Development and validation of a reporter gene assay for the bioactivity determination of anti-TSLP monoclonal antibodies

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis Pub Date : 2025-08-09 DOI:10.1016/j.jpba.2025.117106

Shuting Hou , Bojia Yang , Lan Wang , Chuanfei Yu , Junzhi Wang

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引用次数: 0

Abstract

Thymic stromal lymphopoietin (TSLP) promotes Th2-mediated inflammation via dendritic cell activation and STAT5 signaling and plays critical roles in inflammatory disorders and allergic diseases. While tezepelumab stands as the first and only approved anti-TSLP monoclonal antibody (mAb) with over 10 anti-TSLP mAbs in clinical development, no validated anti-TSLP mAb bioassay was reported yet. Bioactivity determination is essential for ensuring mAb quality. To bridge this gap, we generated a novel HuT78-STAT5-luc reporter cell line through lentiviral transduction of STAT5 response element-driven luciferase into HuT78 cells. This stable cell line expressed luciferase in a TSLP dose-responsive manner. After systematic optimization of cell density, incubation time, TSLP concentration and mAb concentration, we established a reporter gene assay (RGA) with four-parameter regression compliance for the anti-TSLP mAb bioactivity determination. The RGA underwent full validation according to the International Council for Harmonization (ICH) Q2(R2) guideline, namely specificity, linearity, accuracy, precision and robustness. In conclusion, we established a robust and user-friendly RGA that can be applied for the quality control of anti-TSLP mAbs.

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用于测定抗tslp单克隆抗体生物活性的报告基因试验的建立和验证

胸腺基质淋巴生成素（TSLP）通过树突状细胞激活和STAT5信号传导促进th2介导的炎症，并在炎症性疾病和过敏性疾病中发挥关键作用。虽然tezepelumab是第一个也是唯一一个被批准的抗tslp单克隆抗体（mAb），有超过10个抗tslp单克隆抗体处于临床开发阶段，但尚未有报道证实抗tslp单克隆抗体的生物测定。生物活性测定是保证单抗质量的关键。为了弥补这一空白，我们通过慢病毒将STAT5反应元件驱动的荧光素酶转导到hu78细胞中，产生了一种新的hu78 -STAT5-luc报告细胞系。这种稳定的细胞系以TSLP剂量响应的方式表达荧光素酶。在对细胞密度、培养时间、TSLP浓度和单抗浓度进行系统优化后，建立了符合四参数回归的抗TSLP单抗生物活性测定报告基因法（RGA）。根据国际协调委员会（ICH） Q2（R2）指南，即特异性、线性、准确性、精密度和鲁棒性，对RGA进行了全面验证。总之，我们建立了一个鲁棒性和用户友好的RGA，可用于抗tslp单克隆抗体的质量控制。

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

Journal of pharmaceutical and biomedical analysis 医学-分析化学

CiteScore

6.70

自引率

5.90%

发文量

588

审稿时长

37 days

期刊介绍： This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome. Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.