Development of an ELISA with acidification treatment for an antibody conjugate incorporating Exatecans

• Published in: Analytical biochemistry 2024-07, Vol.690, p.115530-115530, Article 115530
• Main Authors: Zhang, Yingying, Yun, Xi, Ouyang, Lu, Zhang, Xianjing, Gong, Likun, Qin, Qiuping
• Format: Article
• Language: English
• Subjects: Antibody-drug conjugate; Bioanalysis; Camptothecin; Exatecan; Pharmacokinetics
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2024.115530

The successful development of Sacituzumab Govitecan and Trastuzumab Deruxtecan has made camptothecin derivatives one of the most popular payloads for antibody-drug conjugates (ADCs). Camptothecin and its derivatives all exist in a pH-dependent equilibrium between the carboxylate and lactone forms. Such transformation may lead to differences in the ratio of the two molecular forms in calibration standards and biological matrix (bio-matrix) samples, thereby leading to inaccurate conjugated antibody results. In this study, we reported an enzyme-linked immunosorbent assay (ELISA) free of the aforementioned influence for the detection of the Exatecans-conjugated antibody (conjugated SM001) in cynomolgus monkey serum. The assay was developed by first acidifying all samples with glacial acetic acid (HAc), then performing neutralization and thereafter capturing conjugated SM001 with anti-Exatecan monoclonal antibody (mAb) and detecting it with biotinylated Nectin4 (hNectin4-Bio) and horseradish peroxidase-labeled streptavidin (SA-HRP). Results showed that all tested performance parameters met the acceptance criteria. The conjugated SM001 concentrations obtained were in parallel to but slightly lower than total antibody (TAb) throughout the pharmacokinetic (PK) study, revealing that the assay strategy implemented for conjugated SM001 measurement worked well for the elimination of interference triggered by the heterogeneous existence of the lactone and carboxylate forms of Exatecan (lactone-Exatecan and carboxylate-Exatecan). [Display omitted] •A novel ELISA for an antibody conjugated with Exatecans was developed using acidification treatment strategy and validated.•The assay strategy adopted for conjugated SM001 eliminated interference caused by lactone- and carboxylate-Exatecan.•The validated assay was applied to a cynomolgus PK study of SM001 and generated accurate results of the conjugated SM001.•The same assay strategy can be applied to measurement methods for other antibodies conjugated with camptothecin derivatives.