Su Yunpeng, Li Shi-Yan, Ghosh Sunil, Ortiz Janelle, Hogge Donna E, Frankel Arthur E
Cancer Research Institute, Scott & White Memorial Hospital, Temple, TX, USA.
Biologicals. 2010 Jan;38(1):144-9. doi: 10.1016/j.biologicals.2009.08.016. Epub 2009 Sep 23.
We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin-interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at -80 degrees C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography-HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC(50) of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC(50) of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 degrees C. Further, the drug was stable at 4 and 25 degrees C in the plastic syringe and administration tubing for 48 h.
我们已制备出临床级别的DTIL3K116W，一种变异的白喉毒素 - 白细胞介素 - 3融合蛋白，用于治疗急性髓系白血病。该产品经滤膜除菌、无菌分装，并储存于-80℃。通过考马斯亮蓝染色的SDS - PAGE、内毒素检测、细胞毒性检测、无菌检测、质谱分析、受体结合亲和力检测、ADP - 核糖基化检测、对正常人CFU - GM的抑制作用、二硫键分析、免疫印迹、稳定性检测、尺寸排阻色谱 - HPLC、测序及免疫组织化学对其进行了表征。分装后的产品在0.25M NaCl/5mM Tris，pH 7.9中无菌，蛋白浓度为1.08mg/ml。SDS - PAGE检测的纯度>99%。HPLC检测的聚集体<1%。内毒素水平为0.296EU/mg。肽图分析和质谱分析证实了其组成和分子量。分装后的药物与抗IL3和DT抗体保持反应活性。效价研究显示在TF1/H - ras细胞上48小时的EC(50)为0.5pM。通过竞争性实验证实其结合特性，IC(50)为1.4nM。ADP - 核糖基化活性与DTGM - CSF相当。通过免疫组织化学检测该药物与测试的冷冻人体组织切片无反应。在-80℃下6个月内没有溶解性丧失、蛋白水解聚集或效价丧失的迹象。此外，该药物在塑料注射器和给药管道中于4℃和25℃下48小时稳定。
