University of Exeter Medical School, Heavitree Road, Exeter, EX1 2LU, UK.
College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
Anal Bioanal Chem. 2019 Sep;411(24):6487-6496. doi: 10.1007/s00216-019-02029-0. Epub 2019 Aug 2.
The fraction of intact monomer in a sample (moles/moles), the monomeric purity, is measured as a quality control in therapeutic monoclonal antibodies but is often unknown in research samples and remains a major source of variation in quantitative antibody-based techniques such as immunoassay development. Here, we describe a novel multiplex technique for estimating the monomeric purity and antigen affinity of research grade antibody samples. Light scattering was used to simultaneously observe the mass of antibody binding to biosensor surfaces functionalised with antigen (revealing Fab binding kinetics) or protein A/G (PAG). Initial estimates of monomeric purity in 7 antibody samples including a therapeutic infliximab biosimilar were estimated by observing a mass deficit on the PAG surface compared to the NISTmAb standard of high monomeric purity. Monomeric purity estimates were improved in a second step by observing the mass of antigen binding to the mass of antibody on the PAG surface. The NISTmAb and infliximab biosimilar displayed tightly controlled stoichiometries for antigen binding of 1.31 ± 0.57 and 1.71 ± 0.16 (95% confidence interval)-within the theoretical limit of 1-2 antigens per antibody depending on avidity. The other antibodies in the panel displayed antigen binding stoichiometries in the range 0.06-1.15, attributed to lower monomeric purity. The monomeric purity estimates were verified by electrospray ionization mass spectrometry (ESI), the gold standard technique for structural characterization of antibodies. ESI data indicated that the NISTmAb and infliximab biosimilar samples had monomeric purity values of 93.5% and 94.7%, respectively, whilst the research grade samples were significantly lower (54-89%). Our results demonstrate rapid quality control testing for monomeric purity of antibody samples (< 15 min) which could improve the reproducibility of antibody-based experiments.
样本中完整单体的分数（摩尔/摩尔），即单体纯度，是治疗性单克隆抗体的质量控制指标，但在研究样本中通常未知，并且仍然是定量抗体技术（如免疫测定开发）中主要的变异性来源。在这里，我们描述了一种用于估计研究级抗体样品单体纯度和抗原亲和力的新型多重技术。使用光散射同时观察与抗原（揭示 Fab 结合动力学）或蛋白 A/G（PAG）功能化生物传感器表面结合的抗体质量（揭示 Fab 结合动力学）或蛋白 A/G（PAG）。通过观察与高单体纯度的 NISTmAb 标准相比 PAG 表面上的质量缺陷，对包括治疗性英夫利昔单抗生物类似物在内的 7 种抗体样品的初始单体纯度进行了估计。通过观察 PAG 表面上的抗原结合量与抗体质量之比，在第二步中提高了单体纯度的估计。NISTmAb 和英夫利昔单抗生物类似物的抗原结合显示出紧密控制的计量比为 1.31 ± 0.57 和 1.71 ± 0.16（95%置信区间）-在 1-2 个抗原/抗体的理论极限内，这取决于亲和力。该组中的其他抗体显示出 0.06-1.15 的抗原结合计量比，归因于较低的单体纯度。单体纯度的估计通过电喷雾电离质谱（ESI）进行验证，ESI 是抗体结构表征的金标准技术。ESI 数据表明，NISTmAb 和英夫利昔单抗生物类似物样品的单体纯度值分别为 93.5%和 94.7%，而研究级样品则明显较低（54-89%）。我们的结果表明，抗体样品单体纯度的快速质量控制测试（<15 分钟）可以提高抗体实验的重现性。
