U.S. patent application number 15/552473 was filed with the patent office on 2018-02-15 for continuous flow microfluidic system.
This patent application is currently assigned to The University of British Columbia. The applicant listed for this patent is THE UNIVERSITY OF BRITISH COLUMBIA. Invention is credited to Timothy LEAVER, Kevin OU, Euan RAMSAY, Robert James TAYLOR, Colin WALSH, Andre WILD.
Application Number | 20180043320 15/552473 |
Document ID | / |
Family ID | 56789492 |
Filed Date | 2018-02-15 |
United States Patent
Application |
20180043320 |
Kind Code |
A1 |
RAMSAY; Euan ; et
al. |
February 15, 2018 |
CONTINUOUS FLOW MICROFLUIDIC SYSTEM
Abstract
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. In certain embodiments, continuous flow
operation and parallelization of microfluidic mixers contribute to
increased nanoparticle production volume.
Inventors: |
RAMSAY; Euan; (Vancouver,
CA) ; TAYLOR; Robert James; (Vancouver, CA) ;
LEAVER; Timothy; (Delta, CA) ; WILD; Andre;
(Vancouver, CA) ; OU; Kevin; (Toronto, CA)
; WALSH; Colin; (Belmont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF BRITISH COLUMBIA |
Vancouver |
|
CA |
|
|
Assignee: |
The University of British
Columbia
Vancouver
BC
|
Family ID: |
56789492 |
Appl. No.: |
15/552473 |
Filed: |
February 24, 2016 |
PCT Filed: |
February 24, 2016 |
PCT NO: |
PCT/US2016/019414 |
371 Date: |
August 21, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62275630 |
Jan 6, 2016 |
|
|
|
62120179 |
Feb 24, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 2219/00873
20130101; B01J 2219/00831 20130101; B01J 2219/00855 20130101; B01F
5/0644 20130101; B01J 2219/00824 20130101; B01F 5/0647 20130101;
B01F 13/1022 20130101; B01J 2219/00833 20130101; B01F 2215/0032
20130101; B01F 2005/0623 20130101; B01J 2219/00894 20130101; B01J
2219/00783 20130101; B01F 5/0619 20130101; B01J 2219/0086 20130101;
B01F 2005/0636 20130101; B01J 2219/00986 20130101; B01J 2219/00858
20130101; B01J 2219/00869 20130101; B01J 2219/00898 20130101; A61K
9/1682 20130101; B01J 19/0093 20130101; B01J 2219/00822 20130101;
G01N 2035/00158 20130101; B01J 2219/00889 20130101; G01N 2015/0038
20130101; B01F 13/0059 20130101 |
International
Class: |
B01F 5/06 20060101
B01F005/06; B01F 13/10 20060101 B01F013/10; B01F 13/00 20060101
B01F013/00; A61K 9/16 20060101 A61K009/16; B01J 19/00 20060101
B01J019/00 |
Claims
1. A system for continuous flow operation of a microfluidic chip,
the system comprising: (1) a microfluidic chip, comprising: (a) a
first inlet configured to receive a first solution; (b) a second
inlet configured to receive a second solution; and (c) a first
mixer, comprising: (i) a first inlet microchannel configured to
receive the first solution from the first inlet; (ii) a second
inlet microchannel configured to receive the second solution from
the second inlet; and (iii) a mixing microchannel configured to mix
the first solution and the second solution to provide a
nanoparticle solution at a mixer outlet; and (d) a chip outlet in
fluid communication with the mixer outlet through a nanoparticle
solution microchannel; (2) 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;
(3) 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; and (4) a
system outlet in fluid communication with the chip outlet, wherein
the system outlet is configured to output the nanoparticle
solution.
2. The system of claim 1, wherein the first solution comprises an
active pharmaceutical ingredient.
3. The system of claim 1, wherein the second solution comprises a
particle-forming material in a second solvent.
4. The system of claim 1, wherein the first solution comprises a
nucleic acid in a first solvent and the second solution comprises
lipid particle-forming materials in a second solvent.
5. The system of claim 1, wherein 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.
6. The system of claim 5, wherein the first mixer is a chaotic
advection mixer.
7. The system of claim 5, wherein the mixing region comprises a
herringbone mixer.
8. The system of claim 4, wherein the mixing region has a
hydrodynamic diameter of about 20 microns to about 300 microns.
9. The system of claim 1, wherein the first mixer is sized and
configured to mix the first solution and the second solution at a
Reynolds number of less than 1000.
10. The system of claim 1, wherein the microfluidic chip is
sterile.
11. The system of claim 1, further comprising 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.
12. The system of claim 11, wherein the plurality of mixers are
within a plurality of microfluidic chips.
13. The system of claim 11, wherein 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.
14. The system of claim 11, wherein the system further comprises 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.
15. The system of claim 11, wherein the 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.
16. The system of claim 11, wherein 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; 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; and 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.
17. The system of claim 11, wherein the plurality of mixers are
within the microfluidic chip.
18. The system of claim 11, wherein at least a portion of the
plurality of mixers are parallelized mixers, arranged in parallel,
wherein each of the portion of plurality of mixers has a mixer
outlet in fluid communication with the system outlet.
19. The system of claim 18, wherein the parallelized mixers are
arranged in a stacked configuration on the microfluidic chip.
20. The system of claim 18, wherein the parallelized mixers are
arranged in a horizontal configuration, in substantially the same
plane, on the microfluidic chip.
21. The system of claim 18, wherein the parallelized mixers are
arranged in both a horizontal configuration and a stacked
configuration on the microfluidic chip.
22. The system of claim 1, further comprising 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.
23. The system of claim 1, further comprising 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.
24. The system of claim 1, wherein the first continuous flow fluid
driver and the second continuous flow fluid driver are
independently selected from the group consisting of a positive
displacement pump, a centrifugal pump, and a pressure driven
pump.
25. The system of claim 1, wherein the system includes a disposable
fluidic path.
26. The system of claim 25, wherein 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.
27. The system of claim 26, wherein 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, polymer, titanium, and ceramic.
28. The system of claim 25, wherein every surface touched by the
first solution, the second solution, and the nanoparticle solution
are disposable.
29. The system of claim 1, wherein the system has a footprint area
of 1 m.sup.2 or less.
30. The system of claim 29, wherein the system has a production
volume of at least 0.76 L of nanoparticle solution per hour.
31. The system of claim 1, further comprising a pulse dampening
mechanism configured to minimize flow pulsation from the first
continuous flow fluid driver, the second continuous flow fluid
driver, or both.
32. A sterile package comprising a sterile microfluidic chip
according to any of the preceding claims sealed within the sterile
package.
33. A method of forming nanoparticles, comprising flowing a first
solution and a second solution through a system according to any of
the preceding claims and forming a nanoparticle solution in the
first mixer of the microfluidic chip.
34. The method of claim 33, wherein 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.
35. The method of claim 34, wherein the plurality of mixers are
contained within a plurality of microfluidic chips.
36. The method of claim 34, wherein the plurality of mixers are
contained within a single microfluidic chip.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Patent
Application No. 62/120,179, filed Feb. 24, 2015, and U.S. Patent
Application No. 62/275,630, filed Jan. 6, 2016, the disclosures of
which are hereby incorporated by reference in their entirety.
BACKGROUND
[0002] The manufacture of pharmaceutical compositions on a
large-scale for clinical development and commercial production has
traditionally been challenging. Often techniques used in the
laboratory for small-scale production of pharmaceuticals are not
amenable to scale-up. These challenges are exacerbated when
manufacturing complex pharmaceutical colloidal systems such as
nanoparticles. 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. Traditional processes for
the manufacture of nanoparticles are batch-based systems and often
results in production of meta-stable nanoparticles where particle
characteristics such as size, polydispersity and encapsulation
efficiency are sensitive to (local) environmental changes within
the batch manufacturing process, including, but not limited to,
temperature, pH, ionic strength, buffer composition, solvent
concentrations. Consequently, traditional batch processes for the
manufacture of nanoparticles are expensive, time consuming, and
difficult to reproduce, which necessitates substantial optimization
with increases in batch sizes leading to increased commercial risk.
Moreover, traditional nanoparticle manufacturing processes
necessitate nanoparticle product contact with the manufacturing
apparatus, which requires costly and time-consuming cleaning and
sterilization validation because it is not economically viable to
dispose of the apparatus after manufacture of each batch.
[0003] In view of these challenges, improved nanoparticle
manufacturing systems that yield greater production volume are
desirable.
SUMMARY
[0004] This summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This summary is not intended to identify
key features of the claimed subject matter, nor is it intended to
be used as an aid in determining the scope of the claimed subject
matter.
[0005] In one aspect, a system for continuous flow operation of a
microfluidic chip is provided. In one embodiment, the system
includes:
[0006] (1) a microfluidic chip, comprising: [0007] (a) a first
inlet configured to receive a first solution; [0008] (b) a second
inlet configured to receive a second solution; and [0009] (c) a
first mixer, comprising: [0010] (i) a first inlet microchannel
configured to receive the first solution from the first inlet;
[0011] (ii) a second inlet microchannel configured to receive the
second solution from the second inlet; and [0012] (iii) a mixing
microchannel configured to mix the first solution and the second
solution to provide a nanoparticle solution at a mixer outlet; and
[0013] (d) a chip outlet in fluid communication with the mixer
outlet through a nanoparticle solution microchannel;
[0014] (2) 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;
[0015] (3) 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; and
[0016] (4) a system outlet in fluid communication with the chip
outlet, wherein the system outlet is configured to output the
nanoparticle solution.
[0017] In one aspect, a method of forming nanoparticles is
provided. In one embodiment, the method 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.
DESCRIPTION OF THE DRAWINGS
[0018] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0019] FIG. 1 is a schematic representation of a continuous flow
system of the present disclosure.
[0020] FIG. 2 is a schematic representation of a continuous flow
system of the present disclosure.
[0021] FIG. 3 is a schematic illustration of a representative
fluidic device of the disclosure.
[0022] 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.
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.times.Manifold), or
four microfluidic mixers arrayed in parallel in a single device
illustrated in FIG. 3 (4.times. On-Chip). The total flow rates
through the microfluidic device are shown in the legend. Error bars
represent the standard deviation of the mean.
[0023] 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
NanoAssemblr.TM. Benchtop Instrument. The NanoAssemblr.TM. 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.
[0024] FIG. 6 is a schematic illustration of a representative
system of the disclosure, a continuous-flow staggered herringbone
(SHM) micromixer. The mixing of two separate streams occurs in the
patterned central channel which grooved walls drive alternating
secondary flows that chaotically stir the fluids injected. The
chaotic mixing leads to exponential increase of the interfacial
area thus reducing the diffusion distances between two fluids.
Rapid interdiffusion of the two phases (aqueous and ethanolic
containing fully solvated lipids) results in the self-assembly of
LNPs, whose size depends primarily on their lipid composition and
aqueous/ethanolic flow rate ratio.
[0025] FIG. 7 is a three-dimensional view of a representative
parallel fluidic structure useful for making limit size lipid
nanoparticles.
[0026] 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.
[0027] FIG. 9 illustrates a second representative parallel fluidic
structure useful for making limit size lipid nanoparticles.
[0028] FIG. 10 is a labeled photograph of an exemplary 4.times.
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.
[0029] FIG. 11 is a labeled photography of an exemplary 8.times.
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.
[0030] 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.
DETAILED DESCRIPTION
[0031] 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. In certain embodiments, 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.
[0032] Microfluidic mixers are microfluidic elements that are
integrated into microfluidic devices on a microfluidic chip. As
used herein, 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. In several exemplary
embodiments disclosed herein, 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.
However, other microfluidic devices are also compatible with the
disclosed systems.
[0033] In particular, 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.
Moreover, the present disclosure employs microfluidics which has
the advantage of exquisite 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. In further embodiments, 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.
[0034] 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.sub.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. The
number of microfluidic mixers (n.sub.2) is scaled depending on
throughput requirements. In one embodiment, 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.
[0035] In certain embodiment, after mixer array 108, one or more
(n.sub.3) post-formation microfluidic mixers 110 are arranged in
sequence so that one or more additional components (n.sub.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.
[0036] Nanoparticles are formed via nanoprecipitation due to rapid
mixing of the fluid streams within the mixer array 108. In one
embodiment, 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.sub.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. Alternatively, dilution can be achieved using a further
microfluidic mixer array as part of the post-formation microfluidic
mixer(s) 110. One additional benefit in the continuous flow process
is that a time delay in diluting particle product can influence
particle quality and stability. Accordingly, 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"). In one embodiment, the distance
between the last mixer (e.g., 108 or 110) and the dilution junction
is about 1 cm to about 50 cm. In one embodiment, 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.
[0037] In another embodiment, the system, including tubing
dimensions and flow characteristics, are configured to produce a
specific hold time. In one embodiment, 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.
[0038] 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.
[0039] In certain embodiments nanoparticle manufacturing is
conducted in a specialized barrier facility that eliminates the
requirement for filtration to ensure a sterile product.
[0040] 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. In
one embodiment, 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.
[0041] FIG. 2 shows apparatus 200, one embodiment of the present
disclosure. In one embodiment, the apparatus provides a system for
the manufacture of lipid nanoparticles containing a nucleic acid.
In another embodiment, the apparatus provides a system for the
manufacture of limit size lipid nanoparticles including, but not
limited to, liposomes and nanoemulsions containing therapeutic
material. In a further embodiment, the apparatus provides a system
for the manufacture of polymer nanoparticles containing therapeutic
material.
[0042] In one aspect, a system for continuous flow operation of a
microfluidic chip is provided. In one embodiment, the system
includes:
[0043] (1) a microfluidic chip, comprising: [0044] (a) a first
inlet configured to receive a first solution; [0045] (b) a second
inlet configured to receive a second solution; and [0046] (c) a
first mixer, comprising: [0047] (i) a first inlet microchannel
configured to receive the first solution from the first inlet;
[0048] (ii) a second inlet microchannel configured to receive the
second solution from the second inlet; and [0049] (iii) a mixing
microchannel configured to mix the first solution and the second
solution to provide a nanoparticle solution at a mixer outlet; and
[0050] (d) a chip outlet in fluid communication with the mixer
outlet through a nanoparticle solution microchannel;
[0051] (2) 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;
[0052] (3) 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; and
[0053] (4) a system outlet in fluid communication with the chip
outlet, wherein the system outlet is configured to output the
nanoparticle solution.
[0054] The systems and methods will now be described in further
detail.
[0055] Continuous Flow
[0056] Continuous flow allows for large volumes of product (e.g.,
nanoparticles) to be produced using microfluidics, which are
traditionally low-volume production systems. The use of
parallelization, described in more detail below, further increases
production capacity when combined with continuous flow.
[0057] As used herein, 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. In one embodiment, the constant
flow rate varies by +/-10% or less at 50 mL/min flow rate and 250
PSI backpressure. In a further embodiment, the constant flow rate
is +/-4% or less at 50 mL/min flow rate and 250 PSI backpressure.
These values are in the absence of any pulse dampener.
[0058] 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.
[0059] In one embodiment, the pulse dampener(s) have a 3:1
reduction in flow pulsation (dependent on pump operating
conditions). In one exemplary embodiment, the pulse dampener
comprises a flexible PTFE membrane and stainless steel and
polyetheretherketone as fluid contacting materials.
[0060] In one embodiment, 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
[0061] The pressures experienced by the microfluidic devices are
relatively high, due to the desire for high throughput and the
necessary high flow rates. In one embodiment, 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.). With regard to microfluidic elements in the
system, the maximum pressure occurs at the inlets of the chip. The
microfluidic chip inlet pressure can reach a maximum of 200 PSI. In
one embodiment, the peak pressure on the microfluidic chip is from
about 100 PSI to about 200 PSI. In one embodiment, the system
operates at pressure of about 5 PSI to about 200 PSI.
[0062] In one embodiment, 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.
[0063] As a result of the miniaturization of production via
microfluidics and the increased capacity afforded by continuous
flow operation, the footprint of the systems disclosed is greatly
reduced compared to known systems capable of producing similar
volumes per unit time. As an example, the smallest commercially
available batch system, the NanoAssemblr (manufactured by Precision
Nanosystems Inc. of Vancouver, BC) is a microfluidic system with a
small footprint. However, due to the slow nature of batch
processing, in order to produce 1 L of nanoparticles in 80 minutes,
40 NanoAssemblrs would be required, which would result in a
footprint of about 2 m.sup.2. This is at least twice the footprint
of even the most basic continuous flow system disclosed herein.
[0064] In one embodiment, the system has a footprint area of 1
m.sup.2 or less. In one embodiment, the system has a footprint area
of 0.8 m.sup.2 or less. A photograph of a representative system
having two pumps driving four microfluidic mixing chips (4.times.),
each with a single mixer, is pictured in FIG. 10. The footprint of
this system is about 0.8 m.sup.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.
[0065] 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.
[0066] Referring to FIG. 11, an 8.times. 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.
[0067] In one embodiment, the system has a production volume of
0.76 L of nanoparticle solution per hour. In embodiments with
multiple mixers (e.g., on the same microfluidic chip or separate
microfluidic chips) the production volume can be increased by the
number of mixers, N. For example, four mixers can produce a volume
of 4.times.0.76 liters/hour. In another embodiment, the system has
a production volume of 0.5 L of nanoparticle solution per hour. In
another embodiment, the system has a production volume of 1.0 L of
nanoparticle solution per hour.
[0068] In one embodiment, the system is scalable to produce a
product solution from 0.025 L to 5000 L.
[0069] In one embodiment, 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.
[0070] Microfluidic Mixers
[0071] 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:
[0072] (1) U.S. patent application Ser. No. 13/464,690, which is a
continuation of PCT/CA2010/001766, filed Nov. 4, 2010, which claims
the benefit of U.S. Ser. No. 61/280,510, filed Nov. 4, 2009;
[0073] (2) U.S. patent application Ser. No. 14/353,460, which is a
continuation of PCT/CA2012/000991, filed Oct. 25, 2012, which
claims the benefit of U.S. Ser. No. 61/551,366, filed Oct. 25,
2011;
[0074] (3) PCT/US2014/029116, filed Mar. 14, 2014 (published as WO
2014/172045, on Oct. 23, 2014), which claims the benefit of U.S.
Ser. No. 61/798,495, filed Mar. 15, 2013;
[0075] (4) PCT/US2014/041865, filed Jul. 25, 2014 (published as WO
2015/013596, on Jan. 29, 2015), which claims the benefit of U.S.
Ser. No. 61/858,973, filed Jul. 26, 2013;
[0076] (5) PCT/US2014/060961, which claims the benefit of U.S. Ser.
No. 61/891,758, filed Oct. 16, 2013; and
[0077] (6) U.S. Patent Application No. 62/275,630, filed Jan. 6,
2016,
[0078] the disclosures of which are hereby incorporated by
reference in their entirety. Furthermore, representative
microfluidic devices are disclosed in further detail herein.
[0079] In certain embodiments, 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. In one embodiment, with reference to FIG.
6, the device includes:
[0080] (a) a first inlet 302 for receiving a first solution
comprising a first solvent;
[0081] (b) a first inlet microchannel 304 in fluid communication
with the first inlet to provide a first stream comprising the first
solvent;
[0082] (c) a second inlet 306 for receiving a second solution
comprising lipid particle-forming materials in a second
solvent;
[0083] (d) a second inlet microchannel 308 in fluid communication
with the second inlet to provide a second stream comprising the
lipid particle-forming materials in the second solvent; and
[0084] (e) 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.
[0085] In one embodiment, the second region of the microchannel
comprises bas-relief structures. In certain embodiments, 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. In other embodiments, the
second region includes a micromixer.
[0086] In the devices and systems, means for varying the flow rates
of the first and second streams are used to rapidly mix the streams
thereby providing the nanoparticles.
[0087] In certain embodiments, the devices of the disclosure
provide complete mixing occurs in less than 10 ms.
[0088] In certain embodiments, one or more regions of the device
are heated.
[0089] In one embodiment, 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.
[0090] In one embodiment, the first mixer is a chaotic advection
mixer.
[0091] In one embodiment, the mixing region comprises a herringbone
mixer. While a SHM mixer is illustrated in certain FIGURES (e.g.,
FIG. 6), it will be appreciated that other mixing configurations
are also contemplated. In one embodiment, the mixer is a dean
vortexing mixer. In another embodiment, the mixer is a Dean vortex
bifurcating mixer (DVBM), which are discussed in greater detail
below. In one embodiment, the microfluidic chip includes two
different types of chaotic advection mixers. In a further
embodiment, the two different types of chaotic advection mixers are
SHM and Dean vortexing. In one embodiment, the microfluidic chip
includes two different types of chaotic advection mixers, wherein
at least one of the two chaotic advection mixers is selected from
the group consisting of SHM and Dean vortexing.
[0092] In one embodiment, 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).
[0093] The mixing region can also be defined using standard width
and height measurements. In one embodiment, the mixing region has a
width of about 100 to about 500 microns and a height of about 50 to
about 200 microns. In one embodiment, the mixing region has a width
of about 200 to about 400 microns and a height of about 100 to
about 150 microns.
[0094] In order to maintain laminar flow and keep the behavior of
solutions in the microfluidic devices predictable and the methods
repeatable, the systems are designed to support flow at low
Reynolds numbers. 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 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.
[0095] In one embodiment, the microfluidic mixer device contains
one micromixer. In one embodiment, 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. In a further
embodiment, 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. In one
embodiment, the second region of the microchannel comprises
bas-relief structures. In certain embodiments, the second regions
each have a fluid flow rate of from 1 to about 50 mL/min. In a
preferred embodiment, 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.
[0096] In other embodiments, 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.
[0097] In one embodiment, 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. In a
further embodiment, 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. In
one embodiment, 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. In a preferred
embodiment, the sealing gaskets are O-rings. In one embodiment, the
device holder and clamping system comprise a solid polycarbonate
plate applying mechanical force through tightening screws. In a
further embodiment the microfluidic mixer device and device holder
are a single disposable plastic piece without the need for a
gasket-based clamping system.
[0098] Solutions and Products
[0099] 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.
[0100] The first solution, also referred to herein as the "aqueous
reagent" herein, is provided in a first solution reservoir. In one
embodiment, the first solution comprises a first solvent. In one
embodiment, the first solution comprises an active pharmaceutical
ingredient. In one embodiment, the first solution comprises a
nucleic acid in a first solvent. In another embodiment, the first
solution comprises a buffer. In one embodiment, the first solution
consists essentially of a buffer.
[0101] The second solution, also referred to herein as the "solvent
reagent" herein, is provided in a second solution reservoir. In one
embodiment, the second solution comprises a second solvent. In one
embodiment, the second solution comprises lipid particle-forming
materials in a second solvent. In one embodiment, the second
solvent is a water-miscible solvent (e.g., ethanol or
acetonitrile). In certain embodiments, the second solution is an
aqueous buffer comprising polymer nanoparticle forming
reagents.
[0102] In one embodiment, the first solution comprises a nucleic
acid in a first solvent and the second solution comprises lipid
particle-forming materials in a second solvent.
[0103] Parallelization
[0104] In the most basic configuration of the disclosed system, a
single microfluidic mixer is contained in one microfluidic device
on one microfluidic chip. However, increased production volume is
achieved by parallelizing the mixers, whether through on-chip
parallelization, using multiple chips, or both.
[0105] In one embodiment, 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. In one embodiment,
the plurality of mixers are all of the same dimensions. In another
embodiment, the plurality of mixers have different dimensions.
[0106] In one embodiment, the plurality of mixers are within a
plurality of microfluidic chips. In another embodiment, the
plurality of mixers are on a single microfluidic chip.
[0107] In one embodiment the microfluidic mixer array incorporates
1-128 microfluidic mixers arrayed in parallel to increase the
throughput of the manufacturing system. As an example, a 128-mixer
system according to the disclosed embodiments is capable of
producing about 1.5 L/min of nanoparticle solution.
[0108] In a further embodiment, the microfluidic mixer device
contains more than one micromixer. In one embodiment, a single
device contains four microfluidic mixers (FIG. 3). In an exemplary
embodiment, the microfluidic mixers are arrayed in parallel in a
single device. FIG. 3 provides an illustration of a representative
device 250, which comprises two inlet channels 255 that feed a
fluid manifold system 260 (i.e., an on-chip manifold). The fluid
manifold system splits the inlet streams equally among the four
microfluidic mixers arrayed in parallel in the single device 260.
The output of the microfluidic mixers is collected in the outlets
265. In a further embodiment, the outlet 265 of each mixer is in
fluid communication with an outlet manifold system (not pictured)
that collects mixed solution from all four devices, in a manner
analogous to the outlet manifold 214 of FIG. 2.
[0109] 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. However, by
minimizing the separation between mixers, 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.
[0110] In another embodiment, the single device has microfluidic
mixers array in the planar and vertical directions of the chip, for
high-density 3-dimensional microfluidic parallelization. In other
embodiments, microfluidic mixer arrays can be arranged in sequence
for multi-step manufacture of complex nanoparticle systems. In
certain embodiment, the system of the present disclosure operates
at a flow rate between 1 mL/min and 50 mL/min per microfluidic
mixer. In another embodiment, at least one microfluidic mixer of
the system operates at a flow rate of about 10 mL/min to about 25
mL/min. In a further embodiment each independent continuous flow
fluid driver operates at a flow rate of 1.0 L/min.
[0111] In one embodiment, at least a portion of the plurality of
mixers are parallelized mixers, arranged in parallel, wherein each
of the portion of plurality of mixers has a mixer outlet in fluid
communication with the system outlet.
[0112] In one embodiment, the parallelized mixers are arranged in a
stacked configuration on the microfluidic chip.
[0113] In one embodiment, the parallelized mixers are arranged in a
horizontal configuration, in substantially the same plane, on the
microfluidic chip.
[0114] In one embodiment, the parallelized mixers are arranged in
both a horizontal configuration and a stacked configuration on the
microfluidic chip.
[0115] In certain embodiments, the disclosure provides devices that
include more than one fluidic mixing structures (i.e., an array of
fluidic structures). In certain embodiments, 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. In
these embodiments, the devices produce from 100 mL to about 400 L
without a change in lipid nanoparticle properties.
[0116] In one embodiment, the microfluidic device includes:
[0117] (a) a first inlet for receiving a first solution comprising
a first solvent;
[0118] (b) a first inlet microchannel in fluid communication with
the first inlet to provide a first stream comprising the first
solvent;
[0119] (c) a second inlet for receiving a second solution
comprising lipid particle-forming materials in a second
solvent;
[0120] (d) a second inlet microchannel in fluid communication with
the second inlet to provide a second stream comprising the lipid
particle-forming materials in the second solvent;
[0121] (e) 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; and
[0122] (f) a fourth microchannel for receiving and combining the
plurality of streams comprising lipid nanoparticle.
[0123] In certain embodiments, each of the plurality of
microchannels for receiving the first and second streams
includes:
[0124] (a) a first microchannel in fluidic communication with the
first inlet microchannel to receive the first stream comprising the
first solvent;
[0125] (b) a second microchannel in fluidic communication with the
second inlet microchannel to receive the second inlet stream
comprising the second solvent; and
[0126] (c) 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.
[0127] In certain embodiments, 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.
[0128] In certain embodiments, the second regions each have a
hydraulic diameter of from about 20 to about 300 .mu.m. In certain
embodiments, the second regions each have a fluid flow rate of from
1 to about 40 mL/min.
[0129] For embodiments that include heating elements, 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. In these embodiments, 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.
[0130] In one embodiment, the disclosure provides a system for
making limit size nanoparticles that includes a parallel
microfluidic structure. In a parallel structure, N single mixers
are arrayed such that a total flow rate of N.times.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.
[0131] 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.
[0132] Referring to FIG. 7, the device 500 includes three fluidic
systems 400a, 400b, and 400c 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.
[0133] Referring to FIGS. 8A and 8B, each fluidic system
includes:
[0134] (a) a first microchannel 402 in fluidic communication via
first inlet 302a with a first inlet microchannel 304a to receive
the first stream comprising the first solvent;
[0135] (b) a second microchannel 406 in fluidic communication via
second inlet 306a with the second inlet microchannel 308a to
receive the second inlet stream comprising the second solvent;
and
[0136] (c) a third microchannel 310a for receiving the first and
second streams, wherein each has a first region 312a adapted for
flowing the first and second streams and a second region 314a
adapted for mixing the contents of the first and second streams to
provide a plurality of streams comprising lipid nanoparticles. The
microchannel 316a conducts one of the plurality of streams from the
mixing region to fourth microchannel 408 via outlet 318a that
conducts the lipid nanoparticles from the device.
[0137] With reference still to FIGS. 8A and 8B, it will be
appreciated that in this embodiment of the device, fluidic system
300a includes a second second solvent inlet 406' and mixing region
310a' with components denoted by reference numerals 302a', 304a',
306a', 308a', 312a', 314a', 316a' and 318a'. These reference
numerals correspond to their non-primed counterparts 302, 304, 306,
308, 312, 314, 316, and 318 in FIG. 8A and FIG. 8B.
[0138] 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. In this representative
embodiment, 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.
[0139] 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).
[0140] FIG. 9 illustrates a parallelized device 500 includes five
fluidic systems 300a, 300b, 300c, 300d, and 300e 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.
[0141] Referring to FIG. 9, fluidic system 500a includes:
[0142] (a) a first microchannel 402 (with inlet 403) in fluidic
communication via first inlet 302a with a first inlet microchannel
304a to receive the first stream comprising the first solvent;
[0143] (b) a second microchannel 406 (with inlet 405) in fluidic
communication via second inlet 306a with inlet microchannel 304a to
receive the second inlet stream comprising the second solvent;
and
[0144] (c) a third microchannel 310a for receiving the first and
second streams, wherein the third microchannel has a first region
312a adapted for flowing the first and second streams and a second
region 314a adapted for mixing the contents of the first and second
streams to provide a third stream compromising lipid nanoparticles.
In FIG. 9, microchannel 316a conducts the third stream from the
mixing region to fourth microchannel 408 via outlet 318a.
Microchannel 408 conducts the lipid nanoparticles from the device
via outlet 409.
[0145] In the fluidic system of FIG. 9, the five fluidic systems
300a-300e 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
300a-300e) 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. 9, 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. Accordingly, in one embodiment, the parallelized device has
n fluidic systems (e.g., 300a 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.
[0146] With reference to FIGS. 8A and 8B, it will be appreciated
that in this embodiment of the device, fluidic system 300a includes
a second second solvent inlet 406' and mixing region 310a' with
components denoted by reference numerals 302a', 304a', 306a',
308a', 312a', 314a', 316a' and 318a'. These reference numerals
correspond to their non-primed counterparts 302, 304, 306, 308,
312, 314, 316, and 318 in FIG. 8A and FIG. 8B.
[0147] In one embodiment, the disclosure provides a device for
producing limit size lipid nanoparticles, comprising n fluidic
devices, each fluidic device comprising:
[0148] (a) a first inlet 302a for receiving a first solution
comprising a first solvent;
[0149] (b) a first inlet microchannel 304a in fluid communication
with the first inlet to provide a first stream comprising the first
solvent;
[0150] (c) a second inlet 306a for receiving a second solution
comprising lipid particle-forming materials in a second
solvent;
[0151] (d) a third microchannel 310a for receiving the first and
second streams, wherein the third microchannel has a first region
312a adapted for flowing the first and second streams and a second
region 314a 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
316a,
[0152] wherein the first inlets 302a-302n of each fluidic device
300a-100n are in liquid communication through a first bus channel
402 that provides the first solution to each of the first
inlets,
[0153] wherein the second inlets 306a-306n of each fluidic device
300a-300n are in liquid communication through a second bus channel
406 that provides the second solution to each of the second inlets,
and
[0154] wherein the outlets 318a-318n of each fluidic device
300a-300n 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.
[0155] In certain embodiments, n is an integer from 2 to 40.
[0156] Vertical parallelization is achieved by forming planar
mixers and stacking them together and connecting the inlets and
outlets through a vertical bus. 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. However, as the distance separating the two mixers is less
than 500 microns, the increased resistance is negligible when
compared to the overall resistance of the mixing structure (which
is identical for each layer). This is confirmed both through the
experimental results and through fluid flow simulations. The
distance separating mixing layers for which this condition is true
is dependent on the width of the bus.
[0157] 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.times. 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.degree. C. for 25 minutes. After baking, both
layers are exposed to oxygen plasma and then aligned. The aligned
chips are then baked at 80.degree. 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.
[0158] Using a custom chip holder, 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. In order to interface with these
holders, 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.
[0159] 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.
[0160] Manifolds
[0161] In one embodiment, 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. In another embodiment, 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.
[0162] As used herein, the term "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.
[0163] In one embodiment, illustrated in FIG. 2, 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.
[0164] In another embodiment, a third manifold 214 is used to
collect the multiple streams emerging from the microfluidic mixer
array into a single output stream. In one embodiment, 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.
[0165] In one embodiment, 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.
[0166] In one embodiment, the 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.
[0167] In one embodiment, the 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.
[0168] In one embodiment, the system further comprises:
[0169] 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;
[0170] 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; and
[0171] 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.
[0172] In one embodiment, the plurality of mixers are within the
microfluidic chip.
[0173] Representative manifold materials include PEEK, stainless
steel, COC/COP, polycarbonate, and Ultem.
[0174] In one embodiment, the manifold device comprises, but not
limited to, 9-Ports interfaced with 0.0625 inch outside diameter
tubing. In another embodiment, 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. In
another embodiment, 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. In another embodiment, 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. In a parallelized microfluidic mixer array, the
microfluidic mixer provides over 95% of the fluidic resistance in
the fluid path. Thus, the equal distribution of flow is attributed
to the microchannel features in the microfluidic mixer device and
the relative difference in tubing length, from the manifold to
microfluidic mixer device, is insignificant.
[0175] Fluid Driver Systems
[0176] In one embodiment, the fluid drivers are pumps. In one
embodiment, the system includes two, or more, independent
continuous flow fluid drivers.
[0177] In the embodiment illustrated in FIG. 2, the solvent
metering pump 206 and aqueous metering pump 208 are independent
continuous flow pumping systems that provide fluid flow in the
apparatus.
[0178] In one embodiment, 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
[0179] In one embodiment, the continuous flow pumping systems are
positive displacement pumps. Examples of positive displacement
pumps include, but are not limited to, peristaltic pumps, gear
pumps, screw pumps, and progressive cavity pumps. In a preferred
embodiment, the independent continuous flow pumps are dual head
reciprocating positive displacement pumps.
[0180] In another embodiment the dual head reciprocating positive
displacement pumps have front mounted interchangeable pump heads.
In a further embodiment the front mounted interchangeable pump
heads enable independent flow rates from 10 mL/min to 1000
mL/min.
[0181] In another embodiment, the dual head reciprocating positive
displacement pumps are Knauer Azura P2.2 L pumps. In a further
embodiment 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.
[0182] Interchangeable pump heads enable simple and rapid scaling
of continuous flow manufacturing system. In certain embodiments,
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. In certain embodiments, 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).
In other embodiments, 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).
[0183] In one embodiment, the dual head reciprocating pump head
provides low pulsation flow. In a further embodiment, 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. In one embodiment, backpressure is achieved by addition of
24 inches of tubing with an internal diameter of 0.02 inches.
[0184] In another embodiment, 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.
[0185] Dilution
[0186] In one embodiment, 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.
[0187] In a further embodiment, referring to FIG. 2, 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. In certain embodiments, the
dilution process is achieved by pumping one or more buffers
continuously into the output stream emerging from the microfluidic
mixer array. In one embodiment, the dilution pumping system is a
positive displacement pump. In a further embodiment the positive
displacement pump is selected from peristaltic pumps, gear pumps,
screw pumps and progressive cavity pumps. In certain embodiments
the dilution pumping system is a peristaltic pump. In certain
embodiments the dilution pumping system is a peristaltic pump with
dual pump heads. In a preferred embodiment, the dilution pumping
system is a Masterflex peristaltic pump with dual pump heads. In
another embodiment 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.
[0188] In certain embodiments, the dilution media is introduced
into the nanoparticle stream by a connector. In one embodiment the
connector is a Tee connector. In another embodiment the connector
is a Y-connector. In certain embodiments, the dilution media
contacts the nanoparticle stream at an angle ranging from
0.1.degree. to 179.9.degree.. The angle of contact moderates the
level of agitation induced onto the nanoparticles in the dilution
process. In a preferred embodiment, the Masterflex peristaltic pump
dual pump heads have roller profiles offset by 30.degree. thereby
reducing the pump's output flow pulsation level by 80-95%.
[0189] In certain embodiments, the dilution media is introduced
into a second, inline microfluidic mixer. In one embodiment, the
second microfluidic mixer is on a second, in-line device. In
another embodiment, the second microfluidic mixer is on the same
microfluidic device.
[0190] 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.
[0191] In apparatus 200 (FIG. 2), there are two reservoirs, solvent
reservoir 202 and aqueous reservoir 204. In one embodiment the
reservoirs are disposable bags. In a further embodiment, the
reservoirs are vessels, including, but not limited to, stainless
steel reservoirs. In one embodiment, the solvent is a
water-miscible solvent such as, but not limited to, ethanol
containing one or more lipids, and the aqueous is a low pH buffer
such as, but not limited to, citrate buffer pH 4.0. In a further
embodiment the low pH buffer such as, but not limited to, citrate
buffer pH 4.0 contains one or more nucleic acids. In a further
embodiment, 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. For the formation of polymer
nanoparticles, a representative buffer is a saline solution.
[0192] In one embodiment 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. In
another embodiment 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.
[0193] In one embodiment the manufacturing apparatus has a
mechanism to dilute the nanoparticle fluid stream emerging from the
microfluidic mixer array. In one embodiment dilution is achieved by
in-line dilution where the aqueous buffer contacts directly with
the output stream.
[0194] In one embodiment 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. In another
embodiment 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. In further embodiment 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.
In one embodiment 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).
[0195] In one embodiment, the system includes dilution in-line in
the absence of the microfluidic device.
[0196] Waste Valve
[0197] In a further embodiment of the present disclosure, 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. In one embodiment, a valving system directs the output
stream to waste collection or sample collection. In one embodiment,
a two-vessel collection (waste and sample) 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. In one embodiment the valve system is a manual
system or an automated system is used to pinch off the soft tubing.
In a further embodiment the manual valve system is a tube clamp. In
a further embodiment the automated system is a solenoid pinch
valve.
[0198] In one embodiment, 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.
[0199] Fully Disposable Fluid Path
[0200] In one embodiment of the present disclosure, the system
incorporates a fully disposable fluid path. As used herein, 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. However, in the present disclosure, 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).
[0201] In the embodiment illustrated in FIG. 2, 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.
[0202] In one embodiment, 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.
[0203] In one embodiment, 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.
[0204] In one embodiment, every surface touched by the first
solution, the second solution, and the nanoparticle solution are
disposable.
[0205] In certain embodiments 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.
[0206] Sterile System Components
[0207] In one embodiment, the microfluidic chip is sterile.
Sterilization is essential for certain production processes. In a
further embodiment, the microfluidic chip is sterilized prior to
integration into the system. In another embodiment, the
microfluidic chip is sterilized in-place within the system.
[0208] Representative sterilization methods include steam
autoclave, dry heat, chemical sterilization (i.e., sodium hydroxide
or ethylene oxide), gamma radiation, gas, and combinations thereof.
In a specific embodiment, the microfluidic chip is sterilized with
gamma radiation.
[0209] Due to the importance of sterilization in certain
applications, in certain embodiments the microfluidic chip is
formed from materials that are compatible with certain types of
sterilization preferred for a particular application.
[0210] In one embodiment, the microfluidic chip is formed from
materials that are compatible with gamma radiation. Materials
compatible with gamma radiation are those that can be irradiated.
For example, polycarbonate, cyclic olefin polymer, cyclic olefin
copolymer, and high- and low-density polyethylene. Materials that
cannot be irradiated include polyamides, polytetrafluoroethylene,
and any metal.
[0211] In addition to providing embodiments that facilitate
sterilization of system components, 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.
[0212] Additionally, certain particles that can be synthesized
using the disclosed systems are incompatible with terminal
sterilization and therefore cannot be sterilized
downstream--therefore, a sterile manufacturing path is required.
Generally, 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.
[0213] Representative particles that are not compatible with
terminal sterilization (e.g., sterile filtering) 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.
[0214] According to the disclosed embodiments, 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. In one embodiment, 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.
[0215] The sterile system parts can be sterilized by any methods
know to those of skill in the art and disclosed herein. For
example, gamma radiation is used in certain embodiments to
sterilize the system parts.
[0216] After sterilization, the sterile system parts are maintained
in a sterile environment and packaged in a sealed manner so as to
maintain sterility until use.
[0217] In certain embodiments nanoparticle manufacturing is
conducted in a specialized barrier facility that eliminates the
requirement for filtration to ensure a sterile product.
[0218] Software Control
[0219] In the system is controlled by software (e.g., FIG. 1 part
102). In further embodiments the entire manufacturing system is
software controlled. Such software controls are generally known to
those of skill in the art. In one embodiment, with reference to
FIG. 2, software controls the first 202 and second 204 fluid
drivers. In a further embodiment, software controls all fluid
drivers in the system 100 or 200.
[0220] Methods for Making Nanoparticles
[0221] In one embodiment the present disclosure provides methods
for scalable production of nanoparticles using the
microfluidic-based continuous flow manufacturing apparatus of the
disclosure.
[0222] In one aspect, a method of forming nanoparticles is
provided. In one embodiment, the method 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.
[0223] In one embodiment, 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.
[0224] In one embodiment, the plurality of mixers are contained
within a plurality of microfluidic chips.
[0225] In one embodiment, the plurality of mixers are contained
within a single microfluidic chip.
[0226] In one embodiment, the apparatus provides a system and
process for the manufacture of lipid nanoparticles containing a
therapeutic material.
[0227] In another embodiment the apparatus provides a system and
process for the manufacture of polymer nanoparticles containing a
therapeutic material.
[0228] In one embodiment the apparatus has two reservoirs: a
solvent reservoir and aqueous reservoir. In one aspect, the solvent
reservoir contains a water-miscible solvent such as, but not
limited to, ethanol containing one or more lipids. In another
aspect, the solvent reservoir contains a water-miscible solvent
such as, but not limited to, acetonitrile containing one or more
polymers, or polymer-drug conjugates. In one aspect, the aqueous
reservoir contains a buffer such as, but not limited to, citrate
buffer pH 4.0. In a further aspect, the aqueous reservoir contains
a buffer such as, but not limited to, citrate buffer pH 4.0 that
contains one or more therapeutic materials.
[0229] In one embodiment, the contents of the reservoirs are drawn
into the fluid path of the apparatus of the disclosure by
independent continuous flow pumping systems. In one aspect the each
independent continuous flow manufacturing pump operates at a flow
rate of 0.1 L/min-1.0 L/min. In one aspect, 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:
[0230] (a) a first stream comprising a first solvent (e.g., an
aqueous buffer) is introduced into the first channel of each
independent microfluidic mixer at a first flow rate;
[0231] (b) 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;
[0232] (c) flowing the first and second streams from the first
region to the second region; and
[0233] (d) mixing the first and second streams in the second region
of the apparatus to provide a third stream comprising lipid
nanoparticles.
[0234] In one embodiment, the apparatus is a microfluidic
apparatus. In certain embodiments, the flow pre-mixing is laminar
flow. In certain embodiment, mixing the first and second streams
comprises chaotic advection. In other embodiments, mixing the first
and second streams comprises mixing with a micromixer.
[0235] In one aspect the microfluidic mixer array incorporates
1-128 microfluidic mixers arrayed in parallel to increase the
throughput of the manufacturing system. In certain aspects a single
microfluidic mixer is contained on one device. In another aspect, a
single device contains multiple microfluidic mixers. In one
embodiment, a single device contains four microfluidic mixers (FIG.
3).
[0236] In one embodiment, the flow rates of the first stream and
second stream flow rate between 1 mL/min and 50 mL/min per
microfluidic mixer.
[0237] In certain embodiments 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).
[0238] In certain embodiments the final nanoparticle product is
dispensed into sterile vials.
[0239] Dean Vortex Bifurcating Mixers ("DVBM")
[0240] As noted above, 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 .mu.m).
The provided DVBM mixers include a plurality of toroidal mixing
elements (also referred to herein as "toroidal mixers." As used
herein, "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.
[0241] 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.
[0242] In one embodiment, 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.
[0243] In one embodiment, the first neck angle is from 0 to 180
degrees.
[0244] In one embodiment, the first neck region has a length of 0.2
mm or greater.
[0245] In one embodiment, the plurality of mixing elements include
channels having a hydrodynamic diameter of about 20 microns to
about 2 mm.
[0246] In one embodiment, the mixer is sized and configured to mix
the first liquid and the second liquid at a Reynolds number of less
than 1000.
[0247] In one embodiment, the mixer includes two or more mixers in
parallel, each mixer having a plurality of toroidal mixing
elements.
[0248] In one embodiment, 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.
[0249] In one embodiment, 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.
[0250] In one embodiment, the first length is greater than the
second length.
[0251] In one embodiment, the third length is greater than the
fourth length.
[0252] In one embodiment, the ratio of the first length to the
second length is about equal to the ratio of the third length to
the fourth length.
[0253] In one embodiment, 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.
[0254] In one embodiment, the first impedance is greater than the
second impedance.
[0255] In one embodiment, the third impedance is greater than the
fourth impedance.
[0256] In one embodiment, the ratio of the first impedance to the
second impedance is about equal to the ratio of the third impedance
to the fourth impedance.
[0257] In one embodiment, the mixer includes 2 to 20 toroidal
mixing elements in series.
[0258] In one embodiment, the mixer includes 1 to 10 pairs of
toroidal mixing elements in series.
[0259] In one embodiment, the toroidal mixing elements have an
inner radius of about 0.1 mm to about 2 mm.
[0260] Also provided are methods of mixing a first liquid with a
second liquid, comprising flowing the first liquid and the second
liquid through a DVBM mixer according to the disclosed
embodiments.
Definitions
[0261] Microfluidic
[0262] As used herein, the term "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).
[0263] Therapeutic Material
[0264] As used herein, the term "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.
[0265] Nanoparticles
[0266] As used herein, the term "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.
[0267] Lipid Nanoparticles
[0268] In one embodiment, lipid nanoparticles, comprise:
[0269] (a) a core; and
[0270] (b) a shell surrounding the core, wherein the shell
comprises a phospholipid.
[0271] In one embodiment, the core comprises a lipid (e.g., a fatty
acid triglyceride) and is solid. In another embodiment, the core is
liquid (e.g., aqueous) and the particle is a vesicle, such as a
liposomes. In one embodiment, the shell surrounding the core is a
monolayer.
[0272] As noted above, in one embodiment, the lipid core comprises
a fatty acid triglyceride. Suitable fatty acid triglycerides
include C8-C20 fatty acid triglycerides. In one embodiment, the
fatty acid triglyceride is an oleic acid triglyceride.
[0273] The lipid nanoparticle includes a shell comprising a
phospholipid that surrounds the core. Suitable phospholipids
include diacylphosphatidylcholines,
diacylphosphatidylethanolamines, ceramides, sphingomyelins,
dihydrosphingomyelins, cephalins, and cerebrosides. In one
embodiment, the phospholipid is a C8-C20 fatty acid
diacylphosphatidylcholine. A representative phospholipid is
1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).
[0274] In certain embodiments, the ratio of phospholipid to fatty
acid triglyceride is from 20:80 (mol:mol) to 60:40 (mol:mol).
Preferably, the triglyceride is present in a ration greater than
40% and less than 80%.
[0275] In certain embodiments, the nanoparticle further comprises a
sterol. Representative sterols include cholesterol. In one
embodiment, the ratio of phospholipid to cholesterol is 55:45
(mol:mol). In representative embodiments, the nanoparticle includes
from 55-100% POPC and up to 10 mol % PEG-lipid.
[0276] In other embodiments, 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] Representative 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,
oxaliplatin, adriamycin, methotrexate, carboplatin, estramustine,
and pharmaceutically acceptable salts and thereof.
[0281] In another embodiment, lipid nanoparticles, are nucleic-acid
lipid nanoparticles.
[0282] The term "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.
[0283] Cationic lipid. The lipid nanoparticles include a cationic
lipid. As used herein, the term "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). As used herein, the term
"cationic lipid" includes zwitterionic lipids that assume a
positive charge on pH decrease.
[0284] The term "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. Such 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 (DMRIE). Additionally, a number of commercial preparations
of cationic lipids are available which can be used in the present
disclosure. These include, for example, LIPOFECTIN.RTM.
(commercially available cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand
Island, N.Y.); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic lipids comprising
dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from
Promega Corp., Madison, Wis.). The following 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).
[0285] In one embodiment, 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 (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanedio (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), and
2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane
(DLin-K-DMA).
[0286] Suitable amino lipids include those having the formula:
##STR00001##
[0287] wherein R.sub.1 and R.sub.2 are either the same or different
and independently optionally substituted C.sub.10-C.sub.24 alkyl,
optionally substituted C.sub.10-C.sub.24 alkenyl, optionally
substituted C.sub.10-C.sub.24 alkynyl, or optionally substituted
C.sub.10-C.sub.24 acyl;
[0288] R.sub.3 and R.sub.4 are either the same or different and
independently optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, or optionally
substituted C.sub.2-C.sub.6 alkynyl or R.sub.3 and R.sub.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;
[0289] R.sub.5 is either absent or present and when present is
hydrogen or C.sub.1-C.sub.6 alkyl;
[0290] m, 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;
[0291] q is 0, 1, 2, 3, or 4; and
[0292] Y and Z are either the same or different and independently
O, S, or NH.
[0293] In one embodiment, R.sub.1 and R.sub.2 are each linoleyl,
and the amino lipid is a dilinoleyl amino lipid. In one embodiment,
the amino lipid is a dilinoleyl amino lipid.
[0294] A representative useful dilinoleyl amino lipid has the
formula:
##STR00002##
[0295] wherein n is 0, 1, 2, 3, or 4.
[0296] In one embodiment, 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).
[0297] Other 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.beta.-(N--(N',N'-dimethylaminoethane)carbamoyl)cholesterol
(DC-Chol),
N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl
carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane
(DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE). Additionally, a number of commercial preparations
of 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).
[0298] 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.
[0299] In one embodiment, the lipid particle includes ("consists
of") only one or more cationic lipids and one or more nucleic
acids.
[0300] Second lipids. In certain embodiments, the lipid
nanoparticles include one or more second lipids. Suitable second
lipids stabilize the formation of nanoparticles during their
formation.
[0301] The term "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.
[0302] Suitable stabilizing lipids include neutral lipids and
anionic lipids.
[0303] Neutral Lipid. The term "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.
[0304] 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-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
[0305] In one embodiment, the neutral lipid is
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
[0306] Anionic Lipid. The term "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.
[0307] Other suitable lipids include glycolipids (e.g.,
monosialoganglioside GM.sub.1). Other suitable second lipids
include sterols, such as cholesterol.
[0308] Polyethylene glycol-lipids. In certain embodiments, 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. In one embodiment, the polyethylene glycol-lipid is
N-[(methoxy poly(ethylene
glycol).sub.2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine
(PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is
PEG-c-DOMG).
[0309] In certain embodiments, 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.
[0310] Nucleic Acids. 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.
[0311] As used herein, the term "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. In the context of this disclosure, the terms
"polynucleotide" and "oligonucleotide" refer to a polymer or
oligomer of nucleotide or nucleoside monomers consisting of
naturally occurring bases, sugars and intersugar (backbone)
linkages. The terms "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.
[0312] In one embodiment, the polynucleic acid is an antisense
oligonucleotide. In certain embodiments, the nucleic acid is an
antisense nucleic acid, a ribozyme, tRNA, snRNA, siRNA, shRNA,
ncRNA, miRNA, mRNA, lncRNA, pre-condensed DNA, or an aptamer.
[0313] The term "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.
[0314] The term "nucleotide", as used herein, generically
encompasses the following terms, which are defined below:
nucleotide base, nucleoside, nucleotide analog, and universal
nucleotide.
[0315] The term "nucleotide base", as used herein, refers to a
substituted or unsubstituted parent aromatic ring or rings. In some
embodiments, the aromatic ring or rings contain at least one
nitrogen atom. In some embodiments, the nucleotide base is capable
of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an
appropriately complementary nucleotide base. Exemplary 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-dihydrothymine, 04-methylthymine, uracil
(U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D);
indoles such as nitroindole and 4-methylindole; pyrroles such as
nitropyrrole; nebularine; base (Y); In some embodiments, nucleotide
bases are universal nucleotide bases. Additional exemplary
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.
[0316] The term "nucleoside", as used herein, 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. Also see, e.g., 2'-O-methyl,
4'-.alpha.-anomeric nucleotides, 1'-.alpha.-anomeric nucleotides
(Asseline (1991) Nucl. Acids Res. 19:4067-74), 2'-4'- and
3'-4'-linked and other "locked" or "LNA", bicyclic sugar
modifications (WO 98/22489; WO 98/39352; WO 99/14226). "LNA" or
"locked nucleic acid" is a DNA analogue that is conformationally
locked such that the ribose ring is constrained by a methylene
linkage between the 2'-oxygen and the 3'- or 4'-carbon. The
conformation restriction imposed by the linkage often increases
binding affinity for complementary sequences and increases the
thermal stability of such duplexes.
[0317] 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.
21:4159-65; Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata,
(1993) Nucleic Acids Symposium Ser. No. 29:69-70). When the
nucleobase is purine, e.g., A or G, the ribose sugar is attached to
the N9-position of the nucleobase. When the 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.).
[0318] One or more of the pentose carbons of a nucleoside may be
substituted with a phosphate ester. In some embodiments, the
phosphate ester is attached to the 3'- or 5'-carbon of the pentose.
In some embodiments, 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.
[0319] The term "nucleotide analog", as used herein, 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. In some embodiments, exemplary
pentose sugar analogs are those described above. In some
embodiments, the nucleotide analogs have a nucleotide base analog
as described above. In some embodiments, 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). Additional descriptions of various nucleic acid
analogs can also be found for example in (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141
91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437
(1991); and U.S. Pat. No. 5,644,048. Other 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. USA 92:6097 (1995); non-ionic backbones
(U.S. Pat. Nos. 5,386,023, 5,386,023, 5,637,684, 5,602,240,
5,216,141, and 4,469,863. Kiedrowshi et al., Angew. Chem. Intl. Ed.
English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470
(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597
(194): Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan
Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett.
4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994);
Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones,
including those described in U.S. Pat. Nos. 5,235,033 and
5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-1'76). Several nucleic acid analogs are also described in
Rawls, C & E News Jun. 2, 1997 page 35.
[0320] The term "universal nucleotide base" or "universal base", as
used herein, refers to an aromatic ring moiety, which may or may
not contain nitrogen atoms. In some embodiments, a universal base
may be covalently attached to the C-1' carbon of a pentose sugar to
make a universal nucleotide. In some embodiments, a universal
nucleotide base does not hydrogen bond specifically with another
nucleotide base. In some embodiments, a universal nucleotide base
hydrogen bonds with nucleotide base, up to and including all
nucleotide bases in a particular target polynucleotide. In some
embodiments, 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.
[0321] As used herein, the terms "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. 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. Unless denoted otherwise, whenever a polynucleotide 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.
[0322] As used herein, "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).
[0323] As used herein, "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. Non-limiting examples of
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.
[0324] As used herein, "polynucleobase strand" means a complete
single polymer strand comprising nucleobase subunits. For example,
a single nucleic acid strand of a double stranded nucleic acid is a
polynucleobase strand.
[0325] As used herein, "nucleic acid" is a nucleobase
sequence-containing polymer, or polymer segment, having a backbone
formed from nucleotides, or analogs thereof.
[0326] Preferred nucleic acids are DNA and RNA.
[0327] As used herein, 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. 5,539,082,
5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571,
5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053
and 6,107,470; all of which are herein incorporated by reference.
The term "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. 7: 687-690
(1997); Krotz et al., Tett. Lett. 36: 6941-6944 (1995); Lagriffoul
et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Diederichsen,
U., Bioorganic & Medicinal Chemistry Letters, 7: 1743-1746
(1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1:
539-546; Lowe et J. Chem. Soc. Perkin Trans. 11: 547-554 (1997);
Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560 (1997); Howarth
et al., J. Org. Chem. 62: 5441-5450 (1997); Altmann, K-H et al.,
Bioorganic & Medicinal Chemistry Letters, 7: 1119-1122 (1997);
Diederichsen, U., Bioorganic & Med. Chem. Lett., 8: 165-168
(1998); Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305
(1998); Cantin et al., Tett. Lett., 38: 4211-4214 (1997); Ciapetti
et al., Tetrahedron, 53: 1167-1176 (1997); Lagriffoule et al.,
Chem. Eur. J., 3: 912-919 (1997); Kumar et al., Organic Letters
3(9): 1269-1272 (2001); and the Peptide-Based Nucleic Acid Mimics
(PENAMS) of Shah et al. as disclosed in WO96/04000.
[0328] Polymer Nanoparticles
[0329] The term "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
[0330] As used herein, the term "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.
[0331] The term "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.
[0332] The term "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.
[0333] Synthetic homopolymers include, but are not limited to,
polyethylene glycol, polylactide, polyglycolide, polyacrylates,
polymethacrylates, poly caprolactone, polyorthoesters,
polyanhydrides, polylysine, and polyethyleneimine.
[0334] "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).
[0335] The term "semi-synthetic polymer" refers to any number of
polymers derived by the
[0336] chemical or enzymatic treatment of natural polymers. Such
polymers include, but are not limited to, carboxymethyl cellulose,
acetylated carboxymethylcellulose, cyclodextrin, chitosan and
gelatin.
[0337] As used herein, the term "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.
[0338] 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.
[0339] "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.
[0340] As used herein, 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.
[0341] The following example is included for the purpose of
illustrating, not limiting, the described embodiments.
EXAMPLES
Example 1: siRNA-Lipid Nanoparticles (siRNA-LNP) Manufactured Using
Four Single Microfluidic Mixer Devices Arrayed in Parallel Using a
Manifold, or Four Microfluidic Mixers Arrayed in Parallel in a
Single Device
[0342] In this example, 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. This example
significantly demonstrates the possibility of using either, or
both, on-device and off-device methods of arraying to significantly
increase the number of mixers in a single system. Using both
on-device and off-device methods of arraying yields a two
dimensional method of arraying microfluidic mixers.
[0343] 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.
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.times. Manifold), or
four microfluidic mixers arrayed in parallel in a single device
illustrated in FIG. 3 (4.times. On-Chip). The total flow rates
through the microfluidic device are shown in the legend. Error bars
represent the standard deviation of the mean.
[0344] In this Example, 0.231 mg/mL siRNA in 50 mM sodium acetate
buffer (pH 4.0) and lipid mix (12.5 mM
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 in ethanol) in separate syringes were loaded into
independent Harvard PHD Ultra syringe pumps (Harvard Apparatus,
Holliston, Mass.). The siRNA-total lipid ratio was 0.06 wt/wt.
Mixing volumes ratios of siRNA to lipid mix was 3:1, with 5 mL
total volume processed per mixer (i.e., total formulation volumes:
1.times.=5 mL, 2.times.=10 mL, 4.times.=20 mL). 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.times.). 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.
[0345] Particle size measurement was performed as follows:
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).
Example 2: siRNA-Lipid Nanoparticles (siRNA-LNP) Manufactured Using
Eight Single Microfluidic Mixer Devices Arrayed in Parallel Using a
Manifold
[0346] In this example, 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.
[0347] 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
NanoAssemblr.TM. Benchtop Instrument. The NanoAssemblr.TM. 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.
[0348] In this Example, 0.231 mg/mL siRNA in 50 mM sodium acetate
buffer (pH 4.0) and lipid mix (12.5 mM
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 in ethanol) loaded in separate Flash 100 metering
pump (Scientific Systems, Inc., State College, Pa.). The
siRNA-total lipid ratio was 0.06 wt/wt. Mixing volumes ratios of
siRNA to lipid mix was 3:1, with 65 mL total volume processed per
mixer (ie: total formulation volumes in 8.times.=520 mL). Flow rate
per mixer was 12 mL/min at siRNA to lipid mix flow rate ratio of
3:1 (total flow rate=96 ml/min, thus flow rates for siRNA and lipid
mix were 72 mL/min and 24 mL/min respectively). 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. After the first 20 mL priming waste
was collected, 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.
[0349] While illustrative embodiments have been illustrated and
described, it will be appreciated that various changes can be made
therein without departing from the spirit and scope of the
invention.
* * * * *