Characterization of recombinant human IL-15 deamidation and its practical elimination through substitution of asparagine 77.
OBJECTIVE
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.
Biopharmaceutical Development Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
David F
DF
Nellis
Dennis F
DF
Michiel
Man-Shiow
MS
Jiang
Dominic
D
Esposito
Richard
R
Davis
Hengguang
H
Jiang
Angela
A
Korrell
George C
GC
Knapp
Lauren E
LE
Lucernoni
Roy E
RE
Nelson
Emily M
EM
Pritt
Lauren V
LV
Procter
Mark
M
Rogers
Terry L
TL
Sumpter
Vinay V
VV
Vyas
Timothy J
TJ
Waybright
Xiaoyi
X
Yang
Amy M
AM
Zheng
Jason L
JL
Yovandich
John A
JA
Gilly
George
G
Mitra
Jianwei
J
Zhu
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D019409:Interleukin-15 / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects
D019409:Interleukin-15; D011994:Recombinant Proteins; D001216:Asparagine