Oxidation and reduction analysis of therapeutic recombinant human interleukin-15 by HPLC and LC-MS.
Interleukin-15
LC-MS
Quality control
RP-HPLC
Redox by-products
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
May 2023
May 2023
Historique:
received:
13
01
2023
accepted:
29
03
2023
revised:
26
03
2023
medline:
8
5
2023
pubmed:
15
4
2023
entrez:
14
4
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 37058229
doi: 10.1007/s00253-023-12508-1
pii: 10.1007/s00253-023-12508-1
doi:
Substances chimiques
Interleukin-15
0
Recombinant Proteins
0
Interleukin-2
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
3217-3227Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
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