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

Abstract

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.bispecific antibody, evolved host, hydrogen peroxide, redox | INTRODUCTIONRecently, an interest in the cellular redox state and its effects on recombinant protein production has emerged (Handlogten et al., 2017(Handlogten et al., , 2020Orellana et al., 2015). Reactive oxygen species (ROS) are partial reduction products of molecular oxygen generated as a result of mitochondrial oxidative phosphorylation and oxidative protein folding within the endoplasmic reticulum (ER; Chevallier

Targeted CHO cell engineering approaches can reduce HCP‐related enzymatic degradation and improve mAb product quality

“…The resulting cell lines were cloned using a ClonePix FL (Molecular Devices). Cell lines were maintained in CD CHO medium (Life Technologies) supplemented with 50 µM MSX in polycarbonate Erlenmeyer flasks with vented caps (Corning), in a humidified orbital shaker at 140 rpm, 25 mm orbit diameter, 36.5°C with 6% CO 2 as described in (Mistry et al, 2021). For fed‐batch cultures, cells were cultured in animal component‐free chemically defined proprietary medium as above without the addition of MSX.…”

Large-Scale Comparative Transcriptomic Analysis of CHO Cell Functional Adaptation to Recombinant Monoclonal Antibody Production

“…This could involve modulated expression of regulators of gene expression such as transcription factors (Haredy et al ., 2013; Berger et al ., 2020, Torres and Dickson, 2021; Kim et al ., 2022) and miRNAs (Raab et al ., 2022) or overexpression of single metabolic genes that have been reported to improve the ER capacity for example (Budge et al ., 2020). Alternatively, directed evolution of CHO cell populations using specific selective environments to create CHO host cells with superior functional characteristics may be a useful strategy (Chandrawanshi et al ., 2020: Mistry et al ., 2021).…”

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway

“…[22] Therefore, methods to control cellular redox, and methods to control oxidative or nitric stress to an advantage have been proposed. [23,24] Beyond oxidative stress, protein production can result in activation of other key cellular homeostatic pathways. For example, the unfolded protein response (UPR) -activated by excess unfolded or misfolded proteins in the ER -has been suggested as a key indicator of enhanced antibody production in CHO cells.…”

“…[ 22 ] Therefore, methods to control cellular redox, and methods to control oxidative or nitric stress to an advantage have been proposed. [ 23,24 ]…”