Orbitally Shaken Single-Use Bioreactor for Animal Cell Cultivation: Fed-Batch and Perfusion Mode.

Increasing the cultivation volume from small to large scale can be a rather complex and challenging process when the method of aeration and mixing is different between scales. Orbitally shaken bioreactors (OSBs) utilize the same hydrodynamic principles that define the success of smaller-scale cultures, which are developed on an orbitally shaken platform, and can simplify scale-up. Here we describe the basic working principles of scale-up in terms of the volumetric oxygen transfer coefficient (kLa) and mixing time and how to define these parameters experimentally. The scale-up process from an Erlenmeyer flask shaken on an orbital platform to an orbitally shaken single-use bioreactor (SB10-X, 12 L) is described in terms of both fed-batch and perfusion-based processes. The fed-batch process utilizes a recombinant variant of the mammalian cell line, Chinese hamster ovary (CHO), to express a biosimilar of a therapeutic monoclonal antibody. The perfusion-based process utilizes either an alternating tangential flow filtration (ATF) or a tangential flow filtration (TFF) system for cell retention to cultivate an avian cell line, AGE1.CR.pIX, for the propagation of influenza A virus, H1N1, in high cell density. Based on two example cell cultivations, processes outline the advantages that come with using an orbitally shaken bioreactor for scaling-up a process. The described methods are also applicable to other suspension cell lines.

Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems.

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 106 cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h-1 (doubling time = 32 h). Maximum virus titers in the range of 3.28-3.73 log10(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000-3500 virions/cell and productivities of 0.5-2.2 × 1012 virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log10(HA units/100 µL), 1286 virions/cell and 1.4 × 1012 virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.