Study On Heterogeneity Of N-termini Of Recombinant Human Follicle Stimulating Hormone

Recombinant human follicle stimulating hormone(rh FSH)consists of two peptide chains,? and ?,through non-covalent bonds.The gene encoding human FSH is introduced into Chinese hamster ovary cells(CHO)by gene recombination technology,after cell culture,separation and purification derived rh FSH,in the treatment of infertility has important medicinal value,can promote the normal growth of follicles and estrogen production,it has important medicinal value in the field of reproductive health.The foreign original research drug recombined follicle stimulating hormone(rh FSH)products are Gonal-F produced by Merck Serono,and Puregon produced by Merck & Co.,Ltd.The only domestic products are Changchun Kinsey Pharmaceuticals.The production of Kinsey Heng was approved for listing.At present,nearly 10 domestic companies are in the clinical or reporting stage.According to reports in the literature,there are N-terminal amino acid deletions in the alpha and beta subunits of the original drug rh FSH.The ?-subunit has 1 to 3 amino acids at the N-terminus,and the N-terminal deletion ? subunit ratio is about 2%,while the ? subunit N There is a deletion of 2 amino acids at the end,and the proportion of ? subunits at the N-terminus is about 45%.This phenomenon is widely present at the N-terminus deletion.This phenomenon may be caused by posttranslational modification of the protein.According to the guiding principles of bio similar drug R&D and technology evaluation,the quality standard of bio similar drugs approved by Chinese enterprises for listing should not be lower than that of foreign original research products.The molecular structure should be consistent with the original research product,and the N-terminus heterogeneity and the original research product should also be Basically the same,at present,only the European Pharmacopoeia 8.0 included the standards of rh FSH products,and other countries such as the Chinese Pharmacopoeia and the United States Pharmacopoeia have not yet included,but the detection methods for rh FSH in domestic enterprises are already mature.However,none of the N-terminal inhomogeneity detection methods are found in the European Pharmacopoeia and domestic corporate standards.In recent years,the literature on N-terminal detection methods is also relatively small.The purpose of this paper is to make up for this gap and provide reference for future inclusion of rh FSH standards in the Chinese Pharmacopoeia.In this paper,a new quantitative peptide map method has been established,which can be used for the study of heterogeneity at the N-terminus to provide basis for evaluating the heterogeneity of the N-termini of rh FSH stocks in different companies.In this paper,through optimization of tryptic digestion conditions,optimization of glycosidase digestion conditions,optimization of reversed-phase chromatography conditions,identification of peptides,and screening of columns,it was found that the column CORTECS C18+ 4.6×150 mm was used.2.7 ?m can effectively separate intact ?L1 and deletion ?L1;glycosidase enzyme digestion for 10 min and enzyme digestion 16 h experimental results,and glycosidase and trypsin digestion sequence has no effect on the experimental results;when trypsin digestion,adding Ca2+ can be Enzyme activity was increased and ?1-2 was completely cleaved after 4 h of enzyme digestion.Finally,we obtained a new quantitative peptide map method: first,reductively alkylate rh FSH,add glycosidase at a volume ratio of 1:50(glycosidase: sample),and add 0.5 mol at a volume ratio of 1:25(Ca Cl2: sample).Ca Cl2 solution,add 1 mol/L trypsin at a mass ratio of 1:40(trypsin: sample),incubate at 37°C water bath for 4 h,and heat at 90°C to stop the reaction.Column CORTECS C18+,size: 150 mm ×4.6 mm,2.7 ?m,RP-HPLC detection.Through MALDI-TOF(flight time mass spectrometry)mass analysis to verify the location of each peptide,the experimental results show that this method can effectively isolate the missing ?L1 and intact ?L1 peptides.Through quantitative integration,it is found that the rh FSH product of the original study drug manufacturer A is missing ?.The proportion of subunits is about 43%.The proportion of ? subunits in the domestic two companies' rh FSH products is similar to that of the original research drug manufacturers,which is about 43%.The peptide maps are consistent with those of the original research companies,indicating the molecular structure of rh FSH products in domestic companies.It is basically consistent with the original research drug and meets the requirements of the guiding principles of R&D and technical evaluation of biosimilar drugs.The peptide map analysis method established in this study shortens the time of the peptide mapping method,simplifies the experimental procedure,and can isolate the N-terminal deletion peptide and intact peptide of the ? subunit,thus verifying the N-terminal heterogeneity of rh FSH.The method validation shows that the method has good stability and high accuracy.It provides technical support for the study of the comparability of recombinant human follicle stimulating hormone(rh FSH)and supplements the quality control of recombinant human follicle stimulating hormone with new quality control methods,quality control project.