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首页> 外文期刊>Protein Expression and Purification >GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHTl) for phase I/II clinical trials
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GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHTl) for phase I/II clinical trials

机译：I / II期临床试验的GMP产生和二价抗人T细胞免疫毒素A-dmDT390-bisFv（UCHTl）的表征

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摘要

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 mu m 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 144.2 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 mu g/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. (c) 2007 Elsevier Inc. All rights reserved.
机译：开发了二价抗T细胞免疫毒素A-dmDT390-bisFv（UCHT1），用于治疗T细胞白血病，自身免疫性疾病和诱导移植耐受。为了获得用于I / II期临床试验的临床级二价抗T细胞免疫毒素，使用表达二价抗T细胞免疫毒素的巴斯德毕赤酵母mutEF2JC307-8（2）菌株进行了单批120 L生物反应器培养。用甲醇诱导培养物162小时后，通过0.1μm的中空纤维微滤步骤收获培养基。通过三步纯化程序（650 M丁基捕获步骤，硼酸根阴离子交换步骤和最终Poros阴离子交换步骤）纯化重组蛋白。将最终材料过滤除菌，无菌瓶装并在-80℃下保存。表达水平为207mg / L的培养上清液，最终产量为69.6％或144.2mg / L的培养上清液。通过多种测定表征最终产物。瓶装产品是无菌的。在150 mM NaCl，5％甘油，1 mM EDTA和5 mM Tris（pH 8.0）中，药物浓度为0.8 mg / mL。通过SDS-PAGE的纯度为98％。通过Superdex 200 HPLC得出的聚集体<1％。效力揭示了Jurkat细胞的20 h IC50为17 fM。内毒素水平为0.02 U / mg。化学和生物学分析证实了该分子的纯度，组成和功能活性。除T细胞外，该药物与经测试的冷冻人体组织切片均无反应。小鼠的LD10为500至750μg/ kg。没有证据表明在-80摄氏度下超过1.5年的溶解度降低，蛋白水解，聚集或效能丧失。这种蛋白质药物的可扩展合成方法应可用于I / II期临床试验的生产，并可用于其他白喉毒素融合药物也用于临床开发。 （c）2007 Elsevier Inc.保留所有权利。

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