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

这项工作的主要目的是更好地了解在最终配制步骤中控制单克隆抗体液-液平衡（LLE）的因素。需要避免相分离，因为它导致乳白色溶液，并且在某些情况下，导致高粘度，这两者都使得制剂不适合患者使用。这种行为的主要原因是有吸引力的蛋白质-蛋白质相互作用（PPI），其敏感地取决于溶剂条件（即pH、离子强度、缓冲液和添加剂类型和浓度）。在这里，我们将测量和理解PPI的抗体结构特性，然后阐明相行为的联系。关键结果将是通过操纵制剂缓冲液来控制液液平衡的能力。更具体地说，该项目的目标是：（1）使用静态光散射，根据渗透第二维里系数（SVC）测量抗体溶液的蛋白质-蛋白质相互作用（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值相关实验计划将包括三种完整抗体的研究，它们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进行测量。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。负值对应于吸引蛋白质-蛋白质相互作用，而正值则与排斥力有关。根据pH值和离子强度进行的研究将用于将PPI与抗体的静电性质联系起来（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制剂。稳定化有时与防止蛋白质-蛋白质吸引的附加能力有关（Valente等人（2005）Biophys. J. 89：4211）。这种行为将被调查的蛋白质-蛋白质的吸引力有一个不同的物理起源的系统，给加性效应的机械见解。PPI将使用结构生物信息学方法与抗体的分子描述符相关联。表面性质（电荷和疏水性的斑块）将由抗体序列（使用同源性建模）或三维结构确定。性质将与SVC对pH和离子强度的依赖性相关，以确定具有大的净电荷和具有相反极性的补丁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用于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，用于预测其模式相对于缓冲液条件。我们还将测量在SVC研究中表现出弱吸引力PPI的系统的LLE。大多数抗体溶液的研究已经确定了温度方面的相行为，这仅与PPI间接相关。我们将使用一种更直接的方法，并根据SVC确定LLE。先前的工作表明LLE曲线对于所有沉淀剂是相同的，表明相行为仅由PPI的净量值控制（Ahamed等人（2009）Biophys J.93：610）。这项研究将被扩展，以确定这种效果是否对所有抗体都是普遍的。如果不是，将通过将LLE曲线与抗体结构描述符和PPI的分子来源相关联来获得更深入的了解。最终产品将是一种根据配方中使用的溶剂条件预测液液平衡的方法。

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.