Elucidating aggregation mechanisms in antibody fragment-based therapeutics to improve their manufacturability

阐明基于抗体片段的疗法中的聚集机制，以提高其可制造性

• 批准号: BB/I017119/1
• 负责人: Paul Dalby
• 金额: $ 57.55万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI017119%2F1
• 关键词: Elucidating; aggregation; mechanisms; antibody; fragment

Recent therapies for age-related diseases such as rheumatoid arthritis, macular degeneration, Crohn's disease, and some cancers are engineered forms of biological protein molecules called antibodies that form part of our own natural immune system. Such therapeutic proteins are being derived increasingly from simpler fragments of these antibodies with the hope that this will improve their behaviour in the body, reduce the frequency of injections required, allow them to target new regions of the body, and also allow them to be combined with other biological molecules without becoming too large or unstable. However the manufacturing of therapeutic proteins is extremely challenging due to their delicate and complex nature. Manufacturing processes aim to separate the protein molecules from the rest of the cellular components in which they were synthesised, to obtain extremely pure therapeutic material that is suitable for use in humans as a therapy. However, the processes available for large-scale manufacture place a great deal of stress on the protein due to changes in temperature or acidity, the addition of salts, the use of mechanical agitation, rapid changes in the rate of flow through machinery, and the interaction of proteins with air bubbles. This frequently causes the protein to deform slightly and to subsequently stick together to form tiny particles called aggregates. While these are often not visible to the naked eye, their presence in therapies can be hazardous to patients as they may cause severe inflammation and potentially more deadly immune responses. Therefore, one of the key challenges that the bioprocess development and therapeutic protein manufacturing industries would like to address is to be able to either predict the conditions that cause a protein to aggregate, or to increase their robustness so that they aggregate less frequently during their manufacture. We aim to carry out and demonstrate a suite of rapid experimental measurement techniques that allow a new therapeutic protein to be evaluated quickly for the conditions in which they have a greater tendency to form aggregates. The conditions to be tested will be same as those used throughout bioprocess manufacturing, and will therefore allow bioprocess engineers to rapidly identify the conditions in which their manufacturing processes will be best operated, or whether the protein is unlikely to be manufacturable. Having quickly determined the conditions at which the protein begins to form small and soluble aggregates, we will also carry out a detailed molecular analysis of the structure of proteins at these conditions and also those either side in which the protein remains in solution as a single molecule, and where it forms larger aggregates. This will allow us to see what changes in the protein structure occur before, during, and after the aggregation is initiated and therefore deduce which events are on the critical path to aggregate formation. Having achieved this we will then be able to target changes to the protein called mutations that will interfere with and suppress the aggregation process. Finally, by comparing a related set of therapeutic antibody fragment proteins, we will gain insight into those factors that are specific to each protein type, and those that occur more generally and hence become useful targets for the future engineering of therapeutic protein designs. It will also allow others to improve their mathematical modelling methods that aim to predict whether proteins will aggregate under certain conditions.

最近的治疗与年龄有关的疾病，如类风湿性关节炎，黄斑变性，克罗恩病，和一些癌症是工程形式的生物蛋白分子称为抗体，形成我们自己的自然免疫系统的一部分。这种治疗性蛋白质越来越多地来源于这些抗体的简单片段，希望这将改善它们在体内的行为，减少所需的注射频率，使它们能够靶向身体的新区域，并使它们能够与其他生物分子结合而不会变得太大或不稳定。然而，由于其微妙和复杂的性质，治疗性蛋白质的制造极具挑战性。制造过程旨在将蛋白质分子与合成它们的其余细胞组分分离，以获得适用于人类治疗的极纯治疗材料。然而，由于温度或酸度的变化、盐的添加、机械搅拌的使用、通过机器的流速的快速变化以及蛋白质与气泡的相互作用，可用于大规模生产的工艺对蛋白质施加了很大的压力。这经常导致蛋白质轻微变形，随后粘在一起形成称为聚集体的微小颗粒。虽然这些通常是肉眼不可见的，但它们在治疗中的存在可能对患者有害，因为它们可能导致严重的炎症和潜在的更致命的免疫反应。因此，生物工艺开发和治疗性蛋白质制造行业想要解决的关键挑战之一是能够预测导致蛋白质聚集的条件，或者增加它们的稳健性，使得它们在制造期间聚集的频率较低。我们的目标是进行和演示一套快速实验测量技术，使一种新的治疗性蛋白质能够快速评估它们形成聚集体的趋势。待测条件将与整个生物工艺生产过程中使用的条件相同，因此将允许生物工艺工程师快速确定其生产工艺最佳运行的条件，或者蛋白质是否不太可能生产。在快速确定了蛋白质开始形成小的可溶性聚集体的条件后，我们还将对这些条件下的蛋白质结构进行详细的分子分析，以及蛋白质作为单个分子保留在溶液中的两侧，以及它形成较大聚集体的情况。这将使我们能够看到蛋白质结构在聚集开始之前、期间和之后发生了什么变化，从而推断出哪些事件处于聚集体形成的关键路径上。实现这一点后，我们将能够靶向蛋白质的变化，称为突变，这些突变将干扰和抑制聚集过程。最后，通过比较一组相关的治疗性抗体片段蛋白质，我们将深入了解那些对每种蛋白质类型具有特异性的因素，以及那些更普遍发生的因素，从而成为未来治疗性蛋白质设计工程的有用目标。它还将允许其他人改进他们的数学建模方法，旨在预测蛋白质是否会在某些条件下聚集。

项目成果

Elucidation of an Expanded Aggregation-Prone Conformation of Fab Using Saxs, Md Simulations and Smfret

使用 Saxs、Md 模拟和 Smfret 阐明 Fab 的扩展易聚集构象

• 发表时间: 2018
• 期刊: PROTEIN SCIENCE
• 影响因子: 8
• 作者: Codina Nuria

An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics, and smFRET Identifies an Aggregation Mechanism

• DOI: 10.1016/j.jmb.2019.02.009
• 发表时间: 2019-03-29
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Codina, Nuria;Hilton, David;Dalby, Paul A.
• 通讯作者: Dalby, Paul A.

An Evaluation of the Potential of NMR Spectroscopy and Computational Modelling Methods to Inform Biopharmaceutical Formulations.

• DOI: 10.3390/pharmaceutics10040165
• 发表时间: 2018-09-21
• 期刊: Pharmaceutics
• 影响因子: 5.4
• 作者: Pandya A;Howard MJ;Zloh M;Dalby PA
• 通讯作者: Dalby PA

Identification of Protein-Excipient Interaction Hotspots Using Computational Approaches.

• DOI: 10.3390/ijms17060853
• 发表时间: 2016-06-01
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Barata TS;Zhang C;Dalby PA;Brocchini S;Zloh M
• 通讯作者: Zloh M

The Fab conformations in the solution structure of human IgG4 restricts access to its Fc region: implications for low complement activity

人 IgG4 溶液结构中的 Fab 构象限制了对其 Fc 区的访问：对低补体活性的影响

• 发表时间: 2014
• 期刊: MOLECULAR IMMUNOLOGY
• 影响因子: 3.6
• 作者: Rayner Lucy E.