异常或功能失调的细胞酶导致多种疾病，包括癌症、神经退行性疾病和代谢紊乱。酶水平或生物功能的缺陷可能导致细胞内底物积累到毒性水平并干扰整体细胞功能，最终导致细胞损伤、疾病和死亡。针对遗传性单基因酶缺乏症的市售治疗干预措施包括酶替代疗法和小分子伴侣。体内基因治疗和离体细胞治疗的新方法正在接受临床评估，并为扩大可用的疾病缓解治疗的数量提供了有希望的机会。为了支持这些不同疗法的开发，量化蛋白酶功能活性的测定在疾病诊断、药代动力学和药效学反应评估以及药物疗效评估中变得越来越重要。在这篇综述中，我们讨论了生物分析背景下酶活性测定的技术方面，包括测定设计和格式以及与测定开发、验证和生命周期管理相关的独特挑战和注意事项。© 2024。作者），经美国药学科学家协会独家许可。

Aberrant or dysfunctional cellular enzymes are responsible for a wide range of diseases including cancer, neurodegenerative conditions, and metabolic disorders. Deficiencies in enzyme level or biofunction may lead to intracellular accumulation of substrate to toxic levels and interfere with overall cellular function, ultimately leading to cell damage, disease, and death. Marketed therapeutic interventions for inherited monogenic enzyme deficiency disorders include enzyme replacement therapy and small molecule chaperones. Novel approaches of in vivo gene therapy and ex vivo cell therapy are under clinical evaluation and provide promising opportunities to expand the number of available disease-modifying treatments. To support the development of these different therapeutics, assays to quantify the functional activity of protein enzymes have gained importance in the diagnosis of disease, assessment of pharmacokinetics and pharmacodynamic response, and evaluation of drug efficacy. In this review, we discuss the technical aspects of enzyme activity assays in the bioanalytical context, including assay design and format as well as the unique challenges and considerations associated with assay development, validation, and life cycle management.© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.