染色质组蛋白在单抗纯化中作用机理的研究及大规模抗体纯化平台的建立

Therapeutic monoclonal antibodies have grown to become the dominant product class within the biopharmaceutical market. Many high-end biotechniques are essentially integrated in antibody production, which is a key factor contributing to the long-term development of a country’s biopharmaceutical industry. In recent years, dramatic increases in cell culture productivity have meanwhile increased the stakes, and challenged purification technology to keep pace. Our latest progress in antibody purification indicates that host contaminant behavior is dominated by a class of contaminants defined by their association with chromatin. Chromatin is the decaying remnant of chromosomal material released from dead host cells. Through the course of its journey from nucleus to cell supernatant, it accumulates a large diversity of non-chromatin species, leading to the formation of chromatin heteroaggregates. Their histone components cause them to bind strongly to protein A affinity chromatography media, where they reduce dynamic capacity for antibody, inflate contamination of the eluted product, promote aggregate formation, and reduce antibody recovery. In this study, research will focus on the crosslinking mechanism between chromatin/histones and various host cell proteins and also the action mechanism of chromatin heteroaggregates on the various purification strategies on monoclonal antibody processing. Microscope technology with high resolution and advanced protein structure analysis will be applied to reveal the pivot role of chromatin in the purification of monoclonal antibody. In addition, based on our new chromatin-directed cell culture clarification, a totally novel approach for antibody processing will be developed, which exploits multiple selectivities simultaneously, with a new chromatographic format. The new system is based on tangential flow filtration and monolith or membrane adsorber. This enables sample introduction without equilibration, sample-adsorbent contact without equilibration, and product “capture” with an adsorbent that does not bind the product. More patients will benefit from these new techniques which can produce higher-quality antibody drugs with even lower-cost.

抗体药物是高投入、高产出的行业，是众多高端生物技术的集成，是衡量一个国家生物制药水平高低的重要指标。抗体上游细胞发酵技术的长足发展也同时为下游抗体的纯化提出了新的挑战。在我们的先期研究中发现抗体纯化中各种宿主杂质蛋白的表现行为是由其与染色质相关联性所决定的。染色质是从死亡的宿主细胞中释放的染色体物质的残留物质，在其由细胞核释放到细胞培养液的过程中，逐渐积累交联了大量、多样的非染色质宿主杂质，进而形成了染色质异质交联体，成为各种宿主杂质富集域。本项目将围绕单抗制备过程中染色质组蛋白与各种宿主蛋白、DNA交联机理以及其在单抗诸多纯化方式中的影响机制，应用具有高分辨能力的显微技术以及先进蛋白质结构分析、鉴定手段揭示染色质组蛋白在单抗纯化中举足轻重的作用；进一步发展以染色质为导向的新一代细胞液澄清技术开发具有大规模应用前景的新型单抗纯化平台，在降低生产成本的同时提高单抗药物品质，为广大病患造福祉。

在利用中国仓鼠卵巢细胞等哺乳动物表达系统生产单克隆抗体的过程中，除了目标IgG抗体分子，诸多宿主细胞污染物如宿主杂质蛋白、宿主DNA和各种分子量的蛋白聚集体的共存在，给后续抗体纯化带来了极大的负担。以蛋白A亲和层析为核心的3-4步层析法是抗体纯化的金标准。但蛋白A固有诸多缺点，如成本高、结合能力相对较低、使用寿命短、蛋白A的浸出以及破坏抗体分子结构等。进一步简化生产工艺并降低成本，是抗体生产的重点。本研究中，我们首先系统地研究了细胞培养液pH和电导率对关键污染物如组蛋白、宿主DNA和其它非组蛋白宿主杂质蛋白组成的染色质异质交联体的协同作用。IgG的损失与这些高分子量的染色质异质交联体的沉淀密切相关，其中DNA/组蛋白随缓冲环境的酸化而显著降低。DNA经pH中和后完全恢复，而组蛋白由于低pH引起的沉淀不能恢复。针对组蛋白的高等电点特性，我们完善了通过添加脂肪酸诱导组蛋白/DNA为核心的染色质异质交联体聚集，实现杂质高效去除的机制模型。脂肪酸分子与碱性IgG、高碱性组蛋白结合，形成疏水性较强的大分子复合物，团聚更多的非组蛋白宿主杂质蛋白，溶解度降低而从溶液中析出。与组蛋白相比，IgG分子既有正电域又有负电域。虽然脂肪酸通过IgG正电结合位点结合，但IgG的负电结构域使IgG在溶液中保持可溶性。基于以上研究，我们建立了多种基于染色质导向细胞液澄清技术的抗体纯化工艺。将染色质导向细胞液澄清发展为一个单独的抗体分离单元，实现各种宿主污染物的高效预去除。使得a）空位排阻阴离子交换色谱成为抗体首步分离纯化的新方法，b）切向流过滤成为抗体浓缩、缓冲液置换的高效途径，c）深层滤膜上实现原位硫酸铵诱导的管式反应抗体沉淀。进一步整合高负载量阳离子交换或复合型阴离子交换层析进行抗体精纯，形成多种三单元抗体纯化新策略。无需缓冲液置换，实现无缝衔接。具有显著提高整体生产效率和节约成本的潜力，为当前基于蛋白A的抗体处理平台提供优良的升级方案，具有良好的应用价值。