'BRIC DOCTORATE PROGRAMME' - Development of single molecule assays for the detection of aggregation within high concentration protein therapeutics

以蛋白质为基础的生物制药占目前正在开发的药物的主要比例。虽然这类产品的数量正在增加，但它们在开发过程中面临着相当大的挑战，防止活跃的产品不受欢迎地聚集仍然是一个主要问题。蛋白质聚集可能是不可预测的，并在生物制药开发的任何阶段造成重大困难，例如从配方到制造和储存。此外，越来越多的人要求在高蛋白浓度(&gt；50 mg/ml)的条件下配制此类药物，这种条件有促进聚集的趋势。目前利用一系列分析技术对蛋白质聚集体进行检测和量化。工业标准是尺寸排除层析(SEC)，尽管现在越来越多地使用正交方法，如分析超速离心法和光散射。通过对暴露在不同储存条件下的各种测试配方中的集料类型和水平进行耗时的评估，确定最佳条件。对于蛋白质浓度较高的配方，样品在分析前也需要稀释，因此获得的聚集态可能不能完全代表配方中的实际存在。因此，在可能的早期阶段开发新的方法来确定高浓度制剂中的聚集物，将大大减少与开发相关的时间、精力和成本。与辉瑞公司合作，这个项目将探索单分子力测量的能力，以满足这一领域对新方法的需求。这种测量是通过记录力传感器(例如，原子力显微镜(AFM)悬臂梁)的表面与相对的样品表面接触和脱离时作用在其上的力来获得的。艾伦博士和威廉姆斯教授在利用这种测量来揭示单个蛋白质或打破一系列生物分子复合体方面拥有相当丰富的经验。单分子水平的测量对于药物研究和开发的早期应用很有吸引力，因为此时材料的数量可能很少。由于测量还包括在被迫进行亲密接触后分离单个蛋白质，我们建议它们提供一个更能反映高蛋白质浓度条件的实验系统。在这里，我们希望探索这一假设，使用单分子强制测量，以检测和预测与蛋白质为基础的生物制药的聚集。最初的实验将侧重于将模型蛋白(例如，单抗)固定到传感器和样品表面的简单形式，以及在一系列配方条件下记录的力(例如，那些已知会加剧和防止聚集的条件)。在以后的研究中，我们将致力于将这种形式扩展到其他生物分子活性物质(例如多肽)，并测试与容器表面、塞子材料等的相互作用和聚集潜力。该项目的长期目标将是开发和扩大已用于单分子蛋白质展开研究的蛋白质结构，用于聚集检测和筛选。这种分子含有重复的蛋白质/蛋白质结构域，由力传感器机械地展开。获得的数据直接提供了有关蛋白质在力作用下展开的倾向的信息，并可能提供与理解蛋白质在配方和/或加工过程中如何对剪应力做出反应有关的信息。在所有这些研究中，将通过与辉瑞公司的合作获得关于蛋白质聚集的补充生物物理数据；工业安置的一个关键目标将是让学生使用传统的聚集分析技术获得这些数据。

Protein based biopharmaceuticals comprise a major proportion of the medicines currently under development. Although the number of such products is increasing, they present considerable challenges during development, and preventing unwanted aggregation of the active remains a major issue. Protein aggregation can be unpredictable and result in significant difficulties at any stage of biopharmaceutical development e.g. from formulation, through to manufacture and storage. This is compounded by an increasingly frequent requirement to formulate such medicines at high protein concentration (>50mg/ml); conditions which have a tendency to promote aggregation. Protein aggregates are currently detected and quantified utilizing a range of analytical techniques. The industry standard is size-exclusion chromatography (SEC) although the increased use of orthogonal approaches, such as analytical ultracentrifugation and light scattering is now common. Optimal conditions are identified through a time consuming evaluation of aggregate type and level in a wide range of test formulations, exposed to different storage conditions. For high protein concentration formulations the samples also require dilution prior to analysis, hence the obtained aggregate profile may not fully represent that actually present within the formulation. The development of new approaches to identify aggregation within high concentration formulations, at its earliest possible stages would therefore significantly reduce the time, effort and costs associated with development. In collaboration with Pfizer, this studentship will explore the ability of single molecule force measurements to fulfil the need for new approaches in this area. Such measurements are obtained by recording forces acting on a force transducer (e.g. atomic force microscopy (AFM) cantilever) as its surface is brought into and out of contact with an opposing sample surface. Dr Allen and Professor Williams have considerable experience in utilizing such measurements to unfold single proteins or break a range of biomolecular complexes. Measurements at the single molecule level are attractive for applications early in pharmaceutical research and development, when amounts of material can be small. As the measurements also involve the separation of individual proteins after they are forced into intimate contact, we propose that they provide an experimental system more reflective of the conditions within high protein concentrations. Here we wish to explore this hypothesis, and the use of single molecule forces measurements for the detection and prediction of aggregation with protein based biopharmaceuticals. Initial experiments will focus on simple formats in which model proteins (e.g. a monoclonal antibody) will be immobilized to the transducer and sample surface, and forces recorded in a range of formulation conditions (e.g. those known to exacerbate and prevent aggregation). In later studies, we will aim to extend this format to other biomolecular actives (e.g. peptides), and to test for interaction and aggregation potential with container surfaces, stopper materials etc. A longer-term goal of the project will be to develop and extend the use of protein constructs, as already employed in single molecule protein unfolding studies, for aggregate detection and screening. Such molecules contain repeats of proteins/protein domains, which are mechanically unfolded by the force transducer. The obtained data directly provide information on the propensity of the protein to unfold under force, and may provide information relevant to understanding how proteins would respond to shear stresses during formulation and/or processing. Throughout these studies complementary biophysical data on protein aggregation will be obtained through collaboration with Pfizer; A key goal of the industrial placement will be for the student to obtain such data using conventional aggregate analysis techniques.