Molecular Understanding of the Effects of Stabilizing Excipient during Lyophilization of Biopharmaceuticals

Lyophilization, which is also referred to as freeze-drying, is the most used technique to prepare solid-state protein formulations. The lyophilized products have a prolonged shelf-life compared to aqueous protein formulations. However, the lyophilization process involves freezing and drying of the formulation, which will inevitably generate stresses on the protein. The stresses can denature the native structure of the protein. Therefore, a stable lyophilized protein formation is prepared with a variety of excipients. The events occurring during the freeze-drying of the protein-excipient formulations are extremely challenging to examine in high-resolution experimental techniques. Therefore, a computational study based on molecular dynamics (MD) simulations can provide valuable insight into the events that are occurring before, during, and after the freeze-drying process. This thesis aimed to investigate the role of excipients during the lyophilization process using advanced MD simulations. The investigation led to two different projects, that resulted in two manuscripts.

The first project was about the pH-dependent aggregation mechanism of granulocyte colony-stimulating factor (GCSF). GCSF is known for its pH sensitivity to the aggregation mechanism. In an attempt to characterize the aggregation behavior of GCSF, we have used state-of-the-art simulation techniques. The pH-dependent conformational stability was accessed via metadynamics simulations. The protein-protein interactions between GCSF monomers were simulated in the coarse-grained (CG) system using the SIRAH force field. Using metadynamics simulations, we could successfully show that the reorientation of Trp residues can occur when the pH value is changed. The CG simulations revealed that the protein-protein interactions between GCSF monomers increased when the pH value is increased more than 4 or salt is added to the system. The outputs from the coarse-grained simulations were directly used as an input for small-angle X-ray scattering (SAXS) guided molecular modeling. The outputs of the SAXS guided modeling shows that the dimer fraction which is obtained from the experiments could reproduce a similar trend that was observed from the level of protein-protein interactions during the CG simulations. The protein-protein interactions from the CG simulations at the different pHs indicated that the deprotonation of the acidic residues leads to the weakening of the electrostatic repulsion between aggregation-prone long loop regions in GCSF.

The second manuscript focuses on the protein stabilization mechanism during the freeze-drying process. An MD simulation protocol of the full cycle of the freeze-drying process, including the reconstitution stage, is presented. The study focused on protein-specific stabilization via excipients. Arginine can provide both cryoprotection and lyoprotection of proteins. However, arginine can act differently on different proteins. The protein-specific effect of arginine was investigated for two different proteins: Reteplase and GCSF. The CG simulations of protein-protein interactions with and without the presence of arginine revealed that arginine can prevent the aggregation of Reteplase via local binding to the protein surface. However, in the case of GCSF, the addition of arginine could not prevent protein-protein interactions between GCSF. The freeze-drying simulations could provide the following protein-specific events of Reteplsae: i) collapse of the protein domain structure, ii) recovery of the freeze-drying induced damages during the reconstitution, and iii) exposure of local aggregation-prone region after the reconstitution. The simulation outcome revealed that the local binding of excipients is necessary for Reteplase to obtain domain separation and local stabilization of the aggregation-prone region.