Abstract
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.
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References
Cheever MAM (2008) Twelve immunotherapy drugs that could cure cancers. Immunol Rev 222:357–368
Chen HH, Li NH, Xie YQ, Jiang H, Yang XY, Cagliero C, Shi SW, Zhu CC, Luo H, Chen JS, Zhang L, Zhao ML, Feng L, Lu HL, Zhu JW (2017) Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli. Appl Microbiol Biot 101(13):5267–5278. https://doi.org/10.1007/s00253-017-8265-x
Conlon KC, Lugli E, Welles HC, Rosenberg SA, Fojo AT, Morris JC, Fleisher TA, Dubois SP, Perera LP, Stewart DM, Goldman CK, Bryant BR, Decker JM, Chen J, Worthy TYA, Figg WD Sr, Peer CJ, Sneller MC, Lane HC et al (2015) Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer. J Clin Oncol 33(1):74–U123. https://doi.org/10.1200/jco.2014.57.3329
Cooley S, He F, Bachanova V, Vercellotti GM, DeFor TE, Curtsinger JM, Robertson P, Grzywacz B, Conlon KC, Waldmann TA, McKenna DH, Blazar BR, Weisdorf DJ, Miller JS (2019) First-in-human trial of rhIL-15 and haploidentical natural killer cell therapy for advanced acute myeloid leukemia. Blood Adv 3(13):1970–1980. https://doi.org/10.1182/bloodadvances.2018028332
Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, Beers C, Richardson J, Schoenborn MA, Ahdieh M, Johnson L, Alderson MR, Watson JD, Anderson DM, Giri JG (1994) Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 264(5161):965–968. https://doi.org/10.1126/science.8178155
Huang XQ, Hamilton MJ, Li CL, Schmidt C, Ellem KA (2006) An extraordinarily high level of IL-15 expression by a cell line transduced with a modified BMGneo vector displays hypoxic upregulation. Mol Biotechnol 33 33(1):49–56. https://doi.org/10.1385/MB:33:1:49
Kim G, Weiss SJ, Levine RL (2014) Methionine oxidation and reduction in proteins. Biochim Biophys Acta 1840(2):901–905. https://doi.org/10.1016/j.bbagen.2013.04.038
Klapoetke S, Xie MHW (2017) Disulfide bond characterization of human factor Xa by mass spectrometry through protein-level partial reduction. J Pharm Biomed Anal 132:238–246. https://doi.org/10.1016/j.jpba.2016.10.005
Liu HC, Ponniah G, Neill A, Patel R, Andrien B (2013) Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS Analysis. Anal Chem 85(24):11705–11709. https://doi.org/10.1021/ac403072w
Lv L, Wang H, Shi W, Wang Y, Zhu W, Liu Z, Chen X, Zheng C, Kong W, Li W, Zhu J, Lu H (2022) A homodimeric IL-15 superagonist F4RLI with easy preparation, improved half-life, and potent antitumor activities. Appl Microbiol Biot. https://doi.org/10.1007/s00253-022-12209-1
Miller JS, Morishima C, McNeel DG, Patel MR, Kohrt HEK, Thompson JA, Sondel PM, Wakelee HA, Disis ML, Kaiser JC, Cheever MA, Streicher H, Creekmore SP, Waldmann TA, Conlon KC (2018) A first-in-human phase I study of subcutaneous outpatient recombinant human IL15 (rhIL15) in adults with advanced solid tumors. Clin Cancer Res 24(7):1525–1535. https://doi.org/10.1158/1078-0432.Ccr-17-2451
Nellis DF, Michiel DF, Jiang MS, Esposito D, Davis R, Jiang H, Korrell A, Knapp GC, Lucernoni LE, Nelson RE, Pritt EM (2012) Characterization of recombinant human IL-15 deamidation and its practical elimination through substitution of asparagine 77. Pharm Res 29(3):722–738. https://doi.org/10.1007/s11095-011-0597-0
Schenkel JM, Fraser KA, Casey KA, Beura LK, Pauken KE, Vezys V, Masopust D (2016) IL-15-independent maintenance of tissue-resident and boosted effector memory CD8 T cells. J Immunol 196(9):3920–3926. https://doi.org/10.4049/jimmunol.1502337
Shi S, Chen H, Jiang H, Xie Y, Zhang L, Li N, Zhu C, Chen J, Luo H, Wang J, Feng L, Lu H, Zhu J (2017) A novel self-cleavable tag Zbasic-Delta I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli. Appl Microbiol Biot 101(3):1133–1142. https://doi.org/10.1007/s00253-016-7848-2
Stadtman ER, Moskovitz J, Levine RL (2003) Oxidation of methionine residues of proteins: biological consequences. Antioxid Redox Sign 5(5):577–582. https://doi.org/10.1089/152308603770310239
Sun W, Lai Y, Li H, Nie T, Kuang Y, Tang X, Li K, Dunbar PR, Xu A, Li P, Wu D (2016) High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris. J Immunol Methods 428:50–57. https://doi.org/10.1016/j.jim.2015.12.002
Vyas VV, Esposito D, Sumpter TL, Broadt TL, Hartley J, Knapp GC, Cheng W, Jiang M-S, Roach JM, Yang X, Giardina SL, Mitra G, Yovandich JL, Creekmore SP, Waldmann TA, Zhu J (2012) Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E.coli with stop codon optimization. Biotechnol Prog 28(2):497–507. https://doi.org/10.1002/btpr.746
Waldmann TA, Miljkovic MD, Conlon KC (2020) Interleukin-15 (dys) regulation of lymphoid homeostasis: implications for therapy of autoimmunity and cancer. J Exp Med 217(1). https://doi.org/10.1084/jem.20191062
Zhu W, Wang Y, Lv L, Wang H, Shi W, Liu Z, Yang W, Zhu J, Lu H (2022) SHTXTHHly, an extracellular secretion platform for the preparation of bioactive peptides and proteins in Escherichia coli. Microb Cell Fact 21(1). https://doi.org/10.1186/s12934-022-01856-8
Funding
The work was partly supported by the Science and Technology Innovation Action Plan of Shanghai Science and Technology Commission (No. 15431907000 to Zhu J and No. 21S11906300 to Lu H).
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YW conceived or designed study, performed research, analyzed data, and wrote the paper. HC performed research, designed study, and analyzed data. MZ and ZL performed research and contributed new methods or models. LF and QZ performed research and analyzed data. WS and WZ analyzed data. LS performed research. JZ and HL conceived or designed study, acquired funding, and wrote and edited the paper. All the authors read and approved the final manuscript.
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Wang, Y., Chen, H., Zhao, M. et al. Oxidation and reduction analysis of therapeutic recombinant human interleukin-15 by HPLC and LC-MS. Appl Microbiol Biotechnol 107, 3217–3227 (2023). https://doi.org/10.1007/s00253-023-12508-1
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DOI: https://doi.org/10.1007/s00253-023-12508-1