GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials

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• 作者：Jung Hee Woo ; Jen-Sing Liu ; Soo Hyun Kang ; Ravibhushan Singh ; Seong Kyu Park ; Yunpeng Su ; Janelle Ortiz ; David M. Neville Jr. ; Mark C. Willingham ; Arthur E. Frankel
• 关键词：Diphtheria toxin ; UCHT1 ; CD3 positive ; Pichia pastoris
• 刊名：Protein Expression and Purification
• 出版年：2008
• 出版时间：March 2008
• 年：2008
• 卷：58
• 期：1
• 页码：1-11
• 全文大小：587 K

文摘

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.