蛋白A色谱中配基固定与过程高效化研究

Monoclonal-antibody based drugs are a large and growing segment of pharmaceuticals worldwide. In order to mitigate the bottleneck in downstream process, the research devotes to develop novel high-throughput protein A (SpA) chromatography based on multi-scale approaches to understanding of antibody-SpA interaction and structure-performance relationship of chromatographic materials. In this research, antibody-SpA interaction on solid interface is investigated to acquire scientific understanding of materials contribution to the antibody interaction; the influence between the structure of the adsorbent for SpA chromatography and performance of antibody adsorption are measured and monitored to reveal the structure-performance relationship in depth during antibody adsorption. On a basis of above understanding, the role of materials, linkers and immobilization strategies are evaluated to improve the utilization of SpA, and thus the optimized strategy for immobilization of SpA is proposed. Then chromatographic materials with controlled pore architecture are fabricated and the high-capacity SpA adsorbent for antibody adsorption is synthesized. Then, the novel high-throughput SpA chromatography is developed and the corresponding theoretical model is proposed to describe the uptake kinetics in adsorbents. Finally, SpA chromatography developed herein is applied to purify antibody from various feedstock to demonstrate the applicability of the multi-scale approaches. The research provides insight into the understanding to antibody-SpA interaction and structure-performance relationship of chromatographic material, and establishes a powerful underpinning in reshaping SpA chromatographic technique and growing monoclonal-antibody based drugs.

单克隆抗体药物是制药行业全球增长速度最快的市场。针对当前单抗药物制备中蛋白A（SpA）色谱的技术"瓶颈"，本项目提出了基于多尺度认知的SpA色谱过程高效化的创新研究思路。围绕抗体－SpA结合作用和色谱介质"结构－性能"关系两个科学问题，开展了固液界面抗体与SpA结合作用的研究，全面地认知了材料在结合过程中作用；观测了SpA色谱介质结构对抗体吸附性能的影响，完善了色谱介质"结构－性能"关系的阐述。基于上述认知，研究了材料、间隔臂以及固定化方法对SpA配基利用率的影响，建立SpA配基高效固定化技术；制备孔道结构可控的快速色谱基质，合成高容量SpA色谱介质，进而开发高效SpA色谱技术；同时，建立并完善SpA色谱过程理论；最终实现SpA色谱技术高效化。本研究对深化抗体－SpA结合作用及介质"结构－性能"关系的认知具有重要的科学意义，将为完善SpA色谱技术，推动单抗药物发展奠定强有力的技术基础。

蛋白A（Staphylococcus aureus protein A，SpA）色谱是当前单克隆抗体药物制备工艺的“金标准”，其发展的基础则在于SpA色谱介质的创新。当前，SpA色谱介质的处理能力饱受诟病。相比于离子交换色谱介质的高抗体吸附容量（~ 200 mg/mL）和良好的环境适应性，SpA色谱介质不仅抗体吸附容量非常低（30 – 60 mg/mL）而且存在耐碱性差、配基易脱落的缺点。针对上述问题，本项目提出了基于抗体－SpA结合作用和色谱介质“结构－性能”相互关系两个科学问题深入认知开发高效的SpA色谱技术的研究目标。这一目标在技术上是通过获得耐碱性SpA配基、合成高容量色谱介质和发展抗体色谱新工艺三个步骤实现的。.本项目基于介质及分子尺度上抗体结合突变域Z（N29A）及衍生物的稳定性及其与抗体Fc片段结合作用的研究，发现了影响Z域稳定性的关键氨基酸残基，进而提出了通过改善氨基酸残基相互作用提高SpA-Z配基耐碱性的新策略，获得了若干新型耐碱性Z域衍生物。在此基础上，进一步研究了SpA配基固定对抗体结合作用的影响，不仅验证了配基密度对抗体吸附有着显著影响，也发现了N端标记生物素的SpA-Z蛋白与抗体分子间的亲和性高于C端标记生物素的SpA-Z蛋白50倍以上，由此为合成高容量的抗体色谱介质提供了科学指导。本项目分别合成了多种高容量的色谱介质，系统地阐述了色谱介质结构、表面特征与色谱性能间的关系，提出了抗体高容量吸附的新策略，抗体的饱和吸附容量接近250 mg/mL介质。基于色谱介质内抗体吸附行为的认知，本项目提出了一种串联连续吸附抗体的色谱工艺及利用该工艺的色谱装置，利用该色谱装置从包含抗体和至少一种杂蛋白的生物原料中分离抗体，有效避免了单柱色谱纯化抗体技术中抗体处理量低等问题；实现了抗体的连续化生产，并能在提高抗体处理能力的同时获得高收率和高纯度的抗体产品。本项目的研究工作为SpA色谱的发展奠定了科学与技术基础。

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