以温敏树枝状聚合物为间隔臂的高容量温敏仿生亲和色谱及对抗体的分离纯化

Antibody drugs play an important role in the treatment of a number of human diseasese, specially in antitumor. The global antibody market has grown exponentially due to increasing applications in research, diagnostics and therapy. Antibodies are present in complex matrices (e.g. serum, milk, egg yolk, fermentation broth or plant-derived extracts). This has led to the need for development of novel platforms for purification of large quantities of antibody with defined clinical and performance requirements. However, the choice of method is strictly limited by the manufacturing cost and the quality of the end product required. Affinity chromatography is one of the most extensively used methods for antibody purification, due to its high selectivity and rapidity. Its effectiveness is largely based on the binding characteristics of the required antibody and the ligand used for antibody capture. Nowadays, Protein A based affinity chromatography is a platform in the antibody purification due its high specificity. However, Protein A chromatography still has some drawbacks, such as high cost, harsh elution condition, protein ligand leakage, etc. Therefore, exploring an economical and efficient purification technique will be of great importance. In terms of the affinity ligands chosen to act as capturers of antibody molecules, the current status involves some general pseudobiospecific ligands that include mercapto-derived molecules and short peptide, the biomimetic chromatography shows the potential to antibody purification. However, it still has the drawbacks, such as low capacity, harsh elution condition and acidic elution leading to denaturation and losing the bioactivity of purified antibody, which can not meet the requirement from production increasing of the upstream for the antibody purification in dowmsteam. . In this research, the cheap, safe and stable small molecules (e.g. 4-Pyridylethylmercaptan (MEP), 2-Mercapto-1-methylimidazole (MMI) and 2-Mercapto-5-benzimidazolesulfonic acid (MBI)) and short peptiedes (e.g. tetrapeptide DGGA, hexamer peptide HWRGWV, octapeptide FYWHCLDE) as the biomimetic ligands with the functions of special selectivity to antibody, the silica gel as the matrix and the thermoresponsive dendrimers PAMAM-PNIPAM as spacer-arms, the thermoresponsive biomimetic anfinity chromatographic stationary phase are prepared. The structure, chemical composition and physical properties of the prepared novel thermoresponsive stationary phase are characterized, and its chromatographic and adsorption charateristics to IgG are evaluated in detail. A kind of efficient, safe, low cost, green, high capacity biomimetic affinity chromatography is built and used for antibody purification from milk and CHO cell culture supernatant. As a result, the problems above will be resolved. This research is important to separate and purify the antibody in industrial scale.

抗体药物在治疗以肿瘤为主的人类多种疾病方面起着重要作用，具有巨大的市场前景。针对抗体亲和色谱存在吸附容量低，洗脱条件苛刻，无法满足上游产能提升对下游抗体分离纯化的要求，本课题选择廉价、安全、稳定、对抗体具有选择性吸附的功能小分子和短肽为仿生配体，以硅胶为基质，以温敏树枝状聚合物PAMAM-PNIPAM为间隔臂，利用点击反应实现载体与配体的高效、选择性偶联，制备以温敏树枝状聚合物PAMAM-PNIPAM为间隔臂的温敏仿生亲和色谱介质，构建一种用于抗体分离纯化的高效、安全、低成本、高容量、绿色环保的温敏型仿生亲和色谱，以解决当前仿生亲和色谱活性位点少、吸附容量低、采用酸性洗脱使抗体变性失活的难题。对其结构及对抗体的分离和吸附性能进行表征，将其应用于牛乳清和CHO细胞培养上清液中抗体的分离纯化，建立高效、稳定和低廉的抗体分离纯化新工艺。该研究对抗体的分离纯化及工业化生产具有重要应用价值。

抗体药物是治疗肿瘤等疑难杂症的特效药，具有巨大的市场。针对抗体亲和色谱存在吸附容量低，洗脱条件苛刻，无法满足上游产能提升对下游抗体分离纯化的要求，本项目选用对抗体具有选择性吸附的功能小分子4-巯乙基吡啶为配体，以硅胶为基质，以温敏树枝状聚合物PAMAM-PNIPAAm为间隔臂，制备了高容量温敏亲和色谱固定相，克服了线型间隔臂活性位点少、吸附容量低等的缺点。通过调节树枝状聚合物PAMAM的枝化代数调控间隔臂的长短和活性位点数目，大大提高了配体的固载量和吸附容量，对抗体蛋白的吸附容量提高了2倍。以此为基础，首次构建一种用于抗体分离纯化的温敏亲和色谱法。该技术以水为流动相，在40C吸附蛋白，在5C解吸附蛋白，仅通过改变流动相温度就可实现抗体的一步分离纯化。系统研究了温度、温敏聚合物链长、流动相的盐浓度和pH对温敏亲和色谱分离BSA和-球蛋白的影响，优化了分离条件；比较了接枝不同代数温敏聚合物PAMAM的温敏亲和色谱固定相对BSA和-球蛋白分离的影响；系统研究了-球蛋白在高容量温敏色谱固定相的吸附性能；对温敏亲和色谱对抗体分离的机理进行了初步探讨，并成功将该技术应用于人血清中IgG、蛋黄中IgY和重组单克隆抗体药物inflixmab分离纯化，一步纯化后IgG、IgY和inflixmab的纯度均大于95%，质量回收率大于90%。该工艺洗脱条件温和，绿色环保，从根本上解决了传统亲和色谱酸性洗脱易使抗体变性失活的难题。该技术操作简单，易于放大，可大大简化抗体药物的生产工艺，显著降低生产成本，对抗体药物的分离纯化及工业化生产具有重要的应用价值。共发表论文14篇，其中SCI论文12篇，申请国家发明专利1项，国内会议邀请报告5次，合作出版著作一部。培养青年科技骨干2人，博士生2名，硕士生6名。