Zamboni A, Giuntini I, Gianesello D, Maddalena F, Rognoni F, Herbst D
Research Department 1, Byk Gulden Italia, Cormano MI.
Cytotechnology. 1994;16(2):79-87. doi: 10.1007/BF00754610.
The production of anti-alpha-fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 milligrams glucose with a protein content of only 780 micrograms/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using 125I labelled alpha-fetoprotein. 1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg-5 g) of monoclonal antibodies for diagnostic use.
用于诊断的抗甲胎蛋白单克隆抗体的生产在配备双膜搅拌器以实现无泡通气和连续培养基灌注的搅拌罐发酵器中进行。生产过程使用了一种无血清培养基，该培养基添加了4 mM L-谷氨酰胺和2.0毫克葡萄糖，蛋白质含量仅为780微克/毫升。收获的抗体使用切向超滤系统浓缩50倍，然后通过蛋白G亲和色谱法在一步纯化过程中进行纯化。最终产品的纯度（90%）通过SDS-聚丙烯酰胺凝胶电泳、凝胶排阻色谱法和等电聚焦进行控制。为了对产品进行进一步的质量控制，测定了免疫球蛋白亚类和等电点，并使用125I标记的甲胎蛋白通过放射免疫分析（RIA）测试了纯化单克隆抗体的特异性。在2周内生产出了1.87克纯化的单克隆抗体（纯度90%）。结果发现，使用这种类型的搅拌罐发酵器结合使用蛋白G亲和色谱法的一步纯化过程，是快速生产中等规模数量（500毫克至5克）用于诊断的单克隆抗体的合适方法。
