Abstract
Protein-based pharmaceutical products are subject to a variety of environmental stressors, during both production and shelf-life. In order to preserve their structure, and, therefore, functionality, it is necessary to use excipients as stabilizing agents. Among the eligible stabilizers, cyclodextrins (CDs) have recently gained interest in the scientific community thanks to their properties. Here, a computational approach is proposed to clarify the role of β-cyclodextrin (βCD) and 2-hydroxypropyl-β-cyclodextrin (HPβCD) against granulocyte colony-stimulating (GCSF) factor denaturation at the air–water and ice–water interfaces, and also in bulk water at 300 or 260 K. Both traditional molecular dynamics (MD) simulations and enhanced sampling techniques (metadynamics, MetaD) are used to shed light on the underlying molecular mechanisms. Bulk simulations revealed that CDs were preferentially included within the surface hydration layer of GCSF, and even included some peptide residues in their hydrophobic cavity. HPβCD was able to stabilize the protein against surface-induced denaturation in proximity of the air–water interface, while βCD had a destabilizing effect. No remarkable conformational changes of GCSF, or noticeable effect of the CDs, were instead observed at the ice surface. GCSF seemed less stable at low temperature (260 K), which may be attributed to cold-denaturation effects. In this case, CDs did not significantly improve conformational stability. In general, the conformationally altered regions of GCSF seemed not to depend on the presence of excipients that only modulated the extent of destabilization with either a positive or a negative effect.
Highlights
Therapeutic protein molecules are becoming increasingly important in the treatment of a large number of diseases, but they are often unstable and tend to undergo chemical or physical degradation.[1]
The formation of a large air−water surface during mixing and shaking has often been shown to promote unfolding and aggregation.[5−9] It is generally believed that the migration of proteins to the interface with air, as well as oil−water interfaces, where the exposure of the hydrophobic core is promoted, is responsible for the observed loss of stability.[10−13] The formation of ice during freezing has been found to be detrimental for proteins,[14−17] but in this case, there still is no widespread agreement in the literature about the underlying mechanism
We used a computational approach to investigate the properties of two eligible excipients, βCD and its derivative HPβCD, against the surface-induced denaturation of Granulocyte colony-stimulating factor (GCSF)
Summary
Therapeutic protein molecules are becoming increasingly important in the treatment of a large number of diseases, but they are often unstable and tend to undergo chemical or physical degradation.[1]. Unfolded or misfolded conformations may enhance aggregation phenomena,[2,3] and this poses serious safety issues, as the formation of aggregates may result in undesired immunogenicity.[4]. While it was first thought that adsorption onto the ice surface may be key for destabilization,[14,18] recent experimental and simulation results indicate that direct interaction with the interface is not needed.[19−23] In contrast, pressure build-up,[21] concentration gradients and pH shifts,[21] accumulation of gas bubbles,[24,25] or cold denaturation phenomena[22] were proposed as possible routes of denaturation upon ice formation. The addition of excipients to the protein formulation is needed to prevent undesired loss of therapeutic potency and preserve the monomeric native
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