Characterization of recombinant human IL-15 deamidation and its practical elimination through substitution of asparagine 77

Pharm Res. 2012 Mar;29(3):722-38. doi: 10.1007/s11095-011-0597-0. Epub 2011 Oct 19.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution*
  • Animals
  • Asparagine / chemistry*
  • Asparagine / genetics
  • Cell Line
  • Cell Proliferation / drug effects
  • Humans
  • Interleukin-15 / chemistry*
  • Interleukin-15 / genetics*
  • Interleukin-15 / pharmacology
  • Mice
  • Molecular Sequence Data
  • Protein Stability
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / pharmacology
  • T-Lymphocytes / drug effects


  • Interleukin-15
  • Recombinant Proteins
  • Asparagine