Developing a novel pipeline to determine monoclonal antibody solution structures and stabilities in bioprocessing conditions to optimise manufacture

背景：单克隆人IgG 1和IgG 4抗体已成为生物技术工业中重要的治疗剂。抗体主要针对特定靶标的功效进行工程化，在下游生物过程环境中对聚集高度敏感，特别是当抗体浓度增加至50-200 mg/ml的最终产物浓度时。这对生物技术行业来说是一个重大挑战，因为聚合物可能严重危及安全性。了解如何管理抗体聚集越来越重要，这是BBSRC组织的每年两次的金砖四国会议的主要主题，提案者参加了会议。直接阐明引发蛋白质聚集的物理参数将允许设计更稳健的聚集较少的先导候选物，这些因素包括缓冲液pH、温度（T）、缓冲液离子强度（I）和抗体浓度（c）。系统研究这四个因素对抗体溶液结构及其形成大寡聚体的倾向性的影响，将揭示其聚集的关键阶段，并提出更合理的蛋白质工程来阻止它的途径。我们已经开发了使用散射和超离心来确定IgG抗体的原子溶液结构的新方法（Rayner等人，2013，2014，2015）。今年，我们的方法得到了进一步改进，在CCP-SAS项目中纳入了先进的结构建模方法。例如，实验上，我们的测试表明，IgG 4抗体在pH 3，然后pH 7的两个步骤中改变其构象，形成沉淀的紧凑结构，我们可以模拟所有这些构象变化（Perkins，未发表）。目的：（1）通过研究两种人单克隆IgG 1和IgG 4抗体的溶液构象与pH、T、I和c的关系，我们将构建抗体构象和聚集倾向的相图。(2)使用精确的原子模型的数据，我们将确定在这些不同的条件下的抗体结构与实验条件相关的主要构象变化。(3)另外四种IgG单克隆抗体的加入将使我们能够开发一个快速评估新抗体产品的管道。实验室：富士胶片将提供克量的单克隆IgG 1和IgG 4用于实验，并提供IgG单克隆抗体生产工艺的现场培训。在法国格勒诺布尔的ESRF同步加速器上使用高通量X射线束线BM 29和96孔微孔板，将快速系统地探索pH、T、I和c的影响，以生成相图。如果需要，法国格勒诺布尔ILL的D22仪器上的高通量中子数据收集将补充X射线数据集。在UCL的分析超离心将揭示在选定的pH、T、I和c值下的构象或寡聚化变化。我们的新CCP-SAS建模软件（Hui et al 2015，2016）通过高性能计算将生成多达700，000个随机但物理准确的构象。将该结构文库与每个数据集进行比较，以确定每个缓冲液中的最佳拟合，从而确定构象。最终，我们将设计一个操作管道，以快速开发新的抗体结构，使其具有可制造性。

BACKGROUND: Monoclonal human IgG1 and IgG4 antibodies have become important therapeutics in the biotechnology industry. Primarily engineered for efficacy against specific targets, antibodies are highly susceptible to aggregation in downstream bioprocess environments, particularly as antibody concentrations increase up to final product concentrations of 50-200 mg/ml. This represents a major challenge for the biotechnology industry where aggregates can seriously compromise safety. It is increasingly important to understand how to manage antibody aggregation, and this is a major theme addressed in the twice-yearly BRIC meetings organised by the BBSRC, attended by the proposers. A direct elucidation of the physical parameters that initiate protein aggregation would allow the design of more robust lead candidates that aggregate less. These factors include buffer pH, temperature (T), buffer ionic strength (I), and antibody concentration (c). A systematic study of the effects of these four factors on antibody solution structures and their prospensity to form large oligomers will reveal the key stages towards their aggregation and suggest avenues for more rational protein engineering to block it. We have developed new methods using scattering and ultracentifugation to determine atomistic solution structures for IgG antibodies (Rayner et al, 2013, 2014, 2015). This year, our methods have been improved further by the incorporation of advanced structural modelling methods in the CCP-SAS project. For example, experimentally, our tests showed that an IgG4 antibody changed its conformation in two steps at pH 3, then pH 7, to form a compact structure that precipitates, and we could model all these conformational changes (Perkins, unpublished). We are thus ideally poised to undertake this timely project within the remit of the BBSRC.AIMS: (1) By studying the solution conformations of two human monoclonal IgG1 and IgG4 antibodies as a function of pH, T, I and c, we will construct phase diagrams of antibody conformation and aggregation propensity. (2) Using accurate atomistic modelling of the data, we will determine antibody structures in these different conditions to correlate major conformational changes with experimental conditions. (3) The inclusion of four other IgG monoclonals will enable us to develop a pipeline for the rapid assessment of new antibody products.EXPERIMENTAL PLAN: Fujifilm will provide gram amounts of monoclonal IgG1 and IgG4 for experiments, and on-site training on the manufacturing processes for IgG monoclonals. Using the high-throughput X-ray beamline BM29 at the ESRF synchrotron, Grenoble, France, with 96-well microplates, the effects of pH, T, I and c will be explored rapidly and systematically to yield phase diagrams. If required, high-throughput neutron data collection on instrument D22 at the ILL, Grenoble, France, will supplement the X-ray data sets. Analytical ultracentrifugation at UCL will reveal conformational or oligomerisation changes at selected pH, T, I and c values. Our new CCP-SAS modelling software (Hui et al 2015, 2016) via high performance computing will generate as many as 700,000 randomised but physically-accurate conformations. This structural library will be compared with each data set to identify the best fit in each buffer, hence identifying the conformation. Ultimately, we will design a pipeline of operations to characterise newly-developed antibody structures rapidly for their manufacturability.