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Protein concentration determination in the pegylation process

Protein concentration determination in the PEGylation proces...

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第三届全国化学工程与生物化工年会

作者： Zheng Chunyang Zhang Peng Zai Yanqin Ma Guanghui Su Zhiguo (National Key Laboratory of Biochemical Engineering, Institute of Process Engineering,Chinese Academy of sciences, P.O. Box 353, Beijing 100080, China Graduate University of Chinese Academy of sciences, Beijing, 100049, China Civil and Environmental Engineering school, University of science and Technology of Beijing, Beijing 100083, China)

＜正＞pegylation technology is increasingly important. The protein concentration determination is close related to the reaction, separation and characterization of the final product in the pegylation process. To ensure the research and quality control of pegylation process, in this study, Human serum albumin （HSA） and recombinant human granulocyte-colony stimulating factor （rhG-CSF） were chosen as model proteins and modified by sc-peg5K, sc-peg10K. and sc-peg20K. respectively, to detenninate the effect of activating method and the chain-length of peg reagent on the determination of protein concentration. The effect of the salts, used in the common purification methods of pegylated proteins was also determined. The results indicated that Lowry method is interfered by N-hydroxysuccinimide （NHS） that produced when using sc-peg as peg reagent, however, NaCl, Na2SO4 and （NH4）SO4 （used in ion-exchange, size-exclusion, and hydrophobic interaction chromatography, respectively） interfered the determination of Bradford method. Therefore, it is proposed that Bradford method is selected to determine the samples in the reaction stage, while Lowry method is selected to study the samples in purification stage. This procedure was used to determination the protein concentration in the practical pegylation process of first modifying rhG-CSF using sc-peg20K and then separating the mono-pegylated rhG-CSF using ion exchange chromatography, by comparison with the Bradford or Lowry method, and satisfactory results were obtained in both the characterization of final product and the evaluation of the separation process.

关键词： pegylation process Bradford method Lowry method sc-peg

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