Stirring the Mix of Single-Use Mixers
Mixing is an essential operation within biopharmaceutical manufacturing and is required for processes involving suspension, dispersion, dissolution, and homogenization. These operations are required for applications ranging from preparation of bacterial and cell-culture media, buffers formulation, adjustments to pH or conductivity, chemical inactivation of viruses, equilibration of thawed or stored solutions, maintaining homogeneity of solutions during product hold or processing steps such as tangential flow filtration (TFF)/diafiltration, all the way to formulation and filling of vaccines, protein intermediates, and final drug products.
As with other steps in biopharmaceutical manufacturing, applying single-use concepts to mixing operations offers many advantages to drug manufacturers. Single-use mixing technologies can eliminate sterilization and cleaning validation efforts by users, eliminate tank and impeller cleaning, reduce contamination risks between batches or between multiple products, as well as reduce turnaround time and provide lower initial capital cost. Generally, media and buffer preparation require solid/liquid mixing and dissolution capability, whereas pH/conductivity adjustment and formulation of product are liquid/liquid mixing applications. It is, therefore, important to consider the application--will mixing be solid/liquid, liquid/liquid, homogenization or multiple different applications? If mixing solids with liquids, is this an easy dissolution or a difficult one? Do the solids tend to sink, float, or clump? If mixing liquids with liquids, is either of high viscosity? How miscible are they? What could be the range and scale of potential future requirements? Can the mixing applications be open to the room environment, or will they require closed service? How critical is the potential for particle generation during mixing? Will filters be used downstream that could control any generated particles? Mixers can also be evaluated for volume range, ease of use and change-out, turnaround time and, for larger-scale applications, even floor space and ceiling height requirements (1).
Single-use mixers can be as simple as a two-dimensional bag on a rocker table, a three-dimensional (3D) bag liner inserted into a mixing drum, or a 3D biocontainer with a tubing loop for hydraulic recirculation via a peristaltic pump or other pump type with an in-line disposable pump head. More sophisticated single-use mixers employ a variety of mechanical drives in closed systems. The latter may be a magnetic or mechanical coupling driving a shaft mounted propeller, a rotating stirrer, a paddle, a stirring rod, or a vibrating surface.
Mixers using shaft-mounted pitched blade turbine impellers on mechanical drives can provide high efficiency mixing suitable for most solid/liquid and liquid/liquid mixing applications due to their direct drive coupling and high torque capabilities. Depending on impeller design and direction of operation, they can provide either up-flow or down-flow, with a combination of axial and radial mixing conditions. They can be top or bottom-mounted, in open or closed systems and can be particularly amenable to automation and incorporation of online sensors. Typical applications include media preparation, virus inactivation, controlled pH/buffer preparation, TFF, final formulation, and filling.
Paddle mixers offer efficient solid/liquid and liquid/liquid mixing in open or closed applications scaled from 5-1000 L. Because they have no moving parts within the liquid (other than the paddle), closed paddle mixers are especially suited to ultra-clean (particle sensitive) applications such as aseptic manufacturing without final sterilizing filters that would otherwise control particle contamination. Typical applications include cell therapy culture media, viscous fluids, chromatography sorbent re-slurrying, as well as suspension/re-suspension during purification and final formulation of aseptic operations.
Magnetic and levitating mixers feature rotating vertical paddle blades that create radial flow patterns only. They provide robust, closed-system mixing for industrial scale solid/liquid and liquid/liquid applications such as media and buffer preparation. Both can be provided as a universal, portable drive for applications from 5-3000L where one drive unit services all vessel volumes. Magnetic mixers are well suited to industrial applications where they provide sufficient power for mixing of heavy power loads, especially dry powder culture media dissolution. Levitating mixers, like paddle mixers, are especially suited for ultraclean requirements such as cell therapy media preparation, late-stage downstream processing, and aseptic vaccine manufacturing applications that are not amenable to final sterilizing filtration (and particle removal). They feature no shafts, seals, or bearings inside the mixing bag, thus eliminating friction between moving parts that could otherwise potentially generate particles.
Stir-rod or wand-type mixers are generally used for small-scale open or closed mixing scalable from <1 L to 200 L, where they offer a balance of economy with mixing ability. They are suitable for liquid/liquid mixing and most solid/liquid mixing applications such as easy media and buffer preparation, purification, suspension/re-suspension, TFF/diafiltration, and final formulation
Jet mixers are light and compact closed systems employing a housing around a magnetic stirrer bar, with the housing provide directional jet exit ports towards the corners of a cubical mixer biocontainer (with fluid being sucked in through the top of the housing). This makes them best suited for liquid/liquid mixing applications from 50-200 L where they couple ease of use, portability, small footprint, and economy with adequate mixing efficiency. These mixers include a magnetic driver and controller positioned on a trolley that fits easily beneath standard cubical containers. The trolley can easily be detached from the container and reinstalled, even while the mixing bag is full. Typical clean applications include homogenization of harvest fluids and re-suspension/re-homogenization of stored fluids in both product development and cGMP environments.
Evaluation and Qualification
Beyond sufficient mixing at desired scales, mixers should be assessed for ease of set-up, filling, powder addition, operation, draining, and change-out, as well as sampling capabilities and any other factors desired for ease of use. One also needs to consider any chemical compatibility requirements for the applicable film and containers and suitability of the film based on biological safety tests (e.g., United States Pharmacopeia <88> Biological Reactivity for Class VI Plastics), presence of any animal-derived additives, applicable solvent extractables data, etc. The mixer supplier should also be able to supply equipment validation data and information on supply reliability of the disposable components.
Mixers can be qualified for liquid dispersion, solid/liquid mixing, and liquid/liquid mixing. For liquid dispersion, visual dye mixing is typically performed by adding a colored water-soluble dye to a water-filled mixing container and mixing to determine whether the mixer container has any dead zones, which are assessed by observation of non-uniformity of dye color after the desired mixing time and conditions.
To evaluate solid-liquid mixing, the mixing container is again filled with the desired quantity of the solvent liquid. Next the impeller is started with only the liquid in the biocontainer. The solid can be added into the container while simultaneously starting a timer. Unless large amounts of solid are used, the time to add the solid to the fluid is generally minimal and can thus be considered zero. The container outlet/drain valve should be closed at this point to avoid any trapping of solids in the recirculation lines.
When no visual presence of particulates is observed in the container, the mixing-and-measurement stage can be initiated, where the solution is monitored using an inline sensor(s) installed within a recirculation loop or directly in the container. Mixing time can be monitored via conductivity, turbidity, or UV280 measurements using inline meters. The fluid properties can also be measured in the drain line during drainage to ensure there are no dips or spikes in the measurement, which would represent a dead zone in the mixing container.
To evaluate liquid/liquid mixing, the container can again be filled with the desired quantity of water and the impeller started with only water in the container. Then the timer and recirculation loop pump can be started as the concentrated liquid solute is added to the container. The start of the liquid addition is considered time = 0. The fluid can be monitored via an inline monitor within the recirculation loop. When the monitor value is stable and reaches ± 5% of the expected value, the solution can be drained and again 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 container.
As single-use mixers are used more in downstream and final formulation, sheer can be a greater adverse factor, especially with sensitive proteins. Shear can be reduced by using lower impeller speeds along with larger impellers. Pitched blade impellers have lower sheer than vertical fin style impellers of the same size, but the latter can have larger surface area and thus be run at a slower speeds, resulting in overall lower sheer for the same mixing efficiency. The ideal impeller shape and size for low sheer applications is a lobed propeller style with a large diameter.
While mixers can be evaluated empirically, it is also helpful to have a basic understanding of the engineering principles affecting mixing. A review of the literature and consultation with knowledgeable mixer suppliers can help reveal predictable performance and assist in proper mixer selection for well-defined applications.
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