Chemostat‐based transcriptional analysis of growth rate change and BCL‐2 over‐expression in NS0 cells

Krampe, Britta; Fagan, Ailís; Gaora, Peadar Ó; Al‐Rubeai, Mohamed

doi:10.1002/bit.23100

Cited by 6 publications

(3 citation statements)

References 59 publications

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“…Specific growth rate, biomass, ER and LC polypeptide were only correlated to specific productivity at certain time points during the growth phase. The positive correlation between HC mRNA, LC mRNA, HC polypeptides and intracellular antibody with specific productivity appeared consistently throughout the growth phase of batch culture which are generally in agreement with previous reports on the cellular mechanisms that dictate relationships between productivity and mRNA levels in various cell lines [9], [12]–[14], [16], [17], [20], [62]. This quantitative correlation suggests that HC and LC mRNA chains is extraordinary stable and that they appear to be translated with equal efficiency during the culture duration.…”

Section: Discussionsupporting

confidence: 91%

Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

2013

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Selection of high producing cell lines to produce maximum product concentration is a challenging and time consuming task for the biopharmaceutical industry. The identification of early markers to predict high productivity will significantly reduce the time required for new cell line development. This study identifies candidate determinants of high productivity by profiling the molecular and morphological characteristics of a panel of six Chinese Hamster Ovary (CHO) stable cell lines with varying recombinant monoclonal antibody productivity levels ranging between 2 and 50 pg/cell/day. We examined the correlation between molecular parameters and specific productivity (qp) throughout the growth phase of batch cultures. Results were statistically analyzed using Pearson correlation coefficient. Our study revealed that, overall, heavy chain (HC) mRNA had the strongest association with qp followed by light chain (LC) mRNA, HC intracellular polypeptides, and intracellular antibodies. A significant correlation was also obtained between qp and the following molecular markers: growth rate, biomass, endoplasmic reticulum, and LC polypeptides. However, in these cases, the correlation was not observed at all-time points throughout the growth phase. The repeated sampling throughout culture duration had enabled more accurate predictions of productivity in comparison to performing a single-point measurement. Since the correlation varied from day to day during batch cultivation, single-point measurement was of limited use in making a reliable prediction.

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Section: Discussionsupporting

confidence: 91%

Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

2013

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“…Unfortunately, this mode of cultivation does not allow to study the effect of reduced culture temperatures on r-protein productivity separately, since a reduction of the specific growth rate is simultaneously caused by mild hypothermia. In this sense, chemostat culture resurgence as the most appropriate tool for obtaining biologically reliable and homogeneous data [32], [33], [34], [35] (based on the advantages offered through environmental control, reproducibility and delivering constant physicochemical conditions), allowing control the specific growth rate of the cells (μ), through the dilution rate (D), after reaching steady state (SS) being valid in mammalian cell technology to high viability (D = μ). Thus, chemostat culture emerges as a relevant option to achieve a correct understanding of the cell behavior to solve the problem of evaluating various culture variables [36].…”

Section: Introductionmentioning

confidence: 99%

Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

et al. 2014

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Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h−1) and low dilution rate (0.012 h−1) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.

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“…Further work examined the effect of Bcl-2 overexpression in NS0 cells expressing a monoclonal antibody cultured in chemostats at two dilution rates. Overall, the Bcl-2 NS0 cells had elevated transcription gene expression levels, highly elevated anti-apoptosis gene expression levels, and slightly elevated pro-apoptosis gene expression levels (Krampe et al, 2011). …”

Section: Applied Genomics In Bioprocessingmentioning

confidence: 99%

Genomics in mammalian cell culture bioprocessing

Wuest

Harcum

Lee

2012

Biotechnology Advances

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Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a $108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised.

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Chemostat‐based transcriptional analysis of growth rate change and BCL‐2 over‐expression in NS0 cells

Cited by 6 publications

References 59 publications

Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

Genomics in mammalian cell culture bioprocessing

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