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.

Keywords: bispecific antibody; evolved host; hydrogen peroxide; redox.

Figure 1

Creation of an H₂O₂ evolved host. A viability plot tracking cell recovery during the H₂O₂ host evolution process (a) (arrows indicate the day and concentration of H₂O₂ addition, black circles represent cell viability counts). The H₂O₂ evolved host were passaged to 29 and 90 PDL, and CHO control host to 7 PDL respectively in CD‐CHO supplemented with 6 mM glutamine after which all hosts were re‐challenged with 37 mM H₂O₂ and viabilities recorded (viability measured on Days 1, 2, 4, 7, 10, 11, and 12) (b). N = 1. CHO, Chinese hamster ovary; PDL, population doubling level

Figure 2

CHO host cell survival in response to redox stressors using Chemstress plates. A comparison of relative host cell viabilities following 72‐h incubation with menadione sodium bisulfite (MSB) (a), buthionine sulfoximine (BSO) (b), mercaptosuccinic acid (MS) (c), and cobalt chloride (CoCl) (d) between CHO control and H₂O₂ evolved host cells. The graphs show the mean ± SD, N = 3 in all cases, statistics determined using an unpaired t‐test. CHO, Chinese hamster ovary. *p < 0.05, **p < 0.005

Figure 3

Characterization of the antioxidant capacity of H₂O₂ evolved host cells. A comparison of total GSH (a) and the ratio of GSH:GSSG (b) between CHO control and H₂O₂ evolved hosts. Relative mRNA expression of glutathione synthetase (GSS) (c), gamma‐glutamylcysteine ligase modulator subunit (GCLM) (d), catalase (e), and xCT in CHO control and H₂O₂ evolved host cells. All qPCR data were normalized to MMADHC mRNA expression and SD calculated on fold change relative to control. The graphs show the mean ± SD, N = 3 in all cases, statistics determined using an unpaired t‐test. CHO, Chinese hamster ovary. **p < 0.005, ***p < 0.0005

Figure 4

Survival of transfected hosts expressing BisAb A and B in response to menadione sodium bisulfite treatment (MSB). Viability plots of CHO control host A and H₂O₂ evolved host A (a) and CHO control host B and H₂O₂ evolved host B (b) in response to 6 µM MSB or H₂O treatment for 72 h. The graphs show the mean + SD, N = 3 in all cases, statistics determined using a one‐way ANOVA and a Tukey's multiple comparison test. CHO, Chinese hamster ovary. *p < 0.05 (compares CHO control host A + 6 µM MSB and H₂O₂ evolved host A + 6 µM MSB)

Figure 5

Characterization of the antioxidant capacity of transfected H₂O₂ evolved host cells. Left panel: Comparison of total GSH (a) and the ratio of GSH:GSSG (b). Relative mRNA expression of glutathione synthetase (GSS) (e), gamma‐glutamylcysteine ligase modulator subunit (GCLM) (f), xCT (g) catalase (h), and glutathione peroxidase1 (GPrx1) (i) in CHO control host A and H₂O₂ evolved host A. Right Panel: Comparison of total GSH (c) and the ratio of GSH:GSSG (d). Relative mRNA expression of GSS (j), GCLM (k), xCT (l) catalase (m), and GPrx1 (n) in CHO control host B and H₂O₂ evolved host B. All qPCR data were normalized to MMADHC mRNA expression and SD calculated on fold change relative to control. The graphs show the mean ± SD, N = 3 in all cases, statistics determined using an unpaired t‐test. CHO, Chinese hamster ovary. **p < 0.005, ***p < 0.0005, **** = p < 0.00005

Figure 6

Recovery of stable pools post‐transfection. Cell viability and viable cell density (VCD) of H₂O₂ evolved host A and CHO control host A (a) and cell viability and VCD of H₂O₂ evolved host B and CHO control host B (b) were monitored after transfection. Three pools were generated for each molecule and each host. Error bars are mean viability or VCN ± SD. N = 3 in all cases, statistics determined for the Day 11‐time point using an unpaired t‐test. CHO, Chinese hamster ovary. *p < 0.05, ***p < 0.0005

Figure 7

Stable pool performance during the fed‐batch process. Left panel: A comparison of viable cell density (VCD) (a), Viability (b), Lactate (c), Titer (d), and cell‐specific productivity (qP) (e) between CHO control A and H₂O₂ evolved host A. Right panel: A comparison of VCD (f) Viability (g), Lactate (h), Titer (I), and qP (j) between CHO control B and H₂O₂ evolved host B. A total of three pools expressing each molecule were evaluated for each host. Error bars are mean ± SD. N = 3 in all cases, statistics determined using an unpaired t‐test for titer and qP and multiple t‐tests on VCD, viability, and lactate time courses comparing each time point individually. CHO, Chinese hamster ovary; ns, not significant. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005