title: GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials.
abstract: 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.
authors.affiliation: Scott and White Memorial Hospital, Scott and White Cancer Research Institute, 5701 South Airport Road, Temple, TX 76502, USA. jwoo@swmail.sw.org
authors.forename: Jung Hee
authors.initials: JH
authors.lastname: Woo
authors.forename: Jen-Sing
authors.initials: JS
authors.lastname: Liu
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Kang
authors.forename: Ravibhushan
authors.initials: R
authors.lastname: Singh
authors.forename: Seong Kyu
authors.initials: SK
authors.lastname: Park
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Su
authors.forename: Janelle
authors.initials: J
authors.lastname: Ortiz
authors.forename: David M
authors.initials: DM
authors.lastname: Neville
authors.forename: Mark C
authors.initials: MC
authors.lastname: Willingham
authors.forename: Arthur E
authors.initials: AE
authors.lastname: Frankel
mesh_terms: 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*
chemical_list: D004167:Diphtheria Toxin; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins