GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials.
The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical grade bivalent anti-T cell immunotoxin for phase I/II clinical trials, a single batch of 120 L bioreactor culture was performed using the Pichia pastoris mutEF2JC307-8(2) strain expressing the bivalent anti-T cell immunotoxin. After 162 h induction of the culture by methanol, the culture medium was harvested by a 0.1 microm hollow-fiber microfiltration step. The recombinant protein was purified by a 3-step purification procedure (Butyl 650 M capturing step, borate anion exchange step and final Poros anion exchange step). The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2mg/L of culture supernatant. The final product was characterized by multiple assays. Vialed product was sterile. The drug concentration was 0.8 mg/mL in 150 mM NaCl, 5% glycerol, 1mM EDTA, and 5mM Tris (pH 8.0). Purity by SDS-PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC(50) of 17f M on Jurkat cells. Endotoxin level was 0.02 U/mg. Chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The drug did not react with tested frozen human tissue sections except for T cells. LD(10) in mice was between 500 and 75 0microg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at -80 degrees C. The scalable synthesis of this protein drug should be useful for production for phase I/II clinical trials and can be applicable for other diphtheria toxin fusion drugs for clinical development.
Woo
Jung Hee
JH
Scott and White Memorial Hospital, Scott and White Cancer Research Institute, 5701 South Airport Road, Temple, TX 76502, USA. jwoo@swmail.sw.org
Liu
Jen-Sing
JS
Kang
Soo Hyun
SH
Singh
Ravibhushan
R
Park
Seong Kyu
SK
Su
Yunpeng
Y
Ortiz
Janelle
J
Neville
David M
DM
Willingham
Mark C
MC
Frankel
Arthur E
AE
D000818:Animals; D019149:Bioreactors; D002460:Cell Line; D017321:Clinical Trials, Phase I as Topic; D017322:Clinical Trials, Phase II as Topic; D004167:Diphtheria Toxin / Q000378:metabolism; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology / Q000592:standards; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*
D004167:Diphtheria Toxin; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins