Pall Allegro 200 L Mixer
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1 Application Note USD 2744 Pall Allegro 200 L Mixer High efficiency, easy to use single-use mixing
2 1. Introduction Single-use technologies offer many advantages to drug manufacturers by eliminating or significantly reducing sterilization or cleaning procedures, reducing risk of contamination between different processes or batches, and providing lower initial capital cost 1,2. A mixer is an essential tool within biopharmaceutical processes for many applications that range from buffer preparation to final formulation. Pall Life Sciences has introduced the Allegro TM 200 L single-use mixer system specifically targeted for biopharmaceutical mixing applications. In this application note, we highlight product features and performance for a range of applications relevant to bioprocessing. The schematic of the Allegro 200 L mixer biocontainer and of the biocontainer within the Allegro tote is shown in Figure 1 and mixer specifications are summarized in Table 1. Table 1: Mixer specifications Specification Details Fill Volume 50 L 200 L Impeller Speed rpm Impeller Design Four angled blades Impeller Rotation Bi-directional Biocontainer Operating Pressure 5-15 mbar Operating Temperature 4-40 C Operating Viscosity Up to 150 cp Baffles Available as option The Allegro 200 L single-use mixer is targeted for mixing applications which include: Buffer preparation Media preparation ph/conductivity adjustment Vaccine formulation Mixing of viscous fluids Protein formulation Final formulation 2 USD 2744
3 Figure 1: Schematic of the Allegro 200 L mixer biocontainer and of the biocontainer within an Allegro tote with baffles Powder Addition Port and Impeller Cover Top Plate SS Shaft Impeller Drain Valve Seal, Seal Housing, Endcap 2. Materials and Methods The mixer was evaluated for three scenarios, visual dye mixing, solid/liquid mixing, and liquid/liquid mixing. For solid/liquid and liquid/liquid mixing, mixing time was monitored via conductivity, turbidity, or UV280 measurements using in-line meters. 2.1 Visual Dye Mixing The initial visual dye mixing was performed to determine whether the mixer had any dead zones. This test was completed by filling the Allegro biocontainer with 200 L of deionized water and adding concentrated Allura Red dye. Mixing was visually monitored to ensure that the entire 200 L was a uniform red color representing no dead zones. 2.2 Solid-liquid Mixing When monitoring solid-liquid mixing, schematics as seen in Figure 2 were used. First, the Allegro biocontainer was filled with the desired quantity of liquid. Next the impeller was started with only liquid in the biocontainer. Then simultaneously the timer was started and the solid was added into the biocontainer. In all cases but one (ammonium sulfate mixing required large amounts of solid for testing), the time to add the solid to the fluid was minimal and thus considered zero. The initial mixing was conducted as the start up mode schematic. In this mode the outlet/drain valve was closed to avoid any trapping of solids in the recirculation lines. When no visual presence of particulates were observed in the biocontainer, the mixing mode was switched to the mixing and measurement step, where the solution was monitored using an in-line sensor installed within the recirculation loop. The recirculation speed was set to 3 L/min, which had negligible effect on the mixing of the solution. Mixing was achieved when the in-line sensor reached within ±5% of the expected value (as compared to a standard) and was stable. Next, the solution was drained as in the drainage and measurement step. The fluid was monitored during drainage to ensure there were no dips or spikes in the measurement, which would represent a dead zone in the mixing biocontainer. USD
4 Figure 2: Mixing methods 1 Start up (powder addition and initial mixing) Flow meter Mixer biocontainer Pump stopped Draining port closed No flow Measurement device conductivity meter/ UV sensor 2 Mixing and measurement step Flow meter Mixer biocontainer Pump Flow direction Measurement device conductivity meter/ UV sensor 3 Drainage and measurement step Flow meter Mixer biocontainer Pump stopped Mixing stopped Measurement device conductivity meter/ UV sensor Gravity/via pump drainage process 4 USD 2744
5 2.3 Liquid-liquid Mixing First, the Allegro biocontainer was filled with the desired quantity of water. Next the impeller was started with only the water in the biocontainer. Then the timer and recirculation loop pump were started as the concentrated solute (see Table 2) was added to the biocontainer, thus mixing was conducted using the experimental setup as shown in the mixing and measurement step. The start of the liquid addition was time = 0. The fluid was being monitored via an in-line monitor within the recirculation loop. When the monitor value was stable and had reached ± 5% of the expected value, the solution was drained as per the drainage and measurement step. The fluid was monitored during drainage to ensure there were no spikes or dips in the sensor measurement values, which would represent the presence of non-uniform mixing in the biocontainer. 2.4 Impact of Baffle Utilization The final experiment was conducted to study the effect of mixing using the three removable baffles. The mixing characteristics were studied by mixing with only water and then altering the fill volume within the biocontainer and the impeller speed. The fill volume was tested at the minimum fill volume, 50 L, and the maximum fill volume, 200 L. The impeller speed was also tested at a low speed, 100 rpm, and the maximum speed, 200 rpm. 3. Results The Allegro 200 L single-use mixer was evaluated by performing mixing studies using solutions that cover various biopharmaceutical applications (see Table 2). 3.1 Visual Mixing Tests were conducted to ensure there were no dead zones within the Allegro single-use mixer. Initially the biocontainer was filled with 200 L of water and the impeller was maintained at 100 rpm speed. Then 8 g of Allura Red dye (concentration 0.04 g/l) was added and mixing was monitored visually (Figure 3). The presence of a homogenous red solution within the biocontainer was achieved within 5 seconds, indicative of the absence of any dead zones. Figure 3: Visual mixing with Allura Red dye (a) Before addition of dye (b) 1 second (c) 2.5 seconds (d) 5 seconds Figure 3 shows the biocontainer was filled to 200 L with water and 8 g of Allura Red dye (concentration 0.04 g/l) was added at time t=0. Complete mixing was achieved within 5 seconds for an impeller rotational speed of 100 rpm. 3.2 Liquid-Liquid Mixing An example of liquid-liquid mixing that was tested was a concentrated solution of sodium chloride (NaCl) mixed into deionized water. In this test, the impeller was spinning before addition of the concentrated liquid salt, thus time = 0 is the start of the liquid addition. Liquid addition took 45 seconds, which represents t = 0 to 0.75 minute. The results in Figure 4 show that complete mixing was achieved in less than one minute (± 5% of the expected conductivity value). For liquid-liquid mixing of two solutions with viscosities close to 1 cp, it was found that neither the impeller speed nor the presence of baffles influence the mixing time. USD
6 Figure 4: Concentrated NaCI solution mixing in 200 L water Normalized Conductivity Time (min) 200 rpm x 200 L baffles 200 rpm x 200 L no baffles 100 rpm x 200 L no baffles Figure 4 shows a graph of a concentrated 200 g/l NaCl solution mixing into 200 L of water. The concentrated salt solution was poured into the mixer during the first 45 seconds (t=0 to 0.75). The mixing time was less than a minute for a range of impeller rotational speeds (rpm). There was no observable difference in mixing time due to the presence or absence of baffles. 3.3 Solid-Liquid Mixing An example of solid-liquid mixing for media preparation applications is shown in Figure g/l of powdered Tryptic Soy Broth (TSB) medium was mixed into 200 L of deionized water. In this experiment, the Allegro biocontainer was filled with 200 L of deionized water and the impeller was set to 200 rpm. Then when the solid was being added into the biocontainer, the timer was started (beginning of solid addition is time = 0). Recirculation of fluid and UV measurement was started when no visual particulates were seen in the biocontainer, and then measurement was continued for about 15 minutes during mixing to ensure a stable reading. UV measurement was also monitored during the draining procedure to ensure there were no dead zones in the biocontainer. Figure 5 shows that the powdered TSB medium was fully mixed within 3 minutes, which was confirmed visually by the absence of particulates. After 3 min, no observable dead zones were noticed during the mixing or the draining process. 6 USD 2744
7 Figure 5: Solid-liquid mixing 30g/L TSB mixing in 200 L water 200 rpm Normalized UV280 Absorbance Time (min) Figure 5 shows the mixing of 30 g/l powdered Tryptic Soy Broth (TSB) medium into 200 L of water. The mixing time is approximately 3 minutes when tested with an impeller speed of 200 rpm. 3.4 Impact of Baffle Utilization Figure 6 shows photographs of mixing demonstrating the effect of baffles. The top pictures show mixing without baffles and the bottom pictures show mixing with 3 baffles. The photographs shows no vortices in any of the mixing cases using baffles, while without baffles show vortices in both the 100 and 200 L fill volume cases. These results show that the baffles break up the circular motion that occurs when mixing with an impeller, and forces a more randomized mixing. The baffles were designed as per standard design guidelines, thus the randomized mixing and break up of circular motion should help break up solid clumps. We recommend use of baffles when mixing solids that tend to clump together, such as fine powders or powdered culture media. Also for applications such as mixing surfactants or protein solutions, the lack of vortices ensures less air entrained into the solutions and thus less foaming during mixing. Figure 6: The effect of baffles No Baffles 50 L 100 rpm 100 L 100 rpm 200 L 200 rpm VORTEX VORTEX 3 Baffles Figure 6 illustrates mixing without baffles shows air bubbles in the 50 L case and shows vortexing in both the 100 and 200 L cases. When baffles are added to these same mixing conditions, there is an absence of vortexing. USD
8 3.5 Summary of Mixing Experiments Table 2: Summary of mixing trials Liquid-Liquid Mixing Liquid Impeller Mixing Mixing Volume Speed Time Solute Application (L) (rpm) (min) 1% (v/v) acetone Final Formulation < < <1 NaCl (final 3 g/l) Conductivity Adjustment <1 NaCl (initial 200 g/l; final 10 g/l) < < <1 200 ml of dye into Final Formulation <1 250 cp corn syrup 1.25% (v/v) Polysorbate Solid- Liquid Mixing 0.17 M NaCl (10 g/l) Conductivity Adjustment < < <1 1 M NaCl (58.44 g/l) <1 Dulbecco s Phosphate Buffer Preparation <5 Buffered Saline (9.6 g/l) 21 g/l Citrate Buffer M Ammonium Sulfate * ( g/l) 5.36 g/l Dulbecco s Media Preparation Modified Eagle s Medium (DMEM) g/l Terrific Broth (high density pellet form) g/l Terrific Broth (low density powder form) 30 g/l Tryptic Soy Broth g/l Aluminum Vaccine Formulation Hydroxide (powder) * In the ammonium sulfate experiment, the mixing time incorporates the powder addition time, which required about 7 minutes to put 26.5 kg of solid into the biocontainer. 8 USD 2744
9 In biopharmaceutical processing, mixing is required for preparation of buffers and culture media, adjustment of solution ph and conductivity, final formulation of product, and maintaining homogeneity of the solution during product hold or a processing step. Generally, buffers and media are prepared by solid-liquid mixing, whereas ph/conductivity adjustment and formulation of product are liquid-liquid mixing. Thus applications could further be classified into two broad categories which are liquid-liquid and solid-liquid mixing. Table 2 summarizes the results of the mixing studies completed for a variety of mixing applications. Both the granular and concentrated salt (NaCl) solutions are typically used for adjusting the conductivity for various buffers and product intermediates. The acetone solution represents mixing low density components as part of final product formulation. The corn syrup and Polysorbate 80 mixing simulate viscous liquid-liquid mixing encountered in final product formulation. Within the solid-liquid mixing section, Dulbecco s phosphate buffered saline, sodium citrate, and ammonium sulfate represent commonly used buffers in pharmaceutical applications. The Terrific Broth (TB), Tryptic Soy Broth (TSB), and Dulbecco s Modified Eagle s Medium (DMEM) are common culture media prepared with low density and slightly hydrophobic powders that are mixed into water to form aqueous solutions. Aluminum hydroxide is commonly used as an adjuvant in vaccine formulation and the single-use mixer was able to re-suspend powdered Aluminum hydroxide into water within 8 minutes. 4. Conclusions The Allegro 200 L single-use mixer has been demonstrated to be capable of mixing a wide variety of liquid-liquid, solid-liquid, and high viscosity solutions. The rectangular shape of the impeller is well-engineered to assist in mixing the solutions. Additionally for some applications, the use of baffles minimizes the vortices created by high impeller speeds, thus enhancing the mixing of powdered material into liquid as well as reducing the air entrapment or foaming. Pall Life Sciences can provide additional support through our Applications R&D team and Scientific and Laboratory Services to support any potential mixing applications. Please contact Pall for further details. 5. References 1 Martin, J. Reducing the Risk of Microbial Contamination. Pharmaceutical Technology Europe. 1 February Boehm, J.; Bushnell, B. Contamination Prevention: How Single-Use Systems Can Ensure a Safe, Clean, and Efficient Bioprocess Environment. Pharmaceutical Technology. 2 June USD
10 AllegroSystems The Single-Use Solution Visit us on the Web at us at United States toll free (USA) phone fax Europe +41 (0) phone +41 (0) fax International Offices Pall Corporation has offices and plants throughout the world in locations such as: Argentina, Australia, Austria, Belgium, Brazil, Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, Mexico, the Netherlands, New Zealand, Norway, Poland, Puerto Rico, Russia, Singapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, the United Kingdom, the United States, and Venezuela. Distributors in all major industrial areas of the world. The information provided in this literature was reviewed for accuracy at the time of publication. Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly. 2011, Pall Corporation. Pall,, Allegro and the Allegro design are trademarks of Pall Corporation. indicates a trademark registered in the USA. Filtration.Separation.Solution.SM is a service mark of Pall Corporation. 5/11, PDF, GN USD 2744
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