Antibody fragments such as antigen-binding fragments (Fabs) are being increasingly investigated as therapeutic molecules in the pharmaceutical industry. They offer multiple advantages relative to full length antibodies such as small sizes and general lack of glycosylation thus enabling low-cost manufacturing using microbial systems. The primary objective of this project was to increase soluble extracellular production of an intact candidate Fab using E. coli as a recombinant production host. Although E. coli is an ideal host because of its rapid and inexpensive production capabilities, expression of eukaryotic proteins requiring the formation of disulfide bonds can pose complications such as the formation of insoluble inclusion bodies resulting in low yields. This is due to the lack of complex protein processing machinery and the presence of an oxidizing intracellular environment in E. coli. As expected, initial studies yielded low titers of the intact Fab product. We aimed to increase titers of the Fab by utilizing a two-fold strategy: (1) Designing plasmids to balance the Heavy chain (HC)-to-Light chain (LC) expression ratio and target more intact Fab to the periplasm and (2) Co-expressing a suite of chaperone proteins to assist in disulfide bond formation or isomerization. For the plasmid design, a plasmid screen with different characteristics such as using promoters of varying strength, using single or dual-promoter systems and changing the HC and LC orientation were tested. The second strategy of chaperone co-expression was executed by first transforming cells with the top Fab plasmid from the screen and then co-transforming an additional helper plasmid that produced the assistor chaperone. Seven different chaperones were screened individually under various induction conditions to select the ones which yielded the highest titer. Co-expressing one of the chaperones within the screen yielded a 63% increase in Fab titer in comparison to when no chaperone protein was present. Initial screening was done in the Micro-24 small scale bioreactor system. Strains with optimal plasmid design and selected chaperone co-expression were scaled to the 1L bioreactor to observe performance of the strain in a continuous feed system. Thus, we have developed a toolbox of plasmids and assistor chaperone proteins to improve soluble Fab expression in E. coli.

Increasing soluble expression of an intact Fab (fragmented antibody) in E. coli by smart plasmid design and chaperone co-expression

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.