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Temperature-sensitive biologics are lyophilized to preserve therapeutic viability, but the process presents complexities and challenges that are as yet not fully understood.
As more biological molecules are explored as therapeutic agents to treat diseases, the use of lyophilization is increasing. Despite its challenges, lyophilization remains the best option to preserve molecular integrity and, therefore, therapeutic efficacy. However, the process is complex and challenging. Optimizing it requires a clear understanding of underlying mechanisms and issues.
At its most basic, lyophilization is a freeze-drying process by which water or liquid is removed from a product after it has been frozen and placed under a vacuum. Lyophilized product can be stored safely for much longer periods than unlyophilized product, avoiding such problems as loss of biological activity and unpredictable, undesirable immune responses. The process also minimizes damage to the material that would occur if it were to be dried only. Living cells, proteins, and other complex molecules survive well with lyophilization and can easily be rehydrated after prolonged storage periods. In the case of a biological therapeutic agent, the biologic is rehydrated and put into solution prior to administering to the patient.
Although costly and time consuming, turning a therapeutic agent into a dried powder formulation via lyophilization has been the industry’s most practical solution to stabilizing the biologic. Lyophilization also offers other advantages, such as providing a desired expiration limit for proteins that are naturally volatile or that easily and quickly break down, providing stability at ambient temperatures, inhibiting proteolytic enzymes from breaking down the protein, and allowing for the potential modification of the biologic formulation after manufacture and prior to administration (e.g., adding preservatives, changing pH or viscosity, or adding diluents for special applications) (1).
Despite the advantages that lyophilization offers, biomanufacturers still struggle with the challenges of the process, which can often be attributed to the complexity of biological molecules, such as therapeutic proteins, antibodies, antibody drug conjugates, and, now, cell and gene therapies. For instance, common components used in the manufacture and formulation of proteins, monoclonal antibodies, and other biologics can destabilize molecules in freeze-dried, or even frozen, formulations. These include such common components as buffers and surfactants used during the manufacturing process (2). Phosphate buffers, such as sodium phosphate, may contain one component that may precipitate during freezing, which could change the pH of the solution in which the biological molecule is carried. The shift in pH could destabilize the molecule during lyophilization, thus it is important to understand how commonly used biomanufacturing components can act during the freeze-drying process. It is recommended that minimum buffer concentration needed to control pH, regardless of buffer type, be used to avoid instability in the biological molecule during lyophilization (2).
The nature of complex biological molecules poses other challenges to the lyophilization process. For one, biologics are sensitive to various interactions, including those that occur at the air/liquid interface, at the liquid/primary packaging interface, and even at the solution/ice interface. These interactions typically cause the protein to unfold, which can result in aggregation and, thus, loss of bioactivity. The end result would be the loss of therapeutic efficacy in the patient. Another challenge is freeze concentration; when ice forms, biologics become more concentrated and molecules come into close contact with each other. This can potentially lead to pH shifts as well as increases in ionic strength. Finally, because protein structure and bioactivity depend on the molecule’s interaction with surrounding water, removing water during lyophilization destabilizes the protein structure; thus, for a biologic, some residual moisture must be retained in the final lyophilized cake to ensure stability (3,4).
In addition to the challenges of dealing with complex, biologically active molecules, there is also a need for specialized knowledge. Because there is no “one-size-fits-all” lyophilization process, each biologic product requires an extensively studied and designed process specific to that molecule. For example, extensive studies may be needed to understand the freezing and drying behavior of components used for the formulation of the biologic, and studies should be done to determine how factors such as formulation strength and/or containers affect the freeze-drying process. Complex formulation components, such as nanoparticles, microparticles, or liposomes (5), would also require study and further knowledge of their behavior and characteristics during freeze-drying. In addition, mechanical factors such as temperature, pressure, and time setting, differ with each type of molecule, and thus these parameters should be taking into consideration when designing a lyophilization process for a specific biologic (6).
Another challenge is that lyophilization presents a high contamination risk because product is exposed for extended periods of time. It is therefore recommended that sterile lyophilization be conducted in an ISO 5 (Class 100) environment (5). Clean-in-place and steam-in-place capabilities should also be in place.
Despite the challenges, time, and costs of lyophilizing biological molecules, the need for lyophilization is growing, driven by the increasing number of innovative biologic product candidates and biosimilars that are now being developed. Industry observers have even noticed growing demand for lyophilization in the small-molecule pharmaceutical sector, as more small-molecule drugs are being produced in an aqueous medium (6).
The biopharmaceuticals market is also seeing the incursion of the first round of biosimilars for blockbuster innovator biologics, and many of them will be lyophilized products. As innovator patents reach expiration over the next decade (4), more biosimilars are expected to reach the market. In this case, biopharmaceutical companies developing biosimilars have an added challenge of demonstrating that the lyophilized biosimilar looks and acts the same as the innovator biologic it references. Regulators will scrutinize any biosimilar formulations, which must closely match the formulation of the innovator products.
1. B.S. Chang, et al., “Lyophilized Biologics,” in Lyophilized Biologics and Vaccines, D. Varshney and M. Singh, Eds. (Springer, New York, NY, 2015), pp. 93–120.
2. Baxter, “Best Practices in Formulation and Lyophilization Development: Proteins, mAbs, and ADCs,” Baxter BioPharma Solutions White Paper, accessed Dec. 20, 2019.
3. C. Challener, BioPharm International 30 (1) 32–35 (2017).
4. L. Wegiel and G. Sacha, “Considerations for Development of a Lyophilized Biosimilar,” Baxter BioPharma Solutions Case Studies, 2018.
5. N. DiFranco, “Lyophilization of Pharmaceuticals: An Overview,” LubrizolCDMO.com, Oct. 8, 2019.
6. E. Price, “What is Driving the Growing Demand for Lyophilization?” PCISynthesis.com, Aug. 15, 2019.
Vol. 33, No. 1
When referring to this article, please cite it as F. Mirasol, “Lyophilization Presents Complex Challenges,” BioPharm International 33 (1) 2020.