Application Of Polyethylene Glycol Precipitation In The Protein Separation Of Biopharmaceuticals From Prokaryotic Expressing System

Background and aim:Biopharmaceuticals including recombinant proteins and monoclonal antibodies are growing rapidly in clinical applications.Prokaryotic Escherichia coli（E.coli）is one of the most commonly used systems in the production of biopharmaceuticals because of its relatively low investment cost,short production cycle,and easy to operate.However,heterogeneous expression of biopharmaceuticals in E.coli often results in formation of insoluble inclusion bodies（IBs）,from which the renatured and biologically active target proteins have to be recovered through denaturation and refolding processes.Since IBs are often contaminated heavily with E.coli cell wall and outer membrane components,purification of IBs before refolding procedures is considerably helpful to improve the recovering yields.Size exclusion chromatography（SEC）has been reported as a useful method to purify IBs,but the relatively low loading capacity and high cost have significantly increased manufacturing cost in industrial application.Development of more efficient,scalable and cost effective processes to purify IBs is of critical importance for biopharmaceutical production from E.coli expression system.Recombinant human interleukin 15（rhIL-15）using in clinical development has been manufactured by E.coli system.In the current process,large quantity of host cell contaminants exists in the raw rhIL-15IBs,and S-200 SEC chromatography under denaturing conditions was performed to remove high molecular weight contaminants.However,sample loading capacity（usually 2-5%of column volume）,extended packing height and slow loading speed led SEC to be a bottleneck of the current processes.Therefore,it would be beneficial to biopharmaceutical industry to develop a novel process to replace SEC for purification of the contaminated IBs.Polyethylene glycol（PEG）precipitation has been widely used in purifying biological molecules including recombinant proteins and antibodies.Considering the advantages of PEG precipitation in recovery and purification of macromolecules,and removable during downstream ion-exchange or hydrophobic interaction columns,it is interesting to explore whether PEG precipitation method is suitable to replace the SEC step in purification of rhIL-15 inclusion bodies from E.coli cells.Method:Using recombinant human interleukin 15（rhIL-15）as an example,inclusion bodies of rhIL-15 were solubilized in 7 M of guanidine hydrochloride and rhIL-15 was precipitated by the addition of PEG 6000.To compare side-by-side,purification of rhIL-15 inclusion body through S-200 column chromatography was performed in parallel.The collected fractions containing rhIL-15 was refolded and purified by sequential Butyl Sepharose HP,Source 15Q,Q_XL and Superdex 75pg.Purified rhIL-15 was characterized by a panel of assays,including ExRP-HPLC,SEC-HPLC,mass spectrometry and CTLL-2 assay to demonstrate high quality and comparable bioactivity as reference standard.Result:A final concentration of 5%（m/v）PEG 6000 was found to be optimal to precipitate target protein in both recovery and purity.Comparing with the previous reported S-200 size exclusion purification method,PEG precipitation was not only easier to scale up,achieving the same high-yield level and quality of the product,but also reducing about50%of time consumption and 95%of material costs.After refolding and further purification,the rhIL-15 product showed a high purity of 97%and a comparable bio-activity of 1×10⁷ IU/mg with the reference material.The data demonstrated that with the two approaches,we can produce rhIL-15 with the similar level of yield and quality,and PEG can accommodate with downstream chromatography steps very well.Our work demonstrated that PEG precipitation in combination with denaturants provides a new strategy for the purification of inclusion bodies that can potentially be applied in large-scale industrial production of biotechnological drugs from E.coli expressing system.