| Characterization of Recombinant Human IL-15 Deamidation and Its Practical Elimination through Substitution of Asparagine 77. | |
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MedLine Citation:
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PMID: 22009587 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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PURPOSE: 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. METHODS: 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. RESULTS: Adding 20% ethanol to buffers or heating at ≥32°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 ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. CONCLUSIONS: 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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Authors:
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David F Nellis; Dennis F Michiel; Man-Shiow Jiang; Dominic Esposito; Richard Davis; Hengguang Jiang; Angela Korrell; George C Knapp; Lauren E Lucernoni; Roy E Nelson; Emily M Pritt; Lauren V Procter; Mark Rogers; Terry L Sumpter; Vinay V Vyas; Timothy J Waybright; Xiaoyi Yang; Amy M Zheng; Jason L Yovandich; John A Gilly; George Mitra; Jianwei Zhu |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-10-19 |
Journal Detail:
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Title: Pharmaceutical research Volume: - ISSN: 1573-904X ISO Abbreviation: - Publication Date: 2011 Oct |
Date Detail:
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Created Date: 2011-10-19 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8406521 Medline TA: Pharm Res Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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Biopharmaceutical Development Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, 21702, USA. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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