Engineering of high performance microbial secreted expression systems for improved antibody expression in the host


The expression of monoclonal antibodies in microbial hosts has gained increasing interest for analytical and therapeutic applications due to their simplicity and speed. In this work, we describe a secreted approach to develop the expression systems with combinations of different promoters and signal peptides for enhanced Fab fragments expression in E. coli. We also develop a high-throughput screening method using ELISA to isolate the high expression clones by screening more than thousand clones. The effects of different hosts in Fab production were also investigated. The top clone Fab products are purified with affinity resin and further characterized by LC/MS and LC/MS/MS for identity, ELISA for antigen affinity, and CD for higher order structure. The best clone was then fermented in a scale-down model by parallel 250ml mini-bioreactors to find out the optimized process conditions for future 2~5 liter bioreactor scale-up process. Our recent result show that more than 70% Fab is secreted into the culture medium. This would be beneficial to the purification process by avoiding cell disruption, cell debris removal, and reduce loading of host cell protein, and host cell DNA impurities. The integrated platform makes us gain high quality product for pre-clinical test in six months possible.


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