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

Jung Hee Woo, Jen Sing Liu, Soo Hyun Kang, Ravibhushan Singh, Seong Kyu Park, Yunpeng Su, Janelle Ortiz, David M. Neville, Mark C. Willingham, Arthur E. Frankel

Research output: Contribution to journalArticle

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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 languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalProtein Expression and Purification
Volume58
Issue number1
DOIs
StatePublished - Mar 2008

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Immunotoxins
Phase II Clinical Trials
Clinical Trials, Phase I
T-cells
T-Lymphocytes
Pharmaceutical Preparations
Anions
Assays
Transplantation Tolerance
T-Cell Leukemia
Diphtheria Toxin
Borates
Jurkat Cells
Pichia
Proteolysis
Bioreactors
Recombinant Proteins
Endotoxins
Edetic Acid
Microfiltration

Keywords

  • CD3 positive
  • Diphtheria toxin
  • Pichia pastoris
  • UCHT1

ASJC Scopus subject areas

  • Biochemistry

Cite this

GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials. / Woo, Jung Hee; Liu, Jen Sing; Kang, Soo Hyun; Singh, Ravibhushan; Park, Seong Kyu; Su, Yunpeng; Ortiz, Janelle; Neville, David M.; Willingham, Mark C.; Frankel, Arthur E.

In: Protein Expression and Purification, Vol. 58, No. 1, 03.2008, p. 1-11.

Research output: Contribution to journalArticle

Woo, Jung Hee ; Liu, Jen Sing ; Kang, Soo Hyun ; Singh, Ravibhushan ; Park, Seong Kyu ; Su, Yunpeng ; Ortiz, Janelle ; Neville, David M. ; Willingham, Mark C. ; Frankel, Arthur E. / GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials. In: Protein Expression and Purification. 2008 ; Vol. 58, No. 1. pp. 1-11.
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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.",
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