| rhGM-CSF is a kind of acid protein which is secreted by activated T-cell, B-cell, macrophages, mast cell, endothelium and fibroblast cell. It contains 127 amino acids with a molecule of 14.5 KD and is applied for leucocytes decrease caused by radiotherapy and chemotherapy for sarcomata cure. It also contributes to bone marrow transplantation, granule cell deficiency, syndrome of hypogenesis of marrow and regenesis anaemia.Clinical application indicated that the rhGM-CSF did not achieve the satisfactory therapeutic efficacy and have many adverse reaction(side-effects). As the rhGM-CSF has short serum half-life and rapid clearance in vivo, it must be daily subcutaneously injected in order to maintain an effective drug concentration, which result in the increasing cost and significant burden on the patient.In order to resolve the above problems, we have adopted an method which a 40kDa monomethoxypolyethylene glycol polymer covalently attached to the rhGM-CSF protein. This method increased the molecular weight of rhGM-CSF, and decreases rate of clearance of renal accordingly. It will achieve a better therapeutic efficacy by changing the character of pharmacokinetics .First, by adjusting molar ratio of PEG and rhGM-CSF, and controlling other reactive condition, for example, reactive pH, temperature, reactive time and purification procedure, it was possible to isolate rhGM-CSF derivatives containing one PEG moiety, and conjugation efficiency is above 40%, in the conjugated mixture, derivative which consistent with one 40kDa PEG to one rhGM-CSF is above 90%.At the same time, we have found that increasing concentration of rhGM-CSF protein and molar ratio of PEG to rhGM-CSF is significant approach which increasing reaction efficiency. In addition, to obtain one PEG to one rhGM-CSFconjugation, decreased reaction pH is essential.Subsequently, we studied on the condition of purification on couple mixed liquor and established the purification technology of couple mixed liquor. The result showed that the process is simple, reproducible and easy control which is suitable for large scale manufacture. In the experiment, we found that anion exchange chromatography is essential to mixed liquor.Matrix-Assisted Laser Desorption Ionization time of fly Mass Spectrometry (MALDI-TOF-MS), peptide mapping are applied to assay of the final derivative. conjugate eventually is a rhGM-CSF with the same of PEG of 40kDa the conjugate, and an a certain and fixed amino that orders the possibility in the rhGM-CSF member up.The bioactivity of PEG-GM-CSF wan evaluated with the TF-1 rat lymphoma cell line in vitro. Although the biggest incitement in 50% density (EC50) value ascension , there is little change in the maximal level of cell proliferation, and no difference to the effectiveness and rhGM-CSF of the cell.In this experiment, a 60Co-γray irradiation mice model were prepared and used to investigate the effect of long-term GM-CSF for injection on periphery blood cell, and bone marrow depression. The result indicated that long-term GM-CSF could significantly increase the value of periphery blood cell (WBC and Gran),stimulatied in vivo hematopoiesis, release the myelosuppression induced by 60Co-γray irradiation.
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