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.

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