The impact of process temperature on mammalian cell lines and the implications for the production of recombinant proteins in CHO cells

Masterton, Rosalyn J.; Smales, C. Mark

doi:10.4155/pbp.14.3

Cited by 26 publications

(27 citation statements)

References 78 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Using a count-based differential expression method the abundance of > 1,300 genes were found to be significantly altered 24hrs after the temperature was reduced from 37°C to 31°C. In addition, Cirbp and Rbm3, well known genes involved in the cellular response to cold shock were both found to be upregulated (Masterton & Smales, 2014). Subsequent enrichment analysis highlighted the overrepresentation of genes involved in GO biological processes related to cellular proliferation and cell cycle consistent with the 24% reduction in cell number of the CHOK1-mAb cell line and previous reports in the literature (Marchant, Al-Fageeh, Underhill, Racher, & Smales, 2008).…”

Section: Discussionsupporting

confidence: 77%

“…A strategy commonly referred to as a "temperature shift" is often utilized to improve the performance and extend the lifetime of mammalian cell culture processes in the biopharmaceutical industry (Masterton & Smales, 2014). In this study, we examined the transcriptomic response to a temperature shift using high resolution RNASeq to not only detect differences in expression at the gene level, but also, for the first time in CHO cells, monitor significant variations in mRNA splicing.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

Tzani

Monger

Motheramgari

et al. 2019

Preprint

View full text Add to dashboard Cite

AbstractRNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes, however alternative mRNA splicing, has thus far, received little attention. In this study, we utilised RNASeq for transcriptomic analysis of a monoclonal antibody producing CHOK1 cell line subjected to a temperature shift. More than 2,365 instances of differential splicing were observed 24hrs after the reduction of cell culture temperature. 1,163 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using qPCR in the monoclonal antibody producing CHOK1 line used for RNASeq and a further 2 CHOK1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilised to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

show abstract

Section: Discussionsupporting

confidence: 77%

Section: Introductionmentioning

confidence: 99%

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

Tzani

Monger

Motheramgari

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Generally, protein production in mammalian cell line is greater in stationary phase of growth when there is no competition with endogenous cellular processes [9]. In this study, Fc-fusion protein production reached its maximum level in the last days of culture, it may increase the chance of improper folding during the final stage of culture.…”

Section: Discussionmentioning

confidence: 69%

“…Amongst all the parameters, temperature has a significant impact on accurate protein folding. In addition to potential benefits on protein folding, applying sub-physiological temperatures during cell culture process especially in production phase has also been reported to increase the cell-specific productivity [9]. Marchant and his colleagues reported the cell-specific productivity (qMab) of IgG4 monoclonal antibody (cB72.3) as increased by 50% after temperature shift to 32°C from 37°C during the stationary phase of growth [10].…”

Section: Introductionmentioning

confidence: 99%

Therapeutic Fc fusion protein misfolding: A three-phasic cultivation experimental design

Aghdam¹,

Moradhaseli²,

Jafari³

et al. 2019

PLoS ONE

View full text Add to dashboard Cite

Cell culture process optimization is a critical solution to most of the challenges faced by the pharmaceutical manufacturing. One of the major problems encountered in large-scale production of therapeutic proteins is misfolded protein production. The accumulation of misfolded therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity of the final product. The aim of this study was the statistical optimization of three-phasic temperature shift and timing for enhanced production of correctly folded Fc-fusion protein. The effect of culture temperatures were investigated using the biphasic culture system. Box–Behnken design was then used to compute temperature and time of shifting optimum. Response surface methodology revealed that maximum production with low level of misfolded protein was achieved at two-step temperature shift from 37°C to 30°C during the late logarithmic phase and 30°C to 28°C in the mid-stationary phase. The optimized condition gave the best results of 1860 mg L−1 protein titer with 24.5% misfolding level. The validation experiments were carried out under optimal conditions with three replicates and the protein misfolding level was decreased by two times while productivity increased by ~ 1.3-fold. Large-scale production in 250 L bioreactor under the optimum conditions was also verified the effectiveness and the accuracy of the model. The results showed that by utilizing two-step temperature shift, productivity and the quality of target protein have been improved simultaneously. This model could be successfully applied to other products.

show abstract

“…Availability of such temperature-dependence modelling would also allow process performance to be assessed at intermediated temperatures to those considered here, i.e., 32 °C < T < 37 °C. Previous work in the literature investigated the effect of temperature on cellular response or productivity (Masterton and Smales, 2014).…”

Section: Commonality Among Two Culture Modesmentioning

confidence: 99%

Dynamic modelling, simulation and economic evaluation of two CHO cell-based production modes towards developing biopharmaceutical manufacturing processes

Shirahata

Diab

Sugiyama

et al. 2019

Chemical Engineering Research and Design

View full text Add to dashboard Cite

Chinese Hamster Ovary (CHO) cells are widely used in fermentation towards biopharmaceutical manufacturing. The present paper presents dynamic mathematical models of two different CHO culture modes: one batch mode for the production of interferon, and one perfusion mode for the production of a monoclonal antibody (mAb). The dynamic models have been used for simulating cell, substrate, by-product and product concentration trajectories, which have been compared against previously published experimental results. A sensitivity analysis of both models has been conducted, in order to analyse the relative importance of operating parameters towards biopharmaceutical process design. An economic analysis has also been subsequently performed: time and net present cost for given target capacities have been evaluated, using the validated dynamic models for the batch and perfusion modes. Economic trends have been discussed for variable initial concentration of viable CHO cells to be used in bioreactors: the latter has been recognized as the most sensitive model parameter for both culture modes.

show abstract

The impact of process temperature on mammalian cell lines and the implications for the production of recombinant proteins in CHO cells

Cited by 26 publications

References 78 publications

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

Therapeutic Fc fusion protein misfolding: A three-phasic cultivation experimental design

Dynamic modelling, simulation and economic evaluation of two CHO cell-based production modes towards developing biopharmaceutical manufacturing processes

Contact Info

Product

Resources

About