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

“金砖四国博士计划”——开发用于检测高浓度蛋白质疗法中聚集的单分子测定法

• 批准号: BB/J003840/1
• 负责人: Stephanie Allen
• 金额: $ 12.22万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Training 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%2FJ003840%2F1
• 关键词: BRIC; DOCTORATE; PROGRAMME; Development; single

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.

基于蛋白质的生物制药占目前正在开发的药物的大部分。尽管此类产品的数量正在增加，但它们在开发过程中存在相当大的挑战，并且防止活性物质的不必要的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（例如从配制到制造和储存）导致重大困难。由于越来越频繁地要求以高蛋白质浓度（> 50 mg/ml）配制此类药物，这一点变得更加复杂;这些条件具有促进聚集的趋势。蛋白质聚集体目前利用一系列分析技术进行检测和定量。工业标准是尺寸排阻色谱法（SEC），尽管正交方法的使用越来越多，如分析超离心和光散射现在很常见。最佳条件是通过对暴露于不同储存条件的各种试验配方中的骨料类型和水平进行耗时评估来确定的。对于高蛋白浓度制剂，样品在分析前也需要稀释，因此获得的聚集体谱可能不完全代表制剂中实际存在的聚集体谱。因此，在尽可能早的阶段开发新的方法来确定高浓度制剂中的聚集，将大大减少与开发有关的时间、精力和成本。与辉瑞公司合作，这个学生将探索单分子力测量的能力，以满足在这一领域的新方法的需要。这样的测量是通过记录当力传感器（例如原子力显微镜（AFM）悬臂）的表面与相对的样品表面接触和不接触时作用在力传感器上的力来获得的。艾伦博士和威廉姆斯教授在利用这种测量来展开单个蛋白质或破坏一系列生物分子复合物方面有相当丰富的经验。在单分子水平上的测量对于药物研究和开发的早期应用是有吸引力的，因为材料的量可能很小。由于测量还涉及单独的蛋白质分离后，他们被迫进入密切接触，我们建议，他们提供了一个实验系统，更能反映高蛋白质浓度的条件。在这里，我们希望探讨这一假设，并使用单分子力测量的检测和预测与蛋白质为基础的生物药物的聚集。最初的实验将集中在简单的格式，其中模型蛋白质（例如单克隆抗体）将被固定到换能器和样品表面，并在一系列的配制条件下记录力（例如已知加剧和防止聚集的那些）。在以后的研究中，我们的目标是将这种形式扩展到其他生物分子活性物质（如肽），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展蛋白质构建体的使用，如已经在单分子蛋白质展开研究中使用的，用于聚集体检测和筛选。这样的分子包含蛋白质/蛋白质结构域的重复，其通过力传感器机械地展开。所获得的数据直接提供关于蛋白质在力作用下展开的倾向的信息，并且可以提供与理解蛋白质在配制和/或加工期间如何响应剪切应力相关的信息。在这些研究中，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;工业布局的一个关键目标是让学生使用传统的聚集分析技术获得这些数据。