BRIC DOCTORATE PROGRAMME - Understanding on-column protein aggregation and its impact on bioprocessing

金砖四国博士项目 - 了解柱上蛋白质聚集及其对生物加工的影响

• 批准号: BB/J003832/1
• 负责人: Daniel Bracewell
• 金额: $ 13.24万
• 依托单位: University College London
• 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%2FJ003832%2F1
• 关键词: BRIC; DOCTORATE; PROGRAMME; Understanding; column

VISION Chromatography is a ubiquitous unit operation in the purification of biopharmaceuticals yet few studies have addressed the biophysical characterisation of proteins at the solution-resin interface. This project aims to use advanced methods to characterise protein aggregation during adsorption/desorption stages under industrially relevant conditions to provide the basis for rational design. SIGNIFICANCE Biopharmaceuticals such as monoclonal antibodies (mAbs) are complex mixtures by nature and are susceptible to modifications during manufacture that can increase costs and development times and may ultimately prevent a molecule from being developed successfully. One pathway which is common to mAbs and other biopharamaceuticals is that of aggregation, which has to be controlled for product quality consistency and has also been linked to immunogenicity. Aggregation is therefore a common concern during development and manufacturing, particularly for liquid dosage forms. Rational design of product processing and formulation conditions would benefit greatly from improved understanding of aggregation phenomena. BACKGROUND Most of the approaches used to date for predicting aggregation rates implicitly assume bulk solution measurements and quantities are relevant e.g. the mid point unfolding temperature. However the importance of the liquid-solid, liquid-liquid, and air-liquid interface for protein aggregation is illustrated by the increasing number of studies that show aggregation can be accelerated by using agitation to entrain air or to increase exposure to solid-liquid interfaces, or insoluble liquids to create two phase systems. In addition to these studies of interfacial phenomena industrial groups report that chromatography elution during biopharmaceutical manufacture is of key concern for aggregation. In one study a leading group has taken the important example of protein A chromatography (used for mAb purification) and shown desorption from the resin exerted an added destabilising influence beyond that of the solution conditions. It is proposed this effect is related to structural changes in the product molecule during the events of adsorption and desorption. This complicates the examination of processing conditions and stabilising agents as they must be studied on-column rather than in simplified solution studies, for example in the same study certain agents were of benefit to product stability in solution but had a negative impact on-column. OBJECTIVES It is specifically these on-column adsorption/desorption phenomena this project is to study. The program aims to investigate this by looking at the following aspects. The first involves controlled variation of the adsorbent surface. This will be subdivided to examine; a) ligand type, using a common base matrix and solution conditions but differing ligands e.g. protein A and ion exchange moieties, b) the base matrix of the adsorbent, e.g. agarose vs. glass vs. methacrylate and c) pore size by using different porosity glass resins with the same ligand. The second facet is systematic manipulation of solution conditions again this can be subdivided into; a) manipulation of pH and salt to understand charge and hydrophobic effects and, b) investigation of stabilising agents (e.g. polysorbate, citrate and arginine). The critical final piece of the study will be the use of a range of protein's that display differing stability profiles. All these studies would be underpinned by the use of appropriate biophysical methods (electrophoresis (CE/IEF), HPLC/UPLC techniques, spectroscopic methods (CD, FTIR, DLS, fluorescence)), to understand the nature of the aggregation and structural changes taking place. When taken as a totality the above dataset will provide a comprehensive new perspective on those parameters critical to on-column aggregation allowing the mechanistic basis of the phenomena to be described and in turn providing the rationale for process design.

在生物药物的纯化中，视觉色谱是一个普遍存在的单元操作，但很少有研究涉及溶液-树脂界面上蛋白质的生物物理特性。本项目旨在使用先进的方法来表征在工业相关条件下吸附/解吸阶段的蛋白质聚集，为合理设计提供依据。意义生物药物，如单抗，本质上是复杂的混合物，在制造过程中容易受到修改，这可能会增加成本和开发时间，并可能最终阻止分子的成功开发。单抗和其他生物药物共有的一个途径是聚集，这必须控制产品质量的一致性，也与免疫原性有关。因此，在开发和制造过程中，聚集是一个普遍关注的问题，特别是对于液体剂型。合理设计产品加工和配方条件将极大地受益于对聚集现象的理解。背景迄今为止，大多数用于预测聚集速率的方法都隐含地假设散体溶液的测量和数量是相关的，例如中点展开温度。然而，越来越多的研究表明，通过使用搅拌来携带空气或增加接触固液界面或不溶性液体来创建两相体系，可以加速蛋白质聚集，这说明了液-固、液-液和气-液界面对蛋白质聚集的重要性。除了这些对界面现象的研究外，工业组织还报告说，生物制药生产过程中的层析洗脱是聚集的关键问题。在一项研究中，一个领导小组以蛋白质A层析为重要例子(用于mAb纯化)，并表明从树脂上解吸产生了超出溶液条件的额外的不稳定影响。认为这种效应与产物分子结构在吸附和解吸过程中的变化有关。这使工艺条件和稳定剂的检查变得复杂，因为它们必须在柱上研究，而不是在简化的溶液研究中，例如在同一研究中，某些试剂有利于产品在溶液中的稳定性，但对柱上有负面影响。目的本课题研究的正是这些柱上吸附/解吸现象。该计划旨在通过以下几个方面来调查这一问题。第一个涉及吸附剂表面的受控变化。这将被细分以检查：a)配体类型，使用相同的碱基基质和溶液条件，但使用不同的配体，例如蛋白质A和离子交换部分，b)吸附剂的碱基质，例如琼脂糖和玻璃，以及甲基丙烯酸酯，以及c)使用具有相同配体的不同孔隙率的玻璃树脂。第二个方面是对溶液条件的系统操作，这可以细分为：a)控制pH和盐以了解电荷和疏水影响，以及b)稳定剂(例如聚山梨酸酯、柠檬酸盐和精氨酸)的研究。这项研究的关键最后一步将是使用一系列显示不同稳定性特征的蛋白质。所有这些研究都将以适当的生物物理方法(电泳法(CE/IEF)、高效液相/超PLC技术、光谱学方法(CD、FTIR、DLS、荧光))为基础，以了解聚集的性质和发生的结构变化。作为一个整体，上述数据集将提供对列上汇总至关重要的参数的全面新视角，从而能够描述现象的机械基础，进而为流程设计提供理论基础。