Glycosylation Characteristic Identification And Quality Control Analysis Of Recombinant Human Chorionic Gonadotropin(hCG)

Infertility has become a common,frequent and serious disease affecting people's quality of life.Human chorionic gonadotropin(hCG),as a natural hormone,is a glycoprotein hormone secreted by human placental trophoblast cells during pregnancy.As a drug,hCG plays an important role in the treatment of female infertility,induction of ovulation and in vitro fertilization and embryo transfer.Traditional commercial production of urinary hCG(u-hCG)is extracted from pregnant women's urine,but it has many shortcomings.As an improvement,with the development of biopharmaceuticals,commercial recombinant hCG(r-hCG)has been put on the market.HCG is linked by subunits alpha and beta through non-covalent way,in which subunits alpha contain two N-glycans,and subunits beta contain two N-glycans and four O-glycans.As a glycoprotein molecule,hCG has a high degree of glycosylation,resulting in many isomers with different glycosylation degrees and glycosylation types.Different glycosylation modifications result in different molecular forms of hCG with different biological functions.Glycosylation modification plays an important role in the biological function of hCG,especially N-glycosylation.Therefore,it is necessary to identify the glycosylation characteristics of hCG and analyze its quality control.In this paper,high performance liquid chromatography(HPLC)and mass spectrometry(MS)were used to characterize and analyze the structural characteristics of hCG N-glycosylation,which was independently developed by Zhuhai Livzon Mabpharm,Inc.This study includes the identification of N-glycosylation sites of hCG,the identification of N-glycosylation types,and the analysis of glycosylation proportions of various types of hCG glycoproteins.The N-glycosylation sites of the ? subunit were all in Asn52 and Asn78,and the N-glycosylation sites of the ? subunit were in Asn13 and Asn30,which were consistent with the theory.More than 17 glycosylation types of hCG were identified through N-sugar type identification experiments,and most of them were NGNA.Through research,it was confirmed that the developed N-glycosylation ratio analysis method could meet the needs of drug research and drug analysis,and the differences of Lishenbao(Urofollitropin for Injection),Ovidrel(Recombinant Human Choriogonadotropin alfa for Injection)and hCG developed by Livzon Mabpharm,were evaluated by N-sugar relative content detection technology.Using hCG as a molecular model,an experimental method was provided for the determination of the relative content of N-glycosylation in Biopharmaceuticals.