Research Of Polyethylene Glycol Chemical Modification Of Recombinant Human Granulocyte Colony-Stimulating Factor

Chemical modification of proteins is of great significance in biomedical area, in which protein PEGylation is one of the most important technologies. After PEGylation, proteins usually have longer circulating half life, better curative effect and fewer side effects. In this thesis, recombinant human Granulocyte colony stimulating factor(rhG-CSF) was modified with monomethoxypolyethylene glycol (mPEG) which activitied by NHS. The process of PEGylation and its influence were investigated .The reaction condition was optimized by orthogonal design of the experiment. The effects of reaction conditions on modification degree and in vitro bioactivity were investigated. Under the optimized reaction condition , the bioactivity of PEG-G-CSF retained 40% while the modification degree was 50%. In order to isolate and purify the PEG-G-CSF conjugate, small molecule was removed by Sephadex-25 gel chromatography. Moreover, two consecutive ion-exchange chromatography steps were used to separate and purify the PEGylated proteins. In the first step, cation-exchange chromatography was used to separate PEGylated rhG-CSF from unPEGylated protein. In the second step, anion-exchange chromatography was used to purify the individual PEGylated species and remove the excess free PEG.The modification degree of PEGylated G-CSF was evaluated by Fluoremetric assay compared with TNBS assay. The molecular weight distribution was identified by MALDI-TOF mass spectrometry ,SEC-HPLC and SDS-PAGE. The result showed that The molecular weight of the conjugates was 23.3Kda, 28.5KDa,33.7Kda and 38.1KDa respectively.Their in vitro cell proliferation activity and the stability were examined by MTT assay using NFS-60 cell. In order to stabilize the conjugate, addition of human serum albumin and mannitol to the solution of conjugate was first investigated. It was turned out that the activity of conjugate decreased. However, its heat stability, the ability to resist protease increased significantly. Addition of mannitol and human serum albumin to the solution of PEG-G-CSF could maintain the bioactivity upto 92% for one month and 51% for three months when stored at 4℃.