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高压对蛋白质的影响在生物过程中有应用潜力。本论文以蛋白质生产的下游过程为对象，探索高压对蛋白质复性、蛋白质修饰和蛋白质装载药物这三个单元的作用，发展了蛋白包涵体高浓度复性、蛋白质的高压聚乙二醇修饰和高压下蛋白纳米笼装载小分子药物技术，主要创新点如下：（1）开发了高压下直接溶解并复性重组人睫状神经营养因子（rhCNTF）包涵体的新过程。通过压力作用，有效抑制了传统稀释复性过程中极易生成的疏水聚集体，当蛋白浓度为4 mg/mL时，200 MPa压力作用16 h可以获得近100%的复性收率，是现有文献报道的最高值，解决了高复性浓度与高复性收率难以兼顾的问题。通过强阴离子交换层析获得了rhCNTF，纯度高于95%，具有与标准品相似的二/三级结构和生物活性, 整体收率为54.1%，高于传统方法（36.7%）。（2）建立了强疏水性的重组人干扰素β-1b（rhIFN β-1b）包涵体的高压复性工艺。使用8 M脲结合1% Triton X-100溶解杂蛋白，提升包涵体中目标蛋白纯度至65%。然后在320 MPa压力下辅以0.4% Zw3-14，0.5 M Arg和5 mM DTT作用rhIFN β-1b包涵体16 h，在4 mg/mL初始蛋白浓度条件下达到100%的质量收率和近80%的复性收率。通过强阳离子交换和丁基硫疏水层析的组合，获得了rhIFN β-1b，纯度大于95%，并且具有与标准品相似的二/三级结构和生物活性，整体收率为37.8%，高于传统制备方法（23.1%）。（3）提出了高压辅助蛋白质聚乙二醇（PEG）修饰策略。利用压力作用提升蛋白表面被修饰位点氨基酸残基的暴露程度，增大PEG修饰试剂与蛋白被修饰位点的可及程度，减弱常规修饰反应参数的影响，增强修饰反应效率。在250 MPa压力，pH 7.0条件下按修饰摩尔比1:1.2 (rhCNTF : mPEG-MAL)使用mPEG-40kDa-MAL修饰rhCNTF蛋白5 h后修饰率达到近90%，相同条件下的常压修饰率不足5%。200 MPa压力下使用mPEG-40kDa-MAL和mPEG-20kDa-MAL定点修饰rhG-CSF时分别能够提升蛋白单修饰率达到32%和45%，而常压下几乎不发生修饰反应。在250 MPa压力下使用mPEG-30kDa-ALD定点修饰rhG-CSF氮末端氨基的修饰率为82%，相较于常压修饰结果（72.4%）提升幅度不明显。（4）尝试了高压下重链铁蛋白（HFn）纳米颗粒装载阿霉素（DOX）药物的方法。在500 MPa压力pH 5.5条件下，DOX可以被载入HFn纳米球中。与此同时，在高压下辅以20 mM Arg几乎可以完全抑制蛋白沉淀，实现HFn蛋白的100%回收。高压处理结束后通过梯度降压策略以及将样品在常压pH 7.4条件下继续透析放置，进一步提升了药物装载比例至32:1(DOX : HFn)。通过高压方法制备的HFn-DOX纳米颗粒中不含有聚集体而且在长期存储过程中药物泄露比例低于20%，在体内外相比单独使用DOX具有更高的抗肿瘤生物活性。使用脱盐层析的方法可以分离并回收未被装载的DOX药物。;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 β-1b (rhIFN β-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 β-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 β-1b prepared by HHP method showed similar advanced structure and in vitro bioactivity to rhIFN β-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.
|王祺. 高压下蛋白质折叠、修饰及装载药物的过程研究[D]. 中国科学院研究生院,2017.|
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