Considerations on Protein Stability During Freezing and Its Impact on the Freeze-Drying Cycle: A Design Space Approach

Abstract

Freezing is widely used during the manufacturing process of protein-based therapeutics, but it may result in undesired loss of biological activity. Many variables come into play during freezing that could adversely affect protein stability, creating a complex landscape of interrelated effects. The current approach to the selection of freezing conditions is however nonsystematic, resulting in poor process control. Here we show how mathematical models, and a design space approach, can guide the selection of the optimal freezing protocol, focusing on protein stability. Two opposite scenarios are identified, suggesting that the ice-water interface is the dominant cause of denaturation for proteins with high bulk stability, while the duration of the freezing process itself is the key parameter to be controlled for proteins that are susceptible to cold denaturation. Experimental data for lactate dehydrogenase and myoglobin as model proteins support the model results, with a slow freezing rate being optimal for lactate dehydrogenase and the opposite being true for myoglobin. A possible application of the calculated design space to the freezing and freeze-drying of biopharmaceuticals is finally described, and some considerations on process efficiency are discussed as well.

Economic Analysis of a Freeze-Drying Cycle

“…Moreover, even though economic evaluations have been presented in the past [9][10][11], they are often too broad or too specific to be used for making comparisons between different process strategies. Whereas little effort has been made in this field to determine the economic impact of freeze-drying quantitatively, the optimization of the process has usually remained product-or quality-specific [12][13][14][15][16][17][18], with no considerations of possible bottlenecks in the expenses. In this work, we aim to create a model to evaluate the actual economic impact of a freeze-drying cycle, and evaluate the impact of process parameters on processing costs and, finally, the benefits of process optimization.…”

Aggregation of protein therapeutics enhances their immunogenicity: causes and mitigation strategies

“…70,93,94 Conformational stability has also been shown to be affected by other stresses, including changes in pH 94 and ionic strength. 95 Furthermore, changes in conformation caused by adsorption onto surfaces, such as ice crystals during freezing, 96 or air bubbles formed by mechanical agitation, 57 is proposed to lead to protein denaturation. 97,98 With regard to colloidal stability, factors that crucially affect repulsive interactions between protein monomers include ionic strength and pH.…”

“…As an example, protein therapeutics are often lyophilized to keep them stable during storage and transportation, a process that can result in aggregation via cold denaturation 69,70 or by surface interaction with the ice-water interface. 96 However, the lyophilization process and/or formulation can be optimized, to minimize aggregate formation. To prevent aggregation during freeze-drying a recent study demonstrated how changing to a fast freezing procedure reduced aggregation and loss of activity of a model protein (myoglobin), which is sensitive to cold denaturation.…”

“…Non-ionic surfactants are often used in protein formulations to prevent aggregation caused by adsorption onto surfaces and are thought to work by out-competing the protein for access to hydrophobic interfaces. 92 Such surfaces include interaction with the vessel holding the formulation, but also adsorption to ice-water interfaces during freezing 96 and to air-water interfaces introduced by for instance mechanical agitation stresses. 125 The most common non-ionic surfactants used in protein formulations are polysorbate 20 (PS20) and polysorbate 80 (PS80).…”

“…128 This maintenance of conformational stability by either PS20 or PS80 has been shown to prevent aggregation caused by mechanical agitation 67,[128][129][130] and during freezing. 96,131 However, commercial polysorbate preparations are often prone to spontaneous autooxidation, resulting in the formation of peroxides, which can damage proteins (see section on Chemical degradation).…”