Purification method for recombinant human follicle-stimulating hormone

Abstract

The present invention relates to a purification method for recombinant human follicle-stimulating hormone (FSH), including anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The method is low in cost, few in steps, simple and feasible, and stable in quality; and no complicated denaturation and renaturation processes are comprised in the method, and the use of reversed phase chromatography, metal chelate chromatography or hydrophobic interaction chromatography, which have a greater impact on protein activity, is prevented. According to the method, firstly, anion exchange of flow-through mode is used for removing a large number of contaminating proteins, pigments and some residual DNA, endotoxin and host proteins, which not only protects the subsequent affinity filler, but also achieves a coarse purification and concentration enrichment; affinity media are conjugated with camel-sourced antibodies, the carrying capacity is high, and only an intact FSH molecule, instead of a single subunit, can be bound specifically, so degraded monomer subunits are better removed; and gel filtration can further remove residual DNA, endotoxin and host proteins, and can also remove protein aggregates and inadequately glycosylated FSH proteins.

Description

technical field [0001] The present invention relates to a protein purification method. Specifically, the invention discloses a new purification process of recombinant human follicle-stimulating hormone. Background technique [0002] Follicle-stimulating hormone (FSH) is the most important endocrine hormone secreted by the anterior pituitary gland that can promote and enhance the development and maturation of follicles in the ovary. It can be used to induce ovulation or superovulation, and can promote the development of sperm in men. FSH, like LH and chorionic gonadotropin (hCG), is a heterodimeric glycoprotein consisting of noncovalently bound α and β subunits, containing 92 and 111 amino acids, respectively. The sugar group is connected to the respective regions of the α and β subunits in the form of N-glycosidic bonds. Sugar accounts for 15% to 30% of glycoprotein hormone molecules, mainly including mannose, fucose and galactose, and N-acetylglucosamine. The end of the ...

AI Technical Summary

Problems solved by technology

However, the heterogeneity of glycoprotein hormones on the sugar chain increases the difficulty of its chromatographic separation, which can be summed up in three aspects: low content, instability and heterogeneity

[0008] Reversed-phase chromatography requires the use of organic solvents as the elution medium for proteins. Long-term exposure of proteins to organic solvents will lead to changes in molecular structure to varying degrees, which can lead to irreversible denaturation and seriously affect the biological activity of proteins.

Metal chelation chromatography will lead to the introduction of high concentrations of metal ions into the protein solution, leading to the reduction of protein activity to varying degrees, and increasing the difficulty of removing metal ions

Hydrophobic chromatography is used for the separation of gonadotropins. The inventors found that the activity recovery rate is low, and the specific inactivation mechanism is unknown. Therefore, it is of great practical significance to develop a FSH purification process that reduces the loss of protein activity.

Images