Improving Single-Use Bioreactor Design and Process Development: New Research Toward Intensifying Seed-Train and Scale-Up Methods Using 5:1 Turn-Down

In an early October webcast, Surendra Balekai (senior global product manager for bioproduction at Thermo Scientific) discussed improving bioreactor design for a 5:1 turn-down ratio. Balekai has worked for over 17 years with biological manufacturers in designing processing equipment for developing vaccines, blood products, and recombinant proteins.

Balekai’s Presentation
When bioreactors operate at a 5:1 ratio (20% volume), the primary challenges are carbon dioxide build-up in the headspace and fluid mixing at low volume. The CO2 produced will significantly affect the cell culture process by reacting to create an acidic environment that affects cell growth and viability. Dynamically in a low-volume system, the positions of the impeller, spargers, and probes, are all as important as temperature control. The impeller could be oversized and create a relatively larger shear zone at low volumes. If the sparger is in the wrong position, it won’t be able to flush gases out effectively. To provide effective heat transfer at low volumes, it is essential that the vessel is bottom jacketed. Also, probes should be well immersed in the process fluid.

Typically, when an overlay is placed on top of the bioreactor, the system will be unable to flush out all gases built up in the headspace. To solve that problem, ThermoFisher patented a new type of sparging technology called “crossflow sparging.” Gases are introduced at the fluid surface level or a little above it, making it possible to flush metabolic gases (including CO2) out of the bioreactor.

Single-use bioreactors use a drilled-hole sparger (film with holes drilled through it) on the bottom. The number and size of those holes vary according to bioreactor size. They are calculated to allow uniform bubbles to travel throughout the whole cell culture column, providing enough oxygenation to cells while stripping CO2 from a culture.

To achieve better bioreactor performance, the impeller has been modified. Its motor angle was changed to ensure that the impeller does not hit the bioreactor wall while it uniformly mixes cultures to limit shear stress. Also, probe locations are positioned near the bottom of the bioreactor.

We considered mass-transfer efficiency, delivery of oxygen to cells, and stripping of CO2 from cultures. We tested the optimum height for the cross flow sparger in stripping CO2 and found that the greatest efficiency came with a position of 10–12 inches (25–30.5 cm) from the liquid surface. We determined the correct parameters for running a bioreactor both at full volume and at low volume to achieve the same cell-viability results.

With this system, you can start with a 750-mL flask and scale directly to a 50-L bioreactor at 5:1 volume. From there, you can move on to a 2,000-L bioreactor at 5:1 volume (400 L), eventually increasing that volume to the full 2,000 L. You can intensify a seed train either by starting with four 50-L vessels and ending with four 2,000-L bioreactors at full volume or by adding the four 50-L vessels together into one 2,000-L bioreactor for 4× seed concentration.

Innovative Thermo Scientific HyPerforma 5:1 single-use bioreactors are now available with all these features. For older HyPerforma bioreactors, retrofit kits can also provide these features.

Questions and Answers
Do problems arise when the crossflow sparger is submerged as the process volume is increased? The crossflow sparger is effective only at smaller volumes. As bioreactor volume increases, the sparger has to be moved up. It cannot function at high volumes.

Can we upgrade a two-year old HyPerforma 2:1 bioreactor to a 5:1 system? Yes. All HyPerforma bioreactors designed in the past two years can be upgraded with a 5:1 kit.

When will this be available for a 2000-L bioreactor? That is something we are working on for a launch in mid-2017.

Is there an advantage to running 5:1 in continuous bioprocessing? It will be beneficial for scale-up. Whether you use it for continuous processing or regular batch processes, you can save a significant number of seed bioreactors as well as flow space. In downstream performance (whether for batch processes or continuous processing), the bioreactors will perform the same way.

Can I use the same BPC in 2:1 as in 5:1 bioreactors? There are a couple of design changes. With shaft extension, for example, the sleeve length in the bioreactor changes, as does the crossflow sparger’s position. A BPC vessel can be modified to accommodate those changes. The exact same bags cannot be used, but a bag can be modified for a 5:1 bioreactor.

Is the fluid-level overlay pinched off on scale-up? If so, how — by a pinched clamp seal? Either a pinch clamp can seal off the crossflow sparger, or the air line can be disconnected and connected to the top.

More Online
The full presentation of this webcast can be found on the BioProcess International website at www.bioprocessintl.com/ask-the-experts/improving-single-use-bioreactor-design-process-development-new-research-towards-intensifying-seed-train-scale-methods-using-51-turn.

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