On May 13 2020 Oliver Hardick presented the webinar Rapid cycling protein A based fiber chromatography to address emerging downstream bottlenecks. Here we highlight and respond to the most common questions raised during the webinar.
Is the profile of product/process related impurities for Fibro chromatography similar to that for a protein A resin?
Yes, the purity data is very similar between MabSelect PrismA Resin and Fibro PrismA. We have demonstrated this in over 50 industrial mAbs analyzing HCP reduction, DNA removal, protein A leakage, and aggregate formation. We have also carried out work to analyze residual HCP from different feeds after MabSelect PrismA and Fibro PrismA which shows strong alignment. This demonstrates the importance of the ligand in affinity chromatography.
Are there any plans to launch Fibro in 96-well filter plates devices for screening purposes?
Not at the moment. HiTrap Fibro PrismA paired up with ÄKTA pure or ÄKTA avant and an autosampler does a great job of HTPD work with the ultrafast purification cycles without the need for robotic handling systems. In addition, Fibro results in full chromatograms delivering another level of process understanding which is not achieved from RoboColumns or PreDictor plates.
Are there recommendations in place for cleaning-in-place and what do those look like? I imagine CIP steps would add significant time to cycling times?
We have seen processes achieve 200 cycles without any CIP steps, though our recommendation is to run a short CIP step of 30 or 60 second with 0.5 or 1 M NaOH every cycle. This is achievable within the 5 to 8 min cycle time Reversing the flow of the CIP step and re-equilibration will also improve the efficiency of the cleaning step.
How does low titer (< 0.1 g/L) impact the binding capacity? Can high volumes be loaded onto the Fibro units, without the material being washed off?
There will be no impact. The DBC exhibited is usually the same and we have demonstrated this with feeds as low as 0.01 g/L. This in combination with high flow rates makes Fibro chromatography a good choice for low titer feed. However, the titer of the feed will have an impact on the volume loaded onto the unit. With lower titer feed meaning larger volumes of feed per cycle, we would recommend a more rigorous CIP regime with reversed flow, a low pH strip, and a longer exposure to 1.0M NaOH is used on every cycle.
Please contact your local sales rep for more information that can be shared from the product presentation on application examples of low titer/high volumes. You can also find the Instructions for use on the web page.
Does the Fibro chromatography technology give large elution pools?
The eluate mAb-concentration vary with mAb and process as it does with resin-based protein A capture. We are aiming for an elution volume of 2 to 3 MV for the large scale GMP compatible Fibro units and achieve eluate mAb concentration of greater than 10 g/L on the large Fibro units. For the small scale Fibro units, the elution pool volume will be larger, 3 to 4 MV for HiScreen Fibro PrismA and 6 to 7 MV for HiTrap Fibro PrismA.
Resin chromatography has column efficiency test such as HETP to monitor the column integrity. Are there such tests for Fibro PrismA?
All GMP compatible Fibro units will be integrity and efficiency tested before dispatch in sanitized form ready for immediate use. The user can also do this with a breakthrough test (surrogate to HETP) details of which will be supplied with purchased units.
Will the buffer consumption be larger than for a resin-based process?
Buffer volume consumption in the chromatography step is highly dependent on binding capacity, so it depends on which resin you compare with. From a column volume/matrix volume per cycle, perspective we expect Fibro chromatography to be as good or better. Recent high capacity resins such as MabSelect PrismA have higher binding capacity and therefore also slightly lower buffer consumption. The buffer usage in Fibro will not be out of line with existing facility infrastructure for buffer production. We are targeting buffer consumption around 0.5 to 0.6 L/g and this has been achieved in our collaborations.
Do you have any data on viral clearance of the Fibro PrismA unit?
Viral clearance data is very similar to that achieved with MabSelect PrismA. Results will be published as we approach the larger GMP compatible Fibro PrismA unit release. This is another demonstration of the importance of the ligand in this step. It is the PrismA ligand that has the largest impact on the product stream in this product capture operation.
How do you see the low pH, viral inactivation treatment performed at process scale with 200 cycles of Fibro in a day?
The low pH viral inactivation process post Fibro can be tackled in a number of ways depending on the preference of the user. Many users will treat the 200 eluate fractions as a single lot collected in a single tank, as the process durations are in-line with current resin column operations. Others will split this into smaller sub lots combining say 50 fractions and either pool after VI or continue to process as individual sublots. Some users implementingcontinuous setups are exploring continuous low pH VI options.
Can you explain how Fibro will be used in a manufacturing scale process? What are the benefits compared to resin based chromatography?
The core benefit of Fibro at manufacturing scale is its flexibility. At larger scales you can choose to utilize the fast purification cycles to shorten processing times for the Protein A step compared to resin based chromatography. This saves time for other operations. Alternatively, you can use the unit’s full lifetime (~ 200 cycles) for a single upstream batch reducing the amount and costs of consumables. This enables cost efficient single batch chromatography that can significantly reduce cost in clinical manufacturing compared to resin-based chromatography. In commercial manufacturing the single-use operation enables reduction of lengthy changeover between campaigns and eliminates cleaning validation making it an attractive format for multiproduct facilities.
Can sample pretreatment effect Fibro unit lifetime.
Cycle lifetime is directly related to the properties and volume of the mAb harvest being loaded on the fibro unit. Multiple examples of >200 cycle lifetimes have been observed with well clarified feeds at titres of 2-5g/L. In general, the same sample preparation procedure used for resins can be applied on the Fibro unit. We recommend mAb harvest to be clarified by centrifugation and filtration. Working with more challenging or low titer feeds, DEPTH filtration can be used to efficiently remove process related impurities. We have seen that for certain challenging feeds adding a step to remove charged particles including DNA/ HCP or chromatin (e.g. charged DEPTH filters, diatomaceous earth filter aid, or selective precipitation) will significantly increase lifetime of the Fibro unit
Does the use of Fibro PrismA set any specific requirements for hardware?
Compatibility with existing hardware has been at the center of development for the first generation of Fibro PrismA. Fibro PrismA uses the same buffer components and volumes, chromatography systems, infrastructure, and ligands as resin chromatography, ensuring a good fit with existing biomanufacturing facilities. The upcoming large-scale Fibro PrismA units are designed to work with ÄKTA ready single-use flowpath system.
Could Fibro units be used in PCC mode?
In most cases there is no advantage to operating Fibro in this manner. Fibro has all the productivity benefits using a traditional single column setup. Due to its fast purification cycles you can utilize the full lifetime (~ 200 cycles) in a single batch as well as minimizing the separation unit footprint. Productivity is measured by g/L/h grams of mAb purified per liter of adsorbent per hour. In a multicolumn set up you multiply both the grams and the liters so the impact is minimal.
Note that for very low titer feeds (< 0.5 g/L) there may still be an advantage to the Fibro approach over the multicolumn resin chromatography, where the load time will be long and allow the completion of the rest of the cycle during the sample load.
How is Cytiva planning to expand its fiber-based offering?
In 2021, the Fibro PrismA portfolio will expand to support the clinical and commercial-scale manufacturing of mAbs. The Fibro technology platform is applicable to a wide range of applications beyond mAb capture. All ligands available for resins may also be attached to Fibro units. The benefits in bind/elute operation are clear but Fibro also has demonstrated interesting opportunities in the separation of closely related species with gradient elutions. It is likely that a large variety of options will become available in the future.
The macroporosity of the base matrix gives Fibro technology a strong potential for use in purify viral vectors for vaccines and cell and gene therapy therapies, such as AAV and Lentivirus. The Fibro platform is planned to include products for other large entities like vesicles, plasmids, and mRNA.
If you have a particular commercial processing challenge that requires a different ligand that may see a significant benefit from Fibro then please reach out to your local Cytiva rep to discuss this further.