Improving the quality of a recombinant rabbit monoclonal antibody against PLXDC2 by optimizing transient expression conditions and purification method

• Published in: Protein expression and purification 2018-06, Vol.146, p.27-33
• Main Authors: Shimizu, Hisayo, Nakagawa, Masataka, Todaka, Nemuri, Imaizumi, Keitaro, Kurosawa, Yasunori, Maruyama, Toshiaki, Okumura, C.J., Shibata, Takashi, Tanaka, Yosuke, Sato, Yoshinori, Ono, Yasuo, Akuta, Teruo
• Format: Article
• Language: English
• Subjects: HEK293; PLXDC2; Rabbit monoclonal antibody
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2018.01.011

Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods. The quality and function of purified antibody preparations were checked by electrophoresis and western blot analysis. The secreted rabbit mAb produced by a combination of culturing at 32 °C, purification by ammonium sulfate fractionation, and diethylaminoethyl resin (DEAE) ion exchange chromatography was of high quality. In contrast, the antibody produced by the cells grown at 37 °C for 6 days after transfection and purified by Protein A/G affinity method was low quality. Hypothermic conditions during production reduced protein heterogeneity probably by favorably affecting the levels of glycosylation and aggregation. In particular, according to western blotting data, CIMmultus DEAE chromatography that utilizes monolithic columns not only excluded inferior charge variants resulting from nonspecific reactions but also yielded rabbit mAb that was of better quality than commercially available rabbit polyclonal antibodies. The combination of techniques suggested by us may be a general approach to enhance product quality of rabbit mAbs produced by transient expression systems. •Optimized method of rabbit monoclonal antibodies by HEK293-6E cells is proposed.•HEK293-6E cells grown hypothermically at 32 °C produced best quality antibodies.•CIMmultus DEAE chromatography was the optimal method of antibody purification.