Effect of continuous feeding of CO2 and pH in cell concentration and product titers in hIFNγ producing HEK293 cells: Induced metabolic shift for concomitant consumption of glucose and lactate

Román, Ramón; Farràs, Mercè; Camps, Marc; Martínez-Monge, I.; Comas, P.; Martínez-Espelt, M.; Lecina, Martí; Casablancas, Antoni; Cairó, Jordi J.

doi:10.1016/j.jbiotec.2018.10.005

Cited by 9 publications

(4 citation statements)

References 26 publications

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“…The online monitoring and control of mammalian cell culture processes are mostly based on bulk signals. Historically, pH or dissolved oxygen (DO) has been related to changes in the cell metabolism and process control strategies (e.g., feeding on OUR) were developed based on these measurements (Hu & Aunins, ; Jiang, Chen, & Xu, ; Romn et al, ). Even novel measurement concepts in the process analytical technology initiative mostly measure bulk compositions and relate this information to cellular regulations and effects.…”

Section: Resultsmentioning

confidence: 99%

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Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

Möller

Bhat

Riecken

et al. 2019

Biotech & Bioengineering

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The influence of process strategies on the dynamics of cell population heterogeneities in mammalian cell culture is still not well understood. We recently found that the progression of cells through the cell cycle causes metabolic regulations with variable productivities in antibody‐producing Chimese hamster ovary (CHO) cells. On the other hand, it is so far unknown how bulk cultivation conditions, for example, variable nutrient concentrations depending on process strategies, can influence cell cycle‐derived population dynamics. In this study, process‐induced cell cycle synchronization was assessed in repeated‐batch and fed‐batch cultures. An automated flow cytometry set‐up was developed to measure the cell cycle distribution online, using antibody‐producing CHO DP‐12 cells transduced with the cell cycle‐specific fluorescent ubiquitination‐based cell cycle indicator (FUCCI) system. On the basis of the population‐resolved model, feeding‐induced partial self‐synchronization was predicted and the results were evaluated experimentally. In the repeated‐batch culture, stable cell cycle oscillations were confirmed with an oscillating G1 phase distribution between 41% and 72%. Furthermore, oscillations of the cell cycle distribution were simulated and determined in a (bolus) fed‐batch process with up to 25×106 cells/ml. The cell cycle synchronization arose with pulse feeding only and ceased with continuous feeding. Both simulated and observed oscillations occurred at higher frequencies than those observable based on regular (e.g., daily) sample analysis, thus demonstrating the need for high‐frequency online cell cycle analysis. In summary, we showed how experimental methods combined with simulations enable the improved assessment of the effects of process strategies on the dynamics of cell cycle‐dependent population heterogeneities. This provides a novel approach to understand cell cycle regulations, control cell population dynamics, avoid inadvertently induced oscillations of cell cycle distributions and thus to improve process stability and efficiency.

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Section: Resultsmentioning

confidence: 99%

“…were developed based on these measurements (Hu & Aunins, 1997;Jiang, Chen, & Xu, 2018;Romn et al, 2018). Even novel measurement concepts in the process analytical technology initiative mostly measure bulk compositions and relate this information to cellular regulations and effects.…”

Section: Red N For Online Cell Cycle Measurementsmentioning

confidence: 99%

Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

Möller

Bhat

Riecken

et al. 2019

Biotech & Bioengineering

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“…All three cell lines displayed a high variation throughout the post‐transfection phase (24 to 72 h) especially for the accumulation of metabolic by‐products that negatively influence productivity such as lactate, glutamate, and ammonia. [ 25 ] Particularly ammonia is associated with cellular toxic effects and can interfere in protein processing and virus replication. [ 26 ] We detected the highest ammonia concentrations in LP cell line, which may indicate an innate metabolic‐based disadvantage for virus production in this cell line.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive mRNA‐sequencing‐based characterization of three HEK‐293 cell lines during an rAAV production process for gene therapy applications

Pistek,

Kahlig,

Hackl

et al. 2023

Biotechnology Journal

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Human embryonal kidney cells (HEK‐293) are the most common host cells used for transient recombinant adeno‐associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub‐cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK‐293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK‐293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in‐process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK‐293 cells.

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“…Finally, the PRT did not change the pH levels between HPL4h and HPL4h-i solutions, but the levels with respect to HPL1 were significantly lower in both cases (Figure 2O), which have a major impact on the cell growth. 34…”

Section: Effect Of Applying a Pathogen Reduction Treatment On Hpl Qua...mentioning

confidence: 99%

Optimization of human platelet lysate production and pathogen reduction in a public blood transfusion center

González¹,

Campos‐Cuerva

Fernández‐Muñoz³

et al. 2022

Transfusion

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Background: Human platelet lysate (HPL) has been proposed as a safe and efficient xeno-free alternative to fetal bovine serum (FBS) for large-scale culturing of cell-based medicinal products. However, the use of blood derivatives poses a potential risk of pathogen transmission. To mitigate this risk, different pathogen reduction treatment (PRT) practices can be applied on starting materials or on final products, but these methods might modify the final composition and the quality of the products.Study Design and Methods: We evaluated the impact of applying a PRT based on riboflavin and ultraviolet irradiation on the raw materials used to manufacture an improved Good Manufacturing Practices (GMP)-grade HPL product in a public blood center. Growth promotion and the levels of growth factors and proteins were compared between an inactivated product (HPL4-i) and a non-inactivated product (HPL4). Stability studies were performed at 4°C, À20°C, and À80°C. Results:The application of a PRT on the starting materials significantly altered the protein composition of HPL4-i as compared with HPL4. Despite this, the growth promoting rates were unaffected when compared with FBS used as a control. While all products were stable at À20°C and À80°C for 24 months, a significant decrease in the activity of HPL4-i was observed when stored at 4°C.

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Effect of continuous feeding of CO2 and pH in cell concentration and product titers in hIFNγ producing HEK293 cells: Induced metabolic shift for concomitant consumption of glucose and lactate

Cited by 9 publications

References 26 publications

Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

Comprehensive mRNA‐sequencing‐based characterization of three HEK‐293 cell lines during an rAAV production process for gene therapy applications

Optimization of human platelet lysate production and pathogen reduction in a public blood transfusion center

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