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 μm 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 °C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2 mg/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, 1 mM EDTA, and 5 mM Tris (pH 8.0). Purity by SDS-PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC50 of 17 fM 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. LD10 in mice was between 500 and 750 μg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at -80 °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.
Original language | English (US) |
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Pages (from-to) | 1-11 |
Number of pages | 11 |
Journal | Protein Expression and Purification |
Volume | 58 |
Issue number | 1 |
DOIs | |
State | Published - Mar 2008 |
Keywords
- CD3 positive
- Diphtheria toxin
- Pichia pastoris
- UCHT1
ASJC Scopus subject areas
- Biotechnology