重组人促卵泡激素(rhFSH)肽图分析方法研究

Study on the method of the peptide mapping analysis of recombinant human follicle stimulating hormone(rh-FSH)

目的建立重组人促卵泡激素(rh-FSH)的标准肽图分析方法。方法通过超滤、透析、冷冻干燥法富集制备rh-FSH对照品。通过尿素变性、二硫苏糖醇(DTT)还原、碘乙酸酰基化制备供试品溶液。采用N型-糖苷酶F(PNGaseF)酶解去除rh-FSH糖侧链,经SDS-PAGE验证切糖结果后,用胰蛋白酶水解,酶解所得肽段通过RP-HPLC分析,得到rh-FSH肽图。结果利用国外原研厂rh-FSH制剂富集制备的rh-FSH结构完整,可用作rh-FSH肽图分析的对照品。国产样品的肽图谱与对照品一致。结论建立的肽图分析方法可作rh-FSH产品的结构确证。

Purpose To establish the method of standard peptide mapping for recombinant human follicle stimulating hormone（rh-FSH）.Methods Enrichment and preparation of the rh-FSH reference sample by ultrafiltration,dialysis and freeze drying.Preparing the sample solution according to the program of urea denaturation,dithiothreitol（DTT）reducing and indoacetic acid alkylation.The oligosaccharide chain was removed from the molecule of rh-FSH by the N-type glycosidase F（PNGaseF）and then the result of treatment was assessed by SDS-PAGE.The peptide map of rh-FSH was obtained by trypsin hydrolysis and RP-HPLC analysis.Results The rh-FSH reference sample prepared from the rh-FSH preparation of a foreign origin manufacture by an enrichment procedure was of integrity in the molecule structure.It is available to be used as the reference for the rh-FSH peptide mapping analysis.The peptide map of the sample for testing of the rh-FSH was conformed to that of the rh-FSH reference.Conclusion The method developed may be served as the routine method in quality controls for the rh-FSH product.