The Research On Pilot Scale Purification Of Recombinant Human Granulocyte Colony-stimulating Factor

rhG-CSF is a kind of recombinant human granulocyte colony stimulating factor which is highly expressed in E.coli.It can stimulate many kinds of HBSC to proliferate and differentiate and is used in neutropenia induced by cytoxic chemotherapy in clinical practice.G-CSF is a kind of multi-function cell factor and used widely physiologically. A simple and stable purification protocol is established,which is very suitable for large-scale purification.The choice of method is based on the characteristics of target proteins. All information concerning properties of the target protein and contaminants will help us to design a reasonable strategy for purification. The strategy usually includes three steps including capturing, intermediate purification and polishing.Chromatography is applied widely in large scale because it is scaleable, reproducible and economic. Based on the different principles, the chromatography techniques have been developed using ion exchange (IEX), hydrophobic interaction(HIC), affinity(AC), gel filtration(GF), reversed phase(RPC) and expanded bed adsorption(EBA).In the study, we found that rhG-CSF was degraded in the process of purification. It has proved that the rhG-CSF in rhG-CSF fusion protein is vulnerable to degradation because of two reasons. The first is that rhG-CSF is degraded by proteolytic enzymes of host cell. The second is that disulfide bonds of rhG-CSF can be degraded itself under certain conditions. We observed if the cell is crushed intensely ,the rhG-CSF is degraded and it can be oxidized under certain condition.So how to avoid the degradation constitutes a difficult task for purifying rhG-CSF from E.coli .To inhibit the activity of proteolytic enzyme, E.coli cells was freeze thawing repeatly to release rhG-CSF and avoid the spill of proteolytic enzyme in cold lysis buffer . rhG-CSF in the supernatant of cell lysate was salting out then desalinizated by G-25 the next step it is captured and intermediately purified byCM pharose. Subsequently, the peak that eluted from CM pharose was loaded on S-100 column and purified to homogeneity. The purity of rhG-CSF is more than 95%. Bisides,we adjust buffer solution to inhibit the activity of proteolytic enzym and add the solution of rhG-CSF.The purification process was scaled up from bench to pilot scale. In production we grading-up the process to manufacture qualitial rhG-CSF.Collectively, rhG-CSF expressed in E.coli could be efficiently purified to homogeneity and a pilot-scale purification scheme was developed. The purified rhG-CSF can be used physiologically.