Production of Pegylated Gcsf From Non-Classical Inclusion Bodies Expressed Inescherichia Coli

Background: the recombinant human granulocyte colony stimulating factor conjugatedwith polyethylene glycol (pegylated gcsf) has currently been used as anefficient drug for the treatment of neutropenia caused by chemotherapy due to itslong circulating half-life. previous studies showed that granulocyte colony stimulatingfactor (gcsf) could be expressed as non-classical inclusion bodies (ncibs), whichcontained likely correctly folded gcsf inside at low temperature. therefore, in thisstudy, a simple process was developed to produce pegylated gcsf from ncibs.methods: bl21 (de3)/pet-gcsf cells were cultured in the liflus gx 1.5 l bioreactorand the expression of gcsf was induced by adding 0.5 mm iptg. after 24 hr offermentation, cells were collected, resuspended, and disrupted. the insoluble fractionwas obtained from cell lysates and dissolved in 0.1% n-lauroylsarcosine solution. thepresence and structure of dissolved gcsf were verified using sds-page, native-page, and rp-hplc analyses. the dissolved gcsf was directly used for the conjugationwith 5 kda peg. the pegylated gcsf was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography.results: pegylated gcsf was obtained with high purity (97%) and was finallydemonstrated as a form containing one gcsf molecule and one 5 kda peg molecule(monopeg-gcsf).conclusion: these results clearly indicate that the process developed in this studymight be a potential and practical approach to produce pegylated gcsf from ncibsexpressed in escherichia coli (e. coli).