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Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor

Significance Statement

The article features a novel bioreactor device, called CellTank, for the culture of animal cells at high cell density. This system is a compact perfusion bioreactor for manufacturing of biologics. It consists of a non-woven fiber matrix continuously perfused with the culture medium, using an integrated centrifugal pump. The matrix, immersed in a reservoir, harbors the cells, which are either anchorage dependent or adapted for suspension.

The purpose of the article study was to evaluate the CellTank potentials to support very high cell densities and biologics production. The work has been performed and supervised in the group of Cell Technology at KTH, Royal Institute of Technology (Sweden) in collaboration with CerCell (Denmark) for the CellTank design and manufacturing, and with Belach (Sweden) for the control system of the culture process. Three perfusion runs of Chinese Hamster Ovary cells producing an IgG1 antibody in CellTank of 150 mL matrix volume are presented. The results include a perfusion run with a cell concentration up to 200 millions cells per mL and two runs with cell arrest by mild hypothermia at densities around 100 to 130 millions cells per mL. The cell density was monitored by dielectric spectroscopy, i.e. bioimpedance.

One of the advantages of the CellTank system is the homogeneous distribution of the liquid flow in the whole bioreactor: homogeneous concentrations of the metabolites in the different parts of the bioreactor were observed despite the very high cell densities. In comparison, other bioreactor technologies, where the cells are immobilized such as hollow fiber bioreactor, are typically subject to nutrient and by-product gradients, creating differential and sub-optimal environments. The other advantages are the compactness, the easiness of operation, and the minimal need of tuning the system for operations.

The 150 mL matrix CellTank has the capacity to support the production of hundreds milligrams protein (for low producing cell lines) to several grams protein (for high producing lines). Larger matrices, up to 2.2 liter using the same flow design, can be used to increase the production capacity if needed. This technology could also be used for virus production. The CellTank is very suitable for the manufacturing of protein for diagnostics, research, early stage development or production. 

About The Author

Dr. Veronique Chotteau [M. Sc. Electrical Engineering, M. Sc. Molecular Biology and Biotechnology, Ph.D. Biotechnology/Automatic Control (Université Catholique de Louvain, Belgium)] has ≥ 25 years of experience in mammalian cell culture including ≥ 10 years in biopharmaceutical industry at Pharmacia Upjohn, Biovitrum (nowadays Swedish Orphan Biovitrum). Her expertise covers process development: perfusion (e.g. recombinant factor VIII ReFacto), fed-batch, stem cell bioprocessing, small-, pilot- and commercial scale, GMP, project management, evaluation of new projects. Since 2008, Dr. Chotteau is Principal Investigator, leading the group of Cell Technology at the Biotechnology Faculty, KTH, Royal Institute of Technology, Stockholm, Sweden. Her group is world-leader in high cell density perfusion of mammalian cell-based processes for biopharmaceutical production, and is highly active in mathematical modeling of culture process, as well as in human stem cell bioprocessing.  

About The Author

Ye Zhang [M. Sc. Biotechnology Eng.] is PhD student in Chotteau’s group.  

About The Author

Per Stobbe [M. Sc. Mechanical Eng.] is CEO of CerCell (Denmark). He has 30 years experience of filtration techniques and mechanical devices.  

About The Author

Christian Orrego Silvander [M. Sc. Biotechnology Eng.], former business and bioprocess application engineer at Belach (Sweden), is currently business developer at Serendipity Innovation, Sweden.

 

Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor. Global Medical Discovery feature

Journal Reference

J Biotechnol. 2015;213:28-41.

Zhang Y1, Stobbe P2, Silvander CO3, Chotteau V4.

Show Affiliations

1School of Biotechnology, Dept. Industrial Biotechnology/Bioprocess Design, Cell Technology Group (CETEG), Royal Institute of Technology, KTH, SE-10691 Stockholm, Sweden. Electronic address: [email protected]

2PerfuseCell, Malmmosevej 19C, DK-2840 Holte, Denmark.

3Belach Bioteknik, Dumpervägen 8, SE-14250 Skogås, Sweden(1).

4School of Biotechnology, Dept. Industrial Biotechnology/Bioprocess Design, Cell Technology Group (CETEG), Royal Institute of Technology, KTH, SE-10691 Stockholm, Sweden. Electronic address: [email protected]

Abstract

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200pF/cm (where 1pF/cm is equivalent to 1×10(6) viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37°C to 29°C was studied during 13 days at cell densities above 100pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31°C was applied at cell density 100pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

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