We developed a large-scale tangential-flow filtration (TFF) process using the ÄKTA readyflux™ XL system, T-series Centrasette™ cassettes, cassette holder, and Omega™ polyethersulfone (PES) filter membranes. This approach enables efficient concentration and diafiltration in downstream processing of biopharmaceutical samples in regulated bioprocessing environments. The process uses a single-use flow path, minimizing product contact with multi-use surfaces, which supports high standards for contamination control.
We achieved a 10-fold reduction in volume (from 500 to 50 L), followed by a four-fold buffer exchange in less than 2 h using 5 m2 of filter surface area.
Why TFF matters in large-scale bioprocessing
Tangential flow filtration is a rapid, efficient method for concentrating and purifying biopharmaceutical products in bioprocesses using ultrafiltration membranes. You can also use TFF for buffer exchange, preparing samples for subsequent purification steps. We designed this process as a complete, disposable solution, using single-use bags for buffer and sample storage. TFF cassettes provide high flux rates, making them suitable for protein concentration applications. You can adapt this process to fit your specific requirements.
We designed the setup as a disposable process, with the filtration system using a disposable flow path, including that of the filter holder.
The system setup is shown in Figure 1. The connection tubing to the filtration system flow kit is shown in Figure 2.
Fig 1. Overview of the single-use process using Xcellerex™ XDM mixer bags, Xcellerex XDUO 100 L mixing system, and ÄKTA readyflux XL filtration system.
Fig 2. T-series Centrasette™ TFF cassettes in a holder connected to the filtration system.
MATERIALS AND METHODS
System components for single-use TFF
- ÄKTA readyflux XL tangential flow filtration system
- T-series Centrasette cassettes
- Omega PES membranes
- Xcellerex XDM mixer bags
- Xcellerex XDUO mixing system
- Single-use tubing flow path and sample distribution plate
Buffers and solutions
- Diafiltration buffer: 2.63 kg citric acid, 4.69 kg glycine, adjusted to pH 4.0 with NaOH in 250 kg distilled water. Buffer was transferred to a 200 L bag
- Equilibration buffer: 525 g citric acid, 938 g glycine, 438 g NaCl, pH 4.0 in 50 kg water.
Sample protein solution
- Sample: 550 g bovine γ-globulin, 5.78 kg citric acid monohydrate, 10.32 kg glycine, 4.82 g NaCl, adj. to pH 4.0 in distilled water to a total weight of 520 kg.
- Sample filtration: ULTA™ Pure SG 30 in. 0.2 µm filter, transferred into a 500 L bag.
System setup
Single-use flow path connecting the tubing to the distribution plate of the cassette holder. There was no contact between where the product passed through the cassettes and the endplates of the holder.
- Recirculation bag: 100 L custom bag for the XDUO 100 mixing system with an added retentate return on top of the XDM bag.
- Bag modification: One of the ¾ in. probe ports was removed from the barbed fitting and was used to mount a 1 in. reinforced silicone tubing long enough to reach the retentate return on the flow kit. This created a submerged return of the retentate to avoid foaming.
- Software: UNICORN™ software methods were used for process control.
Flat sheet cassette and flow path installation
- Installation of cassettes in the holder: On each side of the distribution plate, compressed using the automatic torque functionality of the holder.
- The connection tubing to the filtration system flow kit (Fig 2).
- The recirculation tank was connected to the Feed tank inlet, water to Feed 1, and equilibration buffer to Feed 2. Sample added during the fed-batch operation was connected to Transfer 1 and the diafiltration buffer to Transfer 2.
Flush
- We flushed filters with distilled water at 30% manual feed flow with permeate and retentate fully open, and both directed to waste to 50 L of feed flow.
- The system was drained before conducting the integrity test.
Integrity test
- Installation switch was set to Integrity test and the inlet for integrity testing was pressurized at 2 bar, (29.0 psi, 0.2 MPa).
- The volume of air traveling through the permeate was measured by studying the graduation on the cylinder before and after 1 min. A total of 1500 mL air was collected, which meant the integrity test passed.
Filter equilibration
- Equilibration of the filters as for the flush, but with buffer rather than distilled water, and with a UV zeroing command after 1 min of processing.
- A normalized water permeability (NWP) calculation was also started at the same time and averaged 380 LMH/bar during the final stages of equilibration.
- The system was drained.
Sample filtration process
The aim was a 10-fold concentration at 30 L/min (360 LMH load) constant feed flow with a TMP of 1 bar, 14.5 psi, 0.1 MPa) followed by a four-fold diafiltration at the same filtration parameters. Since the recirculation tank had a 100 L maximum volume, the concentration was run as a fed batch keeping the volume in the feed tank at around 50 L.
The process involved the following steps:
- The XDUO 100 L mixing system weight was tared when the bag was installed.
- The sample connected to Transfer 1 was loaded via the transfer pump at 50% manual flow into the XDUO system until the retentate volume was 60 L.
- The agitator of the XDUO system was started with 50% clockwise stirring.
- Sample was recirculated with permeate closed for 3 min at 25% manual feed flow with the retentate pressure control valve (PCV) fully open.
- The permeate line was opened, feed flow was set to 30 L/min and TMP was set to of 1 bar (14.5 psi, 0.1 MPa); UNICORN software monitored the concentration factor. The feed tank level maintenance setpoint was set to 50 L.
- Concentration phase was manually interrupted at around a concentration factor of 9.8 and the diafiltration commenced under the same running parameters.
- The software monitored the calculated diafiltration factor until it reached 4 and then proceeded to recirculation-only mode with closed permeate and completely open retentate PCV at 30 L/min.
- After 3 min of recirculation, the system was drained—first with feed pump action to drain the recirculation chamber—and then with air assisted draining of the retentate pathway using the transfer pump.
Filter wash
- A 10 L volume of diafiltration buffer was added to the recirculation tank directly after emptying and recirculated for 5 min with the permeate closed.
- Manual feed flow was maintained between 10% and 20% to avoid foam formation, but provide adequate mixing.
Note: No agitator was used because of the low volume.
Results
The results are summarized as follows:
- Achieved a 10-fold reduction in sample volume.
- Completed four-fold buffer exchange.
- Protein yield after concentration and diafiltration: 96.4%.
Table 1 shows the volumetric outputs from the results of the filtration procedure. The permeate was not saved, so its volume is based on the total permeate output from the flow meter. This information was also used to calculate the total amount of sample processed during fed-batch concentration. The volume of the recirculation path was kept the same throughout both concentration and diafiltration.
Table 1. Fraction and volumetric output during the filtration process
| Fraction | Volume (L) |
|---|---|
| Average retentate volume | 53.4 |
| Total permeate flow during concentration | 471.6 |
| Total sample volume loaded during fed-batch | 525 |
| Concentration factor | 9.8 |
| Total permeate flow during diafiltration | 214.0 |
| Diafiltration factor | 4.0 |
Table 2 shows the absorbance at 280 nm (A280) measurements performed, which proportionally corresponds to the protein concentration. Since the permeate volume was not saved, no A280 data is available, but the UV sensor in the permeate flow path detected near zero absorbance.
Table 2. A280 measurements for the different fractions during the TFF process
| Fraction | Volume (L) | A280 | Yield (%) |
|---|---|---|---|
| Start sample | 525 | 1.09 | 100 |
| Concentrated and diafiltered sample | 53.4 | 10.36 | 96.4 |
| Wash sample | 10 | 2.60 | 4.5 |
Fig 3. Concentration and diafiltration during the TFF process. Flux is shown in light blue in LMH (L/m2/h). The feed flow (dark blue) was fixed at 30 L/min and transmembrane pressure (TMP) control (green) remained stable at 1 bar (14.5 psi, 0.1 MPa) throughout.
Figure 3 shows the concentration and diafiltration phases of the TFF process. The initial transfer of sample, recirculation, and drain were omitted. The permeate flux dropped after stabilization of TMP from around 110 LMH to 75 LMH during concentration. At the start of the diafiltration, the flux initially increased and then stabilized. The sharp peaks observed in the middle of the process were due to a process-unrelated artifact. At the start of the diafiltration, the conductivity in the retentate dropped and then leveled out towards the end as NaCl was being washed out. The whole process was finished in approximately 2 h.
Note: We determined the hold-up volume in the flow path, including filters, to be 3.4 L from earlier experiments by measuring the volume that decreased from the recirculation chamber as a result of filling the retentate pathway.
Conclusions
- The filtration application was designed as a single-use process, using XDM mixer bags, XDUO 100 L mixing system, and ÄKTA readyflux XL filtration system.
- The single-use flow path of the filtration system was connected through the tubing connector to the distribution plate of the cassette holder, where the product passes the cassettes without contact with the holder endplates.
- The concentration and diafiltration of bovine γ-globulin were successful, reducing the volume from above 500 to 50 L (10-fold), and changing four buffer volumes via continuous diafiltration in less than 2 h.
- The protein yield was nearly 100% as measured by A280 from samples before and after processing.
CY38038
Selecting Omega PES membranes and cassettes
Choose Omega PES membranes for high flux and protein retention premounted in Centrasette cassettes for scalable, single-use applications.
Frequently asked questions
How do I adapt this process for different proteins?
Adjust buffer composition and pH to match your protein’s requirements. Validate membrane compatibility for your target molecule.
What are the benefits of single-use flow paths?
Single-use flow paths reduce contamination risk and simplify cleaning validation.
How do I monitor protein yield?
Use absorbance at 280 nm to track protein concentration before and after processing.
What factors impact flux decay?
Concentration polarization, membrane fouling, and viscosity. Use appropriate crossflow and periodic flushes.
Next steps and technical support
Contact Cytiva for technical consultation or to discuss optimizing your process design.