High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells

Abstract

Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed‐batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale‐up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next‐generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21‐mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor‐like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine‐enriched cell culture media.

Progress in fed-batch culture for recombinant protein production in CHO cells

“…This glucose feeding strategy also promoted cell growth efficiently. Cysteine greater than 2.5 mM with low cell densities resulted in counteracted oxidative stress, but Komuczki et al presented that high cysteine concentration with a higher cell concentration can counteract oxidative stress (Komuczki et al 2022 ). Wang et al reported that an upturn in terms of the product titer was influenced by temperature shifted to 33℃ in 14-day fed-batch culture, and 100% normalized titer was set as the highest titer on day 14.…”

Exploring metabolic responses and pathway changes in CHO‐K1 cells under varied aeration conditions and copper supplementations using 1H NMR‐based metabolomics

“…[ 44 ] While augmented feeding typically extends culture duration, it can simultaneously result in wasteful metabolism and hinder growth when culminating in high osmolality. [ 45–47 ] In light of these findings, recalibrating the feeding strategy is warranted to optimize overall culture performance. [ 48 ]…”

Developing an ultra‐intensified fed‐batch cell culture process with greatly improved performance and productivity

“…Entering the production stage, the benefit of IFB can only be established on the premise that the status of cells, such as cell density and viability and cell specific productivity, could be sustained till a desirable advantage in volumetric productivity can be discerned. However, maintaining cell status in such a high density culture appeared to be challenging and cell line dependent (Stepper et al, 2020; Xu et al, 2020a; Yang et al, 2014; Yongky et al, 2019), as many studies have observed that some toxic cellular metabolites, such as lactate and ammonia, and metabolic by‐products like reactive oxygen species (ROS), would exert a chain of deteriorating effects on cellular ecosystem and metabolic disorder (Graham et al, 2021; Handlogten et al, 2018; Komuczki et al, 2022; Pereira et al, 2018; Zhou et al, 2023). Consequently, extensive developmental efforts particularly on media or process optimization were often required to achieve a competitive productivity (Stepper et al, 2020; Xu et al, 2020a).…”