The Purification Study Of Human Menopausal Gonadotropin

Human menopausal gonadotropin(HMG),a glycoprotein gonadotropin secreted by the basophilic cells in the anterior pituitary gland,contains an equal proportion of follicle-stimulating hormone(FSH)and luteinizing hormone(LH),both being generally extracted and isolated from the urine of perimenopausal women and the main factor is FSH.Clinically,HMG is mainly used to treat infertility caused by ovulation disorders in women and is the main drug for ovulation induction.In addition,it's also used for Sperm deficiency or low activity caused by low male hormone levels,amenorrhea,irregular menstruation and Sexual dysfunction.In recent years,the difficulty of urine collection is still exist and crude production costs increase,while the purchase price of domestic pharmaceutical companies is low,domestic intermediates inclined to export pharmaceutical raw materials to foreign pharmaceutical companies at the same time,because foreign pharmaceutical companies,on the production process,have no strict restriction,it opens the way for HMG crude product producers to research high-purity HMG and then export for higher profit.At present,the main purification methods of HMG include ethanol extraction,ion exchange,hydrophobic chromatography,multivariate chromatography,dye/antibody affinity chromatography and so on.The purpose of this project is to study the purification of HMG and to establish a high-purity HMG laboratory-scale production process with stable quality.Research contents:The purification effect and ative yield of crude HMG from silica gel,aluminum silicate and macroporous adsorption resin were optimized and compared;The purification effect and ative yield of crude HMG after treated by ammonium acetate-ethanol extraction and ethanol secondary precipitation was optimized and compared.Roughly purified HMG above go on purifying,through the ion-exchange chromatography,hydrophobic chromatography,gel filtration chromatography and other steps to obtain the final high-purity HMG.Research result:The purification effect of crude product by silica gel was not obvious.The purification rate of macroporous adsorption resin was above 1.57,the yield was 58%,the purification rate of aluminum silicate was above 1.58,the yield was 62%;the purification rate of ethanol secondary precipitation was only 1.58,Ammonium acetate-ethanol extraction,the purification fold was 1.7,but the yield was low,only 40%.In this paper,the ammonium acetate-ethanol secondary precipitation of the crude solution was studied.The purification fold was slightly decreased,but the yield was significantly increased,up to 76% or more;In this paper,through the optimization of phenyl hydrophobic column,the purification fold was up to 5 times,the active yield was 70% or more.Comparing anion and anion chromatography purification results,we can see DEAE anion exchange chromatography purification have the best purification effect up to 2.8 times,the activity yield of 81%.After the preliminary experiment,we tried to use the flow through process in the DEAE purification process to avoid the loss of activity as much as possible.After purification,the highest purification rate of HMG could reach about 3.16 times,and the yield was over 85%.Follow-up chromatography used S-200,The purification process was repeated three times and the average FSH immunocompetence of the product was up to 43 IU/mg or more,with biological activity of 35 IU/mg or more.Research conclusion:1?The crude HMG was initially separated to remove some impurities,the activity increased by 1.78 times and the highest yield was 76%.According to a variety of methods,considering the activity and yield,the crude product is treated by ammonium acetate-ethanol secondary precipitation.2?The best chromatographic purification process is: flowthrough of DEAE-sepharose-FF anion column,freeze-dried and concentrated,desalting column replacing buffer,then loading on Phenyl-sepharose-ls hydrophobic column,eluation buffer loading on the desalting column and then on the S-200 column,the final collection of liquid freeze-dried.Under this process,FSH immunocompetence was up to 43 IU/mg or more,with biological activity of 35 IU/mg or more.