Optimization Of RhGH-FC Pilot Test Conditions And Establishment Of Main Index Detection Methods

Recombinant human growth hormone receptor fusion protein(rhGH-Fc)is a fusion protein that connects growth hormone to the Fc segment of the modified human antibody.It retains the biological activity of rh GH molecule and has some properties of antibody.It can promote the growth of bone,viscera and the whole body by promoting protein synthesis,affecting fat and mineral metabolism and plays a key role in the growth and development of human body.It has been widely used in clinical practice in pediatric medical treatment,reproductive field,burn field and anti-aging field.It can bind to the Fc part of the endosome,reducing lysosomal degradation and significantly increasing the biological half-life of rhGH-Fc.Because the fusion protein expression needs processing and glycosylation modification,it cannot be prepared by prokaryotic expression system.At present,the fusion protein prepared by eukaryotic expression system has low expression level and high production cost.In this study,Chinese hamster ovary epithelial cells(CHO)engineered cell line CHO-K1(eukaryotic expression system),which stably expressed rh GH-FC protein,was used as the research target,and the effects of different mixing speed and ventilation mode on the expression level of the fusion protein rh GH-FC were investigated in a 14-L pilot-scale bioreactors,so as to obtain the optimal culture process for rh GH-FC.Rh GH-FC with long half-life was produced by the best culture technology in order to reduce its production cost.Then liquid chromatography-mass spectrometry(GC-MS)was used to establish a preliminary method for the determination of molecular weight,glycosylation modification and disulfide bond of rh GH-FC.First of all,using 14 L bioreactor to original fermentation technology as a control(STD-1),by changing the stirring speed and ventilation way(large bubble aeration and micro bubble aeration)respectively designed three sets of fermentation(STD-2,3,STD-4),by tracking in the process of the fermentation tank culture cell density,osmotic pressure,the ratio of sugar consumption,the concentration of lactic acid and ammonium ion concentration changes,the fusion protein rhGH-Fc training process is optimized.The results showed that the concentration of lactic acid and osmotic pressure in the STD-4 group were significantly better than those in the other groups,and the final protein expression in the STD-4 group reached 0.9 g/L,which was increased by about 34% compared with the original process.The experimental conditions of STD-4 were: temperature: 37 ℃(0-6 d),33 ℃(7-14 d);Dissolved oxygen: 40%;PH: 6.9(0-3 d),6.82(4-14 d),agitator speed: 75 rpm(0-3 d),110rpm(4-7 d),130 rpm(8-14 d);Ventilation: bullae ventilation was 0.02 L/min(0-7 d),0.05 L/min(8-14 d).Secondly,after the purified target protein was pretreated,the method of detecting the molecular weight,glycosylation modification and disulfide bond of rh GH-FC was established by liquid chromatography-mass spectrometry(LC-MS).Results: The method for detecting the molecular weight of rh GH-FC was successfully established,and the detection result of 97348.8 Da was consistent with the theoretical value.The glycosylation modified method of rh GH-FC was successfully established,and the content of sialacidized glycosylation,defucidized glycosylation and high mannose glycosylation of the target protein was 0.53%,8.77% and 5.76% respectively.The detection method of Rh GH-FC disulfide bond was successfully established,and a Rh GH-FC was detected to contain 10 disulfide bonds.There were C51-C165、C182-C189,C245-C305 and C351-C409 on the rh GH segment respectively.C210-C210 and C213-C213 on the Fc chain respectively,which were completely consistent with the results of the published literature.In this study,the production cost of rh GH-FC was reduced by a simple change of culture conditions.In addition,GC-MS was used to establish the detection method for the active function of the related modification group and the three-dimensional structure of the disulfide bond position,which provides a reference for the process optimization of other related fusion proteins,and also lays a foundation for the further analysis of the protein structure and related clinical studies.