Small-volume TFF with ÄKTA readyflux™ system with 1.4” flow kit and flat sheet membrane

We show the efficient filtration of bovine γ-globulin model sample protein starting with the ÄKTA flux S TFF system and scaling up with the single-use ÄKTA readyflux™ instrument, and ¼ inch flow kit designed for use with the system. We used T-series Centrasette™ cassettes with Omega™ flat-sheet membranes for TFF. The process demonstrates fed-batch concentration, and batch concentration followed by diafiltration steps with a 10 L initial sample volume. The ¼ inch flow kit provided the convenience of single-use flow path, pumps, and sensors for pressure, conductivity, temperature, flow, UV, and pH. Using this equipment and the approach described, we succeeded in a 20-fold reduction in volume and performed a 4-fold buffer exchange in only 2 h using 0.5 m² of filter surface area.

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Introduction

Since the discontinuation of flat sheet membrane cassettes from Cytiva, there has been no complete filtration solution for using cassette filters within the Cytiva portfolio. Since joining forces with Pall in 2023, however, Cytiva no longer lacks cassette filters in the offering. Furthermore, the needs of the industry today demand smaller volume TFF solutions in GMP and viral vector spaces — the recently launched ¼ inch flow kit from Cytiva enables the ÄKTA readyflux™ system to work at these lower volumes.

The procedure we describe will be used as a demonstration of a theoretical process at small scale (10 L at 1 g/L bovine γ-globulin protein sample), which could be applied to your process, and features the equipment needed for a complete concentration and diafiltration using Cytiva equipment.

Preceding the application run, the concept was tested at lab scale in ÄKTA flux™ S system. The bovine γ-globulin protein, which ranges from a 150 to 200 kDa (relative molecular mass [Mr] 150 000 to 200 000), is retained on an Omega™ flat-sheet membrane with a 30 kDa molecular weight cutoff (MWCO). We concentrated and diafiltered against the original buffer excluding the original sodium chloride (NaCl) content. Processing of a sample of the protein in question has been used in a large scale application featuring ÄKTA readyflux XL and membrane cassette filters.

A detailed Materials and methods section is found at the end of this document.

Results

A summary of the settings and resulting values from γ-globulin purification on ÄKTA readyflux™ system can be found in Table 1 below.

Table 1. Process settings and resulting values

Table 2 below shows the absorbance at 280 nm (A₂₈₀) measurements we performed, which proportionally correspond to the protein concentration. Since we did not save the permeate volume, no A₂₈₀ data is available, but the UV sensor in the permeate flow path detected near zero absorbance.

Table 2. A₂₈₀ measurements of the protein samples

Figures 1 to 3 below show the equilibration, fed-batch, batch concentration, diafiltration, and recovery phases of the TFF procedure. Figure 1 shows that the TMP control remains stable at 0.3 bar throughout the process. At the start of the diafiltration the conductivity in the retentate clearly drops and levels out towards the end as NaCl is being washed out. The process was finished in a process time of 2 h. Figure 2 shows that the concentration step achieved a concentration factor of 20 and the diafiltration step achieved a diafiltration factor of 4. Figure 3 shows the pressure was stable during each process phase for feed, permeate, and retentate, respectively.

Fig 1. TMP, flux, and conductivity for the phases in the TFF procedure we developed using ÄKTA readyflux™ system, ¼ inch flow kit, and Omega™ flat-sheet membranes: equilibration, fed-batch concentration, batch concentration, diafiltration, and protein recovery phases are shown.

Fig 2. Concentration factor and diafiltration factor for phases in the TFF procedure we developed using ÄKTA readyflux™ system, ¼ inch flow kit, and Omega™ flat-sheet membranes: equilibration, fed-batch concentration, batch concentration, diafiltration, and protein recovery phases are shown.

Fig 3. Permeate, retentate and feed flow pressure for phases in the TFF procedure we developed using ÄKTA readyflux™ system, ¼ inch flow kit, and Omega™ flat-sheet membranes: equilibration, fed-batch concentration, batch concentration, diafiltration, and protein recovery phases are shown.

Conclusions

We demonstrate the application of the ÄKTA readyflux™ ¼ inch flow kit for small volume concentration/diafiltration of a standard process using the ÄKTA readyflux™ and T-series Centrasette™ cassette with Omega™ flat-sheet membrane. The filtration application was designed as a fully single-use process and provided the following:

• The concentration and diafiltration of bovine γ-globulin were successful in reducing the volume from 10 L by 20-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.