Clinical Pharmacology, Pharmacometrics, & Bioanalysis, Bristol Myers Squibb Company, Princeton, New Jersey, 08543, USA.
Translational Sciences and Diagnostics, Bristol Myers Squibb Company, Princeton, New Jersey, 08543, USA.
AAPS J. 2024 Sep 16;26(6):104. doi: 10.1208/s12248-024-00970-2.
Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.
预先存在的抗 AAV 抗体可以使用基于配体结合的测定方法检测。其中一种方法是基于 MSD 的桥接测定法，该方法使用带 sulfo 标签的 AAV 载体作为检测试剂。然而，尚未开发出一种方法来准确测量 AAV 载体上 sulfo 标签的标记程度。为了填补这一空白，我们开发了一种液相色谱-高分辨质谱 (LC-HRMS) 方法来评估 AAV5 载体上 sulfo 标签的标记程度 (DoL)，能够在六种不同的 sulfo 标签挑战比下测量 AAV5 的 DoL。此外，还使用 Biacore 测定法评估了抗 AAV5 单克隆抗体与各种带 sulfo 标签的 AAV5 载体之间的结合亲和力。结果表明，增加 AAV5 上 sulfo 标签标记的 DoL 不会影响其结合亲和力。我们的研究进一步采用 MSD 桥接测定法检测四种抗 AAV5 单克隆抗体 (mAb) 与各种带 sulfo 标签的 AAV5 载体的结合信号/噪声 (S/N) 比值。研究结果表明，sulfo 标签标记的程度与 S/N 比值和 MSD 桥接测定法的灵敏度之间存在很强的相关性。这一结果强调了优化检测试剂标记以提高检测抗 AAV5 抗体的测定灵敏度的重要性。
