Successful Methods for Perfusion Process Optimization
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Hosted by: Brandy Sargent
I began the podcast by asking Dr. Castan what makes perfusion a good manufacturing platform? He identified several reasons including short residence times in the bioreactor through adding fresh media and removal of spent media and as a result is perfect for unstable molecules. Perfusion permits processes to be run with high volumetric productivity by maintaining high cell densities for a long period. With perfusion there are many opportunities for process intensification and it is a good way to quickly get started with production, provided you have a good batch media that cells like.
Next I asked if there were any specific product types or situations that are an especially good fit for perfusion? Andreas described three primary areas – unstable products, integrated continuous manufacturing platforms and process intensifications for removing bottlenecks in manufacturing. Examples would be cell bank manufacturing to create high density cell banks, seed train to remove steps or produce high viable cell density inoculum or hybrid processes of both perfusion and fed batch technologies.
We then discussed the importance of optimization for perfusion processes and the various approaches for optimization. Andreas said it is important to optimize processes to gain the product quality and the process economy that you are aiming for. Cell culture media is the most important thing to optimize in perfusion culture. In order to achieve good throughput for your optimization it needs to be performed in a scale down model then results must be verified under bioreactor conditions.
Then I talked to Andreas about a recent poster he authored and presented at ESACT. The poster outlines development of perfusion specific media and designing medium that supports low cell-specific perfusion rates. I asked him to explain what cell-specific perfusion rate (cspr) is and why a low cell-specific perfusion rate is important? Andreas explained that cell-specific perfusion rate is the volume of media added per cell per day. If you assume your medium supports a cspr of 50 picoliter and you want to run the process at 30 million cells/mL your volumetric perfusion rate would be 1.5 bioreactor volumes per day. If you take the same medium with 100 million cells/mL you would need a volumetric perfusion rate of 5 bioreactor volumes per day, which is not feasible for production. A medium with a better depth of cspr of 10 picoliter at a cell density of 100 million cells/mL would have a volumetric perfusion rate of 1 bioreactor volume per day, which is feasible for production.
His team investigated screening methods to develop medium with a very low cspr. They started with basal media, then screened different feed solutions and Cell Boost feeds in batch mode. The Cell Boosts with a positive impact were taken to a DOE study in spin tubes that replicated perfusion conditions, then promising formulations were moved to perfusion processes in WAVE or the XDR bioreactors.
Poster – Perfusion media development for scalable processes
Next I asked him if you can turn a fed-batch medium into a perfusion medium? He said yes, transferring a fed batch media to a perfusion process is a simple process. He lays out the methodology in the previously mentioned poster and they have done this for two cell lines and three fed-batch media and feeds. The methodology is also described in the Biotechnology Process Journal Article, “Repurposing fed‐batch media and feeds for highly productive CHO perfusion processes.”
I followed up by asking how did the methodology work in the case study presented in the poster? Andreas said it was a very fast method with batch screening taking one week, the DOE study took two weeks and within one month they had the composition for a perfusion media that fit the desired clone. They tested two media developed using this process on their internal Herceptin producing cell line and for both media they reached cspr below 20 picoliter. They were able to reduce one media to only 7 picoliter. The cell specific productivity for this process was comparable to what they saw in fed-batch.
We then discussed another approach to optimizing perfusion processes, optimizing the equipment used in process. In another poster presented at ESACT, Andreas presented the use of hydrocyclones for cell separation. I asked him to describe how hydrocyclones work and why they make an attractive alternative to traditional cell separation devices? He said that hydrocyclone devices are comprised of cylindrical and conical parts that allow centrifugal separation provided by feed suspension introduced tangentially at high flow rates into the device. The absence of rotors or moving parts make it an interesting separation alternative to perfusion in long term operation. A simple device that is not prone to clogging as is usually seen with filtration devices
Poster – Hydrocyclone for mAb production in a perfusion single-use bioreactor
I then asked what his experiences were with the hydocyclone in the case study? Andreas explained that they investigated separation efficiencies at different flow rates and different flow concentrations and found that separation efficiencies of 70-80% could be achieved with the current hydrocyclone. This test was run using a perfusion process in an XDR 50 bioreactor at 50 million cells/mL for more than 2 weeks in very stable conditions. It was a very successful test of the device.
I summarized by asking if companies are limited in time and/or resources for optimizing their perfusion processes, what would he focus on first? He said media should be focused on first because as explained earlier a low cspr is key to reaching high volumetric productivity, low volumetric perfusion rates and high product concentrations, all of which result in good process economies. Next you could think about how you could use perfusion to reduce process scale.
Lastly, I asked if he had anything else that he would like to add for our listeners. He said that before moving into perfusion you should ask yourself what makes the most sense for your process. You need to think about your infrastructure, prerequisites, platform, and previous knowledge. Then you can decide where perfusion makes the most sense in your process. In cell bank manufacturing, seed train, or production bioreactor. Media is the most important factor to work with and it is not difficult to use existing media and cell boosts to develop perfusion media that fits your clone.