Process Investigation Of High Hydrostatic Pressure In Protein Refolding,PEGylation And Drug Encapsulation

High hydrostatic pressure(HHP)is reported to affect the structure of proteins,which may have potential application in bioprocesses.In this study,HHP was applied for downstream process of biopharmaceuticals,including protein refolding,modification,and drug encapsulation.We developed the HHP refolding strategy for protein inclusion bodies(IBs)at high concentration,HHP PEGylation of proteins,and HHP encapsulation of drugs through protein nanocages.The main innovations are as follows:1)We demonstrated that HHP could simultaneously solubilize and refold of IBs of recombinant human ciliary neurotrophic factor(rhCNTF).HHP enabled the refolding yield of rhCNTF up to almost 100%even at 4 mg/mL under 200 MPa for 16 h by inhibiting the hydrophobic aggregation.To our knowledge,both the refolding concentration and refolding yield we achieved are the highest among the cases reported for HHP refolding directly from IBs till now.After purification by Q Sepharose FF anion exchange chromatography,the purity of rhCNTF reached 95%with total process recovery of 54.1%.The purified rhCNTF showed similar advanced structure and in vitro bioactivity to the native species.2)We reported a HHP strategy,combined with cosolutes for facile refolding of strongly hydrophobic recombinant human interferon ?-1b(rhIFN ?-1b).Pretreatment of the IBs with 8 M urea and 1%Triton X-100 could reduce the amount of impurities and facilitate the protein to refold.Under 320 MPa,almost 80%refolding yield and 100%of mass yield can be obtained with the optimal composition of cosolutes even from 4 mg/mL of rhIFN ?-1b IBs suspensions.After purification by SP Sepharose FF cation exchange chromatography and Butyl-S Sepharose FF hydrophobic chromatography,the overall yield of 37.8%was achieved compared with that of only 23.1%for traditional method.The rhIFN ?-1b prepared by HHP method showed similar advanced structure and in vitro bioactivity to rhIFN ?-1b standard.3)The high hydrostatic pressure PEGylation(HHPP)of proteins was firstly proposed.The speculated benefit of HHPP was facilitation of target site exposure,reducing the steric hindrance and making the reaction much easier.PEGylation of rhCNTF with mPEG-40kDa-MAL at molar ratio of 1:1.2(rhCNTF:PEG)at pH 7.0 under normal pressure for 5 hours achieved less than 5%yield.In comparison,when the pressure was elevated,the PEGylation yield was increased dramatically,reaching nearly 90%at 250 MPa.Nearly 45%of monomeric mPEG20k-MAL-rhG-CSF and 32%of monomeric mPEG40k-MAL-rhG-CSF could be obtained with molar ratio of 1:2(rhG-CSF:PEG)under 200 MPa for 16 h at pH 7.5 in PB buffer while negligible of PEGylation was observed at atmospheric pressure.Compared with the significantly enhanced PEGylation efficiency at thiol by the elevated hydrostatic pressure,the yield of PEGylated rhG-CSF at amino group of N-terminus was only raised to some degree,82%for HHPP and 72.4%for conventional method.4)A novel HHP-based strategy was attempted for encapsulation of doxorubicin(DOX)in human H-ferritin(HFn)nanocage.At the pressure of 500 MPa and pH 5.5,DOX molecules were found to be encapsulated into HFn.Meanwhile,addition of 20 mM arginine could completely inhibit precipitation and aggregation,resulting in highly monodispersed nanoparticles(NPs)with almost 100%protein recovery.Furthermore,stepwise decompression and incubation of the complex in atmospheric pressure at pH 7.4 for another period could further increase the DOX encapsulation ratio.HFn-DOX NPs prepared through HHP showed similar morphology and structural features to the hollow cage and no notable drug leakage occurred,as well as significant cytotoxicity in vitro and higher antitumor bioactivity in vivo than naked DOX.Moreover,This HHP encapsulation strategy could economize on DOX that was greatly wasted during the conventional preparation process simply through a desalting column.