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Improving the Quality of a Recombinant Rabbit Monoclonal Antibody Against PLXDC2 by Optimizing Transient Expression Conditions and Purification Method

Improving the Quality of a Recombinant Rabbit Monoclonal Antibody Against PLXDC2 by Optimizing Transient Expression Conditions and Purification Method

Hisayo Shimizu, Masataka Nakagawa, Nemuri Todaka, Keitaro Imaizumi, Yasunori Kurosawa, Toshiaki Maruyama, C J Okumura, Takashi Shibata, Yosuke Tanaka, Yoshinori Sato, Yasuo Ono, Teruo Akuta

Protein Expr Purif. 2018 Jun;146:27-33.

PMID: 29378261

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42417065	Anti-phospho-MAPKAPK-2 (pThr222) antibody produced in rabbit			Price
IAR42417511	Anti-phospho-Trk C (pTyr516) antibody produced in rabbit			Price

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