Freezing of Biologicals Revisited: Scale, Stability, Excipients, and Degradation Stresses

Abstract

Although many biotech products are successfully stored in the frozen state, there are cases of degradation of biologicals during freeze storage. These examples are discussed in the Perspective to emphasize the fact that stability of frozen biologicals should not be taken for granted. Frozen-state degradation (predominantly, aggregation) has been linked to crystallization of a cryoprotector in many cases. Other factors, for example, protein unfolding (either due to cold denaturation or interaction of protein molecules with ice crystals), could also contribute to the instability. As a hypothesis, additional freezingrelated destabilization pathways are introduced in the paper, that is, air bubbles formed on the ice crystallization front, and local pressure and mechanical stresses due to volume expansion during waterto-ice transformation. Furthermore, stability of frozen biologicals can depend on the sample size, via its impact on the freezing kinetics (i.e., cooling rates and freezing time) and cryoconcentration effects, as well as on the mechanical stresses associated with freezing. We conclude that, although fundamentals of freezing processes are fairly well described in the current literature, there are important gaps to be addressed in both scientific foundations of the freezing-related manufacturing processes and implementation of the available knowledge in practice.

Temperature Based Process Characterization of Pharmaceutical Freeze-Thaw Operations

“…Due to their high value and production cost, activity loss of the active pharmaceutical ingredient (API) during shipment and storage has to be limited by selection of suitable formulation agents (Chang et al, 2005;Bauer et al, 2017) and storage conditions. Therefore, many biopharmaceuticals are stored in a frozen state (Singh et al, 2009;Authelin et al, 2020). While freezing slows down and reduces degradation reactions of the API, freezing processes expose the protein to different stresses such as cold denaturation (Privalov, 1990), freeze concentration (Bhatnagar et al, 2007), ice crystal formation (Chang et al, 1996), and potential excipient crystallization.…”

Interfacial Stress and Container Failure During Freezing of Bulk Protein Solutions Can Be Prevented by Local Heating

“…9a). The higher interfacial stress, resulting from this pressure-driven flow through the ice structure, which is rich in entrapped bubbles (air interfaces), enhances protein aggregation, which could also be anticipated from previous reports (10,(14)(15)(16). BSA was more sensitive to interfacial stress than to the concentration gradient, although this should not be generalized because high-concentration gradients have also been consistently reported as a risk factor (10,17).…”

“…This work shows that internal pressure reached 10 bar in 5-L bottles, while 2-L bottles did not show significant pressure variation. Once the top freezes, hydrostatic pressure is released by the percolation of the liquid through the ice crust, thus forming a frozen mound; otherwise, pressure would rise much higher with the decrease of temperature, reaching values near 2 kbar (following water solid-liquid equilibrium) (10). Pressure is therefore expected to rise when the crust becomes thicker, or when the flow rate of liquid towards the top is higher, as for example, for bottles that have larger volume/interface ratio (such as 5 L compared with 2 L).…”

Stability Studies of the Vaccine Adjuvant U-Omp19

“…40,41 Some proteins may undergo unfolding, aggregation or changes in concentration over time in the frozen state. 34,38,39,42 In this work, U-Omp19, was shown to retain its full inhibitory activity, folding, integrity, while no significant aggregation was induced when subjected to freezing (Fig. 5).…”

“…37,38 However, the freezing process and frozen storage can affect the quality of the product. 39 Vaccines containing aluminum salt adjuvants are prone to inactivation following exposure to freeze-thaw stress. 40,41 Some proteins may undergo unfolding, aggregation or changes in concentration over time in the frozen state.…”