Commonly, sample concentration is performed first to reduce the overall sample volume, followed by diafiltration. This approach significantly reduces the amount of diafiltration buffer required. However, if a sample is unstable or too viscous at higher concentration, a partial concentration may be performed first, followed by diafiltration. The final concentration step is then performed in the exchange buffer. This method will use more exchange buffer, but will maintain a greater permeate flux due to lower concentration or viscosity, reducing the process time and ultimately protecting sample integrity.
To demonstrate the utility of the Amicon® Stirred Cell for large volume concentration, a 10x concentration was performed, reducing 500 mL of a 0.1 mg/mL BSA solution with 1 M NaCl to a final volume of 50 mL.
The experiment was performed using:
Figure 1.Continuous diafiltration setup using the Amicon® Stirred Cell Selector Valve and Amicon Stirred Cell Reservoir accessories.
For 10x concentration of 500 mL of 0.1 mg/mL BSA in 1 M NaCl
The concentrated sample (now at 1 mg/mL BSA containing 1 M NaCl) was buffer-exchanged to remove the sodium chloride, using the previously described stirred cell accessories.
As previously described, 0.1 mg/mL BSA solution containing 1M NaCl was concentrated to 1 mg/mL BSA using large volume concentration. The concentrated sample (now at 1 mg/mL BSA containing 1 M NaCl), was then buffer-exchanged to remove the sodium chloride by discontinuous diafiltration using the Amicon® Stirred Cell.
Fewer DVs are required to remove sodium chloride using continuous diafiltration compared to discontinuous diafiltration. Data from the preceding table were plotted to show that continuous diafiltration is more efficient than the discontinuous method.
Discontinuous and Continuous Diafiltration Conclusion
Our results show that continuous diafiltration using the Amicon® Stirred Cells enables more efficient buffer exchange with less diafiltration volumes to reach 99% salt reduction compared to discontinuous diafiltration. Furthermore, in continuous mode, the stirred cell does not have to be disassembled between each diafiltration volume.
During continuous diafiltration, the protein concentration stayed constant at 1 mg/mL throughout the process, while in discontinuous diafiltration, the protein concentration fluctuated significantly between 1 mg/mL and 0.5 mg/mL. Therefore, continuous diafiltration is much more gentle than discontinuous diafiltration, as it maintains product stability by keeping the sample concentration and volume constant during diafiltration.
The flexible, easy-to-use Amicon® Stirred Cells are compatible with a broad range of process volumes (up to 400 mL) that can be further expanded with an addition of an external reservoir, which can be used for large volume concentration as well as batch and constant-volume diafiltration.
The new design of the Amicon® Stirred Cells accommodates a wide range of ultrafiltration and microfiltration disc membranes, which can be used to optimize concentration and diafiltration conditions. Unlike centrifugal devices, the pressure-based format provides a gentler method for concentration, reducing the likelihood of shear stress-induced denaturation.
Further, the inclusion of magnetic stirring at the filtration interface greatly minimizes the risk of concentration polarization and fouling.