Study On Nucleation Of Lysozyme Crystallization

As the direction of future drugs,protein drug's crystal form has many advantages over liquid form as method of delivery.For example,better stability,high concentration of active pharmaceutical ingredients(API)and easy to achieve controlled release of API and so on.However,only insulin has been produced and delivered in crystal form on the market among the about 150 biopharmaceuticals.The main reason is that the crystallization process of protein is much more complex than the crystallization process of small molecules,so it is more challenging.And as the basis of the crystallization process of the protein,nucleation is rarely studied in detail,but the process of nucleation has a decisive role on the quality of crystal product.Therefore,in order to obtain desired structure and size of the protein crystal drugs,full understanding of the nucleation process can help to control protein crystallization better.The purpose of this study is to explore the nucleation process of proteins,mainly to explore a method and technology to accurately measure the rate of nucleation,and investigate the behavior of aggregates in nucleation solutions.On the one hand,this study could be used to guide the crystallization process,which can improve the success rate of protein drug crystallization;on the other hand,this study can enrich the research of protein crystallization.In this paper,the hen egg white lysozyme(HEWL)is used as the research object.The specific research content is as follows:In terms of thermodynamics,the solubility curves of HEWL under different experimental conditions were measured by UV spectrophotometry.Based on the solubility data,the metastable zone of HEWL under different experimental conditions were measured by turbidity method.Solubility and metastable zone,as the basic data of thermodynamics,are the basis of studying the nucleation process.In terms of dynamics,firstly,the nucleation induction time of HEWL under different supersaturation conditions were measured by turbidity method.Then the nucleation free energy map of HEWL was made.These results indicated that higher supersaturation can reduce the nucleation energy,which benefit to nucleation,and also explained forming a stable nucleus must cross the nucleation energy barrier.Secondly,a hot stage integrated with microscope system was applied to study the nucleation rate of HEWL solution.Images of crystals in a certain volume of droplets were continuously recorded with time.The curves,which indicated the changing between nucleation rate and the initial supersaturation of HEWL,were obtained.It is concluded that controlling the initial supersaturation of the HEWL solution can be a way to divide the nucleation from the growth process.And the nucleation rate curve was fitted by the classical nucleation theory model.It was found that heterogeneous nucleation seemed to be dominant at lower supersaturation while at higher supersaturation homogeneous nucleation seemed to play the major role.Finally,the behavior of aggregates in HEWL nucleation solution were measured by dynamic light scattering instrument(DLS)under different experimental conditions.It is used to understand the evolution process of molecular clusters in protein nucleation solution,which was explained by two-step nucleation theory,and further understand the nucleation process.