Analysis of Macromolecular Size Distributions in Concentrated Solutions

IF 6.1 Q1 CHEMISTRY, MULTIDISCIPLINARY
Dr. Sumit K. Chaturvedi, Dr. Peter Schuck
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Abstract

The solution state of macromolecules in concentrated solutions impacts fields ranging from cell biology, to colloid chemistry and engineering of protein pharmaceuticals. Dependent on the interplay between repulsive and weakly attractive forces, proteins may exhibit oligomerization, aggregation, crystallization, liquid-liquid phase separation, or the formation of multiprotein complexes. The particle size-distribution is a key characteristic, but difficult to determine when interparticle distances are on the order of their size and macromolecular motion is coupled through hydrodynamic interactions. Here we extend sedimentation velocity analytical ultracentrifugation to measure macromolecular size distributions under these conditions: We apply results from statistical fluid mechanics for the concentration-dependence of hindered settling and diffusion, embedded in a mean-field approximation that can resolve coupled sedimentation and diffusion processes of different sized species given experimental sedimentation data. This is combined with a description of transient optical aberrations from lensing in the refractive index gradients associated with sedimentation boundaries (Wiener skewing). We demonstrate this approach in the application to protein solutions with macromolecular volume fractions up to ≈10 %, for example, resolving monomers and dimers of albumin at 140 mg/ml. This enables size-distribution analysis of proteins at concentrations of therapeutic antibody formations and close to physiological concentration in serum and cells.

Abstract Image

浓溶液中大分子尺寸分布的分析
大分子在浓缩溶液中的溶液状态影响着细胞生物学、胶体化学和蛋白质药物工程等领域。依赖于斥力和弱引力之间的相互作用,蛋白质可能表现为寡聚化、聚集、结晶、液-液相分离或形成多蛋白复合物。颗粒的大小分布是一个关键的特征,但很难确定当颗粒间的距离是在其大小的顺序和大分子运动是通过水动力相互作用耦合。在这里,我们扩展了沉降速度分析超离心来测量这些条件下的大分子大小分布:我们将统计流体力学的结果应用于阻碍沉降和扩散的浓度依赖,嵌入在平均场近似中,可以解决给定实验沉降数据的不同大小物种的耦合沉降和扩散过程。这是结合描述瞬态光学像差透镜在折射率梯度相关的沉淀边界(维纳歪斜)。我们在大分子体积分数高达≈10%的蛋白质溶液中证明了这种方法的应用,例如,在140 mg/ml的浓度下分辨白蛋白的单体和二聚体。这使得在治疗性抗体形成浓度和接近血清和细胞中的生理浓度下的蛋白质的大小分布分析成为可能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
7.30
自引率
0.00%
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