Hyperosmolality in CHO culture: Effects on cellular behavior and morphology

Romanova, Nadiya; Niemann, Tarek; Greiner, Johannes F. W.; Kaltschmidt, Barbara; Kaltschmidt, Christian; Noll, Thomas

doi:10.1002/bit.27747

Cited by 13 publications

(24 citation statements)

References 60 publications

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“…According to our limited knowledge, this is the first report that the continuous feeding process can increase the maximum viable cell density. These results may be related to the effect of nutrient levels and osmolality on the cell growth cycle [ 20 ]. However, this result did not reproduce on cell line B.…”

Section: Resultsmentioning

confidence: 99%

Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells

Xiao

Ahmed

Mohsin

et al. 2021

Life

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Chinese hamster ovary (CHO) cells are the most important host system used for monoclonal antibody (mAb) expression. Moreover, the fed-batch culture mode is the most widely used method to increase mAb expression in CHO cells by increasing the amount of feed. However, a high amount of culture feed results in the production of metabolic byproducts. In this work, we used a continuous feeding strategy to reduce metabolic byproducts and improve mouse–human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 expression in Chinese hamster ovary-K1 cells. Moreover, the effects of the feeding strategy on the cell culture and monoclonal antibody production were evaluated in chemically defined suspension cultures of recombinant CHO-K1 cells. Compared with bolus feeding methods, the continuous feeding method did not have any advantages when the feeding amount was low, but with a high feeding amount, the continuous feeding method significantly reduced the concentrations of lactate and NH4+ in the later culture stage. At the end of the culture stage, compared with bolus feeding methods, the lactate and NH4+ concentrations under the continuous feeding mode were reduced by approximately 45% and 80%, respectively. In addition, the antibody C12 expression level was also increased by almost 10%. Compared to the bolus feeding method, the antibody C12 produced by the continuous feeding method had a lower content of high-mannose glycoforms. Further analysis found that the osmolality of the continuous feeding method was lower than that of the typical fed-batch bolus feeding method. Conclusively, these results indicate that the continuous feeding method is very useful for reducing metabolic byproducts and achieving higher levels of mAb production.

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Section: Resultsmentioning

confidence: 99%

Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells

Xiao

Ahmed

Mohsin

et al. 2021

Life

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“…A MitoTracker ® Red CMXRos (Cell Signalling Technology Inc., Danvers, MA, USA) staining was performed, as described in [ 11 ]. In brief, 1.5 × 10 6 cells were stained in 750 µL of equiosmotic medium that contained either 100 nM of the dye or the growth medium as a vehicle control.…”

Section: Methodsmentioning

confidence: 99%

“…This leads to gradients in pH value and nutrient concentration inside stirred-tank bioreactors [ 2 , 3 ] and results in zones of different osmotic conditions; therefore, there is distinct stress for the cells passing through these zones [ 4 , 5 ]. Hyperosmolality can affect production rates and growth behaviors, but both significantly increased product titer [ 6 , 7 , 8 , 9 ] and no observable effects [ 10 , 11 ] have been reported. However, studies consistently report a negative influence of hyperosmolality on cellular growth.…”

Section: Introductionmentioning

confidence: 99%

“…Hyperosmotic exposure negatively impacts the viable cell density (VCD) and causes a pronounced increase in cell size [ 5 , 9 , 11 , 12 , 13 , 14 ]. These effects are usually explained as being a dose-dependent response to hyperosmotic stimulus, leading to a rather homogenous increase in cell size.…”

Section: Introductionmentioning

confidence: 99%

“…This leads to the formation of excessive reactive oxygen species (ROS, such as HO • or O 2− ) [ 17 ]. Mitochondria play a central role in the cellular stress response and numerous aspects of their involvement in counteracting osmotic changes have been well documented in the relevant literature [ 8 , 11 , 18 , 19 , 20 , 21 ]. However, consistent observations pinpointing exactly how mitochondrial density and activity change in osmotically stressed CHO cells remain relatively few and far between.…”

Section: Introductionmentioning

confidence: 99%

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Single-Cell Analysis of CHO Cells Reveals Clonal Heterogeneity in Hyperosmolality-Induced Stress Response

Romanova

Schmitz

Strakeljahn

et al. 2022

Cells

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Hyperosmolality can occur during industrial fed-batch cultivation processes of Chinese hamster ovary (CHO) cells as highly concentrated feed and base solutions are added to replenish nutrients and regulate pH values. Some effects of hyperosmolality, such as increased cell size and growth inhibition, have been elucidated by previous research, but the impact of hyperosmolality and the specific effects of the added osmotic-active reagents have rarely been disentangled. In this study, CHO cells were exposed to four osmotic conditions between 300 mOsm/kg (physiologic condition) and 530 mOsm/kg (extreme hyperosmolality) caused by the addition of either high-glucose-supplemented industrial feed or mannitol as an osmotic control. We present novel single-cell cultivation data revealing heterogeneity in mass gain and cell division in response to these treatments. Exposure to extreme mannitol-induced hyperosmolality and to high-glucose-oversupplemented feed causes cell cycle termination, mtDNA damage, and mitochondrial membrane depolarization, which hints at the onset of premature stress-induced senescence. Thus, this study shows that both mannitol-induced hyperosmolality (530 mOsm/kg) and glucose overfeeding induce severe negative effects on cell growth and mitochondrial activity; therefore, they need to be considered during process development for commercial production.

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“Organized stress” for robust scale‐up of intensified production process with fed‐batch seed bioreactor

Yahia

Piednoir

Dahomais

et al. 2023

Biotech & Bioengineering

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Process intensification has been widely used for many years in the mammalian biomanufacturing industry to increase productivity, agility and flexibility while reducing production costs. The most commonly used intensified processes are operated using a perfusion or fed‐batch seed bioreactor enabling a higher than usual seeding density in the fed‐batch production bioreactor. Hence, as part of the growth phase is shifted to the seed bioreactor, there is a lower split ratio, which increases the criticality of the seed bioreactor and could impact production performance. Therefore, such intensified processes should be designed and characterized for robust process scale‐up. This research work is focused on intensified processes with high seeding density inoculated from seed bioreactor in fed‐batch mode. The impact of the feeding strategy and specific power input (P/V) in the seed bioreactor and on the production step with two different cell lines (CL1 and CL2) producing two different monoclonal antibodies was investigated. Cell culture performance in the production bioreactor has been improved due to more stressful conditions for the cells in the seed bioreactor and the impact of the production bioreactor P/V on the production performance was limited. This is the first reported study highlighting a positive impact of cellular stress in seed bioreactors on intensified production bioreactor with the introduction of the “organized stress” concept.

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Hyperosmolality in CHO culture: Effects on cellular behavior and morphology

Cited by 13 publications

References 60 publications

Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells

Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells

Single-Cell Analysis of CHO Cells Reveals Clonal Heterogeneity in Hyperosmolality-Induced Stress Response

“Organized stress” for robust scale‐up of intensified production process with fed‐batch seed bioreactor

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