High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome

Zamani, Leila; Lundqvist, Magnus; Zhang, Ye; Åberg, Magnus; Edfors, Fredrik; Bidkhori, Gholamreza; Lindahl, Anna; Mie, Axel; Mardinoğlu, Adil; Field, Raymond; Turner, Richard; Rockberg, Johan; Chotteau, Véronique

doi:10.1002/biot.201800036

Cited by 22 publications

(15 citation statements)

References 65 publications

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“…Interestingly, we reveal here that high shear stress stimulates apoptosis associated with three pathways associated with ER stress, cytoskeleton reorganization, and extra-cellular pathway. We previously revealed oxidative stress at very high cell densities, which could be due to a higher shear stress ( Zamani et al., 2018 ). This is consistent with the present observation of the Gene set enrichment results, where oxidative stress, folate metabolism, and UPR increased with high shear stress.…”

Section: Discussionmentioning

confidence: 99%

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells

Zhan¹,

Bidkhori

Schwarz³

et al. 2020

iScience

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Summary Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.

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Section: Discussionmentioning

confidence: 99%

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells

Zhan¹,

Bidkhori

Schwarz³

et al. 2020

iScience

Self Cite

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“…The cells consume oxygen so fast that they become hypoxic in less than 10 s without sparging (see supplemental information). Further, there are a few lab or pilot scale examples of perfusion cell culture at 240 M cells/ml (Clincke, Molleryd, Samani, et al, 2013 ; Clincke, Molleryd, Zhang, et al, 2013 ; Zamani et al, 2018 ). If the industry is to expand to this cell density, a k L a of over 100 h −1 , or lower cell‐specific oxygen uptake rates, would be required.…”

Section: Resultsmentioning

confidence: 99%

The design basis for the integrated and continuous biomanufacturing framework

Coffman

Bibbo²,

Brower

et al. 2021

Biotech & Bioengineering

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An 8 ton per year manufacturing facility is described based on the framework for integrated and continuous bioprocessing (ICB) common to all known biopharmaceutical implementations. While the output of this plant rivals some of the largest fed‐batch plants in the world, the equipment inside the plant is relatively small: the plant consists of four 2000 L single‐use bioreactors and has a maximum flow rate of 13 L/min. The equipment and facility for the ICB framework is described in sufficient detail to allow biopharmaceutical companies, vendors, contract manufacturers to build or buy their own systems. The design will allow the creation of a global ICB ecosystem that will transform biopharmaceutical manufacturing. The design is fully backward compatible with legacy fed‐batch processes. A clinical production scale is described that can produce smaller batch sizes with the same equipment as that used at the commercial scale. The design described allows the production of as little as 10 g to nearly 35 kg of drug substance per day.

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“…Frequent medium perfusion is critical for maintaining high concentration in suspension cultures to resolve fast nutrient consumption and waste production (Zamani et al, 2018). Medium change in a small‐scale laboratory and clinical cell culture with tens to hundreds of milliliters culture volume is commonly performed manually.…”

Section: Introductionmentioning

confidence: 99%

Miniature auto‐perfusion bioreactor system with spiral microfluidic cell retention device

Au²,

et al. 2021

Biotech & Bioengineering

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Medium perfusion is critical in maintaining high cell concentration in cultures. The conventional membrane filtration method for medium exchange has been challenged by the fouling and clogging of the membrane filters in long‐term cultures. In this study, we present a miniature auto‐perfusion system that can be operated inside a common‐size laboratory incubator. The system is equipped with a spiral microfluidic chip for cell retention to replace conventional membrane filters, which fundamentally overcomes the clogging and fouling problem. We showed that the system supported continuous perfusion culture of Chinese hamster ovary (CHO) cells in suspension up to 14 days without cell retention chip replacement. Compared to daily manual medium change, 25% higher CHO cell concentration can be maintained at an average auto‐perfusion rate of 196 ml/day in spinner flask at 70 ml working volume (2.8 VVD). The auto‐perfusion system also resulted in better cell quality at high concentrations, in terms of higher viability, more uniform and regular morphology, and fewer aggregates. We also demonstrated the potential application of the system for culturing mesenchymal stem cells on microcarriers. This miniature auto‐perfusion system provides an excellent solution to maintain cell‐favorable conditions and high cell concentration in small‐scale cultures for research and clinical uses.

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High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome

Cited by 22 publications

References 65 publications

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells

The design basis for the integrated and continuous biomanufacturing framework

Miniature auto‐perfusion bioreactor system with spiral microfluidic cell retention device

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