Fibro™ adsorber chromatography offers quick runs for research and high-productivity single-use purification solutions that scale from process development to manufacturing. We have captured frequently asked questions with answers related to Fibro™ adsorbers and rapid cycling protein A-based fiber chromatography.
Is the profile of product/process-related impurities for Fibro™ chromatography adsorbers similar to that for a protein A resin?
Yes, the purity data is very similar between MabSelect PrismA™ resin and Fibro™ PrismA adsorbers, which both use the protein A ligand. We’ve demonstrated this for HCP reduction, DNA removal, protein A leakage, and aggregate formation. We’ve also carried out work to analyze residual HCP from different feeds, and we found strong alignment between the two. This demonstrates the importance of the ligand in affinity chromatography.
Are there any plans to offer Fibro™ adsorbers in 96-well filter plates for screening purposes?
Not at the moment. HiTrap Fibro™ PrismA adsorber paired with an ÄKTA pure™ or ÄKTA™ avant chromatography system and an autosampler does a great job of high-throughput process development (HTPD) with the ultrafast purification cycles without the need for robotic handling systems. In addition, Fibro™ adsorbers give full chromatograms, delivering a good level of process understanding.
What recommendations do you have for cleaning-in-place (CIP)? Do CIP steps add significant time to cycling times?
We have seen mAb purification processes achieve 200 cycles without any CIP steps, though our recommendation is to run a short CIP step of 30 s or 60 s with 0.5 M 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 cleaning.
How does low titer (< 0.1 g/L) impact the binding capacity? Can I load high volumes onto the Fibro™ adsorber without the material being washed off?
Low titer should not affect the binding capacity. We’ve found that the dynamic binding capacity (DBC) is usually the same with feeds as low as 0.01 g/L. This, in combination with high flow rates, makes Fibro™ adsorbers a good choice for low-titer feeds. However, the titer of the feed will have an impact on the volume loaded onto the unit. Lower titers mean larger volumes of feed per cycle. For that we recommend you use a more rigorous CIP regime of reversed flow, a low pH strip, and a longer exposure to 1.0 M NaOH, on every cycle.
Do you have examples of a workflow with a low-titer feed?
Download the poster High-throughput mAb purification with Fibro™ chromatography for a low-titer feed application example. You can also find guidance on setting up a CIP protocol in the Instructions for use for HiTrap Fibro™ and HiScreen Fibro™ PrismA units.
Does Fibro™ adsorber technology give large elution pools?
The eluate mAb concentrations vary with mAb and process, as with resin-based protein A capture. We’re aiming for an elution volume of 2–3 membrane volumes (MV) for the large-scale GMP-compatible Fibro™ units and for eluate mAb concentrations greater than 10 g/L. The elution pool volume will be 3–4 MV for HiScreen Fibro™ PrismA units and 6–7 MV for HiTrap Fibro™ PrismA units.
How does buffer consumption for Fibro™ chromatography compare to a resin-based process?
Buffer consumption in the chromatography step is highly dependent on binding capacity, so the answer depends on what resin you’re comparing with the Fibro™ adsorber. We expect column or membrane volume per cycle to be comparable for Fibro™ chromatography and resin chromatography. Recent high-capacity resins such as MabSelect PrismA™ resin have higher binding capacities than Fibro™ chromatography and therefore slightly lower buffer consumption. In general, buffer usage for Fibro™ adsorbers will align with existing facility infrastructure for buffer production.
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™ resins. Results will be published as we approach the larger GMP-compatible Fibro™ PrismA unit release. In general, our results have shown that the PrismA protein A ligand has the largest impact on the product stream in this capture step.
How do you see the low-pH viral inactivation treatment performed at process scale with 200 cycles of Fibro™ adsorber capture in a day?
You can tackle the low-pH viral inactivation (VI) process (after capture) in a number of ways, depending on your preference. 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 sublots combining, say, 50 fractions, and either pool after VI or continue to process as individual sublots. Some people using continuous setups are exploring continuous low-pH VI options.
Can you explain how to use Fibro™ adsorbers in a manufacturing-scale process? What are the benefits compared to resin-based chromatography?
The core benefit of Fibro™ adsorber units at manufacturing scale is their flexibility. At larger scales you can use 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 can significantly reduce cost in clinical manufacturing compared to resin-based chromatography. In commercial manufacturing, single-use operation enables reduction of changeover time between campaigns and eliminates cleaning validation, making it a suitable format for multiproduct facilities.
Can sample pretreatment affect Fibro™ adsorber unit lifetime?
Cycle lifetime is directly related to the properties and volume of the mAb harvest loaded on the Fibro™ unit. Multiple examples of >200 cycle lifetimes have been observed with well-clarified feeds at titers of 2–5 g/L. In general, you can use the same sample preparation procedures for resins and Fibro™ units. We recommend you clarify mAb harvest by centrifugation and filtration. For more challenging or low titer feeds, you can use depth filtration 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.
Do Fibro™ PrismA adsorbers have specific requirements for hardware?
Fibro™ PrismA adsorber chromatography 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 will require a reusable holder (not in contact with process stream). The units with holders are designed to work with existing ÄKTA™ chromatography systems.
Can Fibro™ units be used in PCC mode (continuous chromatography)?
In most cases there is no advantage operating Fibro™ chromatography adsorbers in a continuous manner. Due to the fast purification cycles, you can utilize the full lifetime (~ 200 cycles) in a single batch while minimizing the separation unit footprint. Productivity is measured by grams of mAb purified per liter of adsorbent per hour (g/L/h). In a multicolumn setup, 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 using Fibro™ adsorbers over multicolumn resin continuous chromatography, where the load time will be long and allow completion of the rest of the cycle during the sample load.
What are the plans for expanding the fiber-based offerings?
We plan to expand the Fibro™ PrismA portfolio to products for clinical and commercial-scale manufacturing of mAbs. The Fibro™ adsorbers technology platform is applicable beyond mAb capture. Ligands available for resins can also be attached to Fibro™ units. The benefits in bind/elute operation are clear, but Fibro™ adsorbers also present opportunities in the separation of closely related species with gradient elutions. Our aim is to make a large variety of options available in the future.