Enhancement Of Lysozyme Crystallization Under Ultrasound Field

With the increasing demand for biopharmaceuticals,macromolecular drugs such as proteins have been paid more and more attention because of their unique functions.Compared with the traditional liquid drug delivery system,the crystal drugs have the advantages of good drug stability,high concentration of active ingredients and easy controllable release.However,most proteins are difficult to crystallize due to their large molecular weight and high molecular flexibility,which limits the development of protein drugs to some extent.Therefore,it is of great significance for the development and large-scale production of protein crystal drugs to fully explore the means of protein crystallization enhancement.Ultrasound has been widely used as a means of process enhancement.However,at present,ultrasound is mainly used in the crystallization process of organic small molecules and inorganic salts,and the related research on protein crystallization en-hancement is still insufficient.In order to investigate the effect of ultrasound on pro-tein crystallization,lysozyme was used as model protein and sodium chloride was used as salting-out agent,the effects of ultrasound on the thermodynamics and kinet-ics of lysozyme salting-out crystallization,batch crystallization and continuous crys-tallization were studied in detail.Firstly,the thermodynamics and kinetics of lysozyme crystallization were studied.The solubility of lysozyme in acetate buffer was determined by ultraviolet spec-trophotometer,and the obtained data were fitted according to the empirical equation.The effects of different ultrasonic power and immersion depth of the horn on the met-astable region of lysozyme and the induction period of nucleation were investigated by turbidity method.The results showed that ultrasound can significantly shorten the metastable zone and induction period of lysozyme and strengthen the nucleation pro-cess.Secondly,the batch crystallization process of lysozyme enhanced by ultrasound was studied.The effects of different ultrasonic modes and ultrasonic power on crystal habit,particle size and particle size distribution of lysozyme crystals were investigated by optical microscope and Image J software.The effect of different ultrasonic modes and ultrasonic power on the yield of lysozyme crystals was investigated by ultraviolet spectrophotometer.The results showed that lysozyme crystals with perfect crystal habit,moderate size and uniform particle size can be obtained in the ultrasonic-stop mode,and the yield of the crystallization process was higher.Finally,the continuous crystallization process of lysozyme under ultrasonic field was investigated based on the conclusion of lysozyme crystallization thermodynamics and kinetics and the data conclusion of batch crystallization process.The continuous crystallizer adopted a plug-flow tubular crystallizer,and was set into a nucleation zone and a growth zone respectively.By investigating the time required for the continuous crystallization process to reach steady state,the effect of ultrasonic field on crystal habit,particle size and particle size distribution of lysozyme crystals and the effect of ultrasound on the yield of continuous crystallization of lysozyme,it was found that ultrasound could significantly improve the crystal quality and process yield.The above research provided a feasible scheme for the strengthening of the protein crystallization process.