Abstract：
We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentration-dependent, falling within the range of 50-2000 μg/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within ±24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.
摘要：
我们开发了一种基于适体的荧光共振能量转移（FRET）测定，能够识别治疗性单克隆抗体贝伐单抗并仅通过一个混合步骤快速定量其浓度。在这个试验中,两种荧光染料（荧光素和四甲基罗丹明）标记的适体与贝伐单抗上的两个Fab区结合，和FRET荧光时观察到两种染料接近。我们以三种不同的形式优化了这个测定，满足广泛的分析需求。当应用于实际环境中的杂交瘤培养样品时，该测定显示出浓度依赖性的信号响应，在50-2000μg/mL范围内。测定系数(r2)范围为0.998~0.999,偏倚和精密度在±24.0%和20.3%以内,分别。此外,在热和UV应力测试期间，该测定证明了以与常规尺寸排阻色谱法相当的方式检测变性样品的能力。值得注意的是,它提供了额外的优点，即检测结合活性的降低而不改变分子量。与许多现有的过程分析技术工具相比，该试验不仅可以识别贝伐单抗，还可以直接测量与mAb功效相关的质量属性，例如绑定活动。因此,作为实时提供高度可靠的质量属性信息的有价值的平台，该检测具有巨大的潜力。我们认为这将在抗体生产的各个方面为高质量治疗性单克隆抗体的全球分布做出重大贡献。包括生产监控，质量控制,商业批量发布，和稳定性测试。
