A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies.

The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. With this in mind, we generated a novel hydrogen peroxide evolved host using directed host cell evolution. Here we demonstrate that this host has heritable resistance to hydrogen peroxide over many generations, displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defense genes such as those involved in glutathione synthesis and turnover, and has improved glutathione content. Additionally, we show that this host has significantly improved transfection recovery times, improved growth and viability properties in a fed-batch production process, and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells. These findings demonstrate that host cell evolution represents a powerful methodology for improving specific host cell characteristics that can positively impact the expression of difficult to express biotherapeutics.

High-throughput quantitation of Fc-containing recombinant proteins in cell culture supernatant by fluorescence polarization spectroscopy.

Measurement of recombinant protein product titer critically underpins all biopharmaceutical manufacturing process development, as well as diverse research and discovery activity. Here, we describe a simple rapid (<2 min per 96 samples) 96-well microplate-based assay that enables high-throughput quantitation of recombinant immunoglobulin G and Fc-containing IgG derivatives in mammalian cell culture supernatant over a wide dynamic range of 2.5-80 mg/L, using microplate fluorescence polarization (FP) spectroscopy. The solution-phase FP assay is based on the detection of immunoglobulin Fc domain containing analyte binding to FITC-conjugated recombinant Protein G ligand to measure analyte concentration dependent changes in emitted FP. For ease of use and maximal shelf life, we showed that air-dried assay microplates containing pre-formulated ligand that is re-solubilized on addition of analyte containing solution did not affect assay performance, typically yielding an across plate coefficient of variation of <1%, and a between-plate standard deviation below 1%. Comparative assays of the same samples by FP and other commonly used IgG assay formats operating over a similar dynamic range (Protein A HPLC and bio-interferometry) yielded a coefficient of determination >0.99 in each case.