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Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.
Excipients must be carefully chosen to ensure optimum protection for vaccines and live biotherapeutic products.
Excipients are widely recognized as vital to the development of effective small-molecule and biologic drugs, with a wide range of approved compounds of varying chemistries providing functionality from taste-masking to stabilization. The latter is also crucial for vaccines and live biotherapeutic products (LBPs), which can suffer degradation during both upstream and downstream processing, fill/finish, packaging, storage, and transport. The properties of different LBPs and vaccines can differ widely, and thus careful selection of the right excipients is essential. Doing so is not easy, however, and requires experience with these challenging products and access to effective screening technologies.
As understanding of the role the human microbiome plays in human health and disease has increased, many academic research groups, startups, and Big Pharma firms have developed microbiome-based diagnostics and therapies, a number of which are advancing through the clinic. In 2018, at least 200 companies were active in the microbiome space, and 2400 clinical trials were underway (1); between January 2016 and July 2019, more than $5.4 billion was invested in partnerships and acquisitions related to LBPs (2).
LBPs by definition comprise living organisms that exhibit a desired activity within the human body. They have, in fact, been incorporated into probiotic foods and dietary supplements for many years, but typically, excess colony forming units (CFUs) are used to compensate for any loss of viability upon storage. That approach isn’t possible for pharmaceutical products, so the goal during manufacture is to maximize the number of cells that are viable throughout the entire production process, according to Aaron Cowley, chief science officer with Arranta Bio.
“One of the key attributes associated with LBPs is the ability to keep the cells alive and healthy over a duration of time, thus allowing them to function as they are designed,” says Synlogic Therapeutics Chief Operating Officer, Tony Awad. The sum of the stresses that the organisms experience during upstream, downstream, fill/finish, storage, and rehydration will determine if the organisms will retain viability and activity, he adds. Each of the processing steps, therefore, needs to be optimized, because they can all have an impact on subsequent stability.
Processes also need to be designed for scalability, Cowley adds. “Because most of the organisms that comprise LBPs tend to be unstable, it is essential to minimize process times in order to minimize die off. Effective small-scale processes that take much longer to complete when performed at large scale are, therefore, not practical for commercial LBP manufacturing,” he explains.
Viability, and thus stability, depends on a number of factors, including not only the general fermentation conditions and harvest technique, but also excipient/cryopreservative selection and the lyophilization process, according to Cowley. He also stresses that development of an optimum cell bank is crucial as well. “Optimizing the master cell bank is necessary to ensure that the organism being produced is optimally healthy, exhibits the right performance behaviors, and is generated in the maximum yield,” he observes.
Whether the strains are delivered as a single organism or consortium can also affect the stability of LBPs, as can the type of organism. “For example, spore-formers can have greater stability allowing for storage at room temperature but may come with side effects as these bacteria have the potential to become pathogenic,” Awad observes. Comprehensive characterization of LBP strains is also essential. “Knowledge of critical parameters and acceptable ranges—redox, osmolality, etc.—and how to control them is necessary to achieve and maintain the proper functionality throughout the production process,” Cowley asserts.
Stability issues can also occur at any stage of a vaccine’s development lifecycle, according to Jee Look, senior director of drug product development for Emergent BioSolutions’ contract development and manufacturing organization (CDMO) business. “In some cases, instability that occurs during upstream/downstream processing can be mitigated during final drug product manufacturing, but it is important to identify and understand the mechanisms of instability early in the pre-formulation stage as instability in the final drug product would have the largest impact on patient safety and overall production efficiency and would be more costly to fix,” he says.
The instability characteristics possible with a given vaccine also depend on the vaccine type, whether it is a live virus, inactivated virus, virus-like particle, or some other type of subunit, recombinant, or conjugate vaccine. The use of aluminum adjuvants can also create different challenges with respect to vaccine stability. “It is important to understand the mechanism of action of each type of vaccine in order to design the formulation and deal with the instability issue,” Look concludes.
Stabilization of LBPs involves different mechanisms and thus different excipients. For LBPs stored at frozen conditions, cryoprotectants are necessary, while those that undergo lyophilization will require lyoprotectants, according to Awad. For these applications, excipients are added to the cell slurry obtained following harvest to maintain viability through the harsh freezing and drying process, which can be damaging to live microorganisms, according to Cowley.
“Both of these options can lead to better shelf life of the LBP,” Awad observes. Excipients are also used in final LBP formulations to influence cellular size and structure and help stabilize LBPs during cryopreservation and/or to preferentially bind moisture to ensure viability and activity are maintained once the product is packaged, Cowley adds.
Many organisms developed as live therapeutics also tend to combine into long chains. “These aggregates can lead to low CFU results because many tens of cells (or more) are detected as just one colony,” Cowley explains. In this case, use of appropriate media can minimize aggregation. “At Arranta Bio, we have found that certain media formulations in particular prevent aggregation regardless of the type of organism, allowing for the development of optimum processes,” Cowley says.
For vaccines, different types of excipients play important roles during vaccine production, formulation, fill/finish, and storage processes, according to Look. “Selection of excipients can be a complex consideration beyond stability issues and can also depend on the target product profile (TPP), logistics, and business market complexities,” he comments.
For instance, if the TPP requires a long shelf life and/or high temperature storage, the vaccine would need to be converted into solid form. In this situation, Look says sugar excipients such as sucrose could play an important role. For live vaccines, which can be susceptible to manufacturing and storage loss, some type of protein stabilizer such as human serum albumin or gelatin may be required.
Antibiotics are also used to prevent growth of bacteria during production and storage, and stabilizers such as monosodium glutamate (MSG) and 2-phenoxyethanol are used to protect vaccines from heat, light, acidity, or humidity during processing and storage.
Cryoprotectants and lyoprotectants are the most critical excipients for LBP stabilization and function by minimizing cell damage caused by ice crystal formation during freezing, according to Awad. Some cryopreservatives penetrate the cells, but all generally influence viscosity, osmolality, surface tension, and other properties, according to Cowley. “The goal is to choose the right mixture that provides the optimum conditions for each specific organism to survive during cryopreservation,” he says.
This type of protection, according to Awad, is important whether the LBP is stored as a frozen liquid or lyophilized to produce a powder for oral suspension. He also notes that additional excipients may be required if the mechanism of instability is known. “For example, antioxidants may be utilized if the strain is susceptible to oxidation or buffering excipients may be utilized if maintaining pH is important,” he explains.
The types of excipients used to formulate a specific vaccine will depend on the type of degradation mechanism of that vaccine. “Degradation mechanisms include aggregation, precipitation, oxidation, deamidation, shear, freeze/thaw, air-water interface, thermal, etc.,” Look states. For instance, different protein stabilizers will be required depending on the causes of aggregation, while antioxidants are important to prevent oxidation. As one example, Look notes that a surfactant such as polysorbate 80 is typically used to deal with aggregation/precipitation due to interactions that occur at the air-water interface. “In this situation,” he observes, “polysorbate 80 would migrate to the air-water interface and prevent the protein from getting through.”
Understanding the mechanisms of instability to identify excipients that have been demonstrated to overcome these mechanisms is the best approach to selecting optimum stabilizing agents for both LBPs and vaccines. For many of these products, however, the role excipients play is still unknown. “Trial and error, empirical approaches, and historical experience are needed when considering excipient options, especially for new gene-therapy vaccines,” Look comments.
For LBPs, the effectiveness of excipients is evaluated by testing for key quality attributes or release criteria, including cell counts, viability, activity, and shelf life, according to Awad. “Excipient screening through compatibility studies is also often required,” he adds.
One of the main challenges for LBPs is a lack of commercially available excipients and media blends that have been specifically developed for use with these new active ingredients, Cowley notes. Based on its more than 10 years of working with LBPs, Arranta Bio has addressed this issue with the development of a number of proprietary Arranta Media Blends and CryoPreservatives, both based only on plant-derived components, that the company uses to rapidly screen against client microorganisms to identify optimum process conditions.
“We have tested both the media blends and cryopreservatives against commercially available formulations for GMP manufacturing with good results. Our media blends provide higher yields and more stable organisms, with additional indirect benefits downstream, including higher resulting viability and shorter lag phases through freeze drying. Our cryopreservative blends also afford higher viabilities and in general help us consistently achieve our minimum viability target of 40%,” Cowley says.
For vaccines, the selection of optimum excipients requires a “risk and benefit” analysis, according to Look. “A vaccine designed for healthy babies would have different considerations than a vaccine designed for terminally ill cancer patients,” he notes. Most vaccines are injectables and applied to large healthy populations, therefore selection of optimum excipients is more rigorous and depends on several factors, Look adds.
“The initial selection of excipients and concentration usually starts with historical information such as those already used in commercial vaccines. From there, the final selection and optimal concentration is done through pre-formulation and formulation development studies with the specific vaccine,” he explains.
Awad recommends that for any drug formulation, it is generally best to use excipients that have been included in prior approved products rather than novel excipients, because the groundwork has been laid and these excipients are likely to have regulatory agency approval. If a novel excipient is needed, though, additional testing, such as an extended animal study (tox study), may be needed, according to Look.
It is also important, Look reiterates, that excipients are not the only means to resolve vaccine—or drug—instability problems. “A more holistic approach is needed, which includes the consideration of processing conditions and any device used for administration. Some instability issues can be mitigated through process optimization and selection of the appropriate device,” he concludes.
1. C.R. Fernandez, “No Guts, No Glory: How Microbiome Research is Changing Medicine,” www.Labbiotech.eu, Jan. 22, 2019.
2. A. Guillermo, “Why Investors Trust Their Gut About the Human Microbiome,” www.ResultsHealthcare.com, June 7, 2019.
Cynthia A. Challener, PhD, is a contributing editor to Pharmaceutical Technology.
Vol. 44, No. 10
When referring to this article, please cite it as C. Challener, “Factoring Stability in the Biologic Drug Mix,” Pharmaceutical Technology 44 (10) 2020.