Freeze-Thaw Technologies

Bulk quantities of biological drug substances such as proteins, especially therapeutic monoclonal antibodies, are usually stored and transported in frozen form because biologics in their solid state exhibit increased stability, longer shelf life, reduced microbial growth and elimination of foaming during transportation. Drug substances are then thawed for further processing or finishing, and may undergo multiple freezing and thawing cycles during the manufacturing process. The freezethaw process induces stresses on the drug substance via several mechanisms, which may include cryoconcetration, pH changes, crystallization of excipient, and interaction of proteins with ice crystals and air bubbles formed at the ice crystallization front. Cryoconcentration, which is unavoidable on the microscopic scale, can also develop macroscopically as an inhomogeneous distribution of protein and excipients throughout the bulk container, due to non-uniform heat transfer, uncontrolled ice nucleation and progression of ice crystallization front, and insufficient mixing during the freezing and thawing processes. The freeze-thaw stresses could lead to activity loss and protein unfolding, aggregation, and chemical degradation. Thus, there is a need to better understand the complex thermophysical phenomenon of freezing and thawing as well as establish guidelines to design freeze-thaw cycles which will maximize protein stability while minimizing the associated stresses.

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There is currently a need to establish a set of practices to measure the amount of cryoconcentration during the freezing and thawing processes within containers of different shapes and sizes. Studies have been performed to study the behavior of protein solutions at the microscale for physical and chemical characterization. However, gaps exist both for the microscale understanding and the application of this knowledge at the macroscale for the bulk processes used in manufacturing. The impact of temperatures, times, and temperature ramp rates are important factors that can impact the quality of the final drug product. At the large-scale, the container geometry, material properties, and heat transfer characteristics play a key role in determining these factors. The risk of inhomogeneity in concentration also necessitates agitation or filtration after thawing which impart mechanical stresses to the drug substance. A fundamental coupling of the macroscale control factors with the existing microscale knowledge is highly desirable to ensure the design of optimal, scalable, and transferrable freeze-thaw cycles with minimal need for agitation after thawing.

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Stresses induced by the freeze-thaw process and by the agitation process can further be reduced with a thorough understanding of the impact of including excipients such as polysorbates and other surfactants during formulation development. Integrating the best practices to be followed during process and formulation development can mitigate undesirable protein aggregation and significantly improve drug product quality and stability.