High shear resistance of insect cells: the basis for substantial improvements in cell culture process design

AbstractMulticellular organisms cultivated in continuous stirred tank reactors (CSTRs) are more sensitive to environmental conditions in the suspension culture than microbial cells. The hypothesis, that stirring induced shear stress is the main problem, persists, although it has been shown that these cells are not so sensitive to shear. As these results are largely based on Chinese Hamster Ovary (CHO) cell experiments the question remains if similar behavior is valid for insect cells with a higher specific oxygen demand. The requirement of higher oxygen transfer rates is associated with higher shear forces in the process. Consequently, we focused on the shear resistance of insect cells, using CHO cells as reference system. We applied a microfluidic device that allowed defined variations in shear rates. Both cell lines displayed high resistance to shear rates up to 8.73 × 105 s−1. Based on these results we used microbial CSTRs, operated at high revolution speeds and low aeration rates and found no negative impact on cell viability. Further, this cultivation approach led to substantially reduced gas flow rates, gas bubble and foam formation, while addition of pure oxygen was no longer necessary. Therefore, this study contributes to the development of more robust insect cell culture processes.

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Reconsider the shear paradigm - stirring and aeration strategies in cell culture processes

10.21203/rs.3.rs-122078/v1 ◽

2020 ◽

Author(s):

Florian Strobl ◽

Mark Dürkop ◽

Dieter Palmberger ◽

Gerald Striedner

Keyword(s):

Cell Culture ◽

State Of The Art ◽

Cell Damage ◽

Shear Resistance ◽

Pure Oxygen ◽

Foam Formation ◽

Rushton Turbine ◽

Stirred Tank Reactors ◽

Shear Rates ◽

Wide Range

Abstract Multicellular organisms cultivated in continuously stirred tank reactors (CSTRs), are more sensitive to the environmental conditions prevalent in suspension culture than to bacteria. Stirring, mandatory for efficiently providing oxygen and simultaneously preventing concentration gradients, is a major shear source. The primary approach for reducing shear-associated cell damage is to employ unique impeller designs, combined with slow stirring speeds, to maintain efficient mixing and aeration. Here, we characterized the shear resistance of insect and CHO cells in suspension. We applied a microfluidic device that allows defined variations in shear rates, and both cell lines displayed resistance to shear rates up to 8.73 × 105 s − 1. The unexpectedly high shear resistance of these cells made it possible to design new cultivation strategies, diametric to state-of-the-art strategies. We applied Rushton turbine-powered CSTRs, typically employed in microbial processes, to both cell cultures, at high revolution speeds and low aeration rates. With this strategy, we significantly reduced aeration and foam formation, and it was no longer necessary to add pure oxygen to ensure efficient aeration. This novel cultivation approach can eliminate a number of existing bottlenecks in state-of-the-art cell culture processes and provide a wide range of possibilities for developing highly efficient, robust next generation cell culture processes.

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Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

Bioresources and Bioprocessing ◽

10.1186/s40643-019-0270-8 ◽

2019 ◽

Vol 6 (1) ◽

Author(s):

Khandaker Siddiquee ◽

Cheng Zhao ◽

Michelle A. Stemler ◽

Bill Zeck ◽

Jeffrey R. Fishpaugh ◽

...

Keyword(s):

Cell Culture ◽

Feeding Strategy ◽

Scale Up ◽

Chinese Hamster ◽

Recombinant Antigen ◽

Small Scale ◽

Cell Culture Process ◽

Antigen Binding ◽

Scale Production ◽

Target Molecule

Abstract Use of Quality-by-Design (QbD) tools is becoming an important part of the bioprocessing industry when developing a process for manufacturing operations to ensure the robustness and reproducibility of the biologic product. In the present study, a QbD tool, Design of Experiments (DOE), was utilized to optimize a bioprocess for the production of a CHO recombinant antigen-binding fragment (rFab) in small-scale bioreactors. DOE studies evaluated percent dissolved oxygen, temperature, and feeding strategy specific to this Chinese Hamster Ovary (CHO) clone. It was determined that these factors influenced cell viability, yield of the recombinant protein, and metabolic byproduct formation. To ensure the quality of the target molecule in the cell-culture process, small-scale purifications and analytical evaluation of the target molecule were completed prior to cell-culture scale-up to ensure that oxidation of the rFab, presence of free light chain, and truncation of thiol group were not observed. Analysis of the purified rFab by mass spectrometry indicated that rFab oxidation occurred under poor cell-culture conditions. PCR profile array results also revealed increased transcription of the oxidative genes Superoxide Dismutase 3, Myeloperoxidase, Dual Oxidase Like 2, Nuclear Receptor Coactivator 7, NADPH Oxidase Organizer 1, Mitochondria Uncouple Protein 3, Eosinophil Peroxidase, Lactoperoxidase Like, Serum Albumin Like, and Glutathione S-Transferase Pi 1 in this CHO strain. The present study suggests a mechanism and pathway for the oxidation of an rFab molecule during cell-culture bioprocess optimization. The present study also demonstrated the importance of utilizing the QbD tool of DOE to optimize the cell-culture bioprocess prior to scaling up into the large-scale production bioreactor.

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Effect of Sialic Acid on Mammalian Cell Culture and Protein Expression: A Potential Productivity Enhancer for Biopharmaceutical Cell Culture Processes

Processes ◽

10.3390/pr8111449 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1449

Author(s):

Xiangsong Chen ◽

Shang Xiao ◽

Jinyong Wu ◽

Jianming Yao

Keyword(s):

Cell Culture ◽

Sialic Acid ◽

Cell Lines ◽

Hek293 Cell ◽

Chinese Hamster ◽

Lactate Production ◽

Production Costs ◽

Cell Culture Process ◽

Cho Cell ◽

Potential Productivity

Improved productivity of the two most commonly used cell lines in the biopharmaceutical industry, such as human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO), could reduce production costs and increase manufacturing capacity. One method for increasing protein productivity is the addition of antioxidants during the cell culture process. In this study, we examined the effect of sialic acid (SA) on one HEK293 cell line and two CHO cell lines. The addition of SA to HEK293 cell led to a higher viable cell density (VCD), viability (Via), and a lower lactate content in the later stage of cultures. Further results showed that SA reduced the reactive oxygen species (ROS), improved cell viability, reduced lactate production, and increased antibody expression by more than 20% in the later stage of the two CHO cell lines cultures. Besides, an optimized dose of SA had no significant effect on acidic variants level aggregation level, N-linked glycosylation pattern, and SA content on antibodies. These results suggest that the addition of SA can improve the productivity of biopharmaceutical cell culture processes.

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Designing a Strategy for pH Control to Improve CHO Cell Productivity in Bioreactor

Avicenna Journal of Medical Biotechnology ◽

10.18502/ajmb.v13i3.6365 ◽

2021 ◽

Author(s):

Zohreh Ahleboot ◽

Mahdi Khorshidtalab ◽

Paria Motahari ◽

Rasoul Mahboudi ◽

Razieh Arjmand ◽

...

Keyword(s):

Cell Culture ◽

Large Scale ◽

Chinese Hamster ◽

Ph Control ◽

Agitation Speed ◽

Cell Culture Process ◽

Co2 Removal ◽

Bioreactor Culture ◽

Product Titer ◽

Culture Ph

Background: Drastic pH drop is a common consequence of scaling up a mammalian cell culture process, where it may affect the final performance of cell culture. Although CO2 sparging and base addition are used as common approaches for pH control, these strategies are not necessarily successful in large scale bioreactors due to their effect on osmolality and cell viability. Accordingly, a series of experiments were conducted using an IgG1 producing Chinese Hamster Ovary (CHO-S) cell culture in 30 L bioreactor to assess the efficiency of an alternative strategy in controlling culture pH. Methods: Factors inducing partial pressure of CO2 and lactate accumulation (as the main factors altering culture pH) were assessed by Plackett-Burman design to identify the significant ones. As culture pH directly influences process productivity, protein titer was measured as the response variable. Subsequently, Central Composite Design (CCD) was employed to obtain a model for product titer prediction as a function of individual and interaction effects of significant variables. Results: The results indicated that the major factor affecting pH is non-efficient CO2 removal. CO2 accumulation was found to be affected by an interaction between agitation speed and overlay air flow rate. Accordingly, after increasing the agitation speed and headspace aeration, the culture pH was successfully maintained in the range of 6.95-7.1, resulting in 51% increase in final product titer. Similar results were obtained during 250 L scale bioreactor culture, indicating the scalability of the approach. Conclusion: The obtained results showed that pH fluctuations could be effectively controlled by optimizing CO2 stripping.

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Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process

Biotechnology and Bioengineering ◽

10.1002/bit.23291 ◽

2011 ◽

Vol 109 (1) ◽

pp. 146-156 ◽

Cited By ~ 100

Author(s):

Jun Luo ◽

Natarajan Vijayasankaran ◽

Jennifer Autsen ◽

Rodell Santuray ◽

Terry Hudson ◽

...

Keyword(s):

Cell Culture ◽

Chinese Hamster Ovary Cell ◽

Chinese Hamster Ovary ◽

Chinese Hamster ◽

Ovary Cell ◽

Cell Culture Process ◽

Lactate Metabolism ◽

Metabolite Analysis ◽

Culture Process

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Mechanistic understanding of CHO cell culture improvement by rosmarinic acid through multi-omics analysis

10.22541/au.162443293.37537837/v1 ◽

2021 ◽

Author(s):

Zhuangrong Huang ◽

Jianlin Xu ◽

Jun Tian ◽

Kathryn Aron ◽

Yueming Qian ◽

...

Keyword(s):

Cell Culture ◽

Cell Cultures ◽

Rosmarinic Acid ◽

Cell Biology ◽

Chinese Hamster ◽

Viable Cell ◽

Cell Culture Process ◽

Cho Cell ◽

Critical Pathways ◽

Omics Analysis

The use of antioxidants in Chinese hamster ovary (CHO) cell cultures to improve monoclonal antibody production has been a topic of great interest. Nevertheless, the mechanisms by which antioxidant pathways are regulated in CHO cells and their effect on metabolism are not fully understood. In this work, we investigated how treatment with the antioxidant rosmarinic acid (RA) improved viable cell density and titer in CHO cell cultures, and attempted to explore the underlying mechanism(s) using transcriptomics and metabolomics. In particular, transcriptomics analysis indicated that RA treatment modified gene expression and strongly affected the MAPK and Akt signaling pathways which regulate cell survival and cell death. Moreover, we observed that these effects did not appear related to an intracellular metabolism change. In summary, this integrated ‘omics analysis has important implications for the role of the antioxidant RA in industrial cell culture processes. The current study also represents an example in the industry of how multi-omics can be applied to gain an in‐depth understanding of CHO cell biology and to identify critical pathways that can contribute to cell culture process improvement and cell line engineering.

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Miniature Bioreactors for Rapid Bioprocess Development of Mammalian Cell Culture

Jurnal Teknologi ◽

10.11113/jt.v59.1569 ◽

2012 ◽

Vol 59 (1) ◽

Author(s):

Mohd Helmi Sani ◽

Frank Baganz

Keyword(s):

Cell Culture ◽

Mammalian Cell ◽

Process Development ◽

Mammalian Cell Culture ◽

Small Scale ◽

Cell Culture Process ◽

Deep Wells ◽

Working Volume ◽

Culture Process ◽

And Control

At present, there are a number of commercial small scale shaken systems available on the market with instrumented controllable microbioreactors such as Micro–24 Microreactor System (Pall Corporation, Port Washington, NY) and M2P Biolector, (M2P Labs GmbH, Aachen, Germany). The Micro–24 system is basically an orbital shaken 24–well plate that operates at working volume 3 – 7 mL with 24 independent reactors (deep wells, shaken and sparged) running simultaneously. Each reactor is designed as single use reactor that has the ability to continuously monitor and control the pH, DO and temperature. The reactor aeration is supplied by sparging air from gas feeds that can be controlled individually. Furthermore, pH can be controlled by gas sparging using either dilute ammonia or carbon dioxide directly into the culture medium through a membrane at the bottom of each reactor. Chen et al., (2009) evaluated the Micro–24 system for the mammalian cell culture process development and found the Micro–24 system is suitable as scaledown tool for cell culture application. The result showed that intra-well reproducibility, cell growth, metabolites profiles and protein titres were scalable with 2 L bioreactors.

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Dependence of the Flue Gas Flow on the Setting of the Separation Baffle in the Flue Gas Tract

Applied Sciences ◽

10.3390/app11072961 ◽

2021 ◽

Vol 11 (7) ◽

pp. 2961

Author(s):

Nikola Čajová Kantová ◽

Alexander Čaja ◽

Marek Patsch ◽

Michal Holubčík ◽

Peter Ďurčanský

Keyword(s):

Particulate Matter ◽

Flue Gas ◽

Gas Flow ◽

Negative Impact ◽

Combustion Process ◽

Cfd Simulations ◽

Piv Measurements ◽

Computational Fluid Dynamics Cfd ◽

Particle Imaging ◽

Combustion Of Solid Fuels

With the combustion of solid fuels, emissions such as particulate matter are also formed, which have a negative impact on human health. Reducing their amount in the air can be achieved by optimizing the combustion process as well as the flue gas flow. This article aims to optimize the flue gas tract using separation baffles. This design can make it possible to capture particulate matter by using three baffles and prevent it from escaping into the air in the flue gas. The geometric parameters of the first baffle were changed twice more. The dependence of the flue gas flow on the baffles was first observed by computational fluid dynamics (CFD) simulations and subsequently verified by the particle imaging velocimetry (PIV) method. Based on the CFD results, the most effective is setting 1 with the same boundary conditions as those during experimental PIV measurements. Setting 2 can capture 1.8% less particles and setting 3 can capture 0.6% less particles than setting 1. Based on the stoichiometric calculations, it would be possible to capture up to 62.3% of the particles in setting 1. The velocities comparison obtained from CFD and PIV confirmed the supposed character of the turbulent flow with vortexes appearing in the flue gas tract, despite some inaccuracies.

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