超大孔生物分离介质的新型制备过程及层析应用中的新特性研究

Recombinant vaccines, pegylated proteins and antibody-drug conjugates are giant biomolecules which have demonstrated excellent preventive and therapeutic effects against infectious diseases and some cancers. These frontier biopharmaceuticals require efficient bioprocess engineering. However, the conventional agarose-based media are not suitable for chromatography purification of these giant biomolecules due to small pore size of the media. In previous study we have developed a reverse-micelle swelling method for preparation of gigaporous microspheres with the pore size greater than 200 nm, and have achieved initial success with these media for purification of the giant biomolecules. In spite of these efforts, the preparation and application of the new media are not well stabilized and established. In this study, we will carry out a systematic study on the process of reverse-micelle swelling method with advanced techniques such as fluorescein labeling, confocal laser scanning microscopy and multi-angel laser light scattering. Attention will be focused on 1) finding out the instability mechanism of the preparation system and proposing a strategy to obtain a stable preparation dispersion, so that scale-up will be easy to carry out; 2) clarifying the formation mechanism of the gigapores, thus the preparation process can be extended from hydrophobic to polar and hydrophilic system for obtaining polar and hydrophilic gigaporous microspheres with various functional groups; 3) investigating the effect of pore size on chromatographic behaviors of the giant biomolecules such like vaccine with multi-subunits to find the underlying principles, and obtaining successful chromatographic purification of recombinant vaccines.

基因工程多聚亚基疫苗、PEG化蛋白质、抗体-药物偶联物的研制是生物技术药物发展前沿且已在传染病和癌症防治中显示了重大作用，但现有生物分离介质孔径小不适合这些超大分子的层析纯化，成为亟待解决的问题。本项目组前期发展了新颖的反胶团溶胀法并制备出孔径大于200纳米的超大孔生物分离介质，将此介质应用于超大生物分子的层析纯化取得了很好的进展，但新分离介质的制备仍存在不稳定性。本项目采用荧光素标记、激光共聚焦显微镜等测量手段，对新发展的反胶团溶胀法开展过程科学基础研究，旨在：1)发现介质制备体系不稳定机理，提出稳定化解决方案；2)阐明超大孔成孔机理，将制备机理从疏水拓展至极性、亲水体系，阐明热力学和动力学因素对超大孔形成的影响规律和机理，使其成为一种普适性强的方法；3)利用孔径可控优势，重点研究孔径对不同尺寸多聚亚基蛋白质在层析过程中的聚集和解聚行为的影响规律并阐明机理，并在其分离纯化中获得成功应用。

分离介质是生物科学与技术发展的重要支撑，随着生物技术的迅速发展，疫苗、抗体、抗体-药物耦合物、PEG化蛋白质药物等复杂超大分子（分子尺寸为15-100 nm，大部分疫苗为多亚基颗粒）给分离介质带来新的挑战。研究发现，具有数百纳米孔径的超大孔微球在生物大分子的分离纯化中具有独特优势。在本团队的前期研究中建立了超大孔微球的新型制备技术，实现了超大孔微球的一步聚合，并且通过反胶团溶胀的方法在微球内形成相互贯通的孔道，方法简单、重复性好。在此基础上，本研究针对超大孔微球制备过程中的体系不稳定性，根据超大孔的成孔机理，提出通过双水相重构油水界面的策略，有效解决了微球间的弱聚集问题。将已有的聚苯乙烯材料拓展到极性（PGMA）和多糖材料（琼脂糖、葡甘聚糖），提高了基质的生物友好性，避免了非特异性吸附，并使该方法成为普适性的方法。获得不同体系黏度、固化方法（聚合、交联）对吸水溶胀行为、超大孔孔径的影响规律并阐明热力学和动力学控制机制。得到介质孔径对不同蛋白质三、四级结构的作用及其对超大生物分子生物活性的影响规律，并在乙肝疫苗、口蹄疫疫苗两类重要的生物制品的分离纯化中获得成功应用。