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Characterization of Recombinant Human IL-15 Deamidation and Its Practical Elimination through Substitution of Asparagine 77

  1. Author:
    Nellis, D. F.
    Michiel, D. F.
    Jiang, M. S.
    Esposito, D.
    Davis, R.
    Jiang, H. G.
    Korrell, A.
    Knapp, G. C.
    Lucernoni, L. E.
    Nelson, R. E.
    Pritt, E. M.
    Procter, L. V.
    Rogers, M.
    Sumpter, T. L.
    Vyas, V. V.
    Waybright, T. J.
    Yang, X. Y.
    Zheng, A. M.
    Yovandich, J. L.
    Gilly, J. A.
    Mitra, G.
    Zhu, J. W.

  2. Author Address

    [Nellis, David F.; Michiel, Dennis F.; Jiang, Man-Shiow; Jiang, Hengguang; Korrell, Angela; Knapp, George C.; Lucernoni, Lauren E.; Nelson, Roy E.; Pritt, Emily M.; Sumpter, Terry L.; Vyas, Vinay V.; Yang, Xiaoyi; Zheng, Amy M.; Gilly, John A.; Mitra, George; Zhu, Jianwei] NCI, Biopharmaceut Dev Program, SAIC Frederick Inc, Frederick, MD 21702 USA. [Esposito, Dominic; Procter, Lauren V.] NCI, Prot Express Lab, Adv Technol Program, Frederick, MD 21702 USA. [Davis, Richard; Rogers, Mark] M Scan Inc, W Chester, PA 19380 USA. [Waybright, Timothy J.] NCI, Lab Prote & Analyt Technol, Adv Technol Program, Frederick, MD 21702 USA. [Yovandich, Jason L.] NCI, Biol Resources Branch, Frederick, MD 21702 USA.;Zhu, JW (reprint author), NCI, Biopharmaceut Dev Program, SAIC Frederick Inc, Frederick, MD 21702 USA;zhujianwei@mail.nih.gov
    1. Year: 2012
    2. Date: Mar
  2. Journal: Pharmaceutical Research
    1. 29
    2. 3
    3. Pages: 722-738
  4. Type of Article: Article
  5. ISSN: 0724-8741
  1. Abstract:

    The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. Adding 20% ethanol to buffers or heating at a parts per thousand yen32A degrees C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation a parts per thousand yen23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.

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External Sources

  1. View Full Text
  2. DOI: 10.1007/s11095-011-0597-0
  3. View record in Web of Science®
  4. WOS: 000300457500009

Library Notes

  1. Fiscal Year: FY2011-2012
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