Single-Use Bioreactors

single-use bioreactors
Image by skeeze from Pixabay

The single-use bioreactor (SUB) has found increasing favour for cell culturing in recent years, mainly in mammalian cell culture but also for early-stage preparations of inocula,  algae and other microbial processes (Hillig et al., 2014; Zhang et al., 2009). The SUB is generally taking over from conventional batch bioreactors.

The basic advantages are a reduction, even absence of microbial risk, a considerable lowering of operational costs because cleaning isn’t necessary, there is a short turn-around in replacement of units and relatively simple validation.

An SUB is typically made from polyethylene or polyester multi-layer films and foils and these are supplied, usually pre-sterilised by gamma irradiation (Singh, 1999). Three layers is common although many more might be used.  Polyethylene terephthalate (PET) or low density polyethylene (LDPE) are prevalent and also provide some degree of mechanical stability. Barrier properties are provided by layers of polyvinylchloride (PVC) and/or polyvinylamide (PVA) which are commonly found in food barrier applications.  Some bags are metallised to restrict light and provide other gas barrier properties.  Stringent regulations define the contact materials used and each regulatory medical authority such as the European Medicines Agency will define this.

The bag is usually supported for rigidity and of glass or stainless steel. The working volume is extensive, from a range of 1 to 2,000 Litres (Brecht, 2009). There are a wide range of suppliers of such units and these can be readily sourced on the internet.

The main applications include early stage mammalian cell culture process development and inoculum generation (Kalmbach et al., 2011; Oncül et al., 2009) at scales up to 500 L. The original SUBs worked on a slowly rocked platform designed to induce a wave-type motion in the culture fluid promoting good mixing and gas mass transfer. They were described by colleagues as a plastic bag on a wave machine  but minimised shearing of the cells.  The Wave™ bioreactor relies on this principle of rocking motion (Wave Bioreactor, 2007).

The most recent SUB designs now resemble conventional stirred tank bioreactors and are now commonly used for therapeutic antibody and vaccine production up to 2000 L scale. In this design, the stirrer is incorporated into the bag and pre-sterilised too. The stirrer is then mechanically connected to a shaft or moved magnetically. This represents an advance on the wave motion approach to stirring although possible shear means it is intrinsically less gentle.

Issues with single-use bioreactors

One of the principal issues that the SUB suffers is overcoming the restriction of poor oxygen gas transfer. Design and construction constraints mean that insufficient power input is possible to improve both mixing and ultimately gas mass transfer. As with all reactors, when cell densities become high, very often oxygen mass transfer becomes limiting and the cells suffer stress. The reactor then becomes one for perfusion only.

Likewise, carbon dioxide removal is hindered to the same extent and can be an even bigger problem for extending the culture. This gas drops the pH of the medium by formation of carbonic acid. A slight acidification can be sufficiently deleterious on the performance of the bioreactor.  To counter this, the pH must be raised which entails the use of buffering salts that might just for instance, raise salinity if sodium salts (Na+) are used.

A reactor which overcomes a number of limitation above is the CELL-tainer® single-use bioreactor. It not only supports higher cell densities than was possible before but also supports perfusion too.

Measurement and Control

Measurement and control is still feasible using conventional  pH and dissolved oxygen probes even though the bag itself operates as a closed, pre-sterilised system. A variety of sensors based on patches, light probes etc. are available however and can be built into the bag and connected prior to use.

It would be interesting to know if there are any food uses that such a system might be put to use. The production of stomach related microbial products might be of interest.

References

Brecht, R., (2009) Disposable bioreactors: maturation into pharmaceutical glycoprotein manufacturing. Adv. Biochem. Eng. Biotechnol. 115, pp. 1–31.  http://dx.doi.org/10.1007/10.

Hillig, F., Porscha, N., Junne, S., Neubauer, P. (2014) Growth and docosahexaenoic acid production performance of the heterotrophic marine microalgae Crypthecodinium cohnii in the wave-mixed single-use reactor CELL-tainer. Eng. Life Sci. 14, pp. 254–263. http://dx.doi.org/10.1002/elsc.201400010.

Kalmbach, A., Bordás, R., Oncül, A.A., Thévenin, D., Genzel, Y., Reichl, U., (2011) Experimental characterization of flow conditions in 2- and 20-L bioreactors with wave-induced motion. Biotechnol. Prog. 27, pp. 402–409. http://dx.doi.org/10.1002/btpr.516.

Oncül, A.A., Kalmbach, A., Genzel, Y., Reichl, U., Thévenin, D., 2009. Characterization of flow conditions in 2 L and 20 L wave bioreactors using computational fluid dynamics. Biotechnol. Prog. 26, pp. 101–110. http://dx.doi.org/10.1002/btpr.312.

Singh, V., 1999. Disposable bioreactor for cell culture using wave-induced agitation. Cytotechnology 30, pp. 149–158. http://dx.doi.org/10.1023/A:1008025016272.

Wave Bioreactor: Nonproprietary Literature. Wave Biotech LLC: Somerset, NJ, (2007) www.wavebiotech.com/products/wave_bioreactor/literature.html

Zhang, Q., Yong, Y., Mao, Z.-S., Yang, C., Zhao, C. (2009) Experimental determination and numerical simulation of mixing time in a gas–liquid stirred tank. Chem. Eng. Sci. 64, pp. 2926–2933 http://dx.doi.org/10.1016/j.ces.2009.03.030.

 

 

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