By Arnaud de Boulard, Principal Scientist
Liquid-liquid mixing is a commonly performed process in biomanufacturing, which requires gentle mixing as biomolecules, such as proteins, tend to aggregate or form foam. We evaluated the Allegro™ 50 L single-use mixer for proper liquid-liquid mixing at low fill volumes. Polysorbate 80 was used as a surfactant, and no foaming nor vortex was observed under the selected mixing speeds and volumes. We also visually assessed homogenization time using methylene blue as a tracer, with all tests reaching homogeneity within 30 s. Finally, we evaluated the residual volume in the jacketed tank.
Introduction
Mixing applications for bioprocessing often require minimal residual volume as well as minimal or no vortex formation and foaming during mixing even at low filling levels. We evaluated the performance of the Allegro™ 50 L single-use mixer in regards to residual volume, foam formation, and homogenization time at different low filling volumes (2 L, 5 L, 10 L, and 15 L) at 39 rpm. A schematic of the documented minimum volumes in the Allegro Mixer is provided in Figure 1. The mixing speed of 39 revolutions per min (rpm) was selected based on this Application note: Liquid-liquid mixing without vortex formation.
Fig 1. Schematic representing the key minimum volumes of the Allegro™ 50 L mixer: minimum volumes for agitation (bottom of impeller blade), sensing (top of probe port), and mixing (top of impeller blade).
Results and discussion
Residual volume determination
The initial and final biocontainer weighed 2.10 kg and 2.18 kg, respectively. The residual liquid weight was calculated as 0.08 kg or approximately 80 mL.
Evaluation of foaming
We did not observe any foam formation during mixing at the different fill volumes (2 L, 5 L, 10 L, and 15 L). Figure 1 shows images from the videos of the 2 L and 5 L fill volume experiments.
Fig 2. Evaluation of foam formation in an Allegro™ 50 L single-use mixer filled with 2 L and 5 L of an aqueous 2% Polysorbate 80 solution at 39 rpm (19.4%).
Evaluation of mixing time
The mixing times for the various fill levels are presented in Table 2. We found that the mixing time required for the solution to become homogenous decreased with increasing fill volume. Images taken from the videos of the mixing process are shown in Figures 3, 4, 5, and 6.
Table 1. Mixing times evaluated visually for the Allegro™ 50 L single-use mixer at 39 rpm and 2 L, 5 L, 10 L, and 15 L fill levels
| Fill volume (L) | Mixing time (s) |
| 2 | 30 |
| 5 | 15 |
| 10 | 10 |
| 15 | 6 |
Fig 3. Mixing of methylene blue at 2 L fill volume at 39 rpm (19.4%). The solution was visually assessed to be homogenous within approximately 30 s.
Fig 4. Mixing of methylene blue at 5 L fill volume at 39 rpm (19.4%). The solution was visually assessed to be homogenous within approximately 15 s.
Fig 5. Mixing of methylene blue at 10 L fill volume at 39 rpm (19.4%). The solution was visually assessed to be homogenous within approximately 10 s.
Fig 6. Mixing of methylene blue at 10 L filling volume at 39 rpm (19.4%). The solution was visually assessed to be homogenous within approximately 6 s.
Conclusions
We have shown that the Allegro™ 50 L single-use mixer can perform proper liquid-liquid mixing at low fill volumes. In addition, we have shown that:
- The methylene blue solutions reached homogeneity within 30 s under the selected mixing speed of 39 rpm (19.4%), with mixing times decreasing with increased fill volume.
- No foaming and significant liquid surface movement was observed under the selected mixing speed of 39 rpm (19.4%).
- The residual volume of the biocontainer was approximately 80 mL.
An Allegro™ 50 L single-use mixer was installed according to the instructions. Polysorbate 80 (Merck) was used as a surfactant to evaluate foaming formation, while methylene blue (VWR Chemicals) was used to evaluate mixing time. A GoPro video camera was used to record the mixing process during the experimental runs.
Residual volume determination
After assembly, the empty biocontainer was weighed, and then filled to the nominal volume. The biocontainer was then emptied by gravity and weighed again. The residual volume was determined by subtracting the initial weight of the biocontainer from the final weight.
Evaluation of foaming
2 L of 2% Polysorbate 80 aqueous solution was added to an empty biocontainer and mixing was started. The mixing process was recorded for at least 15 s. This process was repeated for the 5 L, 10 L, and 15 L fill volumes.
Evaluation of mixing time
First, 2 L of water was added to an empty biocontainer. Then mixing at 39 rpm and video recording were started. Finally, 0.01% methylene blue (1 g/L) was added to the biocontainer. Mixing time was determined from the time that the methylene blue was added until the liquid had become homogenous. The mixing process was evaluated visually for homogeneity and the experiment was repeated for the 5 L, 10 L, and 15 L fill volumes.