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The NanoAssemblr™ Platform: Microfluidics-Based Manufacture of Nanoparticles
Euan Ramsay, Ph.D.
Co-Founder & CSO/COO
July, 2015
2
Presentation Overview
1. Introduction to Microfluidics-Based NanoAssemblr™
Platform for Nanoparticle Manufacture
2. Examples of Nanoparticles Manufactured by the
NanoAssemblr™ Platform
3. Scale-up Manufacture using the NanoAssemblr™
Platform
3
Presentation Overview
1. Introduction to Microfluidics-Based NanoAssemblr™
Platform for Nanoparticle Manufacture
2. Examples of Nanoparticles Manufactured by the
NanoAssemblr™ Platform
3. Scale-up Manufacture using the NanoAssemblr™
Platform
Conceptual Nanomedicine
Scale-up API -
NanoparticleFormulation
Manufacturing Process
Robust Manufacturing
Process
Nanomedicine Product
Nanomedicine Development Process
4
Conceptual Nanomedicine
Scale-up API -
NanoparticleFormulation
Manufacturing Process
Robust Manufacturing
Process
Nanomedicine Product
Nanomedicine Development Process
5
NanoAssemblr™ Benchtop Instrument
Conceptual Nanomedicine
Scale-up API -
NanoparticleFormulation
Manufacturing Process
Robust Manufacturing
Process
NanomedicineProduct
Nanomedicine Development Process
6
NanoAssemblr™ Benchtop Instrument Scale-Up Platform
Conceptual Nanomedicine
Scale-up API -
NanoparticleFormulation
Manufacturing Process
Robust Manufacturing
Process
Nanomedicine Product
Nanomedicine Development Process
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NanoAssemblr™ Benchtop Instrument
Accelerated Development of Nanomedicines
Scale-Up Platform
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The NanoAssemblr™ Benchtop Instrument
Microfluidic Cartridge
NanoAssemblr™ Benchtop Instrument
Proprietary microfluidics-based instrument
Manufacture novel nanoparticles
Nucleic acid-lipid nanoparticles
Polymer nanoparticles
Liposomes
Oil-in-water nanoemulsions
Prepare 1.5 mL – 20 mL nanoparticles / run
Operate at 4 mL/min - 20 mL/min
Make > 30 formulations / day
Software controlled
Easy-to-use
Rapid nanoparticle development
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The NanoAssemblr™ Microfluidic Cartridge
DRAFT COPY UOBC-1-53454
DRAFT COPY UOBC-1-53454
Microfluidic Cartridge – BOTTOM VIEW Microfluidic Cartridge – TOP VIEW
Microfluidic Chip
Easy-to-use consumable cartridge
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The Magic is in the Microfluidics
Solvent (water miscible) aqueous
Rapid & Controlled Mixing
Stroock et al., Science 2002
Channel diameter: ~100 µm
Staggered Herringbone
Mixers
Nanoparticles
Laminar fluid flow
Diffusion mixing
Rapid mixing (< 3 ms-1)
Small reaction volumes (~ 14 nL)
Low energy input
Predictable and reproducible mixing
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Microfluidics Process Parameters – Aqueous:Solvent Flow Rate Ratio
• Ratio of the flow rates (mL/min) of the aqueous and
solvent input streams
• Higher aqueous:ethanol flow rate ratios result in
more rapid increases in polarity
• Rapid change in polarity forces the nanoparticle
components to organize into the most
thermodynamically and energetically favorable
structure
Process parameters dictate nanoparticle biophysical characteristics
Solvent (water miscible)
aqueous
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Microfluidics Process Parameters – Total Flow Rate
Process parameters dictate nanoparticle biophysical characteristics
Solvent (water miscible)
aqueous
• The combined flow rates of the aqueous stream and
the solvent stream
• Ranges from 4 mL/min – 20 mL/min
• Total Flow Rate is a surrogate for mixing speed
• Increased flow rate increases mixing speed
• At high Total Flow Rates nanoparticles reach “limit size”
• Limit size is defined as “The smallest achievable lipid
particles compatible with the packing of the molecular
constituents in an energetically stable structure”
13
Presentation Overview
1. Introduction to Microfluidics-Based NanoAssemblr™
Platform for Nanoparticle Manufacture
2. Examples of Nanoparticles Manufactured by the
NanoAssemblr™ Platform
3. Scale-up Manufacture using the NanoAssemblr™
Platform
The NanoAssemblr™ Platform: Manufacture of Novel Nanoparticles
O/W Nanoemulsions
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Lipid Nanoparticles
Liposomes
Polymer Nanoparticles
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Lipid Nanoparticles
Lipid Nanoparticles for the Delivery of RNA
• Package RNA into nanoparticle core
• Protect RNA from degradation
• Facilitate RNA uptake into cells
• Promote RNA release into the cytoplasm
16 Images courtesy of Prof. Pieter Cullis, University of British Columbia
Ionizable Cationic Lipid
Cholesterol
Phospholipid
PEG-lipid
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RNA-Lipid Nanoparticles are Complex
Manufacture of RNA-Lipid Nanoparticles is challenging
RNA
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Day
Mean
% S
eru
m T
TR
Kn
ockd
ow
n
Rela
tive to B
aselin
e
80
60
40
20
0
-20
-40
10 20 30 40 50 100
Treatment (mg/kg)
Placebo 0.150
0.300 0.050 0.500
siRNA Dose
† Serum TTR levels were measured in separate Phase I study of ALN-PCS, an RNAi
therapeutic targeting PCSK9, which uses identical LNP formulation as ALN-TTR02 B.U.Med.Center, July 2012
RNA-Lipid Nanoparticles Represent the Current Clinical Gold Standard for RNAi
Patisiran (ALN-TTR02) is Currently in Phase 3 clinical trials for treatment of Transthyretin-Mediated Amyloidosis
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Microfluidics Manufactures “Solid-Core” RNA-Lipid Nanoparticles
Images courtesy of Prof. Pieter Cullis, University of British Columbia
Wasan K. M . et al. (2008) Impact of Lipoproteins on the Biological Activity & Disposition of Hydrophobic Drugs: Implications for Drug Discovery. Nat. Rev. Drug Disc. 7: 84- 99
Molecular model:
Neutral, Solid-Core RNA-Lipid Nanoparticles
Neutral, “Solid-Core” RNA-Lipid Nanoparticles Mimic Endogenous Delivery Systems
Low Density Lipoprotein (LDL):
“Endogenous Lipid Nanoparticles”
Wasan K. M . et al. (2008) Impact of Lipoproteins on the Biological Activity & Disposition of Hydrophobic Drugs: Implications for Drug Discovery. Nat. Rev. Drug Disc. 7: 84- 99
Neutral, “Solid-Core” RNA-Lipid Nanoparticles Mimic Endogenous Delivery Systems
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“Solid-core” RNA-Lipid Nanoparticles Associate with ApoE In Vivo
Images courtesy of Prof. Pieter Cullis, University of British Columbia
ApoE
PEG-lipid dissociates and ApoE associates after injection
LDL receptor, scavenging receptor on hepatocytes
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Ionizable Cationic Lipids Mediate Maximum Endosomal Escape
Images courtesy of Prof. Pieter Cullis, University of British Columbia
pH reduced below pKa of cationic lipid
Cationic lipids combine with anionic lipids to induce non-bilayer structures and release of siRNA
Brij 98
0 10 20 300
50
100
0.01 mg/kg
0.05 mg/kg
0.1 mg/kg
0.3 mg/kg
0.5 mg/kg
1 mg/kg
EC 50
EC 90
Day
Resid
ual F
VII (
%)
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“Solid-Core” RNA-Lipid Nanoparticles Mediate Sustained Liver Gene Knockdown In Vivo
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Can ApoE-Medicated Targeting be Used for Other Tissues?
LDL receptor family members:
LDLR, LRP1, VLDLR, ApoER2, LRP4, LRP1B and Megalin
Need to design novel nanoparticles to deliver RNA beyond the liver
Microfluidics Enables Rapid Development of Novel RNA-Nanoparticles
26
Incumbent Technology
NanoAssemblr™
Rapid
Development
Novel
Nanoparticles
Reproducibility
Ease-of-Use Encapsulation Efficiency
Size Speed
Seamless Scale-Up
Multi-Function Nanoparticles
Compositional Space
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RNA-Lipid Nanoparticle Size Dictated by Manufacturing Process
siRNA-LNP (Cationic Lipid:DSPC:Cholesterol:PEG) Changing Process
RNA-Lipid Nanoparticles reach “Limit Size” at high Total Flow Rates
RNA-Lipid Nanoparticle Size Dictated by Lipid Composition
28
“Limit Size” is dependent on RNA-Lipid Nanoparticle composition
0
1
10
100
0 5 10 15 20 25
%TotalInjectedDose
Time(hr)Time (hr)
0 2 25
% T
ota
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d D
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Time (hr)
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14Blood Liver Spleen
siRNA-LNP In Vivo Behavior
Particle Size Influences siRNA-LNP Pharmacokinetics & Biodistribution
siRNA-LNP Particle Diameter:
Red = 43 nm (5% PEG)
Green = 78 nm (5% PEG)
Blue = 140 nm (5% PEG)
Grey = 78 nm (1.5% PEG)
30
31
30 nm siRNA-LNP Enables Liver Gene Knockdown by Subcutaneous Injection
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Targeted siRNA-LNP
34
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In Vivo Gene Knockdown in T-Lymphocytes
Microfluidics for Targeted Nanoparticles
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• Step-wise reactions • Post-insertion for targeting • Programmable mixing
• Targeted medicines • Multi-functional agents • Bespoke medicines
Molecular Assembly Line
Manufacture of Multi-Functional Nanoparticles
Sequential Addition of Cationic (XTC) and Anionic (PS) Lipids
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39
Encapsulation of Gold Nanoparticles
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Liposomes
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Liposome Size Dictated by Manufacturing process
“Limit size” Liposomes are dictated by the Flow Rate Ratio
Liposome Size Dictated by Lipid Composition
42
Liposome size and polydispersity is dependent on cholesterol content
POPC:Cholesterol:PEG-DSPE (3%)
43
http ://informahealthcare.com/lprISSN: 0898-2104 (print), 1532-2394 (electronic)
JLiposome Res, Early Online: 1–7! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2015.1025411
RESEARCH ARTICLE
Production of lim it size nanoliposomal systems with potential utilityas ultra-small drug delivery agents
Igor V. Zhigaltsev, Ying K. Tam, Alex K. K. Leung, and Pieter R. Cullis
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, Canada
Abstract
Previous studies from this group have shown that limit size lipid-based systems – defined as thesmallest achievable aggregates compatible with the packing properties of their molecularconstituents – can be efficiently produced using rapid microfluidic mixing technique. In thiswork, it is shown that similar procedures can be employed for the production ofhomogeneously sized unilamellar vesicular systems of 30–40 nm size range. These vesiclescan be remotely loaded with the protonable drug doxorubicin and exhibit adequate drugretention properties in vitro and in vivo. In particular, it is demonstrated that whereas sub-40 nmlipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcho-lines cannot be produced, the presence of such lipids may have a beneficial effect on theretention properties of limit size systems consisting of mixed lipid components. Specifically,a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phos-phatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylatedlipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with ahalf-life for drug release of 12 h. These results indicate that microfluidic mixing is thetechnique of choice for the production of bilayer LNP systems with sizes less than 50 nm thatcould lead to development of a novel class of ultra-small drug delivery vehicles.
Keywords
Doxorubicin, herringbone micromixer,limit size nanoparticles, liposome,microfluidic mixing
History
Received 9 December 2014Revised 23 February 2015Accepted 1 March 2015Published online 9 April 2015
Introduction
Important features of lipid nanoparticle (LNP) drug carriersystems include ease of preparation, reproducibility, andefficient encapsulation and retention of the biologically activeagent. However the LNP size is a critically importantparameter. The ability to generate small (5 50 nm) LNP canbe highly desirable to optimize the biodistribution of the LNPcarrier following intravenous (i.v.) injection. In this regard,the vast majority of LNP systems being used as drug deliveryagents have primarily utilized particles of 80–100 nm diam-eter, largely because of the availability of formulationmethods that produce LNP in that size range. To ourknowledge, all clinically approved (to date) nanomedicinesrepresent nanoparticulate carriers larger than 80 nm, examplesinclude DoxilÕ (80-100 nm) (Gabizon, 2002), MarqiboÕ
(100 nm) (Silverman & Deitcher, 2013), and AbraxaneÕ
(130 nm) (Green et al., 2006). It is well established that theenhanced permeation and retention (EPR) effect contribute tothe passive tumor-targeting of nanoparticles with the size of80–100 nm (Maeda et al., 2000). However, on one hand,
numerous works have reported that whereas such ‘‘large’’systems can often accumulate in the adjacent blood vesselsand in the peripheral regions of solid tumors, there is limitedpenetration into tumor tissue itself, thus limiting the potencyof the anticancer agent (Dreher et al., 2006; Jain et al., 2010;Kano et al., 2007; Perrault et al., 2009; Unezaki et al., 1996;Uster et al., 1998). On the other hand, it is widely recognizedthat smaller ( 50 nm) delivery agents may substantiallyimprove penetration and retention within the tumor tissue(Cabral et al., 2011; Chauhan & Jain, 2013; Chauhan et al.,2011; Huo et al., 2013), provided that they are larger than10 nm to avoid renal clearance. As particles in the size rangeof 10–50 nm can be expected to be the most promising carriersystem in accessing extravascular target tissues, the synthesisof such systems is of intense interest for biomedicalapplications. A number of techniques aimed at productionof smaller size vesicular LNP are available for decades, mostof them can be described as ‘‘top down’’approaches based ondownsizing of previously formed larger structures (De Kruijffet al., 1975; Hope et al., 1986; Huang, 1969). Those methods(exemplified primarily by sonication) have many limitations,including, most importantly, lack of scalability. Other tech-niques to produce nanovesicular systems include ‘‘bottomup’’approaches whereby LNP are formed by condensation oflipid from solution rather than by disrupting larger particles
Address for correspondence: Dr. Igor V. Zhigaltsev, Department ofBiochemistry and Molecular Biology, Faculty of Medicine, University ofBritish Columbia, 2350 Health Sciences Mall, Vancouver, Canada. Tel:+ 1 604 822 4955. Fax: + 1 604 822 4843. E-mail: igorvj@ mail.ubc.ca
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Cryo-TEM study
Figure 4 shows representative images of ‘‘empty’’ (A) anddoxorubicin-loaded (B) POPC/DPPC/Chol/DSPE-PEG2000(45/20/35/3) LNPs. As seen, doxorubicin loading at a0.1 mol/mol ratio did not affect the predominantly sphericalshape of the pre-loaded vesicles; a characteristic ‘‘coffee-bean’’ appearance of the internally precipitated drug can beobserved that is similar to the appearance of larger liposomaldoxorubicin formulations such as DoxilÕ . The cryo-TEMmicrographs also provide size information that can be used tovalidate the sizes determined by the light scattering technique.A size analysis based on a sample of 120 particles indicatedmean diameters of 33±4 nm (mean±SD) for both empty andloaded LNPs, in good agreement with the number-weightedsize values determined by DLS.
Long-term stability
In a final area of investigation, a long-term (up to 6 months)stability of doxorubicin-loaded POPC/DPPC/Chol/DSPE-PEG2000 (45/20/35/3) systems stored at 4 C has beenstudied. LNPs were loaded with drug at 0.1 mol/mol drug-to-lipid ratio and concentrated to 10mg/ml total lipid. Nosignificant changes in mean LNP size and drug entrapmentwere observed during the course of the study (results notshown).
Discussion
As pointed out in the ‘‘Introduction’’section, two approachesto formation of ultra-small vesicular LNP systems can betaken, namely ‘‘top down’’ size reduction methods whichusually are harsh procedures requiring high energy input such
Figure 4. Cryo-TEM micrographs of LNP composed of POPC/DPPC/Chol/DSPE-PEG2000 (45/20/35/3) prior to (A) and after (B) loadingwith doxorubicin at a drug-to-lipid ratio 0.1 mol/mol. The bar represents100 nm. For details of sample preparation and cryo-TEM protocols, see‘‘Materials and methods’’section.
0 5 10 15 20 25
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Figure 2. Drug retention in 22 nm POPC/Chol/DSPE-PEG2000 65/35/3(squares) and 33 nm POPC/DPPC/Chol/DSPE-PEG2000 45/20/35/3(diamonds) systems determined in vivo. LNP formulations containingtrace amounts of the tritiated lipid [3H]-CHE were loaded with14C-labeled doxorubicin at a drug-to-lipid ratio 0.1 mol/mol and theninjected intravenously into CD1 mice at a lipid dose of 50mg/kg. Plasmasamples taken at the indicated time points were analyzed for lipid anddrug content by liquid scintillation counting as described in ‘‘Materialsand methods’’ section. Each data point represents mean values±SDfrom each group of mice (n ¼ 4).
0 5 10 15 20 25
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Figure 3. Liposomal lipid levels obtained in plasma of CD1 miceinjected with 22 nm POPC/Chol/DSPE-PEG2000 65/35/3 (squares) and33 nm POPC/DPPC/Chol/DSPE-PEG2000 45/20/35/3 (diamonds) sys-tems. Lipids were quantified as indicated in Figure 2 and ‘‘Materials andmethods’’ section. Each point represents mean values±SD from eachgroup of mice (n ¼ 4).
DOI: 10.3109/08982104.2015.1025411 Production of limit size nanoliposomal systems 5
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Drug Retention in Small Liposomes
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Encapsulation of Hydrophobic Propofol
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48
Design of Experiment Studies
49
Polymer Nanoparticles
CellaxTM Polymer-Drug conjugates
50
100nm 100nm
NanoAssemblr TM Vortex
Nanoparticle Size Dictated by Polymer Concentration
51
Particle size is dictated by polymer concentration
CellaxTM Polymer-Drug conjugates
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Polymer Composition Dictates Biophysical Characteristics
54
55
Polymer-mediated Anti-Cancer Activity
O/W Nanoemulsions
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Nanoemulsion Droplet Size Dictated by Manufacturing Process
57
0 1 2 3 4 5 6 7 8 9 10
10
20
30
40
50
60
70
B POPC/triolein (60/40 mol/mol)
Part
icle
siz
e, nm
Aqueous/ethanol flow rate ratio
Zhigaltsev, I.V. Et al., Langmuir 2012, 28, 3633−3640
58
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
10
20
30
40
50
60
70
80
90
Actual diameter
Theoretical diameter
Part
icle
siz
e, n
m
POPC/Triolein ratio, mol/mol
Zhigaltsev, I.V. Et al., Langmuir 2012, 28, 3633−3640
Nanoemulsion Droplet Size Dictated by Emulsion Composition
59
Presentation Overview
1. Introduction to Microfluidics-Based NanoAssemblr™
Platform for Nanoparticle Manufacture
2. Examples of Nanoparticles Manufactured by the
NanoAssemblr™ Platform
3. Scale-up Manufacture using the NanoAssemblr™
Platform
Assessment of the Robustness of the Manufacturing Process
60
Stable Results = Robust Process = Scalable Process
Design of Experiment (DoE) variables
– Lipid Concentration
– Flow Rate
– Mixing Conditions
– Lipid:RNA Ratio
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Continuous Flow Pumps
Parallelized Microfluidic Mixers
RNA -Nanoparticles
Aqueous (RNA)
Solvent (Lipids)
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Dilution
Buffer Exchange &
Nanoparticle Concentration
Microfluidics Enables “Seamless Scale-Up”
62
Continuous Flow Pumps
Parallelized Microfluidic Mixers
RNA -Nanoparticles
Aqueous (RNA)
Solvent (Lipids)
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Dilution
Buffer Exchange &
Nanoparticle Concentration
Microfluidics Enables “Seamless Scale-Up”
Continuous Flow Scale-up Manufacture of RNA-Lipid Nanoparticles Using Single Mixer
63
Seamless transfer of optimized manufacturing parameters
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Cumulative Fraction (mL)
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Continuous Flow Pumps
Parallelized Microfluidic Mixers
RNA -Nanoparticles
Aqueous (RNA)
Solvent (Lipids)
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Dilution
Buffer Exchange &
Nanoparticle Concentration
Microfluidics Enables “Seamless Scale-Up”
4x Parallelized Microfluidic Mixer Manifold System
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-0.02
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1X Mixer 2X Mixer 4X Mixer
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Microfluidic Parallelization Enables RNA-Lipid Nanoparticle Scale-Up
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12 mL/min 24 mL/min 48 mL/min
Parallelization of microfluidic mixers enables greater throughput
On-Chip Microfluidic Parallelization Produces Equivalent RNA-LNP
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Multiple options for increased throughput by parallelization
48 mL/min 48 mL/min 4X Mixer Chip
16x Parallelization Enabled Through 4x Mixer Cartridges in 4x Manifold System
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16x Parallelization Enables > 20L Batches in 2 h
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Continuous Flow Pumps
Parallelized Microfluidic Mixers
RNA -Nanoparticles
Aqueous (RNA)
Solvent (Lipids)
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Dilution
Buffer Exchange &
Nanoparticle Concentration
Microfluidics Enables “Seamless Scale-Up”
High Quality RNA-Lipid Nanoparticle Product
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Amenable to Industry Standard Post-Manufacture Processing
After Buffer Exchange
After RNA Concentration
After Microfluidics
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Continuous Flow Pumps
Parallelized Microfluidic Mixers
RNA -Nanoparticles
Aqueous (RNA)
Solvent (Lipids)
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Microfluidic Mixer
Dilution
Buffer Exchange &
Nanoparticle Concentration
Microfluidics Enables “Seamless Scale-Up”
Design for GMP Manufacturing
• Continuous flow pumps
• 8X parallelized mixers in disposable manifold
• Disposable fluid path for product contacting materials
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Aqueous Metering Pump
Solvent Reagent Bag
Aqueous Reagent Bag
Solvent Metering
Pump
Dilution Pump
Dilution Reagent Bag
Pinch Valve
To Post-processing
Tee
Tee
Pinch Valve
Sample Switch Waste
288 mL/min
384 mL/min
8X Scale-up System
8 x 12 mL/min
24 mL/min
72 mL/min 96 mL/min
Microfluidic Mixer Array
Design for GMP Manufacturing
8X Scale-Up Instrumentation Produces High-Quality RNA-LNP
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8X Scale-up system processes 5.75 L/hr
GMP Program in Development
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Pumping system selected
GMP-compliant Disposable COC microfluidic cartridges under development
Working with drug development partner to transfer technology to CMO for scale-up and GMP manufacturing
Fully disposable fluid path using USP Class 5/6 materials
Targeting a GMP-ready system by end-of-year 2015
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Summary
1. The Microfluidics-Based NanoAssemblr™ Platform
enables simple, rapid & reproducible manufacture of
novel nanoparticles
2. The NanoAssemblr™ Platform can be used to
manufacture several different types of nanoparticles
3. Process parameters can be used to dictate nanoparticle
biophysical characteristics such as particle size
4. The NanoAssemblr™ Platform enables “seamless”
scale-up by parallelization of microfluidic devices
http://www.nanoassemblr.com/resources/
77
Contact Information
Nepa Gene Co., Ltd.
Sales Team
www.nepagene.jp
3-1-6 Shioyaki, Ichikawa, Chiba, 272-0114 JAPAN
phone: +81 47 306 7222 fax: +81 47 306 7333
Extra Slides
78
Reproducible RNA-Lipid Nanoparticles Independent of Operator or Site
79
Automated Instrumentation Removes Operator Variability
Operator
Recommended