Manufacturing Safe and Sustainable Plant Biomolecules using CellMakers

Green Bioactives Ltd’s Quest for Safe and Sustainable Manufacturing Using Cellexus CellMaker.

At Green Bioactives we aim to develop plant cell culture as a biomanufacturing platform of safe and sustainable plant biomolecules. A main limitation in the use of plant cells as a biomanufacturing platform is their higher sensitivity to shear stress compared to microbial and other types of cells, which results in poor plant cell growth rates when growing them in a bioreactor. Our main challenge during our first year of operations was to develop protocols that would allow us to grow our plant cells at scale in an efficient and reproducible manner.

To achieve this, we developed a scale up strategy to increase our biomanufacturing volume from research scale using shaking flasks (50 mL) to a pre-pilot scale using 50 L bioreactors. We wanted to test different bioreactor systems and determine the most suitable option for growing our plant cells before committing to any significant capital investment.

We first tested a 2.5 L stirred glass bioreactor using a standard Rushton impeller. This set up is known to impose significant shear stress on the cells and to compromise the growth of sheer stress susceptible cell types. Also, this system is designed mainly for microbial fermentations, consequently the tank is inoculated via a needle port, which is suitable for microbial cells but not for larger cells, such as plant cells. As a result, the inoculation of the stirred glass bioreactor involved significant manoeuvring of the equipment, the media and the cells increasing the risk for contamination. In this system, our cells grew slowly, and the culture was prone to contamination. We are now planning to test our plant cell cultures using a single use stirred system equipped with a gentler impeller, such as a marine or helical impeller, which is designed to reduce the shear forces during culture agitation to see if we can overcome the limitations associated with the use of Rushton impellers.

We then tried growing our plant cells using a single-use wave bioreactor system. The rocking motion of this type of bioreactor significantly reduces the shear stresses on the cultured cells compared to a stirred system and they have been widely used for mammalian cell culture. Our plant cells managed to grow at acceptable rates (perhaps not as fast as in shaken flasks) and displayed a healthy morphology under the microscope after 12 days of culture. However, on several occasions we spotted some microbial growth in the system between days 13 to 16 in the wave bioreactor.  We set up some controls to pinpoint the source of the contamination and to differentiate between flows in the handling or permeability of the system. To do this, we inoculated a sterile single-use bioreactor bag with medium and a fresh cell inoculum and let it mix in the bioreactor for several minutes. We subsequently pumped out 100 ml of the culture mix in triplicates and cultured them in shaking sterile flasks. Both cultures showed similar growth rate and morphology during the first two weeks of culture. Notably, at the end of the second week of fermentation, we could identify microbial growth in the wave bioreactor but not in the control shake flasks. A possible reason for this contamination in the bioreactor could be a failure of a hermetic seal located between the barbs and the pH/temperature probe connectors. We think that the rocking bioreactor has a good potential to grow shear sensitive cells in shorter culture periods. However, for our application, we required a system that allows for longer culture runs.

We then became interested in evaluating an air-lift bioreactor system because some research groups had reported growing similar plant cell cultures at scale using airlift systems. We wanted to test a single-use air-lift bioreactor system to reduce the workload otherwise associated with preparing and sterilising the system.

When we started using the 8L scale Cellexus CellMaker, we obtained cell growth rates comparable to what we had observe using shaking flask cultures. In this system our plant cells were able to grow to a very high density, and they had a healthy phenotype when observed under the microscope. Notably, we kept our plant cells growing in the Cellexus CellMaker for up to three weeks without experiencing any compromising of system sterility. We obtained similar results on our subsequent runs. We then tested operating the CellMaker system in a semi-continuous manner which allowed us to optimise system use per single-use bioreactor bag. We then scale up the culture to 50 L using a larger CellMaker system. Our cells grew at acceptable rates using this larger set up. However, there is still room for optimising the process further to maximise the plant cell growth rate and the yield of our high-value plant biomolecules to make the most out of the CellMaker system. Our plans now also include using the CellMaker to grow plant cell from different plant species to confirm the transferability and robustness of the system.