Spent Media Analysis with an Integrated CE-MS Analyzer of Chinese Hamster Ovary Cells Grown in an Ammonia-Stressed Parallel Microbioreactor Platform

Elliott, Kathryn; Harris, Geoffrey; Harcum, Sarah W.; Blakeman, Kenion H.; Gavin, Colin; Anderson, Ji Young L

doi:10.12665/j19oa.elliott

Cited by 9 publications

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“…[ 56 ] Recent work described the negative effects of ammonia on growth, product quantity, and productivity for cells with a standard PDL > 50. [ 44 ] In the present work, both young‐aged cells, defined to be within 10 PDL of the original clone, and standard‐aged cells (between 50 and 90 PDL) were cultured in parallel to determine the role cell age played in the response CHO cells had to an ammonia stress. The growth characteristics of these cultures are shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

“…Transcriptome analysis was also used to determine the underlying causes of metabolic shifts in amino acid metabolism observed in this study and elsewhere. [ 44 ]…”

Section: Resultsmentioning

confidence: 99%

“…Further, these observed alanine profiles agree with previous work using this cell line in the ambr250 under ammonia stress with a different feeding protocol (Figure S1) and consistent with other biological systems. [ 3,16,44,57–60 ]…”

Section: Resultsmentioning

confidence: 99%

“…It is unknown if metabolic enzyme gene expressions change in response to elevated ammonia to enhance detoxification reactions. [44] In this study, parallel fed-batch CHO cell cultures of two distinct ages were treated with either a 10 mM ammonia stress with bolus addition of NH 4 Cl or an equal basal media bolus in an ambr250 system. The two-level factorial design was selected to simulate common end of culture ammonia concentrations and evaluate the age-dependent stress response to ammonia.…”

Section: Introductionmentioning

confidence: 99%

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Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

Synoground

McGraw

Elliott

et al. 2021

Biotechnology Journal

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Background Ammonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed‐batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard‐aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post‐transcriptionally, and this is where future research should focus.

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Section: Resultsmentioning

confidence: 99%

“…Transcriptome analysis was also used to determine the underlying causes of metabolic shifts in amino acid metabolism observed in this study and elsewhere. [ 44 ]…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

Synoground

McGraw

Elliott

et al. 2021

Biotechnology Journal

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“…The ambr250 bioreactors are scalable vessels representative of common industrial conditions; allowing the effects of genetic variations to be assessed with respect to the phenome. In addition to general culture performance, the multi-vessel configuration allows for the modified cells to be characterized under stresses, such as elevated ammonia, lactate, or osmolarity, in parallel [31][32][33]. As a well-controlled process was necessary for these genome studies, it was also important that the PID settings were sufficiently robust to provide a consistent and well-controlled environment.…”

Section: Introductionmentioning

confidence: 99%

PID controls: the forgotten bioprocess parameters

et al. 2022

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The ambr250 high-throughput bioreactor platform was adopted to provide a highly-controlled environment for a project investigating genome instability in Chinese hamster ovary (CHO) cells, where genome instability leads to lower protein productivity. Development of the baseline (control) and stressed process conditions highlighted the need to control critical process parameters, including the proportional, integral, and derivative (PID) control loops. Process parameters that are often considered scale-independent, include dissolved oxygen (DO) and pH; however, these parameters were observed to be sensitive to PID settings. For many bioreactors, control loops are cascaded such that the manipulated variables are adjusted concurrently. Conversely, for the ambr250 bioreactor system, the control levels are segmented and implemented sequentially. Consequently, each control level must be tuned independently, as the PID settings are independent by control level. For the CHO cell studies, it was observed that initial PID settings did not resulted in a robust process, which was observed as elevated lactate levels; which was caused by the pH being above the setpoint most of the experiment. After several PID tuning iterations, new PID settings were found that could respond appropriately to routine feed and antifoam additions. Furthermore, these new PID settings resulted in more robust cell growth and increased protein productivity. This work highlights the need to describe PID gains and manipulated variable ranges, as profoundly different outcomes can result from the same feeding protocol. Additionally, improved process models are needed to allow process simulations and tuning. Thus, these tuning experiments support the idea that PID settings should be fully described in bioreactor publications to allow for better reproducibility of results.

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Precision fermentation with mass spectrometry‐based spent media analysis

Dodia

Sunder²,

Borkar³

et al. 2023

Biotech & Bioengineering

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Optimization and monitoring of bioprocesses requires the measurement of several process parameters and quality attributes. Mass spectrometry (MS)‐based techniques such as those coupled to gas chromatography (GCMS) and liquid Chromatography (LCMS) enable the simultaneous measurement of hundreds of metabolites with high sensitivity. When applied to spent media, such metabolome analysis can help determine the sequence of substrate uptake and metabolite secretion, consequently facilitating better design of initial media and feeding strategy. Furthermore, the analysis of metabolite diversity and abundance from spent media will aid the determination of metabolic phases of the culture and the identification of metabolites as surrogate markers for product titer and quality. This review covers the recent advances in metabolomics analysis applied to the development and monitoring of bioprocesses. In this regard, we recommend a stepwise workflow and guidelines that a bioprocesses engineer can adopt to develop and optimize a fermentation process using spent media analysis. Finally, we show examples of how the use of MS can revolutionize the design and monitoring of bioprocesses.

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Spent Media Analysis with an Integrated CE-MS Analyzer of Chinese Hamster Ovary Cells Grown in an Ammonia-Stressed Parallel Microbioreactor Platform

Cited by 9 publications

References 0 publications

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

PID controls: the forgotten bioprocess parameters

Precision fermentation with mass spectrometry‐based spent media analysis

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