Manzke O, Tesch H, Diehl V, Bohlen H
Klinik I für Innere Medizin, University of Cologne, Germany.
J Immunol Methods. 1997 Oct 13;208(1):65-73. doi: 10.1016/s0022-1759(97)00129-4.
A method for large scale production and single-step purification of bispecific antibodies is described. Hybrid-hybridomas were grown in hollow-fibre bioreactors with an average yield of 8 to 12 g of immunoglobulin per month. Bispecific antibodies were purified from the bioreactor supernatant by hydrophobic interaction chromatography which resolves bispecific antibodies, monospecific immunoglobulins, and culture medium supplements in one single chromatographic step. Proteins were analyzed by ELISA, SDS-PAGE, isoelectric focussing, indirect fluorescence staining, CTL-stimulation and T-cell proliferation assays. Finally, antibody preparations were checked for the presence of endotoxin and mouse DNA. Our results suggest that functional bispecific antibodies for use in therapeutic applications can be batch purified from bioreactor harvest by hydrophobic interaction chromatography in a single step. Compared to other methods such as affinity chromatography (protein A/G), ion-exchange or hydroxyapatite chromatography, our protocol offers a substantial reduction in labor time, cost, protein loss, and risk of contamination.
描述了一种大规模生产和单步纯化双特异性抗体的方法。杂交杂交瘤在中空纤维生物反应器中培养，每月平均产量为8至12克免疫球蛋白。通过疏水相互作用色谱法从生物反应器上清液中纯化双特异性抗体，该方法可在一个色谱步骤中分离双特异性抗体、单特异性免疫球蛋白和培养基补充剂。通过ELISA、SDS-PAGE、等电聚焦、间接荧光染色、CTL刺激和T细胞增殖试验对蛋白质进行分析。最后，检查抗体制剂中是否存在内毒素和小鼠DNA。我们的结果表明，用于治疗应用的功能性双特异性抗体可以通过疏水相互作用色谱法从生物反应器收获物中一步批量纯化。与其他方法如亲和色谱法（蛋白A/G）、离子交换或羟基磷灰石色谱法相比，我们的方案大大减少了劳动时间、成本、蛋白质损失和污染风险。
