作为T淋巴细胞和NK细胞的重要免疫刺激剂，重组人白介素-15（rhIL-15）已在肿瘤免疫治疗或疫苗佐剂方面进行了广泛研究。然而，由于缺乏有效和准确的分析方法来表征副产物，尤其是氧化还原和脱酰胺产物，rhIL-15的生产水平远远落后于其不断增长的临床需求。为了提高rhIL-15的生产和质量控制，我们开发了扩展分辨率反相高效液相色谱（ExRP-HPLC）方法，快速准确地分析rhIL-15氧化和还原副产物，这些副产物可能在纯化过程中出现。首先，我们开发了可以分离具有不同氧化或还原水平的rhIL-15组分的RP-HPLC方法，然后通过高分辨率质谱仪（UPLC-MS）测量完整质量来确定每个峰的氧化还原状态。为了进一步阐明特定残基的氧化复杂模式，具有不同氧化水平的峰被消化成片段进行肽段图谱分析，以确定rhIL-15副产物中氧和氢原子的确切变化。此外，我们进行了部分去酰胺rhIL-15的ExRP-HPLC和UPLC-MS分析，以表征它们的氧化还原。我们的工作首次深入表征了rhIL-15的氧化还原副产物，甚至包括脱酰胺杂质。我们报告的ExRP-HPLC方法可促进快速准确地分析rhIL-15质量，这对于简化rhIL-15的工业生产以更好地满足临床应用需求具有实质性帮助。关键点： •首次表征rhIL-15的氧化还原副产物。 •通过UPLC-MS精确确定了rhIL-15氧化还原副产物中氧和氢原子的变化。 •还进一步分析了去酰胺rhIL-15的氧化还原副产物。 ©2023作者，授予Springer-Verlag GmbH德国的独家许可，属于Springer Nature的一部分。

Being an important immune stimulant of T lymphocytes and NK cells, the recombinant human interleukin-15 (rhIL-15) has been extensively researched in tumor immunotherapy or as a vaccine adjuvant. However, the rhIL-15 manufacturing level lags far behind its growing clinical demand due to the lack of efficient and exact analysis methodologies to characterize the trace by-products, typically redox and deamidation. In order to improve the production and quality control of rhIL-15, here we developed an expanded resolution reverse-phase high-performance liquid chromatography (ExRP-HPLC) approach to quickly and accurately analyze the oxidation and reduction by-products of rhIL-15, which may appear during the purification processes. Firstly, we developed RP-HPLC methods which can separate rhIL-15 fractions with different levels of oxidization or reduction, respectively, and the redox status of each peak was then determined by measuring the intact mass with a high-resolution mass spectrometer (UPLC-MS). To further clarify the complex pattern of oxidization of specific residues, the peaks with various oxidation levels were digested into pieces for peptide mapping to pinpoint the exact changes of oxygen and hydrogen atoms in the rhIL-15 by-products. In addition, we performed the ExRP-HPLC and UPLC-MS analysis of partially deamidated rhIL-15 to characterize their oxidation and reduction. Our work is the first in-depth characterization of the redox by-products of rhIL-15, even for deamidated impurities. The ExRP-HPLC method we reported can facilitate the rapid and accurate quality analysis of rhIL-15, which is substantially helpful for streamlining the industrial manufacturing of rhIL-15 to better meet the demands of clinical applications. KEYPOINTS: • The oxidization and reduction rhIL-15 by-products were characterized for the first time. • The changes of oxygen and hydrogen atoms in rhIL-15 redox by-products were accurately determined by UPLC-MS. • Oxidation and reduction by-products of deamidated rhIL-15 were further analyzed.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.