Xcellerex XDR single-use, stirred-tank bioreactors are well-characterized systems that deliver a performance comparable with that of conventional bioreactors from process development to manufacturing scale. The technology has been successfully used in a variety of different applications including mammalian suspension cell culture, microbial fermentation and cultivation of adherent cells using microcarriers.
Hosted by Patrick Guertin, Senior Manager, Upstream Process Development and Pilot Plant, Xcellerex, now part of GE Healthcare Life Sciences. Mr. Guertin has 25 years of experience and significant expertise in upstream process development, pilot plant operations and cGMP manufacturing for recombinant therapeutics, monoclonal antibodies and vaccines. His skill set also includes process optimization and scale-up and down procedures in microcarrier, fed-batch and perfusion modes.
What would you say are the key components to successful implementation of a single use system for manufacturing biologics in CHO cells? Are there important tips you could share?
One of the key components for single-use system manufacturing is the system’s ability to control the critical process parameters such as dissolved oxygen (D.O.), pH, temperature and agitation within your desired target range. Then, what are the appropriate or optimal control strategies that will achieve these critical process parameters. Dissolved oxygen for example: select a gas sparge element with a porosity that will provide an adequate kLa, without generating an excess amount of foam or negative levels of interfacial shear. This can be achieved through an appropriate sparge pore size, along with appropriate agitation rate (rpm). Additionally, the XDR controller, with the capability for gas cascading (i.e. air/oxygen blending), look-up tables and PID tuning can benefit this process greatly.
If you were going to transition vaccine production from roller bottles to bioreactors using microcarriers, what steps would be necessary to both determine the appropriate bioreactor configuration? How would you begin to explore this process?
This would begin with an understanding of the cells and the microcarriers you choose to examine. Preliminary studies in spinner flasks are most often a prelude to a bioreactor application. Keep in mind that the choice of microcarrier bead is critical. The specific gravity and diameter of the bead is an important aspect to scaling-up an attached process in any reactor, Much of the work I have performed in the XDR has been with Cytodex I microcarriers. Choose a bioreactor with the ability to maintain your critical process parameters, even during the different phases of the process i.e growth, infection, post infection etc. Then you want to operate the single-use bioreactor with an agitation rate (rpm) and sparge that will not disrupt the health or attachment. You want an agitation rate such that the beads are uniformly suspended without generating excess shear due to high rpm or excessive sparging. There is a special consideration with attached cultures in that you need to prevent the cells from being stripped off of the beads during normal cultivation (potential result of a small kolmorgorov eddy due too high rpm) in addition to preventing the beads themselves from being damaged.
I have heard that there are cost savings to be had with switching to single-use bioreactors in biopharmaceutical manufacturing. Can you please outline how that is achieved?
Xcellerex GE Healthcare Life Sciences has an in depth analysis outlining this model. Some brief examples include the following: Savings are a result of time savings (system prep, system turnaround, labor) and infrastructure savings. Single-use bioreactor bags come gamma-irradiated, therefore you don’t need to perform steam in place (SIP) sterilization operations as with traditional systems. With SS there would also be the need for clean steam generator as part of the infrastructure and with this comes maintenance. For SU, at the completion of a run, the SU bioreactor bag can be inactivated and removed from the vessel. Compared to traditional SS tanks where you would need to perform clean-in-place (CIP) which you’ll also need a CIP skid system for. A change out of all the elastomers and gaskets throughout the tank. These aspects would also result in difference in labor and hours, less labor for the SU systems. These are just a few examples of broad comparison.
When training staff who have been used to stainless steel bioreactors, what are the differences for operations when running single-use bioreactors? How much training is necessary to switch?
The amount or level of training will depend on the relative skill level of the operator on the stainless steel system. For example: The controller on the Xcellerex™ XDR system has various levels of access and capability. If an individual will be taking readings and monitoring the system, this will require less training compared to an individual who is expected modify PID settings and tuning. Regarding the vessel and sampling, this tends to be straightforward. Steps such as needleless syringe sampling or welding on sample bag tend to be faster compared to steam on and steam off apps steps or techniques. The connections to the single-use vessel are typically based on aseptic connectors and/or tube welding which is often not applicable for stainless steel systems. These are just a few examples of the differences in steps and operations.
What are your best tips for succeeding with scale-up from process development to pilot scale and then to large-scale manufacturing? We have a CHO process that delivers well in small-scale but the cell specific productivity decreases when we scale up to a 50 L single-use bioreactor. We have used conventional glass bioreactors during the development phase. The viability is fine but the cells produce more lactate in the 50 L bioreactor and we typically need to add more base to keep the pH level. Our target manufacturing scale is 1000 L. Your advice would be greatly appreciated. Thanks!
One of the first steps is to establish a reliable scale-down model in the bench-top reactors. We have used the XDR-10 in the scale-down mode to design and predict what we will see in the large scale systems. The control methods and hardware are critical. Special attention should be given to the dissolved gasses, both pO2 and pCO2, as these parameters will affect the amount of base required as well. You should examine a sparge porosity you are likely use at large scale. This way you can get a relative understanding of the volume to volume gas requirements. This will also provide process information on the amount of foam that may be generated. Further, this will give an early look at the potential impact of interfacial shear resulting from the sparging. The agitation rate (rpm) should also be closely considered. Often times stress on the cells/culture can alter metabolite profile.
Can you use these bioreactors in perfusion mode? If so, how do you set it up and what other equipment is necessary.
The XDR reactor can be and is currently used in perfusion processes and there are a variety of perfusion systems that can be applied. There are a couple of features that should be remembered for this application. The first one is the connections between the perfusion apparatus to the bioreactor, specifically draw and return. Aseptic connectors or tube welding can be used to make these connections. A second aspect to consider is the choice of using a pump or an alternative fluid transfer mechanism to withdraw fluid. The fluid control is done by setting the system control for feed or harvest based upon vessel weight. Other than some of these features, the technology and operation are consistent with traditional perfusion mode.
I know that you have 2,000L single-use bioreactors and I think that is the largest available. Do you think that there will be in larger sizes available in the future or is this the limit?
You are correct, the 2000 L single-use system is the largest available at this time. However, we believe the Xcellerex platform technology is capable of larger sizes/volumes, but we have not seen a market demand for it. This is likely a result of increased titers or higher producing cell lines in conjunction with perfusion and/or process intensification with the existing sizes
I have heard that there can be problems with leachables in single-use bags and recently read a paper in which Amgen identified bDtBPP as a culprit in lowering viable cell density. How has the issue of leachables been addressed and what are plans moving forward?
To date, we have not seen an issue with cell growth in Xcellerex products. Nevertheless, we have initiated efforts to interface with raw-material suppliers to better understand actions that might need to be taken to manage the specific issue that you have mentioned. These efforts are part of a larger mission to manage the overall supply chain for our products to assure product quality and availability. This is valid for both single-use products and multi-use products.
At a recent conference they discussed problems of inconsistent bubble size from gas spargers and how it affects cells negatively. I believe this is happening to our culture. How do you control bubble size to ensure consistency?
A high quality sparge element with consistent pore sizes is a key feature. There are a few aspects to consider. It could be the inconsistent bubble size, but it could also be a specific sub-set or particular bubble size that is having an impact on the cell health. The range or variations of sparge element pore sizes will obviously dictate the resulting bubbles characteristics and/or foam. It also helps to have the sparge bubbles coming through the shear field of the impeller.