Catch the WAVE – A discussion on using the WAVE disposable bioreactor for research and manufacturing operations

Perfusion cultures can be run using Cellbag bioreactors with integrated filter for cell retention. Harvest can be withdrawn directly through the filter, using a harvest line that ends in a luer connector at the bag wall. The filter has a nominal pore size of 7 µm and floats on top of the culture liquid. The lateral movement during the rocking of the bioreactor delays cell attachment thus, improving filter lifetime. In WAVE 25 an improved perfusion control is available, which allows continuous harvest and has an auto-calibration function to accurately maintain the perfusion rate. The fact that no recirculation loop is required makes this option quick and simple to set up. The integrated cell retention filter is available for culture volumes from 1 to 25 L.
Such a setup has been tested for insect cells, resulting in very high cell concentrations and recombinant protein production. Details can be found in: Wang L. et al. 2012 Mol. Biotechnol. 52(2):170-179

For an option that allows an exchange of the filter during the perfusion process, an external hollow fiber cartridge can be used to retain the cells. A study on the use of this cell retention can be found in: Clincke M. et al. 2013 Biotechnol. Prog. 29(3):754-767.

To maintain hypoxic culture conditions in the WAVE Bioreactor, it is possible to connect nitrogen instead of compressed air to the gas mixer.

The WAVE Bioreactor can be used for the large scale cultivation of adherent cells and manufacture of viruses. We recently scaled a microcarrier based Vero cell culture from 10 L working volume in WAVE 20/50 to 50 L working volume in WAVE 200. Comparable cell growth was obtained at both working volumes and the virus concentrations reached 10E+09 TCID50 units/mL. The data were presented at the Vaccine Technology Conference in Albufeira in May 2012 and are also available as application note.

The application notes can be downloaded from the GE Healthcare Life Science catalog (www.gelifesciences.com). Alternatively please contact us if you would prefer a copy mailed directly.
"Scale-up of adherent Vero cells grown on Cytodex microcarriers using ReadyToProcess equipment" 29-0435-48 AA and "Downstream scale-up purification of influenza virus using ReadyToProcess equipment" 29-0435-49 AA

The new system was developed with a focus on reliability and ease of use. The controllers for culture parameters like temperature, pH and DO as well as the liquid handling have been updated. They allow for an intelligent process control, taking into account the Cellbag bioreactor size and the culture volume. If desired the user can also directly access and adjust the PID parameters of the controllers. Furthermore motion profiles for the rocking cycle can be selected, depending if the focus is on maximum efficiency for the gas transfer or low shear conditions for more sensitive cell cultures.
Ergonomics for attachment of the Cellbag bioreactor and handling of the lid have been improved over the previous version, allowing easier and quicker access to the sampling port and air filters without removing the lid. Additionally a tilt function was integrated, which facilitates harvesting without removing the cultivation bag from the tray.
The new WAVE 25 also comes with an extensive documentation package and the new UNICORN software, which is 21 CFR Part 11 compliant as required for use in a regulated environment.
Last but not least the system has been tested in numerous real applications for reliable performance to ensure that hardware, consumables and software work together in an integrated manner.

Multiple hybridoma cell lines have been successfully cultivated in WAVE Bioreactor systems. Using different Cellbag bioreactors, cultivation volumes from < 0.5 L to 25 L can be run on the same
instrument. After an initial characterization of the cultivation process in batch mode, a process optimization targeting perfusion is a viable option to increase the volumetric productivity of hybridomacultures.
An example for such a perfusion process can be found in: Tang Y. et al. 2007. Biotechnol. Prog. 23(1):255-264. The study showed a 10-fold increase of the viable cell concentration when switching from batch to perfusion culture. This resulted in a substantially higher volumetric productivity and an overall 7-fold increase in the amount of antibody produced in perfusion culture.

The floating filter is used to retain cells in the Cellbag Bioreactor during perfusion cultivation or medium exchange. It consists of filter material with a nominal pore size of 7 µm, which is laminated to a plastic film covering the back of the filter. Due to the lower density, the filter floats on top of the cultivation medium. It is connected to the wall of the Cellbag Bioreactor via a harvest tube. The outside port of this harvest tube ends in a Luer connector allowing to attach pump tubing and harvest vessel. Operating a peristaltic pump in the harvest line will create suction and draw liquid through the filter into the harvest vessel. The cells are retained by the filter, the surface of which is largely cleared due to its movement across the culture surface. In this setup no recirculation loop is required, which makes the setup easier. Additionally the cells remain in the controlled environment of the bioreactor throughout the cultivation.
The filter comes in two different sizes, a smaller one for Cellbag Bioreactors with 1 and 5 liter working volumes and a larger one for 10 and 25 liter working volumes.

There are certainly clonal differences in e.g. shear sensitivity or general robustness. The type of recombinant protein should not have a major impact except when expressing receptors or other surface proteins. As with other bioreactors, cells may need adaptation to the agitated environment in a WAVE Bioreactor. Taking special care about equilibrating the bioreactor before inoculum transfer, use of relatively high inoculum concentrations e.g. 4E+05 c/mL and the addition of shear protectants like Pluronic F-68 may help during this phase. Hydrodynamic conditions in the WAVE Bioreactor have been characterized and information is available in the product literature. When transferring from a characterized stirred tank, it is possible to select agitation conditions in the WAVE Bioreactor that result in comparable mixing time and gas transfer rates. This should also be indicative for the shear that cells are exposed to.
When scaling up from a static or shake flask culture and the shear sensitivity of the cell line is not well understood, it is also an option to investigate it in a WAVE Bioreactor by starting at low agitation and increasing it periodically e.g. every two days while cell growth and viability is closely monitored.

The efficiency of transient transfection is very much dependent on the method used for DNA transfer. Most commonly polyethylenimine (PEI) is used as transfection reagent. The optimum DNA:PEI ratio is dependent on the cultivation medium used and has to be established first. Also the maximum PEI concentration the cells tolerate and the requirement for media exchanges before and possibly after transfection need to be tested. An example procedure can be found in: Fliedl L. et al. 2011 J Biotechnol 153:15-21
Transient transfections have been successfully performed in WAVE Bioreactors e.g. Geisse S et al. 2005 J Struct Funct Genomics 6:165-170. Cell cultivation in a bioreactor may be able to increase the expression level due to the improved culture control and also improved options for feeding of the culture. Additionally larger cultivation volumes are handled more easily in a bioreactor than in multiple parallel shake flasks.

When transferring a clone to a new cultivation system, it is important to make the most out of the characterization data generated in the previous one. E.g. what is the optimum seeding concentration, are the cells very sensitive to the cultivation pH, what is the limiting nutrient in the cultivation medium used, how long can the exponential growth phase be maintained, what is the maximum cell concentration reached etc.
As mentioned in an earlier question, it is beneficial for cell adaptation to any bioreactor to inoculate at relatively high cell concentrations and put special emphasis on equilibrating the bioreactor (T, pH, DO) before cell transfer.
A parallel culture in the previously established cultivation system using the same inoculum and cultivation medium should be run. This will facilitate the decision if lack of cell growth or recombinant protein production is to be attributed to the inoculum quality, the cultivation medium or the bioreactor.

CO2 control of the pH in Cellbag Bioreactors occurs via the headspace. Depending on gas flow, bag size and agitation conditions some delay will occur until the gas composition in the headspace is exchanged and the CO2 has dissolved in the cultivation medium. Suggested gas flows and agitation conditions for specific Cellbag Bioreactor sizes can be found in the user manual.
The concentration of CO2 that is required to reach a certain pH, is dependent on the bicarbonate concentration in the medium and the agitation conditions. For a specific set of conditions it can easily be determined experimentally. Once the appropriate CO2 concentration is established, the Cellbag should already be inflated using this gas composition. Starting any controller near the set point of the target variable minimizes the risk for overshooting and ensures that stable operating conditions are reached quickly.
The PID controller parameters for pH and DO control are accessible in WAVEPOD II and WAVE 25. Suggested settings are listed in the user manuals. Experienced users have the option to further tweak the controller settings. One way to speed up the controller response is to increase the P factor. This should be done carefully to avoid oscillation of the controller output. Further information can be found in technical literature about PID tuning.

The problem of cholesterol delivery to NS0 cells was investigated when differences in cell growth were observed in serum containing culture and serum free medium supplemented with lipids and synthetic cholesterol. The conclusion of the authors was that the interaction of the lipid carrier, in this case methyl-beta-cyclodextrin, with the bag film led to cholesterol depletion of the culture. This problem was solved by reducing the excess ratio of lipid carrier to cholesterol, which allowed successful cultivation of NS0 cells in the WAVE Bioreactor. Details: Okonkowski J et al. 2007 J. Biosci Bioeng 103(1):50-59.

Answer: In principle a similar approach can also be applied to Vero cells. The difference is that the cells are adherent and require microcarriers for cultivation in the WAVE Bioreactor. A microcarrier often used for Vero cultivation is Cytodex, please see literature reference in my answer regarding the use of the WAVE Bioreactor for the large scale manufacturing of virus for a detailed description of such a process.
The microcarrier culture could be used for the preparation of a cellbank. The cells either need to be detached form the microcarriers or can be frozen directly. Cryoconservation of cells on Cytodex and other microcarriers has been done successfully (Nie Y et al. 2009 Biotechnol. Prog.25(1):20-31).
Cells adherent to the microcarriers can be concentrated by settling, at the same time this also gives the option to exchange the culture medium to a cryoconservation medium.