Section for Biologics, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
Eur J Pharm Sci. 2013 Jun 14;49(3):400-10. doi: 10.1016/j.ejps.2013.04.019. Epub 2013 Apr 26.
The purpose of this work was to explore the relation between protein-protein interactions (PPIs) and solution viscosity at high protein concentration using three monoclonal antibodies (mAbs), two of the IgG4 subclass and one of the IgG1 subclass. A range of methods was used to quantify the PPI either at low concentration (interaction parameter (kD) obtained from dynamic light scattering, DLS) or at high concentration (solution storage modulus (G') from ultrasonic shear rheology). We also developed a novel method for the determination of PPI using the apparent radius of the protein at either low or high protein concentration determined using DLS. The PPI measurements were correlated with solution viscosity (measured by DLS using polystyrene nanospheres and ultrasonic shear rheology) as a function of pH (4-9) and ionic strength (10, 50 and 150 mM). Our measurements showed that the highest solution viscosity was observed under conditions with the most negative kD, the highest apparent radius and the lowest net charge. An increase in ionic strength resulted in a change in the nature of the PPI at low pH from repulsive to attractive. In the neutral to alkaline pH region the mAbs behaved differently with respect to increase in ionic strength. Two mAbs (A and B) showed little or no effect of increasing ionic strength, whereas mAb-C showed a remarkable decrease in attractive PPI and viscosity. Previous studies have mainly investigated mAbs of the IgG₁ and IgG₂ subclass. We show here, for the first time, that mAbs of the IgG₄ subclass behave similar as the other subclasses. By comparison of the three tested mAbs with mAbs investigated in other studies a clear linear trend emerges between the pH of strongest attractive PPI and highest solution viscosity. The determination of PPI using either kD or apparent radius is thus a useful prediction tool in the determination of solution conditions that favors low solution viscosity at high protein concentration of therapeutically used mAb molecules. The novel methodology using apparent radius is a simple and rapid alternative to determine relative PPI directly under formulation conditions. The method can potentially serve as a high-throughput screening tool in formulation development.
这项工作的目的是使用三种单克隆抗体（mAbs）——两种 IgG4 亚类和一种 IgG1 亚类——探索蛋白质-蛋白质相互作用（PPIs）与高浓度蛋白质溶液粘度之间的关系。我们使用了一系列方法来定量测定低浓度下的 PPI（通过动态光散射，DLS 获得的相互作用参数（kD））或高浓度下的 PPI（通过超声剪切流变学获得的溶液储能模量（G'））。我们还开发了一种新的方法，用于通过 DLS 确定蛋白质的表观半径来测定低浓度或高浓度下的 PPI。将 PPI 测量值与溶液粘度（使用聚苯乙烯纳米球通过 DLS 测量和超声剪切流变学测量）相关联，作为 pH 值（4-9）和离子强度（10、50 和 150 mM）的函数。我们的测量结果表明，在具有最负 kD、最高表观半径和最低净电荷的条件下，观察到最高的溶液粘度。随着离子强度的增加，在低 pH 值下，PPI 的性质从排斥变为吸引。在中性到碱性 pH 区域，mAbs 对离子强度增加的反应不同。两种 mAb（A 和 B）的离子强度增加几乎没有或没有影响，而 mAb-C 的吸引性 PPI 和粘度则显著降低。以前的研究主要集中在 IgG₁和 IgG₂亚类的 mAb 上。我们在这里首次表明，IgG₄ 亚类的 mAb 表现与其他亚类相似。通过比较三种测试的 mAb 与其他研究中测试的 mAb，可以清楚地看出，最强吸引力 PPI 的 pH 值与最高溶液粘度之间存在明显的线性趋势。因此，使用 kD 或表观半径测定 PPI 是一种有用的预测工具，可用于确定在高浓度治疗性 mAb 分子下有利于低溶液粘度的溶液条件。使用表观半径的新方法是在配方条件下直接测定相对 PPI 的简单快速替代方法。该方法有可能成为配方开发中的高通量筛选工具。
