The transition from traditional to disposable bioreactors is moving with pace but challenges are faced when mechanical action is required. Dr Kevin Auton, ceo at Cellexus Biosystems shows how such challenges have been overcome with one of the latest bioreactors
The growth of interest in single-use or disposable components for biopharmaceutical production is being driven by the need to reduce the downtime between processes, the ever growing burden of meeting regulatory approval and the need to reduce the costs associated with the validation of each component of a production process.1-5
In many areas, it is relatively straightforward to replace stainless steel and glass systems that must be cleaned- and steamed-in-place (CIP/SIP), with single-use systems.
Other transitions to single-use systems are not so easily achieved. Key challenges are often found when a mechanical operation must be performed within a vessel, particularly if the contents of that vessel must remain isolated from the environment. In traditional bioreactor systems, propellers, valves and probes can all be inserted through seals and bearings in the vessel's bulk head and these can be used to good effect to create high rates of oxygen mass transfer. The internalised components in traditional bioreactors can be sterilised along with the rest of the vessel.
But when replacing the vessel with a disposable system, it is much harder to make these arrangements through the wall of plastic material or without adding complex, magnetically-driven stirrers.
In the field of disposable bioreactors, the application of rocking platforms, particularly for insect and mammalian cell cultures, is well accepted. The use of a gentle agitation process in such systems eliminates the need for the inclusion of a means of agitation by stirring. However, this comes at a cost: the mass transfer of oxygen into solution is related to the energy of the agitation process. For example, oxygen transfer in a shaker flask is 15-30 mmol L-11h-1 and in a stirred bioreactor, it approaches 200-400 mmol L-1h-1.6
The ideal bioreactor is capable of delivering the high rates of oxygen mass transfer achieved in a stirred bioreactor, but without the shear forces generated by stirred systems, so that it can be used for all cell types.
In many laboratories, E. coli is the organism of choice for protein production - it is fast, inexpensive and easy to undertake compared with protein expression in insect or mammalian cells. However, E. coli often produce insoluble proteins in the form of inclusion bodies - a major disadvantage. Many groups routinely express soluble proteins in E. coli by inducing protein expression at a low temperature. Initially cells are grown at a temperature of 37°C.
Prior to induction, the temperature is decreased to 18-20°C once the culture has achieved an appropriate density. Protein expression is then induced with Isopropyl (ß)-D-1-thiogalactopyranoside (IPTG) with the result that a greater percentage of the protein that is expressed is in the soluble form.
In many of these laboratories, researchers grow several shaker flasks in parallel to produce the quantity of cells they require. In small volume flasks (100ml for example), high cell densities are achieved due to the large surface to volume ratio. However, with larger cell culture volumes in a shaker flask (1 litre of culture in a 2 litre flask), the rate of transfer of oxygen is decreased due to the decreased surface area / volume ration and lower densities are achieved. A bioreactor that has the simplicity of a shaker flask but which can achieve higher yields of soluble protein than a flask would be a significant benefit.
Expressing proteins at 18°C is fine if you work in an air-conditioned laboratory. In many laboratories, the presence of incubators, freezers and other devices raises the temperature in unpredictable ways - sometimes to temperatures in excess of 30°C. This makes it hard to control the temperatures of the cultures below ambient without some form of refrigeration.
A system such as the CellMaker Lite developed by Cellexus Biosystems (Figure 1) is available with an accessory to allow the reduction of temperature of the culture from 37°C to 20°C in 50 minutes. This can be achieved even if the laboratories are not air-conditioned. In addition to the heater pad that comes as standard with the CellMaker Lite, the system is available with a powerful refrigeration unit. By working in opposition, these two control processes can be managed to provide very stable temperatures that are as much as 15°C below ambient.
The performance of the low temperature accessory at higher environmental temperatures was assessed by heating a room to 30°C and operating the CellMaker Lite at ambient temperatures and then at 12-18°C below ambient. Temperature readings were taken over a period of several days.
The instrument once fitted with the low temperature accessory was able to reduce the temperature of an 8 litre culture from 30°C to 18°C within 50 minutes, even working against an environmental temperature, which varied from 28-33°C. The CellMaker Lite was able to control the temperature of the culture under these conditions to within + 0.2°C of the set temperature.
The ability of the CellMaker Lite to culture a recombinant E. coli cell line and express the soluble form of a recombinant protein was compared with a 2 litre baffled shaker flask, with a 1 litre working volume. The working volume of the CellMaker Lite was 8 litres and it was fitted with the low temperature accessory.
Both the flask and CellMaker Lite were inoculated with the same seed culture grown over night in LB media, at a ratio of 18 ml of seed culture for each litre of media. Cells were cultured in TB media (Merck), which was pre-warmed in the flask and CellMaker Lite to 37°C prior to inoculation. The antibiotic marker was Kanamycin. The media was supplemented with SE-15 antifoam (Sigma Aldrich) at a final concentration of 0.0005% (v/v). This antifoam was found to have no effect on cell growth up to the highest concentration assessed (0.01% v/v). SE-15 is a silicone-based antifoam that is water miscible and withstands autoclaving. It has been found highly suitable for use with microbial and mammalian cell culture systems. The progress of the culture was assessed by taking samples and measuring the oxygen demand (OD) at 600nm.
Cells were initially grown at 37°C. On reaching the OD of approximately 2, the temperature was lowered to 20°C in both the CellMaker Lite and the shaker flask. Protein expression was induced with IPTG on reaching the target temperature. The CellMaker Lite took approximately 50 minutes to reach the target temperature; the shaking flask took approximately 2 hours. The environmental temperature ranged from 22-28°C throughout.
The CellMaker Lite was initially aerated at 2 litres per minute (0.25 litre / litre culture) until the density began to increase, giving the cells time to adapt to the new media and increased aeration (Figure 2). Thereafter, the aeration was set to 8 litres per minute (1 litre / litre of culture) and the pressure increased from atmospheric (LOW setting) to 50 mBar (HIGH setting). This caused the foam head to diminish significantly, even at this high rate of aeration.
The CellMaker Lite and the shaker flask cultures showed growth profiles until they reached an OD of approximately 2, after which the flask culture's growth rate slowed - possibly due to oxygen limitation. At this point, the temperature was re-set to 20°C and the cultures were induced when they reached this target temperature. The decrease in temperature took approximately 55 minutes in the CellMaker Lite fitted with the low temperature accessory.
Final OD's of 6.5 and 14 were achieved with the shaker flask and the CellMaker Lite cultures respectively, over the same incubation period.
Some 50 ml of each culture were centrifuged. The cell pellets were re-suspended in 4 ml of lysis buffer to release soluble protein (termed total lysate), then centrifuged to remove cellular debris and insoluble protein. The supernatant (termed the soluble fraction) was subjected to Ni NTA purification to isolate the HIS-tagged recombinant protein*; the unbound fraction was collected. The bound fraction contained the HIS-tagged recombinant protein and was eluted from the column (termed the bound fraction). Total lysate, the soluble fraction, unbound fractions (1:20 dilution), and the bound fraction (undiluted and 8x dilution), were analysed by SDS-PAGE (Figure 3)**.
The overall protein yield per litre of culture was four-fold higher in the CellMaker Lite compared with the shaker flask (determined after purification using Ni NTA). The total protein expression was slightly higher in the shaker flask culture, but the yield of the soluble target protein per litre of culture was greater in the CellMaker Lite.
Aliquots of the two final cultures were plated to evaluate loss of plasmid from the cells during culture:
Shaker flask: diluted 1:7
CellMaker Lite: diluted 1:14
Each was diluted further to 10-4 and 10-5. 0.1ml of each was spread on three types of plates: LB plates, LB + Kanamycin, and LF + chloramphenicol.
The results and colony counts are shown in table 1. There was no significant loss of the expression plasmid over the 24-hour incubation. Some of the chloramphenicol resistance may have been lost but the significance of this is questionable.
The study demonstrated the following comparatives outcomes between a disposable reactor and a baffled shaker flask when using an E. coli expression system:
- The yields of cells per litre was more than two-fold higher in the CellMaker Lite with a working volume of 8 litres than the baffled shaker flask with a working volume of 1 litre.
- The yield of soluble protein was four-fold higher in the CellMaker Lite culture than in the shaker flask culture.
- The CellMaker Lite with low temperature accessory was able to reduce the temperature of the 8 litre culture from 37°C to 20°C in less than one hour.
- There was no difference in the molecular weight of the proteins as determined by SDS PAGE analysis.
- There was no appreciable difference in plasmid stability between the two cultures.