Research Department, Byk Gulden Italia S.p.A., Via Giotto 1, I- 20032, Cormano (Mi), Italy.
Cytotechnology. 1999 Jan;29(1):11-25. doi: 10.1023/A:1008075423609.
Anti PSA monoclonal antibodies for diagnostic use were produced in an in vitro system. After purification using Protein G affinity chromatography a percentage of about 10% of antibody aggregates remained. The use of monoclonal antibodies containing aggregates as a capture antibody in a diagnostic kit reduces the performance of the test making it often unacceptable. The aggregates could be eliminated using gel filtration chromatography but, in that way, the final recovery of the whole production process was only about 50%. Aggregation is favoured when the working pH is near to the isoelectric point of the antibody. We varied the culture medium composition, modifying pH and osmolarity. We tested different values of pH and osmolarity: 7.1, 7.5, 8.0, 8.5 for pH, and 300, 340, 367, 395 mOsm/kg H2O for osmolarity. By modification of the cell culture medium we obtained a significant decrease of monoclonal antibody aggregates in the production cycle. In this way we achieved higher recovery rate and could avoid gel filtration polishing step. The experiments were performed in two stages: first in culture flasks changing one parameter in each experiment, and then in spinner bottle using the best conditions obtained in the first stage. During scale up we used the modifications achieved from the experiment showed in this paper in our production by hollow fibre bioreactor with positive results.
用于诊断用途的抗 PSA 单克隆抗体是在体外系统中产生的。使用 Protein G 亲和层析进行纯化后，仍有约 10%的抗体聚集物残留。在诊断试剂盒中使用含有聚集物的单克隆抗体作为捕获抗体，会降低测试的性能，使其通常不可接受。可以使用凝胶过滤层析来去除聚集物，但这样会导致整个生产过程的最终回收率仅为约 50%。当工作 pH 值接近抗体的等电点时，聚集物就会增多。我们改变了培养基的组成，调整了 pH 值和渗透压。我们测试了不同的 pH 值和渗透压值：pH 值为 7.1、7.5、8.0、8.5，渗透压值为 300、340、367、395 mOsm/kg H2O。通过修改细胞培养基，我们在生产周期中获得了单克隆抗体聚集物的显著减少。这样，我们实现了更高的回收率，并可以避免凝胶过滤抛光步骤。实验分两个阶段进行：首先在培养瓶中改变每个实验的一个参数，然后在搅拌瓶中使用第一阶段获得的最佳条件。在放大规模时，我们在生产中使用了本文实验中获得的改进，使用中空纤维生物反应器取得了积极的结果。
