液相肽图法推断聚乙二醇化重组人生长激素的聚乙二醇修饰位点研究

 目的 利用液相肽图法对聚乙二醇（PEG）化重组人生长激素(rhGC)（PEG-rhGH）的聚乙二醇修饰位点进行测定。方法 通过优化酶切条件，建立肽段分离度高、信息完整的液相肽图方法。继而通过引入高分辨率质谱，对重组人生长激素的液相肽图准确定性。最后通过比较发生改变的修饰前后的肽段，推断聚乙二醇的修饰位点。结果 3个厂家的聚乙二醇化重组人生长激素中发生较为明显修饰的肽段有5个，分别为T1（N-end）、T4（lys38）、T13（lys140）、T14（lys145）及T15（lys158）。结论液相肽图法可用于对结构复杂、不均一性较高的多位点异构体聚乙二醇修饰药物的位点测定。

RP-HPLC Peptide Mapping to Determine Pegylation Sites of PEG-rhGH

OBJECTIVE To determine pegylation sites of PEG-rhGH by RP-HPLC peptide mapping.METHODS Firstly,a RP-HPLC method was set up to get high resolution and detailed information of peptides by optimizing the separating and digestion conditions;secondly,MALDI-TOF mass was used to identify each separated peptide;finally,pegylation sites were determined through comparing the k values of changed peptides in the modified and unmodified digestion map.RESULTS There were five major peptides modified by PEG in the digestion of PEG-rhGH from three pharmaceutical companies.They were T1(N-end),T4(lys38),T13(lys140),T14(lys145)and T15(lys158),respectively.CONCLUSION RP-HPLC peptide mapping can be used to determine pegylation sites of complicated and inhomogeneous positional isomers of PEGylated pharmaceuticals.