| Hepatitis B,caused by Hepatitis B virus(HBV),is one of the most common infectious diseases in the world.The number of deaths due to hepatitis B infection in China each year accounts for nearly 50%of global HBV infection-related deaths.Hepatitis B vaccination is the most important strategy for the prevention and control of hepatitis B.Hepatitis B surface antigen(HBsAg)is the main active substance of hepatitis B vaccine.HBsAg is spherical virus-like particles(VLPs)formed by multiple monomers through disulfide bonds.As a multi-subunit protein,HBsAg stability is poor,especially during the purification process,because it is easy to cause structural changes such as depolymerization or aggregation to cause loss of activity,resulting in a decrease in yield,which has a great negative effect on the large-scale production of hepatitis B vaccine.Protecting the integrity of antigens is one of the important means to solve this problem..Based on this,the preliminary adsorption/desorption experiments with silica gel were carried out,and it was found that it can enhance the stability and integrity of antigen particles.The stability of antigen particles under different pH conditions was further explored.Based on this,the adsorption and desorption steps in the silica gel experiment were optimized to obtain the best purification scheme.Then it was used in combination with combined chromatography to further purify the antigen,and the penetrating ion exchange chromatography was used to protect the antigen structure and improve the yield of antigen activity in the chromatography purification.As follows:1.In the analysis of the stability and integrity of antigen particles by silica gel,Silica gel adsorbs and desorbs the cell crushing fluid.Next,the silica gel desoiption solution was purified by hydrophobic chromatography,The results showed that the particle integrity was 85.79%and the active yield was 49.73%.The integrity of the traditional hydrography method is 64.89%,and the active yield is 17.74%.The purification method of silicone and Hydrophobic chromatography was used to improve the particle integrity by 20.90%,and the active yield increased by 31.99%.The two methods showed that the stability of the silica gel group was 57.60cent,and the other group was 34.73%,and the other group was 22.93%.The experiment shows that the concrete steps of the silicone gel before the Hydrophobic chromatography can help to improve the particle's particle integrity and the activity recovery,and maintain its stability.2.In the study on the influence mechanism and process optimization of HBsAg purification by silica gel adsorption/desorption,the effects of five factors including antigen concentration,adsorption pH,adsorption time,desorption pH and desorption temperature on the yield of activity and protein were first investigated by single factor experiment.High performance liquid gel filtration chromatography(HPSEC)was used to analyze the effects of different factors on the integrity of antigen particles.The effective adsorption concentration of antigen on 1 g of dry silica gel is 47.85 mg.The adsorption experiment of the small system only takes 30 min to reach the adsorption equilibrium.The adsorption pH is around 8,the activity yield and protein yield are higher.Both the yield and the protein yield will gradually increase,and the pH is best around pH10,while the desorption temperature is 55?,the yield is the best.Based on the above five effected factors,HPSEC analysis found that the proportion of medium and large particles showed a positive correlation with the activity yield.At the same time,static light scattering,fluorescence spectroscopy and dynamic light scattering were used to study the stability of antigen particles under different pH conditions from the perspective of particle integrity.The results show that under acidic solutions,when the pH is close to the isoelectric point of HBsAg,the antigen.The electrostatic repulsion between the particles is reduced,and the particles are easy to aggregate;under alkaline conditions,the hydrophobic groups inside the antigen particles are exposed,causing the particles to depolymerize.Comprehensive single-factor experimental results and analysis of static light scattering,fluorescence spectroscopy and dynamic light scattering results to guide silica gel adsorption/desorption purification of HBsAg,establish a response surface model of key factors in the process At the value,the optimal purification process is adsorption pH=7.43,elution pH=10.48,elution temperature 55.4?,the activity yield is at most 39.1%;when the purification multiple is the response value,adsorption pH=7.16,elution The pH=10.52,the elution temperature is 55.1?,and the purification factor is at most 1.90 at this time.3.Explore a combined chromatography method based on silica gel adsorption and desorption to purify HBsAg.Initially explored the pure effect of hydrophobic chromatography and ion exchange chromatography on silica gel desorption solution,and found that hydrophobic chromatography has the characteristics of large load,high nucleic acid removal rate,and high product purity.Therefore,hydrophobic chromatography was selected as the first step of combined chromatography.In the hydrophobic chromatography experiment,the pH and ammonium sulfate stability experiments were first performed on the silica gel desoiption liquid.It was found that the silica gel desorption liquid maintained the best activity under the conditions of pH7.5 and 6%ammonium sulfate;The adsorption and elution of silica gel desorption liquid by butyl sulfur medium,butyl medium,low-density phenyl medium,and high-density phenyl medium were investigated.,The lower the chromatographic yield;the column experiment purification of butyl sulfur hydrophobic medium and butyl hydrophobic medium using dynamic experiments found that the butyl sulfur hydrophobic chromatography activity yield was 54%,which was 9 times that of butyl hydrophobic chromatography;However,in the eluate obtained by butylsulfide hydrophobic chromatography,there are HBsAg particles that are not completely assembled,which needs to be removed by DEAE ion exchange chromatography.In the DEAE ion exchange chromatography experiment,it was found that the protein yield obtained by the elution formula was 4%,the activity yield was 45%,and the HBsAg yield was low;the penetration purification was proposed,and the activity yield was found to be increased by 51%,and the protein recovery Increase by 33%,and finally obtain a route of silica gel-hydrophobic chromatography-penetrating ion exchange chromatography to purify HBsAg,and achieve the ultimate ideal effect of removing incompletely assembled HBsAg particles while improving vaccine yield.After preliminary amplification of the purification route,the final protein yield was 3.94%,and the vaccine activity yield was 40%,which achieved electrophoretic purity(SDS-PAGE)and chromatographic purity(HPSEC).The particle size of the antigen particles was observed by transmission electron microscopy to be 20?40.nm. |
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