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

最近对类风湿性关节炎、黄斑变性、克罗恩病和一些癌症等与年龄相关的疾病的治疗方法都是由称为抗体的生物蛋白分子改造而成的，这些抗体构成了我们自己的自然免疫系统的一部分。越来越多的此类治疗性蛋白质是从这些抗体的更简单片段中提取出来的，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，还允许它们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是非常具有挑战性的。制造过程旨在将蛋白质分子从合成蛋白质的其余细胞成分中分离出来，以获得非常纯粹的治疗材料，适合用于人类的治疗。然而，由于温度或酸度的变化、盐的添加、机械搅拌的使用、机械流动速度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极大的压力。这通常会导致蛋白质轻微变形，然后粘在一起形成称为聚集体的微小颗粒。虽然肉眼通常看不到这些物质，但它们在治疗中的存在对患者可能是危险的，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，以便它们在制造过程中不那么频繁地聚集。我们的目标是实施和展示一套快速实验测量技术，使一种新的治疗蛋白质能够在它们更容易形成聚集体的条件下被快速评估。被测试的条件将与整个生物过程制造过程中使用的条件相同，因此将使生物过程工程师能够迅速确定他们的制造过程将在什么条件下运行得最好，或者蛋白质是否不太可能被制造。在快速确定蛋白质开始形成小而可溶的聚集体的条件后，我们还将对这些条件下蛋白质的结构以及蛋白质作为单分子保持在溶液中的两侧以及在哪里形成更大的聚集体进行详细的分子分析。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，从而推断哪些事件处于聚集形成的关键路径上。实现这一点后，我们将能够针对被称为突变的蛋白质的变化，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，我们将深入了解每种蛋白类型特有的因素，以及那些更普遍出现的因素，从而成为未来治疗性蛋白设计的有用目标。它还将允许其他人改进他们的数学建模方法，这些方法旨在预测蛋白质在特定条件下是否会聚集。

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.