Investigation On Active Protection Of Protection Of Monoclonal Antibody Etanercept With Molecular Simulation

Recently,with the improvement of monoclonal antibody technology,monoclonal antibody drugs are applied to clinical diagnosis and treatment of disease widely.It possesses better specificity and uniform property of structure,however,they have poor thermal stability compared with the small molecule drug.Generally,the content and type of protectants,which are used for protecting the bioactivity of antibody protein,is critical to improve the bioactivity of antibody protein.Thus,it is important to study the function and mechanism of protectants protecting the antibody protein.In the paper,the study of object was etanercept,and the protectants were sucrose,trehalose and mannitol.The dissociation process of etanercept dimer in different environment was simulated by approaches of umbrella sampling and steered molecular dynamic simulation using Gromacs software with amber99sb-ildn united atomic force field.The dissociation process of etanercept in vacuum and aqueous solution indicates that the dissociation free energy of etanercept in aqueous solution is 223.75 kJ·mol^-1,while it is 2250.14 kJ·mol^-1 in vacuum which is about ten times than that in aqueous solution.And the dissociation pulling force of etanercept in aqueous solution is963.46 kJ·mol^-1·nm^-1,while it is 3154.17 kJ·mol^-1·nm^-1 in vacuum which is three times than that in aqueous solution.The results show that the etanercept structural stability in vacuum is better than that in aqueous solution.Since water molecular can be gradually embeded in the interface between the two monomers,which weakens the molecular interaction between the two monomers and makes dissociation free energy and pulling force of etanercept in aqueous solution less than that in vacuum.The dissociation process of etanercept in aqueous solution with different trehalose concentration demonstrates that the dissociation free energy and the dissociation pulling force of etanercept respectively increase by 173.17 k J·mol^-1 and524.42 kJ·mol^-1·nm^-1 in the system which the molar ratio of etanercept and trehalose is about 1:50 compared with control system which the molar ratio of etanercept and trehalose is about 1:0.While,the dissociation free energy and the dissociation pulling force of etanercept significantlly increase by 407.63 kJ·mol^-1 and 1430.08kJ·mol^-1·nm^-1 respectively in the system that the protectants are selected and their ratio is well optimized.Apparently,the trehalose can improve etanercept structural stability for trehalose playing an significant role in protecting antibody protein by selectively weak affinity adsorption.In addtion,the bioactive protection of antibody protein need a minimal concentration of trehalose since a execessive concentration can not improve obviously protective effect.The dissociation process of etanercept in aqueous solution with trehalose/mannitol or sucrose/mannitol shows that the dissociation pulling force of etanercept in complex protective system is much higher than 550 kJ·mol^-1·nm^-1 compared with single protective system.The result turns out that the prescription of complex protectant is better than that of single protectant as complex protectant can be synergistically absorbed on the surface of antibody protein to strengthen its protective effect.The dissociation free energy of etanercept in complex protective system is less than in single protective system,for mannitols in complex protective system have less contribution to dissociation free energy.According to the results,kinetics is more suitable to characterize the structural stability of etanercept than thermodynamics.In summary,the protective mechanism of protectant onto antibody protein and the advantages of complex protection are explained by analyzing pulling force,PMF dissociation free energy,protectant adsorption surface of antibody protein,non-bond interaction between protectant and antibody protein.This method provides technical support for rapidly selecting protectants for protein drug,design and optimization of complex protective prescription.We are sure that the application and promotion of the method will shorten development period,save experimental cost,improve efficiency of research and development.