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.
Aberrant protein folding leading to the formation of characteristic cross-β-sheet-rich amyloid structures is well known for its association with a variety of debilitating human diseases. Often, depending upon amino acid composition, only a small segment of a large protein participates in amyloid formation and is in fact capable of self-assembling into amyloid, independent of the rest of the protein. Therefore, such peptide fragments serve as useful model systems for understanding the process of amyloid formation. An important factor that has often been overlooked while using peptides to mimic full-length protein is the charge on the termini of these peptides. Here, we show the influence of terminal charges on the aggregation of an amyloidogenic peptide from microtubule-associated protein Tau, implicated in Alzheimer's disease and tauopathies. We found that modification of terminal charges by capping the peptide at one or both of the termini drastically modulates the fibrillation of the hexapeptide sequence paired helical filament 6 (PHF6) from repeat 3 of Tau, both with and without heparin. Without heparin, the PHF6 peptide capped at both termini and PHF6 capped only at the N-terminus self-assembled to form amyloid fibrils. With heparin, all capping variants of PHF6, except for PHF6 with both termini free, formed typical amyloid fibrils. However, the rate and extent of aggregation both with and without heparin as well as the morphology of aggregates were found to be highly dependent on the terminal charges. Our molecular dynamics simulations on PHF6 capping variants corroborated our experiments and provided critical insights into the mechanism of PHF6 selfassembly. Overall, our results emphasize the importance of terminal modifications in fibrillation of small peptide fragments and provide significant insights into the aggregation of a small Tau fragment, which is considered essential for Tau filament assembly.
Molecular dynamics is here used to elucidate the mechanism of protein stabilization by carbohydrates and other additives during freezing. More specifically, we used molecular dynamics simulations to obtain a quantitative estimation of the capability of various cryoprotectants to preserve a model protein, the human growth hormone, against freezing stresses. Three mechanisms were investigated, preferential exclusion, water replacement, and vitrification. Model simulations were finally validated upon experimental data in terms of the ability of excipients to prevent protein aggregation. Overall, we found that the preferential exclusion and vitrification mechanisms are important during the whole freezing process, while water replacement becomes dominant only toward the end of the cryoconcentration phase. The disaccharides were found to be the most efficient excipients, in regard to both preferential exclusion and water replacement. Moreover, sugars were in general more efficient than other excipients, such as glycine or sorbitol.
In the pharmaceutical industry, much attention has been given to the problem of predicting the average size of solvent crystals formed during freezing of pharmaceutical solutions. All the methods proposed in the literature to respond to this request are based on empirical laws, while the present work focuses on the development of a mechanistic model. This model was found to give physical theoretical background on the relationship between solvent crystal size, velocity of the freezing front, and temperature gradients within the product being frozen, which was postulated by the empirical laws previously proposed. Model simulations were validated upon experimental observations obtained by scanning electron microscopy. More specifically, the average size of solvent crystals was determined from that of pores formed after lyophilization of the frozen solutions, provided that no matrix collapse occurred. The developed model was demonstrated to be valid over a wide range of freezing conditions, and for solutions containing both amorphous and crystalline solutes. As a further step, a simplified model for dilute solutions has also been developed and experimentally validated.
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