US20180043320A1 - Continuous flow microfluidic system - Google Patents

Continuous flow microfluidic system Download PDF

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Publication number
US20180043320A1
US20180043320A1 US15/552,473 US201615552473A US2018043320A1 US 20180043320 A1 US20180043320 A1 US 20180043320A1 US 201615552473 A US201615552473 A US 201615552473A US 2018043320 A1 US2018043320 A1 US 2018043320A1
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solution
mixers
mixer
microfluidic
outlet
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US15/552,473
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English (en)
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Euan Ramsay
Robert James Taylor
Timothy Leaver
Andre Wild
Kevin Ou
Colin Walsh
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University of British Columbia
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University of British Columbia
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Assigned to THE UNIVERSITY OF BRITISH COLUMBIA reassignment THE UNIVERSITY OF BRITISH COLUMBIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RAMSAY, EUAN, OU, KEVIN, LEAVER, Timothy, TAYLOR, ROBERT JAMES, WALSH, COLIN, WILD, ANDRE
Publication of US20180043320A1 publication Critical patent/US20180043320A1/en
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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Definitions

  • Nanoparticles comprise multiple components, including, but not limited to, lipids, polymers, low molecular weight compounds, nucleic acids, proteins, peptides, and imaging agents, including inorganic molecules.
  • a system for continuous flow operation of a microfluidic chip includes:
  • a microfluidic chip comprising:
  • a first continuous flow fluid driver configured to continuously drive the first solution from a first solution reservoir into the first inlet of the microfluidic chip
  • a second continuous flow fluid driver configured to continuously drive the second solution from a second solution reservoir into the second inlet of the microfluidic chip
  • a method of forming nanoparticles comprises flowing a first solution and a second solution through a system according to the disclosed embodiment and forming a nanoparticle solution in the first mixer of the microfluidic chip.
  • FIG. 1 is a schematic representation of a continuous flow system of the present disclosure.
  • FIG. 2 is a schematic representation of a continuous flow system of the present disclosure.
  • FIG. 3 is a schematic illustration of a representative fluidic device of the disclosure.
  • FIG. 4 shows particle diameter (nm) and polydispersity index (PDI) for representative siRNA-Lipid Nanoparticles (siRNA-LNP) as a function of four single microfluidic mixer devices arrayed in parallel using a manifold, or four microfluidic mixers arrayed in parallel in the representative single device illustrated in FIG. 3 .
  • PDI polydispersity index
  • the siRNA-LNP were composed of 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate/DSPC/Chol/PEG-c-DMA at mole ratios of 50:10:38.5:1.5 and a siRNA-total lipid ratio of 0.06 wt/wt, and the nanoparticles were produced using the illustrative continuous flow system shown in FIG. 2 with either four single microfluidic mixer device arrayed in parallel using a manifold (4 ⁇ Manifold), or four microfluidic mixers arrayed in parallel in a single device illustrated in FIG. 3 (4 ⁇ On-Chip). The total flow rates through the microfluidic device are shown in the legend. Error bars represent the standard deviation of the mean.
  • FIG. 5 shows particle diameter (nm) and polydispersity index (PDI) for representative siRNA-Lipid Nanoparticles (siRNA-LNP) as a function of the manufactured volume.
  • the siRNA-LNP were composed of 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate/DSPC/Chol/PEG-c-DMA at mole ratios of 50:10:38.5:1.5 and a siRNA-total lipid ratio of 0.06 wt/wt, and the nanoparticles were produced using the illustrative continuous flow system shown in FIG. 2 with eight single microfluidic mixer device arrayed in parallel using a manifold.
  • Nanoparticles were sampled every 100 mL from 0 mL to 500 mL and the results compared to a 2 mL preparation of the same siRNA-LNP prepared using the NanoAssemblrTM Benchtop Instrument.
  • the NanoAssemblrTM Benchtop Instrument is commercially available laboratory apparatus that uses microfluidics to manufacture fixed volume batches of nanoparticles. Error bars represent the standard deviation of the mean.
  • FIG. 6 is a schematic illustration of a representative system of the disclosure, a continuous-flow staggered herringbone (SHM) micromixer.
  • SHM herringbone
  • FIG. 7 is a three-dimensional view of a representative parallel fluidic structure useful for making limit size lipid nanoparticles.
  • FIG. 8A shows a top view and FIG. 8B shows a side view of the representative parallel fluidic structure shown in FIG. 7 .
  • the top view of FIG. 8A shows two planar herringbone structures in parallel.
  • the side view of FIG. 8B shows that the fluidic parallel fluidic structure has three layers, to give a total of six herringbone structures.
  • FIG. 9 illustrates a second representative parallel fluidic structure useful for making limit size lipid nanoparticles.
  • FIG. 10 is a labeled photograph of an exemplary 4 ⁇ parallel microfluidic system with “vertical” connections (i.e., extending perpendicular to the major plane of the devices) that includes two continuous flow pumps and four microfluidic mixing chips coupled to inlet and outlet manifolds.
  • FIG. 11 is a labeled photography of an exemplary 8 ⁇ parallel microfluidic system with “horizontal” connections (i.e., extending parallel to the major plane of the devices) that includes two continuous flow pumps and eight microfluidic mixing chips coupled to two inlet manifolds and one outlet manifold.
  • FIG. 12A illustrates a toroidal pair Dean vortex bifurcating mixers (DVBM) in accordance with the disclosed embodiments.
  • FIG. 12B is a photograph of an exemplary toroidal DVBM mixer in accordance with the disclosed embodiments.
  • the present disclosure is directed towards improved systems and methods for large-scale production of nanoparticles used for delivery of therapeutic material.
  • the apparatus can be used to manufacture a wide array of nanoparticles containing therapeutic material including, but not limited to, lipid nanoparticles and polymer nanoparticles.
  • continuous flow operation and parallelization of microfluidic mixers contribute to increased nanoparticle production volume. While the present description is primarily directed to the manufacture of nanoparticles through mixing solutions, it will be appreciated that the devices, systems, and methods, are generally applicable beyond these applications. Therefore, the mixing of two or more solutions of any composition are contemplated by the disclosed embodiments.
  • Microfluidic mixers are microfluidic elements that are integrated into microfluidic devices on a microfluidic chip.
  • a microfluidic chip is defined as a platform comprising one or more microfluidic devices disposed therein, as well as inlets and outlets for connecting fluid inputs and outputs to the microfluidic devices.
  • the microfluidic devices are defined as microfluidic elements that include at least one inlet, one outlet, and one portion that performs a fluidic function, such as mixing, heating, filtering, reacting, etc.
  • the microfluidic devices described are microfluidic mixing devices configured to mix a first solution with a second solution in a mixer to provide a mixed solution.
  • other microfluidic devices are also compatible with the disclosed systems.
  • the present disclosure provides a continuous flow apparatus for the manufacture of nanoparticles, which enables the simple, rapid and reproducible manufacture of nanoparticles from small-scale (e.g., less than 50 mL) production for pre-clinical development, to large-scale production (e.g., greater than 1000 L) for clinical development and commercial supply.
  • the present disclosure employs microfluidics which has the advantage of tiny control over environmental factors during manufacture, and microfluidics possesses the further advantage that increased output is enabled by parallelization of the microfluidic mixers without the need for further process optimization.
  • the number of microfluidic mixers in parallel is dictated by the batch size requirements, and the desired time frame for manufacture of the batch.
  • the present disclosure provides a continuous flow scale-up apparatus for the manufacture of nanoparticles with a fully disposable fluid path.
  • the fully disposable fluid path enables a user eliminating expensive and time-consuming cleaning validation protocols for GMP manufacture.
  • FIG. 1 is a schematic representation of the scope of the present disclosure, a continuous flow microfluidic-based manufacturing apparatus for large-scale nanoparticle production.
  • the representative system 100 uses software systems 102 to control manufacturing parameters such as, but not limited to, fluid flow rate, the ratio of the flow rate for the independent fluid streams, pressure within the apparatus, and temperature control.
  • the apparatus 100 includes two or more (n 1 ) independent fluid inlet streams driven by fluid drivers 106 (e.g., pumps) to provide flow of nanoparticle and therapeutic materials from reservoirs 104 into manifold systems 107 that split the continuous flow streams and equal flow is driven to the inlets of each microfluidic mixer contained within the parallelized microfluidic mixer array 108 .
  • fluid drivers 106 e.g., pumps
  • the number of microfluidic mixers (n 2 ) is scaled depending on throughput requirements.
  • the number of mixers is 2 or greater. In one embodiment, the number of mixers is 2. In one embodiment, the number of mixers is 3 or greater. In one embodiment, the number of mixers is 3. In one embodiment, the number of mixers is 4 or greater. In one embodiment, the number of mixers is 4. In one embodiment, the number of mixers is 8 or greater. In one embodiment, the number of mixers is 8. In one embodiment, the number of mixers is 10 or greater. In one embodiment, the number of mixers is 10. In one embodiment, the number of mixers is 20 or greater.
  • one or more (n 3 ) post-formation microfluidic mixers 110 are arranged in sequence so that one or more additional components (n 4 ) (e.g., targeting ligands) can be added to the nanoparticles emerging from the initial microfluidic mixer array 108 or so that rapid buffer exchange or dilution can occur directly following nanoparticle manufacture.
  • the post-formation microfluidic mixer(s) 110 can also be in parallel, as with mixer array 108 , and are fed materials from reservoir 114 via continuous flow from a fluid driver 112 .
  • Nanoparticles are formed via nanoprecipitation due to rapid mixing of the fluid streams within the mixer array 108 .
  • the outlet streams from the microfluidic mixer array 108 , or post-formation microfluidic mixer(s) 110 is merged back into a single stream (e.g., using a manifold) and the resulting nanoparticles/aqueous/organic mixture is subsequently diluted with one or more (n 5 ) streams of aqueous reagent 118 delivered via fluid driver 116 to stabilize the nanoparticle product before further processing.
  • the dilution step can be achieved by in-line dilution where the aqueous buffer contacts directly with the output stream.
  • dilution can be achieved using a further microfluidic mixer array as part of the post-formation microfluidic mixer(s) 110 .
  • a time delay in diluting particle product can influence particle quality and stability.
  • the tubing length and hold-up volume between mixer and dilution can be tuned to allow for adequate time for particles to “mature” before being diluted.
  • Tubing acts as an accumulator to collect and hold product prior to dilution (for a “hold time”).
  • the distance between the last mixer (e.g., 108 or 110 ) and the dilution junction is about 1 cm to about 50 cm.
  • the distance between the last mixer (e.g., 108 or 110 ) and the dilution junction is about 1 cm to about 10 cm. In one embodiment, the distance between the last mixer (e.g., 108 or 110 ) and the dilution junction is about 10 cm to about 50 cm.
  • the system including tubing dimensions and flow characteristics, are configured to produce a specific hold time.
  • the hold time is about 5 seconds to about 1 hour. In one embodiment, the hold time is about 30 minutes to about 1 hour. In one embodiment, the hold time is about 5 seconds to about 60 seconds. In one embodiment, the hold time is about 5 seconds to about 10 seconds. In one embodiment, the hold time is greater than about 5 seconds. In one embodiment, the hold time is greater than about 10 seconds. In one embodiment, the hold time is greater than about 60 seconds. In one embodiment, the hold time is greater than about 10 minutes. In one embodiment, the hold time is greater than about 30 minutes. In one embodiment, the hold time is greater than about 45 minutes.
  • a valve directs flow to waste collection 119 prior to the system reaching steady state when the valve is configured such that flow is directed to final nanoparticle product collection 128 .
  • nanoparticle manufacturing is conducted in a specialized barrier facility that eliminates the requirement for filtration to ensure a sterile product.
  • FIG. 2 is a schematic of a representative system of the present disclosure, a continuous flow microfluidic-based manufacturing apparatus for large-scale nanoparticle production.
  • the representative system 200 includes two fluid drivers 206 , 208 to provide a continuous flow of aqueous buffer 204 and water-miscible organic containing dissolved lipids streams 202 through tubing 226 that connects the whole system.
  • Manifolds 210 and 211 split the continuous flow streams and equal flow is driven to the inlets of each parallelized mixer.
  • the number of microfluidic mixers is scaled depending on throughput requirements, and there are 8 mixers in the example 212 ). Nanoparticles are formed via nanoprecipitation due to rapid mixing of the aqueous and organic streams within the microfluidic mixers.
  • the outlet streams from the 8 parallelized mixers are merged back into a single stream using a manifold 214 and the resulting nanoparticles/aqueous/organic mixture is subsequently diluted with aqueous reagent 216 pumped 218 through a tee connector 220 to stabilize the nanoparticle product before further processing.
  • the nanoparticles formed off each microfluidic mixer are analyzed for quality and desired characteristics prior to being merged into a final output stream.
  • a valve at the tail end of the system 222 directs flow from waste collection 224 prior to the system reaching steady state when the flow is directed to sample collection 228 ).
  • Fluid contacting materials in the scenario described may be re-used, or be a single-use disposable. Single-use disposable eliminates the need to perform cleaning and cleaning validation on fluid contacting parts thus saving significant time and resources.
  • FIG. 2 shows apparatus 200 , one embodiment of the present disclosure.
  • the apparatus provides a system for the manufacture of lipid nanoparticles containing a nucleic acid.
  • the apparatus provides a system for the manufacture of limit size lipid nanoparticles including, but not limited to, liposomes and nanoemulsions containing therapeutic material.
  • the apparatus provides a system for the manufacture of polymer nanoparticles containing therapeutic material.
  • a system for continuous flow operation of a microfluidic chip includes:
  • a microfluidic chip comprising:
  • a first continuous flow fluid driver configured to continuously drive the first solution from a first solution reservoir into the first inlet of the microfluidic chip
  • a second continuous flow fluid driver configured to continuously drive the second solution from a second solution reservoir into the second inlet of the microfluidic chip
  • Continuous flow allows for large volumes of product (e.g., nanoparticles) to be produced using microfluidics, which are traditionally low-volume production systems.
  • product e.g., nanoparticles
  • microfluidics which are traditionally low-volume production systems.
  • parallelization described in more detail below, further increases production capacity when combined with continuous flow.
  • the terms “continuous” and “continuously” refer to system flow operations of relatively constant flow rate over a long duration.
  • the constant flow rate is not unvarying, but varies very little over extended operation. Variations in flow are referred to as “pulses” or “pulsation.”
  • the level of pulsation depends on the operating conditions (e.g., flow rate and backpressure) of the fluid driver.
  • the constant flow rate varies by +/ ⁇ 10% or less at 50 mL/min flow rate and 250 PSI backpressure.
  • the constant flow rate is +/ ⁇ 4% or less at 50 mL/min flow rate and 250 PSI backpressure.
  • a pulse dampener is incorporated into the system in certain embodiments in order to minimize flow pulsation from one or more of the continuous flow fluid drivers.
  • the pulse dampener(s) have a 3:1 reduction in flow pulsation (dependent on pump operating conditions).
  • the pulse dampener comprises a flexible PTFE membrane and stainless steel and polyetheretherketone as fluid contacting materials.
  • the volume produced during continuous operation is at least 100 mL completed within a 10-minute duration. In a further embodiment, the volume produced during continuous operation is at least 100 mL completed within a 2.5-minute duration. In a further embodiment, the volume produced during continuous operation is at least 100 mL completed within a 1.3-minute duration
  • the system operates at pressures up to 500 PSI.
  • the peak pressure of the system is at the outlet of the pump.
  • the backpressure at the pump outlet is the sum of the backpressure of each downstream component (tubing, manifold, chips, etc.).
  • the maximum pressure occurs at the inlets of the chip.
  • the microfluidic chip inlet pressure can reach a maximum of 200 PSI.
  • the peak pressure on the microfluidic chip is from about 100 PSI to about 200 PSI.
  • the system operates at pressure of about 5 PSI to about 200 PSI.
  • the system is capable of producing greater than 500 mL of product per hour per microfluidic mixer. In one embodiment, the system is capable of producing greater than 750 mL of product per hour per microfluidic mixer. These production rates can be multiplied via parallelization in order to yield multiple liters of product per hour for a single system.
  • the footprint of the systems disclosed is greatly reduced compared to known systems capable of producing similar volumes per unit time.
  • the NanoAssemblr manufactured by Precision Nanosystems Inc. of Vancouver, BC
  • the NanoAssemblr is a microfluidic system with a small footprint.
  • 40 NanoAssemblrs would be required, which would result in a footprint of about 2 m 2 . This is at least twice the footprint of even the most basic continuous flow system disclosed herein.
  • the system has a footprint area of 1 m 2 or less. In one embodiment, the system has a footprint area of 0.8 m 2 or less.
  • the footprint of this system is about 0.8 m 2 and it can produce over 1 L of nanoparticle solution per hour. Further parallelization can improve this production rate even further while maintaining essentially the same footprint.
  • the system of FIG. 10 includes a flow ratio of 3:1 (Pump#2:Pump#1) and “vertical” connections (perpendicular to the major surface of the chips) between the manifolds and microfluidic chips.
  • FIG. 11 an 8 ⁇ system is illustrated that is driven by two pumps. Two manifolds distribute the solutions to be mixed on the eight chips, each containing a single SHM mixing device similar to that illustrated in FIG. 6 . A single third manifold receives the mixed solutions from the eight chips and concentrates them in a single stream for post-mixing processing, dilution, and/or product capture.
  • the system of FIG. 11 is also distinct from that of FIG. 10 in that connections from the manifolds to the chips are in a “horizontal” configuration, parallel to the major surface of the chips.
  • the horizontal configuration for connections is unexpectedly beneficial compared to vertical configuration. The vertical configuration would seem to be superior because each mixer assembly takes up less space. However, manipulation of the connections by a user becomes difficult in the confined space created by the vertical connectors. By using horizontal connectors, the connections are easily operable and more user-friendly.
  • the system has a production volume of 0.76 L of nanoparticle solution per hour.
  • the production volume can be increased by the number of mixers, N.
  • the system has a production volume of 0.5 L of nanoparticle solution per hour.
  • the system has a production volume of 1.0 L of nanoparticle solution per hour.
  • the system is scalable to produce a product solution from 0.025 L to 5000 L.
  • the output of the system is limited only by the amount of starting material. That is, the system can produce product from the starting solutions as long as the starting solutions are available. Therefore, production volume in a single operating session is essentially unlimited by system constraints, due to the use of continuous flow.
  • the microfluidic chips are configured to mix the first solution with the second solution through a mixing region. Many methods for this mixing process are known. In one embodiment, the mixing is chaotic advection. Compatible microfluidic mixing methods and devices are disclosed in:
  • devices are provided for making nanoparticles of the type disclosed herein.
  • the microfluidic devices are incorporated into the continuous flow systems and methods disclosed herein.
  • the device includes:
  • a third microchannel 310 for receiving the first and second streams, wherein the third microchannel has a first region 312 adapted for flowing the first and second streams and a second region 314 adapted for mixing the contents of the first and second streams to provide a third stream comprising limit size lipid nanoparticles.
  • the lipid nanoparticles so formed are conducted from the second (mixing) region by microchannel 316 to outlet 318 .
  • the second region of the microchannel comprises bas-relief structures.
  • the second region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction.
  • the second region includes a micromixer.
  • the devices of the disclosure provide complete mixing occurs in less than 10 ms.
  • one or more regions of the device are heated.
  • the first mixer comprises a mixing region comprising a microfluidic mixer configured to mix the first solution and the second solution to provide the nanoparticle solution formed from mixing of the first solution and the second solution.
  • the first mixer is a chaotic advection mixer.
  • the mixing region has a hydrodynamic diameter of about 20 microns to about 300 microns. In one embodiment, the mixing region has a hydrodynamic diameter of about 113 microns to about 181 microns. In one embodiment, the mixing region has a hydrodynamic diameter of about 150 microns to about 300 microns. As used herein, hydrodynamic diameter is defined using channel width and height dimensions as (2*Width*Height)/(Width+Height).
  • the mixing region can also be defined using standard width and height measurements.
  • the mixing region has a width of about 100 to about 500 microns and a height of about 50 to about 200 microns.
  • the mixing region has a width of about 200 to about 400 microns and a height of about 100 to about 150 microns.
  • the systems are designed to support flow at low Reynolds numbers.
  • the first mixer is sized and configured to mix the first solution and the second solution at a Reynolds number of less than 2000. In one embodiment, the first mixer is sized and configured to mix the first solution and the second solution at a Reynolds number of less than 1000. In one embodiment, the first mixer is sized and configured to mix the first solution and the second solution at a Reynolds number of less than 900. In one embodiment, the first mixer is sized and configured to mix the first solution and the second solution at a Reynolds number of less than 500.
  • the microfluidic mixer device contains one micromixer.
  • the single mixer microfluidic device has two regions: a first region for receiving and flowing at least two streams (e.g., one or more first streams and one or more second streams). The contents of the first and second streams are mixed in the microchannels of the second region, wherein the microchannels of the first and second regions has a hydrodynamic diameter from about 20 to about 500 microns.
  • the second region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction (e.g., a staggered herringbone mixer), as described in US 2004/0262223, expressly incorporated herein by reference in its entirety.
  • the second region of the microchannel comprises bas-relief structures.
  • the second regions each have a fluid flow rate of from 1 to about 50 mL/min.
  • the mixing channel of the microfluidic device is 300 microns wide and 130 microns high.
  • the herringbone structures are 40 microns high and 50-75 microns thick.
  • the first and second streams are mixed with other micromixers.
  • Suitable micromixers include droplet mixers, T-mixers, zigzag mixers, mulitlaminate mixers, or other active mixers.
  • microfluidic mixer devices are mounted in a device holder incorporating a clamping system.
  • the device holder and clamping system provides mechanical forces on the microfluidic mixer devices to seal the device inlet and outlet ports.
  • the device holder and clamping system comprise sealing gaskets to provide a tight seal between the inlet and outlet ports of the microfluidic mixer device and device holder.
  • the sealing gasket acts as a spring to evenly distribute forces on the planar microfluidic mixer device when mounted in the device holder by the clamping system.
  • the sealing gaskets are O-rings.
  • the device holder and clamping system comprise a solid polycarbonate plate applying mechanical force through tightening screws.
  • the microfluidic mixer device and device holder are a single disposable plastic piece without the need for a gasket-based clamping system.
  • One function of the systems and methods disclosed herein is to form nanoparticles in solution (the “product”).
  • the product One function of the systems and methods disclosed herein is to form nanoparticles in solution (the “product”).
  • Previous disclosures by the present inventors relate to generating nanoparticles compatible with the present system, such as those applications previously incorporated by reference.
  • Known and future-developed nanoparticle methods can be performed on the disclosed systems to the extent that the methods require the controlled combination of a first solution with a second solution to form a nanoparticle product, as disclosed herein.
  • the first solution also referred to herein as the “aqueous reagent” herein, is provided in a first solution reservoir.
  • the first solution comprises a first solvent.
  • the first solution comprises an active pharmaceutical ingredient.
  • the first solution comprises a nucleic acid in a first solvent.
  • the first solution comprises a buffer.
  • the first solution consists essentially of a buffer.
  • the second solution also referred to herein as the “solvent reagent” herein, is provided in a second solution reservoir.
  • the second solution comprises a second solvent.
  • the second solution comprises lipid particle-forming materials in a second solvent.
  • the second solvent is a water-miscible solvent (e.g., ethanol or acetonitrile).
  • the second solution is an aqueous buffer comprising polymer nanoparticle forming reagents.
  • the first solution comprises a nucleic acid in a first solvent and the second solution comprises lipid particle-forming materials in a second solvent.
  • a single microfluidic mixer is contained in one microfluidic device on one microfluidic chip.
  • increased production volume is achieved by parallelizing the mixers, whether through on-chip parallelization, using multiple chips, or both.
  • the system further includes a plurality of mixers, each including a first inlet, a first inlet microchannel, a second inlet, a second inlet microchannel, a mixing microchannel, a mixer outlet, and a chip outlet, wherein the plurality of mixers includes the first mixer.
  • the plurality of mixers are all of the same dimensions. In another embodiment, the plurality of mixers have different dimensions.
  • the plurality of mixers are within a plurality of microfluidic chips. In another embodiment, the plurality of mixers are on a single microfluidic chip.
  • the microfluidic mixer array incorporates 1-128 microfluidic mixers arrayed in parallel to increase the throughput of the manufacturing system.
  • a 128-mixer system according to the disclosed embodiments is capable of producing about 1.5 L/min of nanoparticle solution.
  • the device in FIG. 3 is an example of planar parallelization of microfluidic mixers in a single device.
  • Planar parallelization refers to placing one or more mixers on the same horizontal plane. These mixers may or may not be connected by a fluidic bus channel (e.g., connecting the four outlets 265 ). Equal flow through each mixer is assured by creating identical fluidic paths between the inlets and outlets.
  • Vertical parallelization is achieved by forming planar mixers and layering them together in such a way as to share common inlets. Theoretically, fluid flowing from the inlets to the lower mixer encounters a higher resistance than that flowing to the top mixer, therefore leading to a lower flow rate.
  • the increased resistance is negligible when compared to the overall resistance of the mixing structure (which is identical for each layer). Additionally, increasing the diameter of the fluidic bus leading to the microfluidic mixer inlets reduces the impendence of the bus and the resulting impedance differences between individual mixers.
  • the single device has microfluidic mixers array in the planar and vertical directions of the chip, for high-density 3-dimensional microfluidic parallelization.
  • microfluidic mixer arrays can be arranged in sequence for multi-step manufacture of complex nanoparticle systems.
  • the system of the present disclosure operates at a flow rate between 1 mL/min and 50 mL/min per microfluidic mixer.
  • at least one microfluidic mixer of the system operates at a flow rate of about 10 mL/min to about 25 mL/min.
  • each independent continuous flow fluid driver operates at a flow rate of 1.0 L/min.
  • the parallelized mixers are arranged in a stacked configuration on the microfluidic chip.
  • the parallelized mixers are arranged in a horizontal configuration, in substantially the same plane, on the microfluidic chip.
  • the parallelized mixers are arranged in both a horizontal configuration and a stacked configuration on the microfluidic chip.
  • the disclosure provides devices that include more than one fluidic mixing structures (i.e., an array of fluidic structures).
  • the disclosure provides a single device (i.e., an array) that includes from 2 to about 40 parallel fluidic mixing structures capable of producing lipid nanoparticles at a rate of about 2 to about 2000 mL/min.
  • the devices produce from 100 mL to about 400 L without a change in lipid nanoparticle properties.
  • the microfluidic device includes:
  • a plurality of microchannels for receiving the first and second streams, wherein each has a first region adapted for flowing the first and second streams and a second region adapted for mixing the contents of the first and second streams to provide a plurality of streams compromising lipid nanoparticles;
  • each of the plurality of microchannels for receiving the first and second streams includes:
  • a third microchannel for receiving the first and second streams, wherein each has a first region adapted for flowing the first and second streams and a second region adapted for mixing the contents of the first and second streams to provide a plurality of streams compromising lipid nanoparticles.
  • the device includes from 2 to about 40 microchannels for receiving the first and second streams. In these embodiments, the device has a total flow rate from 2 to about 1600 mL/min.
  • the second regions each have a hydraulic diameter of from about 20 to about 300 ⁇ m. In certain embodiments, the second regions each have a fluid flow rate of from 1 to about 40 mL/min.
  • the heating element is effective to increase the temperature of the first and second streams in the first and second microchannels to a pre-determined temperature prior to their entering the third microchannel.
  • the inlet fluids are heated to a desired temperature and mixing occurs sufficiently rapidly such that the fluid temperature does not change appreciably prior to lipid nanoparticle formation.
  • the disclosure provides a system for making limit size nanoparticles that includes a parallel microfluidic structure.
  • N single mixers are arrayed such that a total flow rate of N ⁇ F is achieved, where F is the flow rate used in the non-parallelized implementation.
  • Representative parallel microfluidic structures are illustrated schematically in FIGS. 7-9 .
  • FIG. 7 A perspective view of a representative parallel microfluidic structure is illustrated in FIG. 7 ; a plan view is illustrated in FIG. 8A ; and a side elevation view of the device of FIG. 8A is illustrated in FIG. 8B .
  • the device 500 includes three fluidic systems 400 a , 400 b , and 400 c arranged vertically with each system including one first solvent inlet 402 , two second solvent inlets 406 and 406 ′, two mixing regions 410 and 410 ′, and a single outlet 408 .
  • Each system includes microchannels for receiving the first and second streams 402 and 406 and 406 ,′ respectively.
  • each fluidic system includes:
  • a third microchannel 310 a for receiving the first and second streams, wherein each has a first region 312 a adapted for flowing the first and second streams and a second region 314 a adapted for mixing the contents of the first and second streams to provide a plurality of streams comprising lipid nanoparticles.
  • the microchannel 316 a conducts one of the plurality of streams from the mixing region to fourth microchannel 408 via outlet 318 a that conducts the lipid nanoparticles from the device.
  • fluidic system 300 a includes a second second solvent inlet 406 ′ and mixing region 310 a ′ with components denoted by reference numerals 302 a ′, 304 a ′, 306 a ′, 308 a ′, 312 a ′, 314 a ′, 316 a ′ and 318 a ′.
  • reference numerals 302 a ′, 304 a ′, 306 a ′, 308 a ′, 312 a ′, 314 a ′, 316 a ′ and 318 a ′ correspond to their non-primed counterparts 302 , 304 , 306 , 308 , 312 , 314 , 316 , and 318 in FIG. 8A and FIG. 8B .
  • This structure produces vesicles at higher flow rates compared to the single mixer chips and produces vesicles identical to those produced by single mixer chips.
  • six mixers are integrated using three reagent inlets. This is achieved using both planar parallelization and vertical parallelization as shown in FIGS. 7, 8A, and 8B .
  • Planar parallelization refers to placing one or more mixers on the same horizontal plane. These mixers may or may not be connected by a fluidic bus channel. Equal flow through each mixer is assured by creating identical fluidic paths between the inlets and outlets, or effectively equal flow is achieved by connecting inlets and outlets using a low impedance bus channel as shown in FIG. 9 (a channel having a fluidic impedance significantly lower than that of the mixers).
  • FIG. 9 illustrates a parallelized device 500 includes five fluidic systems 300 a , 300 b , 300 c , 300 d , and 300 e arranged horizontally with each system including one first solvent inlet, one second solvent inlet, one mixing region, and a single outlet 408 .
  • Device 500 includes microchannels for receiving the first and second streams 402 and 406 and a microchannel 408 for conducting lipid nanoparticles produced in the device from the device.
  • fluidic system 500 a includes:
  • microchannel 316 a conducts the third stream from the mixing region to fourth microchannel 408 via outlet 318 a .
  • Microchannel 408 conducts the lipid nanoparticles from the device via outlet 409 .
  • the five fluidic systems 300 a - 300 e are arranged in a single plane that is not the plane of the first microchannel 402 , second microchannel 406 , or fourth microchannel 408 . Therefore, the fluidic busses (embodied by 402 , 406 , and 408 ) are in one plane and the mixers (embodied by 300 a - 300 e ) are in a separate plane. While this configuration adds an additional layer of fabrication complexity to the device (e.g., by requiring an additional layer of lithography to define the layer with the mixers), such a parallelized device provides dramatically enhanced throughput without a proportional amount of device area required. For example, in the device of FIG.
  • the footprint of the device 500 on a chip is much less than the combined footprints of five individual devices with comparable total mixed-volume output.
  • the parallelized device has n fluidic systems (e.g., 300 a et al.) and is configured to produce a mixed output volume that is the same or greater than the output of n standalone mixers (e.g., as illustrated in FIG. 6 ), and in a smaller total device area (i.e., the parallelized device has a device area that is less than the total combined area of the n standalone mixers.
  • fluidic system 300 a includes a second second solvent inlet 406 ′ and mixing region 310 a ′ with components denoted by reference numerals 302 a ′, 304 a ′, 306 a ′, 308 a ′, 312 a ′, 314 a ′, 316 a ′ and 318 a ′.
  • reference numerals 302 a ′, 304 a ′, 306 a ′, 308 a ′, 312 a ′, 314 a ′, 316 a ′ and 318 a ′ correspond to their non-primed counterparts 302 , 304 , 306 , 308 , 312 , 314 , 316 , and 318 in FIG. 8A and FIG. 8B .
  • the disclosure provides a device for producing limit size lipid nanoparticles, comprising n fluidic devices, each fluidic device comprising:
  • a third microchannel 310 a for receiving the first and second streams, wherein the third microchannel has a first region 312 a adapted for flowing the first and second streams and a second region 314 a adapted for mixing the contents of the first and second streams to provide a third stream comprising limit size lipid nanoparticles conducted from the mixing region by microchannel 316 a,
  • first inlets 302 a - 302 n of each fluidic device 300 a - 100 n are in liquid communication through a first bus channel 402 that provides the first solution to each of the first inlets
  • each fluidic device 300 a - 300 n are in liquid communication through a second bus channel 406 that provides the second solution to each of the second inlets
  • each fluidic device 300 a - 300 n are in liquid communication through a third bus channel 408 that conducts the third stream from the device.
  • the reference numerals refer to representative device 500 in FIG. 9 .
  • n is an integer from 2 to 40.
  • Parallelized devices are formed by first creating positive molds of planar parallelized mixers that have one or more microfluidic mixers connected in parallel by a planar bus channel. These molds are then used to cast, emboss or otherwise form layers of planar parallelized mixers, one of more layers of which can then be stacked, bonded and connected using a vertical bus channels. In certain implementations, planar mixers and buses may be formed from two separate molds prior to stacking vertically (if desired). In one embodiment positive molds of the 2 ⁇ planar structure on a silicon wafer are created using standard lithography. In an exemplary manufacturing method, a thick layer of on-ratio PDMS is then poured over the mold, degassed, and cured at 80 C for 25 minutes.
  • the cured PDMS is then peeled off, and then a second layer of 10:1 PDMS is spun on the wafer at 500 rpm for 60 seconds and then baked at 80° C. for 25 minutes. After baking, both layers are exposed to oxygen plasma and then aligned. The aligned chips are then baked at 80° C. for 15 minutes. This process is then repeated to form the desired number of layers. Alignment can be facilitated by dicing the chips and aligning each individually and also by making individual wafers for each layer which account for the shrinkage of the polymer during curing.
  • this chip has been interfaced to pumps using standard threaded connectors. This has allowed flow rates as high as 72 ml/min to be achieved. Previously, in single element mixers, flows about 10 ml/min were unreliable as often pins would leak eject from the chip.
  • chips are sealed to on the back side to glass, and the top side to a custom cut piece of polycarbonate or glass with the interface holes pre-drilled.
  • the PC to PDMS bond is achieved using a silane treatment. The hard surface is required to form a reliable seal with the O-rings. A glass backing is maintained for sealing the mixers as the silane chemistry has been shown to affect the formation of the nanoparticles.
  • the devices and systems of the disclosure provide for the scalable production of limit size nanoparticles.
  • the following results demonstrate the ability to produce identical vesicles, as suggested by identical mean diameter, using the microfluidic mixer illustrated in FIGS. 7-8B .
  • the present disclosure includes a manifold system that splits the fluid streams from the two, or more independent continuous flow pumping systems into multiple fluid streams and directs the multiple fluid streams into a microfluidic mixer array.
  • a single device contains multiple microfluidic mixers with on-device or off-device fluid distribution scheme, such as, but not limited to a fluid bus.
  • manifold is referred to as any fluid conduit that splits or merges liquid flow.
  • a manifold can be external to a microfluidic device (e.g., interfaced with a microfluidic chip via an inlet or outlet port, such as illustrated in FIG. 10 ) or integrated into the microfluidic chip, as illustrated in FIG. 3 .
  • fluid flow driven by the independent continuous flow pumping systems enters two manifolds, a first manifold 210 connected to the aqueous fluid driver 208 and a second manifold 211 connected to the solvent fluid driver 206 .
  • the manifolds 210 and 211 split the solutions flowing therein into multiple fluid streams that flow to parallelized microfluidic mixing devices 212 .
  • a third manifold 214 is used to collect the multiple streams emerging from the microfluidic mixer array into a single output stream.
  • the 8 separate fluid streams containing nanoparticles emerging from the microfluidic mixer array consisting of 8 parallelized microfluidic mixer devices containing a single microfluidic mixer are merged through a manifold to form a single output stream.
  • the system further comprises a first manifold configured to receive the first solution from the first solution reservoir and distribute the first solution to the first inlets of the plurality of mixers.
  • system further includes a second manifold configured to receive the second solution from the second solution reservoir and distribute the second solution to the second inlets of the plurality of mixers.
  • system further comprises a third manifold configured to receive and combine the nanoparticle solution from the chip outlets of the plurality of mixers and direct it in a single channel towards the system outlet.
  • system further comprises:
  • a first manifold configured to receive the first solution from the first solution reservoir and distribute the first solution to the first inlets of the plurality of mixers;
  • a second manifold configured to receive the second solution from the second solution reservoir and distribute the second solution to the second inlets of the plurality of mixers;
  • a third manifold configured to receive and combine the nanoparticle solution from the chip outlets of the plurality of mixers and direct it in a single channel towards the system outlet.
  • the plurality of mixers are within the microfluidic chip.
  • Manifold materials include PEEK, stainless steel, COC/COP, polycarbonate, and Ultem.
  • the manifold device comprises, but not limited to, 9-Ports interfaced with 0.0625 inch outside diameter tubing.
  • the interface between the 0.0625 inch outside diameter tubing and ports of the manifold are made using 10-24 threaded fittings in the form of a single piece.
  • the interface between the 0.0625 inch outside diameter tubing and ports of the manifold are made using 10-24 threaded fittings as a nut and ferrule.
  • a 9-Port 0.0625 inch outside diameter tubing manifold is used to split fluid flow driven by the independent continuous flow pumping systems into 8 separate fluid streams that feed into 8 parallelized microfluidic mixer devices containing a single microfluidic mixer.
  • the relative flow rates of the multiple output streams generated by the manifold are governed by the relative fluidic resistance of each output stream.
  • the microfluidic mixer provides over 95% of the fluidic resistance in the fluid path.
  • the fluid drivers are pumps.
  • the system includes two, or more, independent continuous flow fluid drivers.
  • the solvent metering pump 206 and aqueous metering pump 208 are independent continuous flow pumping systems that provide fluid flow in the apparatus.
  • the first continuous flow fluid driver and the second continuous flow fluid driver are independently selected from the group consisting of positive displacement fluid drivers (such as: reciprocating piston, peristaltic, gear, diaphragm, screw, progressive cavity); centrifugal pumps; and pressure driven pumps
  • the continuous flow pumping systems are positive displacement pumps.
  • positive displacement pumps include, but are not limited to, peristaltic pumps, gear pumps, screw pumps, and progressive cavity pumps.
  • the independent continuous flow pumps are dual head reciprocating positive displacement pumps.
  • the dual head reciprocating positive displacement pumps have front mounted interchangeable pump heads.
  • the front mounted interchangeable pump heads enable independent flow rates from 10 mL/min to 1000 mL/min.
  • the dual head reciprocating positive displacement pumps are Knauer Azura P2.2 L pumps.
  • the Knauer Azura P 2.1 L pumps are modified to have the pressure sensor mounts external to the pump body allowing easy exchange of the pump's pressure sensor.
  • Interchangeable pump heads enable simple and rapid scaling of continuous flow manufacturing system.
  • the front mounted interchangeable pump heads control the ratio of fluid flow rate from the aqueous reservoir 204 to fluid flow rate from the solvent reservoir 202 .
  • the ratio of the flow rate from the aqueous reservoir 204 to the flow rate from the solvent reservoir 202 is greater than 1:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, including intermediate ratios).
  • the ratio of the flow rate from the solvent reservoir 102 to the flow rate from the aqueous reservoir 204 is greater than 1:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, including intermediate ratios).
  • FIG. 10 illustrates a system operating with a ratio of 3:1 (Pump#2:Pump#1).
  • the dual head reciprocating pump head provides low pulsation flow.
  • the pulsation is further dampened through the addition of 10-500 PSI backpressure.
  • Preferred embodiments of backpressure systems include, but are not limited to, a backpressure regulator, or tubing of extended length, added to the outlet of the pumping systems.
  • backpressure is achieved by addition of 24 inches of tubing with an internal diameter of 0.02 inches.
  • independent continuous flow pumping systems are chosen from centrifugal pumps, and pressure driven pumps.
  • the independent continuous flow pumping system provides easy interchange of components and reduces the time needed to replace single-use fluid contacting components.
  • the system further includes a dilution element, wherein the dilution element comprises a third continuous flow fluid driver, configured to continuously drive a dilution solution from a dilution solution reservoir into the system, via a dilution channel, in between the chip outlet and the system outlet.
  • the dilution element comprises a third continuous flow fluid driver, configured to continuously drive a dilution solution from a dilution solution reservoir into the system, via a dilution channel, in between the chip outlet and the system outlet.
  • the present disclosure includes one or more additional pumps 218 to dilute the nanoparticles emerging from the microfluidic mixer array with a buffer 216 ), or other suitable media.
  • the dilution process is achieved by pumping one or more buffers continuously into the output stream emerging from the microfluidic mixer array.
  • the dilution pumping system is a positive displacement pump.
  • the positive displacement pump is selected from peristaltic pumps, gear pumps, screw pumps and progressive cavity pumps.
  • the dilution pumping system is a peristaltic pump.
  • the dilution pumping system is a peristaltic pump with dual pump heads.
  • the dilution pumping system is a Masterflex peristaltic pump with dual pump heads.
  • the pumping system is selected from centrifugal pumps and pressure driven pumps. The choice of pumps listed here is representative and should not limit the scope of the present disclosure. A person of ordinary skill will recognize other alternative pumping systems that may be used with the present disclosure.
  • the dilution media is introduced into the nanoparticle stream by a connector.
  • the connector is a Tee connector.
  • the connector is a Y-connector.
  • the dilution media contacts the nanoparticle stream at an angle ranging from 0.1° to 179.9°. The angle of contact moderates the level of agitation induced onto the nanoparticles in the dilution process.
  • the Masterflex peristaltic pump dual pump heads have roller profiles offset by 30° thereby reducing the pump's output flow pulsation level by 80-95%.
  • the dilution media is introduced into a second, inline microfluidic mixer.
  • the second microfluidic mixer is on a second, in-line device. In another embodiment, the second microfluidic mixer is on the same microfluidic device.
  • Diluting the nanoparticle solution reduces the percentage of solvent present in the solution. For certain nanoparticles, diluting the solvent below 50% increases particle stability. In other embodiments, nanoparticle stability is promoted by diluting solvent below 25%. In further embodiments, nanoparticles are stable below 10% solvent content.
  • the reservoirs are disposable bags.
  • the reservoirs are vessels, including, but not limited to, stainless steel reservoirs.
  • the solvent is a water-miscible solvent such as, but not limited to, ethanol containing one or more lipids
  • the aqueous is a low pH buffer such as, but not limited to, citrate buffer pH 4.0.
  • the low pH buffer such as, but not limited to, citrate buffer pH 4.0 contains one or more nucleic acids.
  • the solvent is a water-miscible solvent such as, but not limited to, acetonitrile containing one or more polymers, or polymer-drug conjugates and the aqueous is a buffer such as, but not limited to, citrate buffer pH 4.0.
  • a representative buffer is a saline solution.
  • the tubing 226 is a microfluidic channel on the microfluidic chip. In one embodiment the tubing 226 is metal tubing external to the microfluidic chip. In an embodiment the tubing is plastic tubing. In another embodiment the internal diameters of the tubing ranges from 0.01 inches to 0.25 inches.
  • the fittings 220 , 222 include, but are not limited to, barbed, compression, sanitary and threaded. In a further embodiment the fittings are metal or plastic. In a preferred embodiment the fittings are plastic.
  • the manufacturing apparatus has a mechanism to dilute the nanoparticle fluid stream emerging from the microfluidic mixer array.
  • dilution is achieved by in-line dilution where the aqueous buffer contacts directly with the output stream.
  • a fluid driver 218 flows dilution reagent from a reservoir 216 and the dilution reagent stream joins the nanoparticle fluid stream at a junction where flow of dilution reagent stream is controlled by a tee connector 220 .
  • a fluid driver 218 flows dilution reagent from a reservoir 216 and the dilution reagent stream enters a microfluidic mixer device through one inlet and the nanoparticle fluid stream enters the microfluidic mixer device through a second inlet and the two streams flow into a microfluidic mixer region where the nanoparticle fluid stream is diluted by mixing with the dilution reagent fluid stream.
  • a fluid driver 218 flows dilution reagent from a reservoir 216 and the dilution reagent fluid stream enters a manifold where the dilution reagent fluid stream is divided into multiple dilution reagent fluid streams and the nanoparticle fluid stream enters a manifold where the nanoparticle fluid stream is divided into multiple nanoparticle fluid streams.
  • the multiple dilution reagent fluid streams and the multiple nanoparticle fluid streams enter multiple microfluidic mixer devices arrayed in parallel where the nanoparticle fluid stream is diluted by mixing with the dilution reagent fluid stream.
  • the ratio of flow rate of the nanoparticle fluid stream to the flow rate of the dilution reagent fluid stream is greater than 1:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, including intermediate ratios). In other embodiments, the ratio of flow rate of the dilution reagent fluid stream to the flow rate of the nanoparticle fluid stream greater than 1:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, including intermediate ratios).
  • the system includes dilution in-line in the absence of the microfluidic device.
  • the microfluidic-based continuous flow manufacturing apparatus for scalable production of nanoparticles includes a mechanism to separate waste collection 224 from sample collection 228 as shown in FIG. 2 .
  • a valving system directs the output stream to waste collection or sample collection.
  • a two-vessel collection is achieved by splitting the output stream from the manufacturing system into two and opening/closing valves on the independent lines to collect in the desired vessel.
  • the valve system is a manual system or an automated system is used to pinch off the soft tubing.
  • the manual valve system is a tube clamp.
  • the automated system is a solenoid pinch valve.
  • the system further includes a waste outlet in fluid communication with a waste valve in between the chip outlet and the system outlet, wherein the waste valve is configured to controllably direct fluid towards the waste outlet.
  • the waste valve is used to eliminate waste from priming the system or other non-production operations. As illustrated in FIG. 2 , the waste valve 222 directs flow to a waste vessel 224 that is separate from the sample vessel 228 .
  • the system incorporates a fully disposable fluid path.
  • the term “disposable fluid path” refers to a system where every element that touches a liquid is “disposable.” Given the precious nature of certain products of the system (e.g., nano-medicines), and the related value of such products, the term “disposable” as used herein refers to a component that has relatively low cost in relation to the product produced.
  • Typical disposable components include tubing, manifolds, and reservoirs that may be made of plastic.
  • disposable also refers to such components as pump heads and microfluidic chips. Therefore, disposable components include those that are made from metal (e.g., pump heads) and/or are finely manufactured (e.g., microfluidic devices).
  • the fully disposable fluid path includes the reagent bags 202 , 204 , 216 ), the tubing 226 ), the interchangeable pump heads 206 , 208 ), the fittings 220 , 222 ), the manifolds 210 , 211 214 ), and the microfluidic devices 212 .
  • the disposable fluidic path includes a disposable microfluidic chip, a disposable first pump head of the first continuous flow pump, a disposable second pump head of the second continuous flow pump, and a disposable system outlet.
  • the disposable first pump head and the disposable second pump head are made of a material independently selected from the group consisting of stainless steel, polymers (e.g., polyetheretherketone (PEEK)), titanium, and ceramic. In one embodiment, at least one of the disposable first pump head or the disposable second pump head comprise a metal.
  • PEEK polyetheretherketone
  • every surface touched by the first solution, the second solution, and the nanoparticle solution are disposable.
  • the apparatus fluid path is fully disposable. In certain embodiments the apparatus is GMP compliant. All fluid contacting materials in these embodiments can be reused, or be single-use disposable.
  • the microfluidic chip is sterile. Sterilization is essential for certain production processes.
  • the microfluidic chip is sterilized prior to integration into the system.
  • the microfluidic chip is sterilized in-place within the system.
  • Representative sterilization methods include steam autoclave, dry heat, chemical sterilization (i.e., sodium hydroxide or ethylene oxide), gamma radiation, gas, and combinations thereof.
  • the microfluidic chip is sterilized with gamma radiation.
  • the microfluidic chip is formed from materials that are compatible with certain types of sterilization preferred for a particular application.
  • the microfluidic chip is formed from materials that are compatible with gamma radiation.
  • Materials compatible with gamma radiation are those that can be irradiated.
  • Materials that cannot be irradiated include polyamides, polytetrafluoroethylene, and any metal.
  • further embodiments include sterile packages containing sterile systems and/or system parts. These sterile packages allow a user to reduce time and cost by avoiding in-place sterilization procedures.
  • sterile filtering is a technique used for terminal sterilization and particles having a dimension (e.g., diameter) greater than about 200 nm are not compatible with terminal sterilization. Accordingly, in one embodiment, the systems and methods are configured to provide sterile synthesis of particles having a dimension of 200 nm or greater. Accordingly, in one embodiment, the systems and methods are configured to provide sterile synthesis of particles without terminal sterilization.
  • Representative particles that are not compatible with terminal sterilization include DNA/RNA lipoplexes manufactured by mixing DNA/RNA with pre-formed liposomes, which can result in particle structures that are larger than 200 nm, which cannot be terminally sterilized.
  • Certain drug-conjugated polymer nanoparticles with high polydispersity are also large enough to make terminal sterilization impractical.
  • a sterile package can be opened in the production environment, the contents of the package implemented into the system, and the system operated without a pre-sterilization step. Accordingly, in one embodiment, sterile package is provided.
  • the sterile package includes a sterile microfluidic chip according to the present disclosure sealed within the sterile package. In a further embodiment, the sterile package includes an entire sterile disposable fluidic path, including pump heads, sealed within the sterile package.
  • the sterile system parts can be sterilized by any methods know to those of skill in the art and disclosed herein.
  • gamma radiation is used in certain embodiments to sterilize the system parts.
  • the sterile system parts are maintained in a sterile environment and packaged in a sealed manner so as to maintain sterility until use.
  • nanoparticle manufacturing is conducted in a specialized barrier facility that eliminates the requirement for filtration to ensure a sterile product.
  • the entire manufacturing system is software controlled.
  • software controls are generally known to those of skill in the art.
  • the present disclosure provides methods for scalable production of nanoparticles using the microfluidic-based continuous flow manufacturing apparatus of the disclosure.
  • a method of forming nanoparticles comprises flowing a first solution and a second solution through a system according to any of the disclosed embodiment and forming a nanoparticle solution in the first mixer of the microfluidic chip.
  • the system comprises a plurality of mixers and the method further comprises flowing the first solution and the second solution through the plurality of mixers to form the nanoparticle solution, wherein the plurality of mixers includes the first mixer.
  • the plurality of mixers are contained within a plurality of microfluidic chips.
  • the plurality of mixers are contained within a single microfluidic chip.
  • the apparatus provides a system and process for the manufacture of lipid nanoparticles containing a therapeutic material.
  • the apparatus provides a system and process for the manufacture of polymer nanoparticles containing a therapeutic material.
  • the apparatus has two reservoirs: a solvent reservoir and aqueous reservoir.
  • the solvent reservoir contains a water-miscible solvent such as, but not limited to, ethanol containing one or more lipids.
  • the solvent reservoir contains a water-miscible solvent such as, but not limited to, acetonitrile containing one or more polymers, or polymer-drug conjugates.
  • the aqueous reservoir contains a buffer such as, but not limited to, citrate buffer pH 4.0.
  • the aqueous reservoir contains a buffer such as, but not limited to, citrate buffer pH 4.0 that contains one or more therapeutic materials.
  • the contents of the reservoirs are drawn into the fluid path of the apparatus of the disclosure by independent continuous flow pumping systems.
  • the each independent continuous flow manufacturing pump operates at a flow rate of 0.1 L/min-1.0 L/min.
  • the present disclosure includes a manifold system that splits the fluid streams from the two, or more independent continuous flow pumping systems into multiple fluid streams such that:
  • a first stream comprising a first solvent e.g., an aqueous buffer
  • a first solvent e.g., an aqueous buffer
  • a second stream comprising a second solvent (e.g., an water-miscible solvent) into the second channel of each independent microfluidic mixer at a second flow rate to provide first and second adjacent streams, wherein the first and second solvents are not the same, and wherein the ratio of the first flow rate to the second flow rate is greater than 1.0;
  • a second solvent e.g., an water-miscible solvent
  • the apparatus is a microfluidic apparatus.
  • the flow pre-mixing is laminar flow.
  • mixing the first and second streams comprises chaotic advection.
  • mixing the first and second streams comprises mixing with a micromixer.
  • the microfluidic mixer array incorporates 1-128 microfluidic mixers arrayed in parallel to increase the throughput of the manufacturing system.
  • a single microfluidic mixer is contained on one device.
  • a single device contains multiple microfluidic mixers.
  • a single device contains four microfluidic mixers ( FIG. 3 ).
  • the flow rates of the first stream and second stream flow rate between 1 mL/min and 50 mL/min per microfluidic mixer.
  • the ratio of the first flow rate to the second flow rate is greater than 1:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, including intermediate ratios).
  • the final nanoparticle product is dispensed into sterile vials.
  • DVBM are useful as mixers in the disclosed continuous flow systems.
  • DVBMs of the type disclosed herein act as efficient mixers and whose injection molding tooling can be produced by an end mill with a radius of R (for example 300 ⁇ m).
  • the provided DVBM mixers include a plurality of toroidal mixing elements (also referred to herein as “toroidal mixers.”
  • toroid refers to a generally circular structure having two “leg” channels that define a circumference of the toroid between an inlet and an outlet of the toroidal mixer.
  • the toroidal mixers are circular in certain embodiments. In other embodiments, the toroidal mixers are not perfectly circular and may instead have oval or non-regular shape.
  • FIG. 12A illustrates a pair of toroidal DVBM mixers in accordance with the disclosed embodiments.
  • FIG. 12B is a photograph of an exemplary toroidal DVBM mixer in accordance with the disclosed embodiments.
  • the DVBM mixer is configured to mix at least a first liquid and a second liquid, the mixer comprising an inlet channel leading into a plurality of toroidal mixing elements arranged in series, wherein the plurality of toroidal mixing elements includes a first toroidal mixing element downstream of the inlet channel, and a second toroidal mixing element in fluidic communication with the first toroidal mixing element via a first neck region, and wherein the first toroidal mixing element defines a first neck angle between the inlet channel and the first neck region.
  • the first neck angle is from 0 to 180 degrees.
  • the first neck region has a length of 0.2 mm or greater.
  • the plurality of mixing elements include channels having a hydrodynamic diameter of about 20 microns to about 2 mm.
  • the mixer is sized and configured to mix the first liquid and the second liquid at a Reynolds number of less than 1000.
  • the mixer includes two or more mixers in parallel, each mixer having a plurality of toroidal mixing elements.
  • the first toroidal mixing element and the second toroidal mixing element define a mixing pair, and wherein the mixer includes a plurality of mixing pairs, and wherein each mixing pair is joined by a neck region at a neck angle.
  • the first toroidal mixing element has a first leg of a first length and a second leg of a second length; and wherein the second toroidal mixing element has a first leg of a third length and a second leg of a fourth length.
  • the first length is greater than the second length.
  • the third length is greater than the fourth length.
  • the ratio of the first length to the second length is about equal to the ratio of the third length to the fourth length.
  • the first toroidal mixing element has a first leg of a first impedance and a second leg of a second impedance; and wherein the second toroidal mixing element has a first leg of a third impedance and a second leg of a fourth impedance.
  • the first impedance is greater than the second impedance.
  • the third impedance is greater than the fourth impedance.
  • the ratio of the first impedance to the second impedance is about equal to the ratio of the third impedance to the fourth impedance.
  • the mixer includes 2 to 20 toroidal mixing elements in series.
  • the mixer includes 1 to 10 pairs of toroidal mixing elements in series.
  • the toroidal mixing elements have an inner radius of about 0.1 mm to about 2 mm.
  • microfluidic refers to a system or device for manipulating (e.g., flowing, mixing, etc.) a fluid sample including at least one channel having micron-scale dimensions (i.e., a dimension less than 1 mm).
  • therapeutic material is defined as a substance intended to furnish pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, understanding, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions.
  • Therapeutic material includes but is not limited to small molecule drugs, nucleic acids, proteins, peptides, polysaccharides, inorganic ions and radionuclides.
  • Nanoparticles is defined as a homogeneous particle comprising more than one component material (for instance lipid, polymer etc.) that is used to encapsulate a therapeutic material and possesses a smallest dimension that is less than 250 nanometers. Nanoparticles include, but are not limited to, lipid nanoparticles and polymer nanoparticles.
  • lipid nanoparticles comprise:
  • the core comprises a lipid (e.g., a fatty acid triglyceride) and is solid.
  • the core is liquid (e.g., aqueous) and the particle is a vesicle, such as a liposomes.
  • the shell surrounding the core is a monolayer.
  • the lipid core comprises a fatty acid triglyceride.
  • Suitable fatty acid triglycerides include C8-C20 fatty acid triglycerides.
  • the fatty acid triglyceride is an oleic acid triglyceride.
  • the lipid nanoparticle includes a shell comprising a phospholipid that surrounds the core.
  • Suitable phospholipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.
  • the phospholipid is a C8-C20 fatty acid diacylphosphatidylcholine.
  • a representative phospholipid is 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).
  • the ratio of phospholipid to fatty acid triglyceride is from 20:80 (mol:mol) to 60:40 (mol:mol).
  • the triglyceride is present in a ration greater than 40% and less than 80%.
  • the nanoparticle further comprises a sterol.
  • Representative sterols include cholesterol.
  • the ratio of phospholipid to cholesterol is 55:45 (mol:mol).
  • the nanoparticle includes from 55-100% POPC and up to 10 mol % PEG-lipid.
  • the lipid nanoparticles of the disclosure may include one or more other lipids including phosphoglycerides, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lyosphosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, di stearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.
  • Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are useful. Triacylglycerols are also useful.
  • Representative nanoparticles of the disclosure have a diameter from about 10 to about 100 nm.
  • the lower diameter limit is from about 10 to about 15 nm.
  • the limit size lipid nanoparticles of the disclosure can include one or more therapeutic and/or diagnostic agents. These agents are typically contained within the particle core.
  • the nanoparticles of the disclosure can include a wide variety of therapeutic and/or diagnostic agents.
  • Suitable therapeutic agents include chemotherapeutic agents (i.e., anti-neoplastic agents), anesthetic agents, beta-adrenaergic blockers, anti-hypertensive agents, anti-depressant agents, anti-convulsant agents, anti-emetic agents, antihistamine agents, anti-arrhytmic agents, and anti-malarial agents.
  • chemotherapeutic agents i.e., anti-neoplastic agents
  • anesthetic agents i.e., beta-adrenaergic blockers, anti-hypertensive agents, anti-depressant agents, anti-convulsant agents, anti-emetic agents, antihistamine agents, anti-arrhytmic agents, and anti-malarial agents.
  • antineoplastic agents include doxorubicin, daunorubicin, mitomycin, bleomycin, streptozocin, vinblastine, vincristine, mechlorethamine, hydrochloride, melphalan, cyclophosphamide, triethylenethiophosphoramide, carmaustine, lomustine, semustine, fluorouracil, hydroxyurea, thioguanine, cytarabine, floxuridine, decarbazine, cisplatin, procarbazine, vinorelbine, ciprofloxacion, norfloxacin, paclitaxel, docetaxel, etoposide, bexarotene, teniposide, tretinoin, isotretinoin, sirolimus, fulvestrant, valrubicin, vindesine, leucovorin, irinotecan, capecitabine, gemcitabine, mitoxantrone hydrochloride
  • lipid nanoparticles are nucleic-acid lipid nanoparticles.
  • nucleic acid-lipid nanoparticles refers to lipid nanoparticles containing a nucleic acid.
  • the lipid nanoparticles include one or more cationic lipids, one or more second lipids, and one or more nucleic acids.
  • the lipid nanoparticles include a cationic lipid.
  • cationic lipid refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides).
  • nucleic acids e.g., oligonucleotides
  • cationic lipid includes zwitterionic lipids that assume a positive charge on pH decrease.
  • cationic lipid refers to any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide
  • cationic lipids are available which can be used in the present disclosure. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).
  • LIPOFECTIN® commercially available cationic liposomes compris
  • lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).
  • DODAP 1,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA 1,2-dilinolenyloxy-N,N-dimethylaminopropane
  • the cationic lipid is an amino lipid.
  • Suitable amino lipids useful in the disclosure include those described in WO 2009/096558, incorporated herein by reference in its entirety.
  • Representative amino lipids include 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (
  • Suitable amino lipids include those having the formula:
  • R 1 and R 2 are either the same or different and independently optionally substituted C 10 -C 24 alkyl, optionally substituted C 10 -C 24 alkenyl, optionally substituted C 10 -C 24 alkynyl, or optionally substituted C 10 -C 24 acyl;
  • R 3 and R 4 are either the same or different and independently optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, or optionally substituted C 2 -C 6 alkynyl or R 3 and R 4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
  • R 5 is either absent or present and when present is hydrogen or C 1 -C 6 alkyl
  • n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0;
  • q 0, 1, 2, 3, or 4;
  • Y and Z are either the same or different and independently O, S, or NH.
  • R 1 and R 2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.
  • a representative useful dilinoleyl amino lipid has the formula:
  • n 0, 1, 2, 3, or 4.
  • the cationic lipid is a DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).
  • Suitable cationic lipids include cationic lipids, which carry a net positive charge at about physiological pH, in addition to those specifically described above, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt (DOTAP.Cl); 3 ⁇ -(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)-N
  • cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECTAMINE comprising DOSPA and DOPE, available from GIBCO/BRL
  • the cationic lipid is present in the lipid particle in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the lipid particle in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the lipid particle in an amount from about 40 to about 60 mole percent.
  • the lipid particle includes (“consists of”) only one or more cationic lipids and one or more nucleic acids.
  • the lipid nanoparticles include one or more second lipids. Suitable second lipids stabilize the formation of nanoparticles during their formation.
  • lipid refers to a group of organic compounds that are esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • Suitable stabilizing lipids include neutral lipids and anionic lipids.
  • neutral lipid refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH.
  • Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.
  • Exemplary lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O
  • the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids includephosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanol-amines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • POPG palmitoyloleyolphosphatidylglycerol
  • Suitable lipids include glycolipids (e.g., monosialoganglioside GM 1 ).
  • suitable second lipids include sterols, such as cholesterol.
  • the second lipid is a polyethylene glycol-lipid.
  • Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
  • Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.
  • the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol) 2000 )carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG).
  • the second lipid is present in the lipid particle in an amount from about 0.5 to about 10 mole percent. In one embodiment, the second lipid is present in the lipid particle in an amount from about 1 to about 5 mole percent. In one embodiment, the second lipid is present in the lipid particle in about 1 mole percent.
  • the lipid nanoparticles of the present disclosure are useful for the systemic or local delivery of nucleic acids. As described herein, the nucleic acid is incorporated into the lipid particle during its formation.
  • nucleic acid is meant to include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are generally termed oligonucleotides, and longer fragments are called polynucleotides. In particular embodiments, oligonucleotides of the present disclosure are 20-50 nucleotides in length.
  • polynucleotide and oligonucleotide refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages.
  • polynucleotide and oligonucleotide also includes polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides.
  • a deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyhbose joined covalently to phosphate at the 5′ and 3′ carbons of this sugar to form an alternating, unbranched polymer.
  • a ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.
  • the nucleic acid that is present in a lipid particle according to this disclosure includes any form of nucleic acid that is known.
  • the nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNA, and triplex-forming oligonucleotides.
  • the polynucleic acid is an antisense oligonucleotide.
  • the nucleic acid is an antisense nucleic acid, a ribozyme, tRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, lncRNA, pre-condensed DNA, or an aptamer.
  • nucleic acids also refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single stranded, double stranded, or contain portions of both double stranded and single stranded sequence, as appropriate.
  • nucleotide as used herein, generically encompasses the following terms, which are defined below: nucleotide base, nucleoside, nucleotide analog, and universal nucleotide.
  • nucleotide base refers to a substituted or unsubstituted parent aromatic ring or rings.
  • the aromatic ring or rings contain at least one nitrogen atom.
  • the nucleotide base is capable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an appropriately complementary nucleotide base.
  • nucleotide bases and analogs thereof include, but are not limited to, purines such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-2-isopentenyladenine (6iA), N6-2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG) hypoxanthine and 06-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6
  • nucleotide bases can be found in Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla., and the references cited therein. Further examples of universal bases can be found for example in Loakes, N. A. R. 2001, vol 29:2437-2447 and Seela N. A. R. 2000, vol 28:3224-3232.
  • nucleoside refers to a compound having a nucleotide base covalently linked to the C-1′ carbon of a pentose sugar. In some embodiments, the linkage is via a heteroaromatic ring nitrogen.
  • Typical pentose sugars include, but are not limited to, those pentoses in which one or more of the carbon atoms are each independently substituted with one or more of the same or different —R, —OR, —NRR or halogen groups, where each R is independently hydrogen, (C1-C6) alkyl or (C5-C14) aryl.
  • the pentose sugar may be saturated or unsaturated.
  • Exemplary pentose sugars and analogs thereof include, but are not limited to, ribose, 2′-deoxyribose, 2′-(C1-C6)alkoxyribose, 2′-(C5-C14)aryloxyribose, 2′,3′-dideoxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose, 2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose, 2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose, 2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose.
  • ribose 2′-deoxyribose, 2′-(C1-
  • LNA locked nucleic acid
  • Sugars include modifications at the 2′- or 3′-position such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo.
  • Nucleosides and nucleotides include the natural D configurational isomer (D-form), as well as the L configurational isomer (L-form) (Beigelman, U.S. Pat. No. 6,251,666; Chu, U.S. Pat. No. 5,753,789; Shudo, EP0540742; Garbesi (1993) Nucl. Acids Res.
  • nucleobase is purine, e.g., A or G
  • the ribose sugar is attached to the N9-position of the nucleobase.
  • nucleobase is pyrimidine, e.g., C, T or U
  • the pentose sugar is attached to the N1-position of the nucleobase (Kornberg and Baker, (1992) DNA Replication, 2.sup.nd Ed., Freeman, San Francisco, Calif.).
  • One or more of the pentose carbons of a nucleoside may be substituted with a phosphate ester.
  • the phosphate ester is attached to the 3′- or 5′-carbon of the pentose.
  • the nucleosides are those in which the nucleotide base is a purine, a 7-deazapurine, a pyrimidine, a universal nucleotide base, a specific nucleotide base, or an analog thereof.
  • nucleotide analog refers to embodiments in which the pentose sugar and/or the nucleotide base and/or one or more of the phosphate esters of a nucleoside may be replaced with its respective analog.
  • exemplary pentose sugar analogs are those described above.
  • nucleotide analogs have a nucleotide base analog as described above.
  • exemplary phosphate ester analogs include, but are not limited to, alkylphosphonates, methylphosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoroamidates, boronophosphates, and may include associated counterions.
  • Other nucleic acid analogs and bases include for example intercalating nucleic acids (INAs, as described in Christensen and Pedersen, 2002), and AEGIS bases (Eragen, U.S. Pat. No. 5,432,272).
  • nucleic analogs comprise phosphorodithioates (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci.
  • universal nucleotide base refers to an aromatic ring moiety, which may or may not contain nitrogen atoms.
  • a universal base may be covalently attached to the C-1′ carbon of a pentose sugar to make a universal nucleotide.
  • a universal nucleotide base does not hydrogen bond specifically with another nucleotide base.
  • a universal nucleotide base hydrogen bonds with nucleotide base, up to and including all nucleotide bases in a particular target polynucleotide.
  • a nucleotide base may interact with adjacent nucleotide bases on the same nucleic acid strand by hydrophobic stacking.
  • Universal nucleotides include, but are not limited to, deoxy-7-azaindole triphosphate (d7AITP), deoxyisocarbostyril triphosphate (dICSTP), deoxypropynylisocarbostyril triphosphate (dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP), deoxyImPy triphosphate (dImPyTP), deoxyPP triphosphate (dPPTP), or deoxypropynyl-7-azaindole triphosphate (dP7AITP). Further examples of such universal bases can be found, inter alia, in Published U.S. application Ser. No. 10/290,672, and U.S. Pat. No. 6,433,134.
  • polynucleotide and “oligonucleotide” are used interchangeably and mean single-stranded and double-stranded polymers of nucleotide monomers, including 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, e.g., 3′-5′ and 2′-5′, inverted linkages, e.g., 3′-3′ and 5′-5′, branched structures, or internucleotide analogs.
  • DNA 2′-deoxyribonucleotides
  • RNA ribonucleotides linked by internucleotide phosphodiester bond linkages, e.g., 3′-5′ and 2′-5′, inverted linkages, e.g., 3′-3′ and 5′-5′, branched structures, or internucleotide analogs.
  • Polynucleotides have associated counter ions, such as H+, NH4+, trialkylammonium, Mg2+, Na+, and the like.
  • a polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof.
  • Polynucleotides may be comprised of internucleotide, nucleobase and/or sugar analogs. Polynucleotides typically range in size from a few monomeric units, e.g., 3-40 when they are more commonly frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units.
  • nucleotide sequence is represented, it will be understood that the nucleotides are in 5′ to 3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytosine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted.
  • nucleobase means those naturally occurring and those non-naturally occurring heterocyclic moieties commonly known to those who utilize nucleic acid technology or utilize peptide nucleic acid technology to thereby generate polymers that can sequence specifically bind to nucleic acids.
  • Non-limiting examples of suitable nucleobases include: adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methlylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).
  • Other non-limiting examples of suitable nucleobase include those nucleobases illustrated in FIGS. 2(A) and 2(B) of Buchardt et al. (WO92/20702 or WO92/20703).
  • nucleobase sequence means any segment, or aggregate of two or more segments (e.g. the aggregate nucleobase sequence of two or more oligomer blocks), of a polymer that comprises nucleobase-containing subunits.
  • suitable polymers or polymers segments include oligodeoxynucleotides (e.g. DNA), oligoribonucleotides (e.g. RNA), peptide nucleic acids (PNA), PNA chimeras, PNA combination oligomers, nucleic acid analogs and/or nucleic acid mimics.
  • polynucleobase strand means a complete single polymer strand comprising nucleobase subunits.
  • a single nucleic acid strand of a double stranded nucleic acid is a polynucleobase strand.
  • nucleic acid is a nucleobase sequence-containing polymer, or polymer segment, having a backbone formed from nucleotides, or analogs thereof.
  • Preferred nucleic acids are DNA and RNA.
  • nucleic acids may also refer to “peptide nucleic acid” or “PNA” means any oligomer or polymer segment (e.g. block oligomer) comprising two or more PNA subunits (residues), but not nucleic acid subunits (or analogs thereof), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in U.S. Pat. Nos.
  • PNA peptide nucleic acid
  • peptide nucleic acid or “PNA” shall also apply to any oligomer or polymer segment comprising two or more subunits of those nucleic acid mimics described in the following publications: Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4: 1081-1082 (1994); Petersen et al., Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996); Diderichsen et al., Tett. Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett.
  • polymer nanoparticles refers to polymer nanoparticles containing a therapeutic material.
  • Polymer nanoparticles have been developed using, a wide range of materials including, but not limited to: synthetic homopolymers such as polyethylene glycol, polylactide, polyglycolide, poly(lactide-coglycolide), polyacrylates, polymethacrylates, poly caprolactone, polyorthoesters, polyanhydrides, polylysine, polyethyleneimine; synthetic copolymers such as poly(lactide-coglycolide), poly(lactide)-poly(ethylene glycol), poly(lactide-co-glycolide)-poly(ethylene glycol), poly(caprolactone)-poly(ethylene glycol); natural polymers such as cellulose, chitin, and alginate, as well as polymer-therapeutic material conjugates
  • polymer refers to compounds of usually high molecular weight built up chiefly or completely from a large number of similar units bonded together. Such polymers include any of numerous natural, synthetic and semi-synthetic polymers.
  • natural polymer refers to any number of polymer species derived from nature. Such polymers include, but are not limited to the polysaccharides, cellulose, chitin, and alginate.
  • synthetic polymer refers to any number of synthetic polymer species not found in Nature. Such synthetic polymers include, but are not limited to, synthetic homopolymers and synthetic copolymers.
  • Synthetic homopolymers include, but are not limited to, polyethylene glycol, polylactide, polyglycolide, polyacrylates, polymethacrylates, poly caprolactone, polyorthoesters, polyanhydrides, polylysine, and polyethyleneimine.
  • Synthetic copolymer refers to any number of synthetic polymer species made up of two or more synthetic homopolymer subunits. Such synthetic copolymers include, but are not limited to, poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol), poly(lactide-co-glycolide)-poly(ethylene glycol), and poly(caprolactone)-poly(ethylene glycol).
  • polystyrene resin refers to any number of polymers derived by the
  • Such polymers include, but are not limited to, carboxymethyl cellulose, acetylated carboxymethylcellulose, cyclodextrin, chitosan and gelatin.
  • polymer conjugate refers to a compound prepared by covalently, or non-covalently conjugating one or more molecular species to a polymer.
  • Such polymer conjugates include, but are not limited to, polymer-therapeutic material conjugates.
  • Polymer-therapeutic material conjugate refers to a polymer conjugate where one or more of the conjugated molecular species is a therapeutic material.
  • Such polymer-therapeutic material conjugates include, but are not limited to, polymer-drug conjugates.
  • Polymer-drug conjugate refers to any number of polymer species conjugated to any number of drug species.
  • Such polymer drug conjugates include, but are not limited to, acetyl methylcellulose-polyethylene glycol-docetaxol.
  • the term “about” indicates that the associated value can be modified, unless otherwise indicated, by plus or minus five percent (+/ ⁇ 5%) and remain within the scope of the embodiments disclosed.
  • siRNA-Lipid Nanoparticles Manufactured Using Four Single Microfluidic Mixer Devices Arrayed in Parallel Using a Manifold, or Four Microfluidic Mixers Arrayed in Parallel in a Single Device
  • the siRNA-LNP produced using four single microfluidic mixer devices in parallel using a manifold is compared to the siRNA-LNP produced using four microfluidic mixers arrayed in parallel in a single device ( FIG. 3 ).
  • the purpose of this example is to demonstrate that there are on-device and off-device methods of arraying microfluidic mixer.
  • the fluid driving pumps were operated under the same process conditions, with identical nanoparticle forming materials, and tests were conducted on the each method of arraying.
  • the results in FIG. 4 show similar siRNA-LNP produced using the two methods of arraying, and the siRNA-LNP is not affected by the method of arraying.
  • FIG. 4 shows particle diameter (nm) and polydispersity index (PDI) for representative siRNA-Lipid Nanoparticles (siRNA-LNP) as a function of four single microfluidic mixer devices arrayed in parallel using a manifold, or four microfluidic mixers arrayed in parallel in the representative single device illustrated in FIG. 3 .
  • PDI polydispersity index
  • the siRNA-LNP were composed of 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate/DSPC/Chol/PEG-c-DMA at mole ratios of 50:10:38.5:1.5 and a siRNA-total lipid ratio of 0.06 wt/wt, and the nanoparticles were produced using the illustrative continuous flow system shown in FIG. 2 with either four single microfluidic mixer device arrayed in parallel using a manifold (4 ⁇ Manifold), or four microfluidic mixers arrayed in parallel in a single device illustrated in FIG. 3 (4 ⁇ On-Chip). The total flow rates through the microfluidic device are shown in the legend. Error bars represent the standard deviation of the mean.
  • siRNA-total lipid ratio was 0.06 wt/wt.
  • Flow rate per mixer was 12 mL/min at siRNA to lipid mix flow rate ratio of 3:1 (i.e., 9 mL/min siRNA and 3 mL/min lipid mix in 1 ⁇ ).
  • the first 2 mL of volume collected from the mixer outlet at the beginning of each formulation run was discarded as waste, and the remaining volume was collected as the sample.
  • the 1 mL of the collected sample was further diluted into 3 mL of Dulbecco's Phosphate Buffered Saline (without calcium and without magnesium) before particle sizing.
  • siRNA-LNP were diluted to appropriate concentration with Dulbecco's Phosphate Buffered Saline (without calcium and without magnesium) and mean particle size (intensity-weighted) was determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS two angle particle sizer (Malvern Instruments Ltd., Malvern, Worcestershire, UK).
  • DLS dynamic light scattering
  • siRNA-Lipid Nanoparticles (siRNA-LNP) Manufactured Using Eight Single Microfluidic Mixer Devices Arrayed in Parallel Using a Manifold
  • siRNA-LNP 520 mL volume of siRNA-LNP was produced using eight single microfluidic mixer devices arrayed in parallel using an external manifold. Each mixer in the array was identical, thus the process conditions for forming siRNA-LNP in each mixer was identical.
  • the purpose of this experiment was to demonstrate the effect of a large number of parallel mixers on siRNA-LNP size and quality.
  • This example significantly demonstrates the successful utilization of a large number of microfluidic mixers used in parallel in the same system to produce a large volume batch of siRNA-LNP using an exemplary system as disclosed herein.
  • FIG. 5 shows particle diameter (nm) and polydispersity index (PDI) for representative siRNA-Lipid Nanoparticles (siRNA-LNP) as a function of the manufactured volume.
  • the siRNA-LNP were composed of 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate/DSPC/Chol/PEG-c-DMA at mole ratios of 50:10:38.5:1.5 and a siRNA-total lipid ratio of 0.06 wt/wt, and the nanoparticles were produced using the illustrative continuous flow system shown in FIG. 2 with eight single microfluidic mixer device arrayed in parallel using a manifold.
  • Nanoparticles were sampled every 100 mL from 0 mL to 500 mL and the results compared to a 2 mL preparation of the same siRNA-LNP prepared using the NanoAssemblrTM Benchtop Instrument.
  • the NanoAssemblrTM Benchtop Instrument is commercially available laboratory apparatus that uses microfluidics to manufacture fixed volume batches of nanoparticles. Error bars represent the standard deviation of the mean.
  • siRNA-total lipid ratio was 0.06 wt/wt.
  • the first 20 mL of volume collected from the mixer outlet at the beginning the formulation run was discarded as waste, and the remaining volume was collected as the sample.
  • the particle formulation was diluted in-line with Dulbecco's Phosphate Buffered Saline (without calcium and without magnesium), 1 part particle solution to 3 part DPBS, driven by a Masterflex L/S peristaltic pump with dual Easy-load II pump heads (Cole-Parmer Instrument Company, Montreal, QC, Canada). The resulting particle formulation was collected in aliquots and sized.

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