Effect of iron addition on mAb productivity and oxidative stress in Chinese hamster ovary culture

Abstract

Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro‐oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria‐generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron‐induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.

Exploring the limits of conventional small-scale CHO fed-batch for accelerated on demand monoclonal antibody production

“…Additionally, two supplements, copper acetate and ferric citrate, were added to boost the q P in medium seeding supplemented fed-batch [ 23 , 26 ]. Copper acetate was added by bolus addition during the early phase, whereas ferric citrate was added in the late production phase [ 21 – 23 , 26 , 27 ]. The medium seeding supplemented fed-batch reached a higher peak VCD of 37.6 ± 3.7 × 10 6 cells/mL as compared to the 31.2 ± 3.6 × 10 6 cells/mL reached in the standard fed-batch.…”

“…Copper acetate (Sigma Aldrich, USA) supplement was added during the early cell growth phase [21][22][23][24]. Ferric citrate (Sigma Aldrich, USA) supplement was added during the late cell production phase [21,[25][26][27]. Initial working volume and process additives volumes (commercial feeds and metal supplements) were adjusted all along the process duration according to biomass and culture phase.…”

Trace metal optimization in CHO cell culture through statistical design of experiments

“…The effects of trace metals on cell culture performance can also depend on the availability of other nutrients in the cell culture medium, including other trace metals 11 . Previous studies have explored the impact of individual metals, such as iron, zinc, copper, or manganese on CHO cell metabolism, productivity, and product quality 5,8,10,12,13 . However, few studies to date explore the benefits of balancing the relative levels of trace metals within the media at certain times during cell growth to establish superior performance.…”

Characterization of Chinese hamster ovary cell culture feed media precipitate

“…As such, gas chromatography-mass spectrometry (GC-MS) 5 and especially liquid chromatography-mass spectrometry (LC-MS) 6,7 have been incorporated into various metabolomic studies to discover critical markers in CHO extracellular environment capable of improving growth and output titer productivity and further provides quantitative assessments of medium additives on cellular metabolism. On the other hand, different elemental analysis techniques have also been used in various studies of cellular physiology, including inductively coupled plasma-optical emission spectroscopy (ICP-OES) 8,9 for quantitative analysis of calcium, phosphorous and silicon to evaluate the level of ion release, and inductively coupled plasma-mass spectroscopy (ICP-MS) 10 for unraveling the effect of iron addition on both antibody productivity and oxidative stress in This work aims to characterize media precipitate formed in an early version of AMBIC's feed media, and by doing so, summarize the analytical techniques available for characterization. The characterization steps are critical for understanding the problem and developing solutions.…”

“…Later in the study, ICP-MS was also incorporated into the study as an alternative to ICP-OES due to the lack of fundamental source of continuum background in ICP-MS that allows detection of analyte at lower concentration range 15,16. In this study, ICP-MS analysis was conducted based on a protocol previously developed 10. ICP-MS Agilent 5110 ICP-OES (Agilent Technologies) and Agilent 7900 ICP-MS with SPS4 Autosampler was used for single element detection.…”