Short communicationUse of glucose consumption rate (GCR) as a tool to monitor and control animal cell production processes in packed-bed bioreactors
Introduction
The biotech industry makes an extensive use of mammalian cells for the manufacturing of recombinant proteins for human therapy. Today, fed-batch and perfusion cultures are the two dominant technologies for the industrial operation of mammalian cell culture processes that require large amount of proteins (Hu and Aunins, 1997). Whatever the production technology of choice, development efforts are continuously invested in order to obtain production processes that deliver: high volumetric productivity, batch consistency, and homogenous product quality at competitive costs.
The decision between fed-batch and perfusion is mainly dictated by the biology of the clone and properties of the product, and has to be done case-by-case during the course of the development of a new drug product (Kadouri and Spier, 1997). When the choice is made for a perfusion process, one of the useful culture systems is a stationary packed-bed bioreactor in which cells are immobilized onto solid carriers. This system is easy to operate and with appropriate carriers and culture conditions very high cell densities (107–108 cell mL−1) can be achieved. Since the cells are the source of the biosynthesis of the recombinant protein, cell concentration is usually one key parameter to monitor in order to control the process, and so it is of primary importance to be able to successfully control cell proliferation in order to maximize production efficiency and reproducibility.
When the cells are directly accessible in the bioreactor (e.g. suspension cultures), then direct methods can be applied. The most straightforward and widely used approach is to sample the bioreactor and determine the cell number and viability off-line, by manual or automated cell counts (DePalma, 2002, DePalma, 2003). In order to monitor the same parameter on-line, several direct methods have been more recently developed (Konstantinov et al., 1994, Olsson and Nielsen, 1997). These techniques encompass various approaches, such as: optical techniques based on light absorbance and/or light scattering (Wu et al., 1995, Mac Michael et al., 1987), or in situ microscopy combined with real-time image analysis (Camisard et al., 2002, Guez et al., 2004, Joeris et al., 2002).
However, most of these techniques are not applicable to immobilized cell culture systems (e.g. packed-bed bioreactors) from which information on cell density cannot be directly retrieved during the cultures without disturbing and/or dismantling the packed-bed. In this case direct cell counts are generally not possible and indirect methods are generally preferred. The principle is to measure on-line/off-line a parameter that can be used during the process as a surrogate of cell number, to control process operations.
A variety of strategies have been developed and tested, reviewed in (Ducommun, 2001), with the aim to correlate a measurement of any kind to the cell number, and a non-exhaustive list is provided hereafter for (a) metabolic rates of key nutrients or by-products: glucose consumption and lactate production (Gorenflo et al., 2003, Konstantinov et al., 1996, Wang et al., 2002, Dowd et al., 2001, Ozturk et al., 1997, Riehl et al., 2002), glutamine and amino acids consumption (Sugiura and Kakuzaki, 1998, Racher and Griffiths, 1993, Stoll et al., 1996, van der Pol et al., 1994), oxygen consumption (Eyer et al., 1995, Preissmann et al., 1997, Ruffieux et al., 1998, Yoon and Konstantinov, 1994), carbon dioxide production, or (b) intensity of a signal that can be acquired on-line: culture fluorescence, nuclear magnetic resonance spectroscopy, or dielectric spectroscopy (Olomolaiye et al., 2002, Cannizzaro et al., 2003).
In this study we used a perfusion process based on the high cell density culture of recombinant CHO cells in a pilot-scale packed-bed bioreactor. The cell culture process comprises two distinct phases: a cell growth phase at 37 °C, and a production phase at lower temperature to prevent further cell growth and to promote protein production. Since the cells entrapped in the packed-bed are not accessible by direct sterile sampling, another method was required to monitor the growth phase, to forecast the end of the growth phase, and to define the appropriate timing to switch to a lower temperature and start the production phase. In a previous study (Ducommun et al., 2002a, Ducommun et al., 2002b) it was shown that dielectric spectroscopy could be used as an on-line, in situ direct technique to monitor cell proliferation in this system. The dielectric spectroscopy technique was then used to characterize the metabolism and growth properties of CHO cells cultured in a packed-bed bioreactor. But, for the control of routine manufacturing runs, a simpler, more reliable, and cost-effective tool was needed. One of the most common parameters measured in cell culture (glucose consumption rate) was found to meet these requirements. This paper describes the steps undertaken to verify this approach of implementing the glucose consumption rate of the culture as a tool to monitor and control manufacturing runs. This paper will present results obtained in routine operations for an industrial process at pilot-scale.
Section snippets
Cell culture—experimental system
An external packed-bed bioreactor was used to culture recombinant CHO cells expressing a secreted human glycosylated monomeric protein in a serum free medium (Sigma, C-9486). Fibra-Cel® disk carriers (Bibby Sterilin, UK) were used as matrix to immobilize cells in the packed-bed bioreactor compartment. In the pilot-scale system that was used for this study, the reservoir and external packed-bed had a working volume of 40 and 20 L, respectively (Fig. 1). In order to supply the cells with oxygen
Results and discussion
The glucose consumption rate of the culture (in g kg−1 day−1, Fig. 2) was determined daily based on the residual glucose concentration (g L−1, Fig. 4A), the incoming fresh medium glucose concentration and the medium perfusion rate through the bioreactor (vvd, Fig. 3). The glucose consumption rate (GCR) of the culture increased rapidly during the cell growth phase at 37 °C, and after 12–14 days every bioreactor run reached a GCR level of at least 300 g of glucose per kilogram of Fibra-Cel® disks per
Conclusion
This study illustrated that glucose consumption rate (GCR) can be successfully applied as an indirect method to monitor and control high-density perfusion cultures of CHO cells in packed-bed bioreactors. In this system, we have determined that a GCR value of 300 g of glucose per kilogram of Fibra-Cel® disks per day could be used as a criterion to switch the process from the cell growth phase (at 37 °C) to a production phase performed at a lower temperature in the range of 33.5–32.5 °C, with the
Acknowledgment
The authors wish to thank Dr. Paul Ducommun for his previous work done at Serono on packed-bed bioreactors.
References (34)
- et al.
Real-time in-situ microscopy for animal cell concentration monitoring during high density culture in bioreactor
J. Biotechnol.
(2004) - et al.
Large-scale mammalian cell culture
Curr. Opin. Biotechnol.
(1997) - et al.
Real-time biomass concentration monitoring in animal cell cultures
Tibtech
(1994) - et al.
On-line and in-situ monitoring of biomass in submerged cultivations
Tibtech
(1997) - et al.
Measurement of volumetric (OUR) and determination of specific () oxygen uptake rate in animal cell cultures
J. Biotechnol.
(1998) - et al.
On-line simultaneous monitoring of ammonia and glutamine in a hollow-fiber reactor using flow injection analysis
J. Biotechnol.
(1996) - et al.
Dynamics of recombinant protein production by mammalian cells in immobilized perfusion culture
Enzyme Microb. Technol.
(1998) - et al.
On-line monitoring of an animal cell culture with multi-channel flow injection analysis
J. Biotechnol.
(1994) - et al.
Validated online fermentation monitoring
Genet. Eng. News
(2002) - et al.
In-line characterization of cell concentration and cell volume in agitated bioreactors using in-situ microscopy: application to volume variation induced by osmotic stress
Biotechnol. Bioeng.
(2002)