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

视觉色谱法是生物制药纯化中普遍存在的单元操作，但很少有研究解决了溶液-树脂界面上蛋白质的生物物理特性。本项目旨在利用先进的方法在工业相关条件下表征吸附/解吸阶段的蛋白质聚集，为合理设计提供依据。单克隆抗体（mab）等生物药品本质上是复杂的混合物，在生产过程中容易受到修饰，从而增加成本和开发时间，并可能最终阻止分子的成功开发。单克隆抗体和其他生物制药常见的一种途径是聚集途径，必须对产品质量一致性进行控制，也与免疫原性有关。因此，在开发和制造过程中，特别是液体剂型，聚合是一个普遍关注的问题。提高对聚集现象的认识将大大有利于产品加工和配方条件的合理设计。背景：迄今为止用于预测聚集率的大多数方法都隐含地假设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，液-固、液-液和气-液界面对蛋白质聚集的重要性得到了证明，这些研究表明，通过使用搅拌来携带空气或增加暴露于固-液界面或不溶性液体来创建两相系统，可以加速聚集。除了这些界面现象的研究外，工业团体报告说，生物制药生产过程中的色谱洗脱是聚集的关键问题。在一项研究中，一个领先的研究小组以蛋白a层析（用于单抗纯化）为例，表明树脂的解吸对不稳定的影响超出了溶液条件。这种效应可能与产物分子在吸附和解吸过程中的结构变化有关。这使得工艺条件和稳定剂的检查变得复杂，因为它们必须在柱上而不是在简化的溶液研究中进行研究，例如，在同一研究中，某些剂对溶液中的产品稳定性有益，但对柱上有负面影响。目的本项目研究的就是这些柱上吸附/解吸现象。该计划旨在通过查看以下方面来调查这一点。第一种方法是控制吸附剂表面的变化。这将被细分来检验；a)配体类型，使用共同的碱基质和溶液条件，但不同的配体，如蛋白质a和离子交换部分；b)吸附剂的碱基质，如琼脂糖、玻璃和甲基丙烯酸酯；c)孔径，使用不同孔隙率的玻璃树脂与相同的配体。第二个方面是对溶液条件的系统操纵这又可以细分为；a)操纵pH和盐以了解电荷和疏水效应，b)研究稳定剂（如聚山梨酸酯、柠檬酸盐和精氨酸）。这项研究的最后一个关键部分将是使用一系列具有不同稳定性特征的蛋白质。所有这些研究都将通过使用适当的生物物理方法（电泳（CE/IEF）， HPLC/UPLC技术，光谱方法（CD， FTIR， DLS，荧光））来支持，以了解聚集的性质和发生的结构变化。当将上述数据集作为一个整体时，将为列上聚合的关键参数提供一个全面的新视角，允许描述现象的机制基础，进而为流程设计提供基本原理。

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.