slow cooling, at a rate of one degree Kelvin per second, says Bill Williams, a professor at the University of Texas and inventor of the thin-film freezing process, which he developed years ago at the Dow Chemical Co.
TFF Pharma licensed his technology and commercialized it in 2019. Williams and his team, with corporate and US government funding, are now focusing on research designed to optimize use of thin-film freezing to improve the processing and delivery of biologics, including vaccines, along with the cold chain. Some of the problems that can occur with lyophilization are due to ingredients within vaccine formulations. As Williams notes, 80% of all existing vaccines use aluminum hydroxide adjuvants. If these ingredients freeze and then melt, particles can then aggregate, affecting both vaccine immunogenicity and activity, he says.
However, Williams notes, slow freezing is another challenge with lyophilization that affects vaccine activity. “When the freezing rate is 100 to 1000 times faster than it is for traditional lyophilization, as it is in thin-film freezing, the integrity of protein materials is maintained,” he says. Using thin-film freezing, the vaccine can either be reconstituted at the point of use, or administered via nasal or pulmonary delivery, he says.
Currently, Williams’ lab is working with TFF Pharma and more than 25 different pharma companies on research that is formulating liquid mAbs,mRNA lipid nanoparticles, and other bioformulations into thin-film powders that can be delivered to the lungs. The company recently worked on a reformulation of remdesivir as an inhaled dry powder, and in July 2020, completed Phase I of a clinical study that studied formulation of voriconazole as an inhaled powder.
Protein destabilization is another issue with lyophilization. Although research has studied the mechanics of chemical destabilization, which occurs as the result of reactions such as deamidation, oxidation, and chain cleavage or “clipping,” the science behind physical destabilization processes such as aggregation is not yet fully understood, says Topp.
“Aggregation is hard to control because it is part of the protein folding problem. We don’t really understand how proteins fold, and we don’t understand how they aggregate either,” she says.
Solid-state hydrogen deuterium exchange (HDX), an analytical method that is being used in research at Purdue and NIBRT, allows proteins to be studied in the solid state at high resolution, and is improving understanding of protein stability, says Topp.“A lot of what seems to drive the performance of these materials has to do with the hydrogen bonds that help make up the structure of the proteins, and the hydrogen bonds between the protein and the matrix that surrounds it. HDX interrogates the hydrogen bond network, and, so far, results suggest that the better the network, the more stable the material,” she says.
Evidence suggests that aggregation may be driven by partial protein unfolding. In the future, Topp says, this research might yield a metric that would reveal how well folded each individual protein is, which could help determine whether a particular biopharmaceutical formulation will perform well or not, whether it is made bylyophilization or another process.
In addition, Topp notes, research suggests that measurements made with HDX correlate closely with the stability of the protein in storage. “If we can characterize these materials and the environment that the protein sees with sufficient resolution, then maybe we can start to replace time consuming 18-month to two-year stability studies with advanced analytical characterization,” she says.
This approach would likely start with comparability protocols and then move to other parts of regulatory filings. “Stability studies impede all kinds of process changes and improvements. We hope that we can put tools in place to change that,” Topp says.
Not only HDX but nuclear magnetic resonance (NMR) spectroscopy and neutron back scattering can be advanced to study stability. “In order for progress to be made, companies need to adopt newer technolgies and include them in regulatory filings, to put the technology in front of regulators and the industry,” she says.
In a lighter vein, Topp envisions a “solid-state protein characterization smackdown,” in which the three methods would be used to analyze a blinded sample of a lyophilized protein such as an mAb, and results then compared. The method or combination of methods that best correlated with storage stability would then be deemed the winner(s).
On a smaller scale, similar work evaluating solid-state HDX has already beendone with Roche Genentech. In this case, however, HDX measurements were taken for blinded samples of four different mAb formulations, and the results were then compared to results of 2.5 years of stability data that had already been gathered for the same formulations.In two weeks, the HDX came up with the same rank order of results as stability testing, Topp says. “HDX measurement metrics showed nearly perfect correlation with aggregation during mAb storage,” she recalls.
Purdue and NIBRT have also been working on projects supported by the National institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) that use HDX. This research has evaluated the impact of process changes as well as the use of alternative drying methods on stability.
LyoHUB was never intended to be a research consortium. Its first goalwas technology road mapping (i.e., discerning ways to revitalize the technology), and another primary activity has been disseminating best practices to improve use and design of lyophilization equipment. “The fact is that there is a lot of poor practice going on in the industry, due to a lack of understanding of the fundamentals of heat and mass transfer,” says Nail. LyoHUB’s latest best practices paper focuses on the formulation of lyophilized products, but validation and equipment qualification are areas that will be updated soon, says Alexeenko. LyoHUB is actively facilitating research and the adoption of new technologies and advanced analytics and equipment among its 25 member companies. To advance this goal, the organization opened up a new demonstration laboratory in 2019 to allow its members to work with new process equipment and analytical systems.
Process analytical technology (PAT) remains an area of focus. One technology being developed is smart vials, wireless sensors that would go into each individual vial being lyophilized and measure such data as temperature, pressure, and extract lyophilization rate, and communicate the information back to the freeze drier, says Alexeenko.
“This is important because, in lyophilization, batches are qualified, and a whole batch can be trashed if something goes wrong in just one or two vials. This sensor allows individual vials to be monitored, providing more control,” says Topp. The vials can also be used to measure and control ice nucleation and to understand its dynamics. “This is the one area in lyophilization where things happen very quickly, and we need to better understand and control that process,” Alexeenko says. Another method that is being studied in projects at LyoHUB’s new demonstration facility is in-situ residual gas analyzer mass spectroscopy, a technology that Purdue has been using since 2018. The method enables the study of problems that can occur in freeze drying when components in a formulation decompose and a pH shift or other issues result.It can detect parts-per-millon-level changes in the composition of the gas during lyophilization, and then allow them to be correlated to process changes.The first applications of the technology used ammonia, but Nail is working on a project that uses organic co-solvents, typically required when processing poorly soluble drugs. This work will study the time course release of ethanol vapor from product, which has been shown to have a critical influence on product reject rates.
Other projects are focusing on improving sensors and process modeling. “You can’t do one well without the other,” says Alexeenko. In process modeling, LyoHUB members are working on a virtual thermocouple, which uses high speed computing and high-fidelity methods to model heat transfer, not only in porous materials but also in and between glass containers, and between glass and plastics containers (e.g., SiO2-type vials). The project combines noninvasive measurement and process modeling so that the temperature within the vial can be reconstructed with a much higher level of detail, says Alexeenko.
A new area of interest is microwave heating using volumetric electromagnetic rather than conductive heat transfer. So far, the method has been found to reduce impact on large biomolecules.
Nail notes increased industry interest in applying lyophilization technology to human stem cells. In 2020, Alexeenko says, Purdue researchers developed lyophilization processes for use with the mRNA and CRISPR Cas9 reagents that are used in some COVID-19 diagnostic testing kits.
Vol. 34, No. 4
When referring to this article, please cite it as: A. Shanley, “Beyond Lyophilization,” BioPharm International 34 (4) 2021.