Beyond the Freezing Point: The Science of Biopharmaceutical Preservation

How Cooling Rate Affects Crystallization

Freezing is widely used to preserve biopharmaceuticals, but maintaining stability involves more than simply keeping products below 0°C. The rate of cooling, the behavior of dissolved excipients, and the temperature at which formulations become fully immobilized can all influence product quality and therapeutic performance. Biopharmaceutical solutions consist of distilled water and solutes, and the latter can be ions, proteins (such as enzymes), lipids, and sugars. This complex mixture makes the cooling rate different from that of simple water. In the case of water, the cooling rate is usually applied to water-to-ice conversion, also referred to as primary crystallization; in biopharmaceuticals, a secondary crystallization also occurs — that of solute and water. For several solutes — including glycine, NaCl, and mannitol — a critical cooling rate has been identified, below which the solute would crystallize during cooling. Above this critical cooling rate, the solute would remain amorphous during cooling, but it could crystallize during subsequent storage.

Critical cooling rate depends on the composition of the solution, such as the presence of different ions. Dissolved sales can alter the way water molecules interact with one another, disrupting hydrogen bonding and influence how those molecules rearrange themselves as the solution cools¹. Cooling may also be influenced by other factors, such as the volume of the solution and heterogeneous surfaces²—a consideration that directly influences nucleation, the first step in any crystallization process. Nucleation is a random and highly sensitive process, specific to experimental conditions, including concentration and nature of impurities and mechanical agitation, which are often difficult to control and reproduce. For example, ice nucleation temperatures in GMP manufacture are usually lower than those in laboratory experiments as extraneous or environmental particles (which can serve as centers of heterogeneous ice nucleation) are tightly controlled under GMP conditions².

The Risk Zone — Understanding Glass Transition in Frozen Biopharmaceuticals

Most of the water present in a biopharmaceutical solution will freeze at approximately -2°C to -5°C. However, aqueous solutions of biopharmaceuticals are not completely frozen even at −20°C. Such materials consist of at least two phases: ice, and the freeze-concentrated solution (FCS), which contains all the solutes including the active ingredient (for example protein), as well as an unfrozen fraction of water. The FCS is distributed and squeezed between ice crystals, and remains mobile at −20°C. Solidification of the FCS takes place below its corresponding glass transition temperature (Tg’), which in a typical formulation is below −35°C. Some chemical reactions, such as oxidation and hydrolysis, can still take place and may even be accelerated in the partially frozen systems2. Storage of biologicals at temperatures higher than Tg’ can alter biologic behavior when thawed and impact therapeutic properties or potency. The partially frozen state between the water liquids, i.e., the minimum temperature at which water can be liquid (slightly below 0°C), and Tg’ is called the “Risk Zone”, and the time a sample spends in the Risk Zone can be quantified².

Temperature Range: Approximately 0°C to Tg’ (Typically -35°C to -53°C Depending on Formulation)

In this partially frozen state:

What the Data Shows: The Role of Cryoprotectant in Defining the Risk Zone

Studies have shown glass transitions of biopharmaceutical solutions ranging between -35°C and -53°C, depending on the utilized cryoprotectant. Two transitions are commonly observed for frozen sucrose and trehalose solutions³: one transition appears at about −33 °C, while the other, weaker transition, appears at about −53°C (quench cool) or −44°C (slow cool). For a binary sucrose-water solution, in particular, the temperature was determined to be −32°C^4,5. A similar observation was made in an earlier study of aqueous sucrose solutions⁶, in which specific viscosity was associated with the characteristic temperature determined to be −35°C for sucrose-water mixtures. The glass transition (Tg’) of a sorbitol formulation was found to be −45°C. Formation of the crystalline sorbitol at −30°C deprived the protein of cryoprotectant and resulted in aggregation over time, while corresponding material stored at −70°C was stable.

From Science to Storage, and What This Means in Practice

Understanding the interplay between cooling rate, crystallization, and glass transition temperature is essential for ensuring biopharmaceutical stability and therapeutic efficacy. The ‘Risk Zone’ is not an abstract concept but a measurable and manageable variable that directly informs temperature selection, formulation design, and solute usage. Controlling the time a sample spends in the Risk Zone, and selecting solutes with appropriate Tg’ values, are practical tools for protecting biologics throughout their lifecycle.

In practice, minimizing time spent in the Risk Zone may involve:

As biologics become increasingly complex, understanding frozen-state behaviour is critical for maintaining product quality throughout manufacturing, storage, and distribution. By controlling crystallization dynamics and minimizing exposure to the Risk Zone, organizations can reduce stability risks and better protect therapeutic efficacy.

Cooling and crystallization are just the beginning — see how the science turned into strategy in The Evolution of Sample Management and Automation in Drug Discovery eBook, by Sue Holland-Crimmon, Ph.D.

References

1. Qiu, M. et al. Tailoring water structure with high-tetrahedral-entropy for antifreezing electrolytes and energy storage at −80 °C. Nat. Commun. 14, 601 (2023)..
2. Authelin, J.-R. et al. Freezing of Biologicals Revisited: Scale, Stability, Excipients, and Degradation Stresses. J. Pharm. Sci. 109, 44–61 (2020).
3. Chang, L. (Lucy) et al. Using modulated DSC to investigate the origin of multiple thermal transitions in frozen 10% sucrose solutions. Thermochim. Acta 444, 141–147 (2006).
4. Piedmonte, D. M., Summers, C., McAuley, A., Karamujic, L. & Ratnaswamy, G. Sorbitol Crystallization Can Lead to Protein Aggregation in Frozen Protein Formulations. Pharm. Res. 24, 136–146 (2007).
5. Carpenter, J. F., Pikal, M. J., Chang, B. S. & Randolph, T. W. Rational Design of Stable Lyophilized Protein Formulations: Some Practical Advice. Pharm. Res. 14, 969–75 (1997).
6. Yu. Shalaev, E. & Franks, F. Structural glass transitions and thermophysical processes in amorphous carbohydrates and their supersaturated solutions. J. Chem. Soc. Faraday Trans. 91, 1511–1517 (1995).