Process for purification of rHu-GCSF

A scalable industrial process for isolation and purification of granulocyte colony stimulating factor (G-CSF) comprising:

• i. isolating inclusion bodies comprising G-CSF from E. coli cells produced by high cell density fermentation, comprising lysing the E. coli cells in a lysis buffer comprising Tris, EDTA and NaCl, wherein the isolating inclusion bodies is performed by instruments selected from the group consisting of laboratory scale sonicator, high pressure cell homogenizer, centrifuge and filtration device;
• ii. solubilizing the G-CSF by incubating the isolated inclusion bodies obtained from step (i) in a solubilization reaction, which comprises Tris, EDTA and Urea, wherein a reducing agent comprising cysteine is added to the solubilization reaction after 1 hour of incubation;
• iii. refolding the solubilized G-CSF obtained from step (ii) by diluting the solubilized G-CSF into a refolding buffer to create a refolding reaction and incubating the refolding reaction for 12 to 28 hours at room temperature, wherein the refolding buffer comprises Tris, urea, sorbitol and an oxidizing agent selected from the group cystine, oxidized glutathione (GSSH) and cystamine;
• iv. acidifying the refolded G-CSF solution obtained from step (iii) by adding to the refolding reaction sodium acetate and an acidifying agent selected from the group of acetic acid, hydrochloric acid and orthophosphoric acid in an amount sufficient to arrive at a pH in the range of 3.0 to 5.5;
• v. clarifying the acidified G-CSF solution obtained from step (iv);
• wherein the clarification is performed by one or both of filtration and centrifugation, and
• vi. purifying G-CSF from the clarified G-CSF solution obtained from step (v) by cation exchange chromatography followed by hydrophobic interaction chromatography (HIC);
• wherein each of the cation exchange chromatography and the hydrophobic interaction chromatography comprise a stationary phase and a mobile phase,
• wherein the G-CSF is recombinant human granulocyte colony stimulating factor, and wherein the yield of the process ranges from 50% to 70%.