BRIC DOCTORATE PROGRAMME: Controlling liquid-liquid phase separation in antibody formulations

这项工作的主要目标是更好地了解在最终配方步骤中控制单抗的液-液平衡(LLE)的因素。需要避免相分离，因为它会导致乳白色溶液，在某些情况下，会产生高粘度，这两种情况都使配方不适合患者使用。这种行为的主要原因是具有吸引力的蛋白质-蛋白质相互作用(PPI)，它敏感地依赖于溶剂条件(即pH、离子强度、缓冲液和添加剂的类型和浓度)。在这里，我们将从抗体结构属性的角度来衡量和理解PPI，然后阐明其与阶段行为的联系。关键的结果将是通过操纵配方缓冲液来控制LLE的能力。更具体地说，该项目的目标将是：(1)利用静态光散射以渗透第二维里系数(SVC)的形式测量抗体溶液中蛋白质与蛋白质的相互作用(2)使用结构生物信息学方法开发PPI的半预测模型(3)测量LLE并与SVC值相关联实验计划将包括对三种完整抗体的研究，这三种抗体表现出不同形式的相行为。最初，PPI将根据SVC作为溶剂条件的函数来衡量。SVC对应于在一对蛋白质之间的分离和相对取向上平均的相互作用。负值对应于有吸引力的蛋白质-蛋白质相互作用，而正值与排斥力有关。作为pH和离子强度函数的研究将被用来将PPI与抗体的静电特性联系起来(见下文)。我们还将研究含有不同缓冲物质和添加剂(氨基酸、糖)的溶液，因为这些通常用于稳定配方。这种稳定性有时与阻止蛋白质-蛋白质吸引的添加能力有关(瓦伦特等人)。(2005)生物物理学。J.89：4211)。对于蛋白质-蛋白质吸引力具有不同物理来源的系统，将对这种行为进行研究，以提供对加性效应的机械洞察。PPI将使用结构生物信息学方法与抗体的分子描述符相联系。表面属性(电荷斑块和疏水性)将根据抗体序列(使用同源建模)或三维结构来确定。性质将与SVC对pH和离子强度的依赖相关联，以确定具有大的净电荷和具有相反极性的斑块对静电相互作用的符号和大小的竞争效应。抗体和添加剂表面的形状和极性之间的互补性可以用来寻找与描述添加剂效果的实验数据的相关性。计算研究将深入了解PPI的分子起源，并提供一种工具来预测其相对于缓冲条件的模式。我们还将测量在SVC研究中显示出弱吸引力PPI的系统的LLE。大多数对抗体溶液的研究都是根据温度来确定相行为的，这只是间接地与PPI有关。我们将使用更直接的方法，根据SVC确定LLE。先前的一项工作表明，所有沉淀剂的液液平衡曲线都是相同的，这表明相行为只受PPI净值的控制(Amed等人。(2009)BiPhys J.93：610)。这项研究将扩大到确定这种效应是否对所有抗体都适用。如果不是，通过将LLE曲线与抗体结构描述符和PPI的分子起源相关联将获得更深层次的了解。最终产品将是一种根据配方中使用的溶剂条件来预测LLE的方法。

The main objective of this work is to develop a better understanding of factors that control liquid-liquid equilibrium (LLE) of monoclonal antibodies during the final formulation steps. Phase separation needs to be avoided as it leads to opalescent solutions and, in some instances, high viscosities, both of which make the formulation unsuitable for patient use. The main causes of this behavior are attractive protein-protein interactions (PPIs), which depend sensitively on the solvent conditions (i.e. pH, ionic strength, buffer and additive types and concentrations). Here, we will measure and understand PPIs in terms of antibody structural properties and then elucidate the link to phase behavior. The key outcome will be the ability to control the LLE by manipulating formulation buffers. More specifically, the aims of the project will be to: (1) Measure protein-protein interactions for antibody solutions in terms of the osmotic second virial coefficient (SVC) using static light scattering (2) Develop semi-predictive models for PPIs using a structural bioinformatics approach (3) Measure the LLE and correlate with SVC values The experimental program will include studies of three intact antibodies, which exhibit different forms of phase behavior. Initially, the PPIs will be measured in terms of the SVC as a function of solvent conditions. The SVC corresponds to an interaction averaged over the separation and relative orientations between a pair of proteins. Negative values correspond to attractive protein-protein interactions, whereas positive values are linked to repulsive forces. Studies made as a function of pH and ionic strength will be used to relate PPIs to electrostatic properties of antibodies (see below). We will also study solutions with different buffer species and additives (amino acids, sugars) as these are often used to stabilize formulations. The stabilization is sometimes linked to the additive ability to prevent protein-protein attraction (Valente et al. (2005) Biophys. J. 89: 4211). This behaviour will be investigated for systems where the protein-protein attraction has a different physical origin to give mechanistic insights of additive effects. The PPIs will be linked to molecular descriptors of the antibodies using a structural bioinformatics approach. Surface properties (patches of charge and hydrophobicity) will be determined from the antibody sequence (using homology modeling) or three-dimensional structure. Properties will be correlated with the dependence of the SVC on pH and ionic strength to determine the competitive effects of having a large net charge and patches with opposite polarity on the sign and magnitude of electrostatic interactions. Complementarity between the shape and polarity of antibody and additive surfaces can be used to look for correlations with experimental data describing the effects of additives. The computational studies will give insight into the molecular origin of PPIs and provide a tool for predicting their patterns with respect to buffer conditions. We will also measure the LLE for systems that exhibited weak attractive PPIs in the SVC studies. Most studies of antibody solutions have determined the phase behavior in terms of temperature, which is only indirectly linked to PPIs. We will use a more direct approach and determine the LLE in terms of the SVC. A previous work indicated the LLE curve is the same for all precipitants indicating the phase behaviour is controlled only by the net magnitude of PPIs (Ahamed et al. (2009) Biophys J. 93: 610). That study will be extended to determine whether this effect is universal for all antibodies. If not, deeper insight will be gained by correlating the LLE curve with antibody structural descriptors and the molecular origin of the PPIs. The end product will be a method for predicting LLE in terms of solvent conditions used in formulation.