The Research On Pilot Scale Purification Of HSP-MUC1

HSP-MUC1 is a recombinant fusion protein constructed by fusing BCG derived HSP65 with MUC1 VNTR peptide. We show that immunization with HSP-MUC1 induces growth inhibition of MUC1-expressing tumors both before and after tumor challenge and prolong the survival of mice with MUC1-expressing tumors. The anti-tumor activity was correlated with induction of MUC1-precific CTL as well as non-specific anti-tumor immunity. Thus, exogenously applied HSP-MUC1 fusion protein can stimulate anti-tumor immunity by inducing the epitope-specific CTL as well as non-specific anti-tumor immunity mediated by the HSP part of the fusion protein. All these results suggest that HSP-MUC1 could be applied to treat MUC1 position tumors.To obtain sufficient amount of HSP-MUC1 purified, we design a suitable scheme which is reproducible, efficient, economical and scaleable.The choice of method is based 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 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 HSP-MUC1 was degraded in the process of purification. It has proved that the HSP65 in HSP65 fusion protein is vulnerable to degradation because of two reasons. The first is that HSP65 is degraded by proteolytic enzymes of host cell. The second is that HSP65 is a proteolytic enzymes that can degrade itself under certain conditions. We observed that the purified HSP-MUC1 is very stable when stored in 4℃, suggesting that HSP-MUC1 is degraded by host derived proteolytic enzymes rather than itself. Therefore, how to avoid the degradation constitutes a difficult task for purifying HSP-MUC1 from E.coli .To inhibit the activity of proteolytic enzyme, E.coli cells was disrupted in cold lysis buffer (1.5 M NaCl, 50 mM Tris, pH 9.0). HSP-MUC1 in the supernatant of cell lysate was captured and intermediately purified by phenyl-Sepharose. Subsequently, the peak that eluted from phenyl-Sepharose was loaded on Q-sepharose FF column and purified to homogeneity. The purity of HSP-MUC1 is more than 95%.The purification process was scaled up from bench to pilot scale. Up to 100 g wet cell mass was disrupted by high-pressure homogenizer to release the expressed proteins that were then purified by 400 ml of phenyl-sepharose and 80 ml of Q-sepharose. 435 mg of purified HSP-MUC1 was obtained and the endotoxin in the sample was lessthan 10 Eu/mg.Collectively, HSP-MUC1 expressed in E.coli could be efficiently purified to homogeneity and a pilot-scale purification scheme was developed. The purified HSP-MUC1 can be used for the further pre-clinical and clinical studies.