A Molecular Dynamics Simulation Study of the Protein Stability of Reteplase in Formulation with Various Excipients during Freeze-Drying Processing

Sammanfattning: The purpose of this project was to investigate protein stabilization by formulation excipients during the process of freeze-drying using Molecular Dynamics (MD) simulation. A recombinant therapeutic protein, Reteplase, was used as a model and the study focused on a comparative analysis of two excipients, arginine and tranexamic acid (TXA), as included in commercially available formulations of Reteplase. The simulation of freeze-drying was divided into five consecutive steps based on the conditions applied to the system: room temperature, freezing, primary drying, secondary drying and reconstitution at room temperature. Protein conformational changes and the existence of aggregation-prone regions (APRs) were investigated, as to assess the conformational stability during freeze-drying. The relevant stabilization mechanisms were analyzed with respect to protein-excipient interactions and the nature of preferential interaction. The study found a modest degree of conformational instability of Reteplase, which was mainly attributed to the tertiary structure level and effectively reduced in the presence of either arginine or TXA. In this regard, arginine was effective at a lower concentration than TXA. The stabilization mechanism was associated with preferential binding to exposed protein residues, mainly mediated via ionic interactions. A higher strength of interaction was observed for TXA, which was suggested as a consequence of its high accessibility to the protein surface and its chemical character, which enables multiple types of interactions. A correlation was suggested between the stabilization of certain APRs and selective protein-excipient interactions. The results from a parallel study of another protein, Granulocyte Colony-Stimulating Factor (G-CSF), was used provide basic level support for the impact of protein surface charge on the proposed modes of stabilization. The conformational space of Reteplase was explored through the application of cluster analysis to Accelerated Molecular Dynamics (aMD) simulation, which provided further support for the good stability of secondary structure elements. The colloidal stability of Reteplase was investigated in a preliminary small-scale study using Coarse-grained (CG) MD simulation. Initial results suggest a tendency of arginine to reduce the strength of unique proteinprotein interactions, as to prevent the experimentally observed aggregation-propensity of Reteplase.