Yueming Qian

Cell culture and gene transcription effects of copper sulfate on Chinese hamster ovary cells

This study reports the effects of varying concentrations of copper sulfate on the metabolic and gene transcriptional profile of a recombinant Chinese hamster ovary (CHO) cell line producing an immunoglobulin G (IgG)‐fusion protein (B0). Addition of 50 μM copper sulfate significantly decreased lactate accumulation in the cultures while increasing viable cell density and protein titer. These changes could be seen from day 6 and became increasingly evident with culture duration. Reducing the copper sulfate concentration to 5 μM retained all the above beneficial effects, but with the added benefit of reduced levels of the aggregated form of the B0 protein. To profile the cellular changes due to copper sulfate addition at the transcriptional level, Affymetrix® CHO microarrays were used to identify differentially expressed genes related to reduced cellular stresses and facilitated cell cycling. Based on the microarray results, down‐regulation of the transferrin receptor and lactate dehydrogenase, and up‐regulation of a cytochrome P450 family‐2 polypeptide were then confirmed by Western blotting. These results showed that copper played a critical role in cell metabolism and productivity on recombinant CHO cells and highlighted the usefulness of microarray data for better understanding biological responses on medium modification.

Sialylation enhancement of CTLA4-Ig fusion protein in Chinese hamster ovary cells by dexamethasone.

The importance of glycoprotein sialic acid levels is well known, as increased levels have been shown to increase in vivo serum half‐life profiles. Here we demonstrate for the first time that dexamethasone (DEX) was capable of improving the sialylation of a CTLA4‐Ig fusion protein produced by Chinese hamster ovary (CHO) cells. DEX was shown to enhance the intracellular addition of sialic acid by sialyltransferases as well as reduce extracellular removal of sialic acid by sialidase cleavage. We illustrated that DEX addition resulted in increased expression of the glycosyltransferases α2,3‐sialyltransferase (α2,3‐ST) and β1,4‐galactosyltransferase (β1,4‐GT) in CHO cells. Based upon our previous results showing DEX addition increased culture cell viability, we confirmed here that cultures treated with DEX also resulted in decreased sialidase activity. Addition of the glucocorticoid receptor (GR) antagonist mifepristone (RU‐486) was capable of blocking the increase in sialylation by DEX which further supports that DEX affected sialylation as well as provides evidence that the sialylation enhancement effects of DEX on recombinant CHO cells occurred through the GR. Finally, the effects of DEX on increasing sialylation were then confirmed in 5‐L controlled bioreactors. Addition of 1 µM DEX to the bioreactors on day 2 resulted in harvests with average increases of 16.2% for total sialic acid content and 15.8% in the protein fraction with N‐linked sialylation. DEX was found to be a simple and effective method for increasing sialylation of this CTLA4‐Ig fusion protein expressed in CHO cells. Biotechnol. Bioeng. 2010;107: 488–496.

A mechanistic study on the effect of dexamethasone in moderating cell death in Chinese Hamster Ovary cell cultures

Dexamethasone (DEX) was previously shown (Jing et al., Biotechnol Bioeng. 2010;107:488‐496) to play a dual role in increasing sialylation of recombinant glycoproteins produced by Chinese Hamster Ovary (CHO) cells. DEX addition increased sialic acid levels of a recombinant fusion protein through increased expression of α2,3‐sialyltransferase and β1,4‐galactosyltransferase, but also decreased the sialidase‐mediated, extracellular degradation of sialic acid through slowing cell death at the end of the culture period. This study examines the underlying mechanism for this cytoprotective action by studying the transcriptional response of the CHO cell genome upon DEX treatment using DNA microarrays and gene ontology term analysis. Many of those genes showing a significant transcriptional response were associated with the regulation of programmed cell death. The gene with the highest change in expression level, as validated by Quantitative PCR assays with TaqMan® probes and confirmed by Western Blot analysis, was the antiapoptotic gene Tsc22d3, also referred to as GILZ (glucocorticoid‐induced leucine zipper). The pathway by which DEX suppressed cell death towards the end of the culture period was also confirmed by showing involvement of glucocorticoid receptors and GILZ through studies using the glucocorticoid antagonist mifepristone (RU‐486). These findings advance the understanding of the mechanism by which DEX suppresses cell death in CHO cells and provide a rationale for the application of glucocorticoids in CHO cell culture processes.

Hypoxia influences protein transport and epigenetic repression of CHO cell cultures in shake flasks.

Shake flasks and bench-top bioreactors are widely used for cell culture process development, however, culture performances significantly differ between them. In order to apply the results received from small-scale cultures to production scale, it is important to understand the mechanisms underlying the differences between various culture systems. This study analyzes the expression patterns of Chinese hamster ovary (CHO) cells producing IgG-fusion protein B0 cultured in shake flasks and 5-L bench-top bioreactors by CHO-specific DNA microarrays. The data show that hypoxia was present in shake flask cultures but not in controlled, bench-top bioreactors. Hypoxic conditions appeared to be associated with epigenetic repression resulting in decreased cell culture performance and protein productivity, which is also present during large-scale bioreactor operations due to oxygen gradients. High protein productivity was associated with increased cellular machinery for protein transport and secretion in conjunction with decreased epigenetic repression in bench-top bioreactor cultivation. Metal ions could improve cell growth and protein production under hypoxia and this condition could be mimicked in small-scale bioreactors to facilitate cell culture process scale-up.

Hypoxia and transforming growth factor-beta1 pathway activation promote Chinese Hamster Ovary cell aggregation

Suspension cultivation is the preferred mode of operation for the large‐scale production of many biologics. Chinese Hamster Ovary (CHO) cells are anchorage‐dependent in origin, but they have been widely adapted to suspension culture. In suspension culture, formation of CHO cell aggregates is a common phenomenon and compromises cell culture performance in multiple ways. To better understand the underlying mechanisms that regulate cell aggregation, we utilized CHO‐specific transcriptome profiling as a screening tool and demonstrated that many genes encoding extracellular matrix (ECM) proteins were upregulated in the cultures with increased cell aggregation. Significantly, hypoxia was identified to be a cause for promoting CHO cell aggregation, and transforming growth factor beta1 (TGFβ1) pathway activation served as an intermediate step mediating this biological cascade. These transcriptomics findings were confirmed by cell culture experiments, and it was further demonstrated that adding recombinant TGFβ1 to the culture significantly increased ECM protein fibronectin expression and cell aggregation. The results of this study emphasize the importance of adequate mixing and oxygen supply for suspension cultures from a new angle, and regulating the TGFβ1 pathway is proposed as a new strategy for mitigating cell aggregation to improve cell culture performance.

WITHDRAWN: Aminoglycoside phosphotransferase II gene as primary selection marker for Pichia pastoris producing full-length monoclonal antibody.

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