Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

Abstract

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.

Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

“…Supporting the notion that the limiting factor is the correct assembly of LC and HC, higher molecular weights protein bands above 200 kDa were observed. Previous studies on expression and purification of IgG from SHuffle cells have observed similar mis-assembled IgG species that are mis-oxidized, as these high-molecular bands collapse into LC or HC, under reducing conditions (Leith et al 2019 ; Lobstein et al 2012 ; Reddy et al 2018 ). Binding capacity to Humira IgG’s cognate antigen TNFα was measured by ELISA and IgG concentration of the purified extracts were calculated (Fig.…”

“…This was the case for the artificial PDI-GPx7 coupled redox pathway, engineered to improve IgG folding in SHuffle cells. Although great success was achieved in improving the folding of Humira IgG, little to no improvement was observed for two other IgG molecules tested, NIST mAb (Reddy et al 2018) and anti-MBP (Lobstein et al 2012) (data not shown). Many other IgG molecules and derivatives need to be evaluated under various expression conditions, to discern the specificity of the PDI-Gpx7 coupled disulfide bond formation in SHuffle cells.…”

“…Redox engineered E. coli SHuffle cells have been previously demonstrated to be an attractive platform for the expression of various antibody formats, such as full-length IgG (Reddy et al 2018 ; Robinson et al 2015 ), Fab’ fragments (Abe et al 2014 ; Mori et al 2018 ; Yusakul et al 2018 ), scFv chains (Ahmadzadeh et al 2020 ; Liu et al 2019 ; Vermeulen et al 2018 ) and VHH domains (Eliseev et al 2018 ; Ta et al 2015 ; Zarschler et al 2013 ). Although SHuffle cells lack the eukaryotic glycosylation machinery, by engineering mutations in the Fc region of antibodies, the requirement for glycosylation for efficient binding of the IgG to its cognate receptor was bypassed (Robinson et al 2015 ).…”

“…Although SHuffle cells lack the eukaryotic glycosylation machinery, by engineering mutations in the Fc region of antibodies, the requirement for glycosylation for efficient binding of the IgG to its cognate receptor was bypassed (Robinson et al 2015 ). Furthermore, unlike mammalian or yeast cells, expression of antibodies in SHuffle cells permits the efficient labelling of antibodies with heavy isotopes for structural studies (Reddy et al 2018 ). A microbial platform for the design, selection, and production of antibodies in a rapid manner, especially in response to an emerging infection is therefore essential.…”

Disulfide Bond Formation in the Periplasm of Escherichia coli

“…SHuffle cells are the first example of how the Dsb system can be engineered for biotechnological applications. Using SHuffle cells, our group was able to express various antibody fragments and full-length antibodies in the cytoplasm (171), as well as isotopically labeled antibodies, by expressing them in minimal media (172). DsbB has also been engineered to improve protein production in the cytoplasm.…”