Finite Element Method (FEM) Modeling of Freeze-drying: Monitoring Pharmaceutical Product Robustness During Lyophilization.

Lyophilization is an approach commonly undertaken to formulate drugs that are unstable to be commercialized as ready to use (RTU) solutions. One of the important aspects of commercializing a lyophilized product is to transfer the process parameters that are developed in lab scale lyophilizer to commercial scale without a loss in product quality. This process is often accomplished by costly engineering runs or through an iterative process at the commercial scale. Here, we are highlighting a combination of computational and experimental approach to predict commercial process parameters for the primary drying phase of lyophilization. Heat and mass transfer coefficients are determined experimentally either by manometric temperature measurement (MTM) or sublimation tests and used as inputs for the finite element model (FEM)-based software called PASSAGE, which computes various primary drying parameters such as primary drying time and product temperature. The heat and mass transfer coefficients will vary at different lyophilization scales; hence, we present an approach to use appropriate factors while scaling-up from lab scale to commercial scale. As a result, one can predict commercial scale primary drying time based on these parameters. Additionally, the model-based approach presented in this study provides a process to monitor pharmaceutical product robustness and accidental process deviations during Lyophilization to support commercial supply chain continuity. The approach presented here provides a robust lyophilization scale-up strategy; and because of the simple and minimalistic approach, it will also be less capital intensive path with minimal use of expensive drug substance/active material.

Protein folding: the stepwise assembly of foldon units.

Equilibrium and kinetic hydrogen exchange experiments show that cytochrome c is composed of five foldon units that continually unfold and refold even under native conditions. Folding proceeds by the stepwise assembly of the foldon units rather than one amino acid at a time. The folding pathway is determined by a sequential stabilization process; previously formed foldons guide and stabilize subsequent foldons to progressively build the native protein. Four other proteins have been found to show similar behavior. These results support stepwise protein folding pathways through discrete intermediates.

How cytochrome c folds, and why: submolecular foldon units and their stepwise sequential stabilization.

Native state hydrogen exchange experiments have shown that the cytochrome c (Cyt c) protein consists of five cooperative folding-unfolding units, called foldons. These are named, in the order of increasing unfolding free energy, the nested-Yellow, Red, Yellow, Green, and Blue foldons. Previous results suggest that these units unfold in a stepwise sequential way so that each higher energy partially unfolded form includes all of the previously unfolded lower free energy units. If this is so, then selectively destabilizing any given foldon should equally destabilize each subsequent unfolding step above it in the unfolding ladder but leave the lower ones before it unaffected. To perform this test, we introduced the mutation Glu62Gly, which deletes a salt link in the Yellow unit and destabilizes the protein by 0.8 kcal/mol. Native state hydrogen exchange and other experiments show that the stability of the Yellow unit and the states above it in the free energy ladder are destabilized by about the same amount while the lower lying states are unaffected. These results help to confirm the sequential stepwise nature of the Cyt c unfolding pathway and therefore a similar refolding pathway. The steps in the pathway are dictated by the concerted folding-unfolding property of the individual unit foldons; the order of steps is determined by the sequential stabilization of progressively added foldons in the native context. Much related information for Cyt c strongly conforms with this mechanism. Its generality is supported by available information for other proteins.