Methods for refolding G-CSF from inclusion bodies

摘要

New methods for the refolding of recombinant granulocyte colony stimulating factor (G-CSF) from inclusion bodies are disclosed. The methods comprise two refolding steps. In particular, the methods comprise the solubilizing of G-CSF with a solubilizing agent, the oxidative refolding (first refolding step) of G-CSF in the presence of the solubilizing agent and an oxidizing agent, the efficient removal of the solubilizing agent, and a second refolding step to complete the folding of G-CSF in the absence of the solubilizing agent. Various methods are described for the efficient removal of the solubilizing agent from partially refolded G-CSF.

主权项

1. A method for refolding granulocyte colony stimulating factor (G-CSF) from inclusion bodies, comprising:
a) solubilising G-CSF in the presence of a solubilising agent selected from the group consisting of a detergent, a tenside and a surfactant; b) performing an oxidation and first refolding step, comprising incubating the solubilised G-CSF in the presence of an oxidizing agent and the solubilising agent; c) removing the solubilising agent by ion exchange resin adsorption and/or ion exchange chromatography, and optionally performing an acid precipitation; and d) performing a second refolding step at a pH value above pH 7, comprising diluting the G-CSF of step (c) with a low conductivity buffer or water and incubating the diluted G-CSF at a conductivity below 2 mS/cm for more than 12 hours, wherein the solubilizing agent after step c) is below 0.01 mg/ml.