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生物医药领域的蓬勃发展推动着单克隆抗体下游纯化技术的不断进步。目前用于抗体纯化的蛋白A亲和配基，存在价格昂贵、配基脱落造成二次污染、配基稳定性差等问题。探索新型高品质亲和配基已成为抗体纯化亟待突破的瓶颈。多肽亲和配基可以弥补蛋白A的缺陷性，成为近些年的研究热点。但其设计方法属于虚筛，存在漏选、假阳性等问题。因此建立一种新型的配基精准筛选技术具有重要意义。核磁共振饱和转移差谱（Saturation transfer difference nuclear magnetic resonance spectroscopy, STD-NMR）具有可在真实吸附环境中实现筛选、蛋白大小不受尺寸限制等优点。因此，本文将该方法用于仿生多肽配基特异性的筛选方法学建立，并以等温滴定量热法（Isothermal titration calorimetry, ITC）和石英晶体微天平 （Quartz crystal microbalance with dissipation, QCM-D）从热力学和动力学对STD-NMR方法学进行了验证，最后将所筛选出的多肽亲和配基应用于抗体亲和介质的制备和抗体的纯化。本论文开展的研究工作如下：1. 建立了区分配基和蛋白间特异性和非特异性吸附的方法学。通过STD-NMR、ITC和QCM-D等一系列方法来对特异性和非特异性结合间的差异性进行描述。结果表明，特异性吸附时，STD信号强度（international signal intensity, ISTD）随配基浓度增加呈线性增长，并伴随着较高的? H、较低的? S和相对少的吸附量，而非特异性吸附则随配基浓度增加呈非线性增长，并伴随着较低的? H、较高的? S和相对多的吸附量。利用已建立的STD-NMR评价方法学，对实验室前期设计出的仿生多肽配基RGWLC以及FYEILHC进行了筛选和评价，结果表明，FYEILHC较RGWLC具有更好的特异性，更适宜用于后续亲和介质的开发研究。2. 以筛选出的仿生多肽配基FYEILHC为亲和配基，以葡聚糖修饰的聚甲基丙烯酸缩水甘油酯 (Dextran-Poly (glycidyl methacrylate)，Dextran-PGMA)超大孔微球为基质，制备出用于单克隆抗体纯化的仿生多肽超大孔Dextran-PGMA微球。以扫描电镜表征微球表面形貌，以AKTA蛋白纯化系统对Dextran-PGMA超大孔亲和介质进行柱上评测。结果表明，偶联仿生多肽配基的超大孔Dextran-PGMA微球具有传质性能好、化学稳定性强以及可从复杂生物样品中纯化抗体的巨大潜力。 综上所述，本文建立了一种可以用于筛选特异性和非特异性配基的新型STD-NMR方法学，并在此基础上制备出偶联特异性仿生配基的Dextran-PGMA超大孔亲和介质，为抗体材料的设计和发展提供了新的方法学和制备思路。;Biological medicine attentions greatly stimulate the development of downstream purification technology of monoclonal antibodies. Protein A, the current affinity ligand for antibody purification, still has several inherent shortcomings, such as high cost, harsh elution conditions and low stability, which lead to the secondary contamination and biological toxicity in purification. Discovery of the novel high-quality affinity ligands has become a bottleneck for antibody purification. In recent years, a group of peptide ligands have become the research hotspot. However, the designs of them were mostly based on the virtual screening approaches and still suffered from drawbacks on false positive result and low hit rate. Therefore, establishment of a direct screening protocol aiming to the actual binding environment is greatly needed. STD-NMR has advantages in that the size of protein is not limited and most importantly it can screen the ligand in actual binding environment. Therefore, here we established a novel STD-NMR approach for the selective ligand screening, and meanwhile we evaluate this approach by isothermal titration thermography (ITC) and quartz crystal microbalance (QCM).Then the screened biomimetic peptide was used for the antibody affinity media preparation and antibodies purification. The following results were obtained:1. Firstly, a none-competitive titration approach was established to distinguish the specificity and nonspecific ligand. We evaluated a series of approaches from multiple scales，such as，STD-NMR，ITC，QCM，and investigated their possibilities in mining the difference between two selective interaction modes. The results showed that the specific ligand tended to a linear STD signal intensity, with relatively higher ?H, lower ?S and lower adsorption amount, while the nonspecific ligand was completely the opposite. Then, the STD none-competitive titration experiment was used to screen two biomimetic ligands, RGWLC and FYEILHC, previously designed by our group. The results showed that FYEILHC had better specificity than RGWLC and was more suitable for the affinity media design.2. Finally, we utilized this biomimetic peptide as ligand by grafting it on the supermacroporous dextran-poly (glycidyl methacrylate) microspheres (dextran-PGMA) and developed it into an efficient IgG chromatographic media. The scan electronic microscopy (SEM) demonstrated that the macroporous structure of microspheres can be mostly maintained after coupling with the biomimetic peptide. AKTA protein purification system was used to evaluate the column with macroporous affinity media. The results showed that the biomimetic peptide ligand grafted dextran-PGMA microsphere had high-throughput separation, chemical stability and great potential to purify IgG from the complex biological samples.To sum up, we established a method for screening specific and non-specific ligands, and prepared an affinity media by screening the biomimetic peptide ligand and grafting them on gigaporous dextran-PGMA microsphere. The study provides a new methodology and preparation method for the design and development of antibody purification materials.
|葛佳. 针对抗体亲和配基筛选的STD-NMR方法学构建[D]. 中国科学院大学,2019.|
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