APPLICATION NOTE

Process optimization and pre-clinical production oflipid nanoparticles using Sunshine
optimizing the conditions for producing nanoparticles
can be laborious and resource intensive. For instance,
ethanol injection is commonly employed to synthesize
mRNA-LNPs, in which lipids suspended in ethanol are
injected drop-by-drop into aqueous mRNA solutions
from a syringe. This can take a long time and can leadto inconsistent results from run to run. Other processes such as impinging jet mixing (where two fluid
streams collide at high pressure in a jet mixing chamber) apply significant shear force to the particles,
which can accelerate degradation and potentiallycause particles to rupture. These techniques (alongwith traditional bulk methods such as thin film rehydration) can be challenging to scale, and struggle togenerate highly homogeneous nanoparticles in termsof size, morphology, lipid composition, and mRNA
payload quantity. This can result in significant variation in the in vivo behavior of mRNA-LNPs.

Introduction
In the field of drug delivery and medicine development, nanoparticles including lipid nanoparticles(LNPs), liposomes, and polymer nanoparticles areat the forefront of a rapidly expanding class of drugdelivery products termed ‘nanomedicines’. They candeliver active pharmaceutical ingredients (APIs) to
specific action sites while protecting the API from premature enzymatic degradation, preventing immune
system overstimulation, and improving intracellular
penetration and retention time. For researchers toaccelerate their nanomedicine development, particle
synthesis solutions are needed that are consistent,
low cost, and efficient with materials. Unchained Labs
provides nanoparticle synthesis solutions underpinned by microfluidics and automation, offeringsystems at every stage of the nanomedicine development journey; from screening and early-stage
R&D, through process optimization and ultimately
into manufacturing and commercialization of drug
products. This application note presents example
use cases for Sunshine, which offers both automatedprocess optimization and continuous manufacturing
capabilities in a single system.

1

After the success of COVID-19 mRNA vaccines (andother drugs before that such as the hATTR amyloidosis treatment Onpattro), LNPs have become
widespread, leading to the rapid development ofmRNA-LNPs for further vaccines, genetic therapies,
and the treatment of many other diseases. LNPs arederived from well-established liposome technology,
with the addition of ionizable lipids for encapsulationof negatively charged genetic materials, such as
siRNA, mRNA, miRNA, and DNA, via electrostaticinteractions. LNPs are set to transform the future ofmedicine, but there is a lot of research that still needs
to be done for LNPs to function as optimal carriers inall their varied roles.

2Figure 1: An example Sunny (microfluidic chip) setup. Sunny 1
shows the mixing of lipids and API (such as mRNA) by hydrodynamic flow focusing to assemble LNPs. Sunny 2 shows optional
in-line dilution, which can be used to adjust pH in-line, and reduce
solvent concentration in collected samples.

In contrast, microfluidic technology is based onhydrodynamic flow focusing and rapid mixing at thejunction of a Sunny from separate channels to form
nanoparticles using stable and predictable laminarflow (Figure 1). Consequently, microfluidic technology maintains consistent conditions for nanoparticle
self-assembly and offers greater process control. Thisapproach makes it easier to determine the formulation that optimizes the critical quality attributes ofparticles.

It is well-known that the nanoparticle assembly process is complex and challenging to control, and that
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PROCESS OPTIMIZATION AND PRE-CLINICAL PRODUCTION OF LIPID NANOPARTICLES USING SUNSHINE

Once the reagent is collected, the system completes
a wash cycle, and then the next experiment starts
with a new set of flow parameters. This processrepeats until all the planned experiments arecompleted, and samples can be collected fordownstream purification and analysis.

Process optimization withSunshine
Most nanomedicine development processes startwith screening a large number of formulations inorder to seek “hits”, or lead candidates for furtherdevelopment. For this process, Unchained Labs offers
Sunscreen, a high-throughput automated system
for screening low volumes of various formulations in
a 96-well plate format. Sunscreen could be used forscreening the ionizable lipid component or lipid andcargo, ratios, and so on, to find a successful formulation. After such a formulation has been selected using
Sunscreen, scientists require a rapid, low-cost, andpreferably automated particle production platform
to optimize the size, shape, and structure of thesenanoparticles ahead of final production/commercialization. With Sunshine (Figure 2), researchers
can automatically optimize process parameters andgenerate test samples.

Using the Sunny Suite Software accompanying thesystem, users can optimize the flow rate ratio (FRR),
total flow rate (TFR), NP precursor volume, sample
collection volume and allow optional in-line dilution
to synthesize controlled, reproducible, and monodispersed nanoparticles. For example, a series of 10
low-volume experiments with a fixed pair of reagents
testing at various conditions of FRR, TFR, and dilution
ratio could be executed automatically in 15 minutes inthe standard protocol mode as shown by the systempressure data in Figure 3.

Figure 3: Pressure profile of Sunshine during a protocol running
10 repeat experiments, showing consistent pressure during thesystem washing and experiment steps.

Once a desired set of experimental conditions has
been identified, the user can switch to continuousmode to create liters of nanoparticles for pre-clinical trials and extensive characterization. Uniquein this dual capability, Sunshine bridges the gap
between formulation screening and commercial
scale manufacturing in a single system solution.

Figure 2: Sunshine

Sunshine may be used initially in “protocol mode”,
where the system can sequentially execute a tableof automated and low-volume experiments from afixed pair of injected reagents, using as little reagent
as 320 µL per input per experiment. In this mode,enough reagent for several experiments is injected
into sample loops within the system. As the protocolruns, aliquots of these reagents are released fromsample loops controlled by careful valve timings andare combined on a Sunny of your choice (UnchainedLabs reusable Sunnies). To move these aliquots ofreagent around the system, driving fluids are used(typically a solvent and buffer like ethanol and PBS),
which push the reagent from the loops, through theSunny and to the Automated Collector.

Herein, this application note describes how
Sunshine can be used to optimize typical formulations with a view to identifying suitable process
conditions ahead of continuous manufacturing.
As a starting point for this study, an example LNP
formulation will be used to represent a successful“hit” formulation which may have been identified
using Sunscreen. In this case, we will use SM-102
as the ionizable lipid, with Luciferase mRNA with
N/P ratio of 6 (see Table 1).
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PROCESS OPTIMIZATION AND PRE-CLINICAL PRODUCTION OF LIPID NANOPARTICLES USING SUNSHINE

Parameter

Description

Aqueous Phase (A)Luciferase mRNA inacetate buffer (50 mM,pH 4)

Organic Phase (O)SM-102: DSPC:
Cholesterol: DMG-PEG
2000 (50:10:38.5:1.5%
molar) in ethanol

N/P Ratio

6Dilution (D)No dilution

Flow Rate Ratio(FRR)
Total Flow Rate (TFR)

Variable (A:O)

Figure 4: mRNA-LNPs produced by various FFR (aqueous toorganic phase) at a fixed TFR of 5 mL/min with no dilution. LNPs
ranged from 58 nm to 68 nm post-dialysis, with an average PDIof 0.12. Particle size distribution and polydispersity index (PDI)
were determined by dynamic light scattering (DLS). The error bars
represent standard deviation of the mean n=3.

5 mL/min unlessotherwise stated

Table 1: Parameters for the synthesis of mRNA-LNPs utilizingSunshine.

Using Sunshine, both flow rate ratio (FRR) andtotal flow rate (TFR) were investigated to identify
an optimal process for forming particles with thisformulation. The target outcome of such work is
to identify an optimized set of process conditions
with which the target formulation might be efficiently manufactured in continuous flow. In thiscase, example criteria for an ideal particle may be
a particle size around 60 nm, with a PDI <0.1, andan EE% of >90%. Using Sunshine, the nanoparticle
synthesis process can be optimized towards thesekey criteria.

Using the RiboGreen assay, the encapsulation efficiency (EE%) of mRNA was evaluated. While the TFR
was maintained at 5 mL/min and the N/P ratio waskept constant at 6, the data in Figure 5 shows theencapsulation efficiency (EE%) was consistently above
90%, and reached 96% for an FFR of 6:1 (aqueous toorganic phase).
Factoring in a desire to keep ethanol concentration
below 30% in the collected sample, and an example
target size of 50–60 nm, a flow rate ratio of 3:1 wasselected for further exploration based on the data
shown in Figure 4 and Figure 5.

Optimizing Flow Rate Ratio
Using a Sunny 190 XT, a series of experiments withvarious FRRs were examined to show how theyaffect the size and polydispersity of the LNPs. Figure 4 illustrates that the size of mRNA-LNPs wascontrollably altered from 68 nm to 58 nm post-dialysis by varying the flow rate ratio between theaqueous and organic phases from 2:1 to 6:1 while
maintaining the constant total TFR of 5 mL/min.
Alongside that, these experiments gave a consistently low PDI, with an average of 0.12 across all
flow rate ratios.
Figure 5: The encapsulation efficiency (EE%) of mRNA-LNPs wasobtained by the RiboGreen assay. mRNA-LNPs were produced bySunshine at a fixed TFR of 5 mL/min and various FRR (aqueous toorganic phase) with no dilution. The N/P ratio was fixed at 6. Theerror bars represent standard deviation of the mean n=3.

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PROCESS OPTIMIZATION AND PRE-CLINICAL PRODUCTION OF LIPID NANOPARTICLES USING SUNSHINE

Optimizing Total Flow Rate
The effect of TFR on mRNA-LNP particle size andEE% was examined (Figure 6). Increasing the TFRfrom 2.5 to 7.5 mL/m showed a reduction in the sizeof mRNA-LNPs, from 94 to 57 nm, with consistently
decreasing PDI. There was a small increase in size
when the TFR reached 10 mL/min. Overall, the best
PDI and low particle size was achieved at a TFR of 7.5mL/min, with a size of 57.7 nm and a PDI of 0.089,
which also coincided with a high encapsulation efficiency of 94.7%, thus meeting all the initially desired
criteria for a nanoparticle production process.

Figure 7: The encapsulation efficiency (EE%) of mRNA-LNPs wasobtained by the RiboGreen assay. mRNA-LNPs were producedby Sunshine at a fixed FRR of 3:1 and various TFRs were investigated with no dilution. The N/P ratio was fixed at 6. The error bars
represent standard deviation of the mean n=3.

Transitioning to continuousproduction
After optimization of a formulation using protocolmode in Sunshine, (producing a typical minimum of
1 mL of sample), users can then switch to a continuous mode of system operation, where Sunshine
can produce up to tens of liters of sample perday (depending on the optimized flow conditions
being used). To demonstrate the simple transition
between protocol mode and continuous mode, aliposomal formulation was prepared using Sunshine in both operation modes at multiple flowrate ratios.

Figure 6: mRNA-LNPs produced by various TFR (aqueous toorganic phase) at a fixed FFR of 3:1 (Aqueous: Organic) with no dilution. The hydrodynamic size ranged from 58 nm to 94 nm, withaverage PDI of 0.12. The error bars represent standard deviationof the mean (n=3).

Given the high encapsulation efficiency, low particle
size and low PDI was observed at 7.5 mL/min (Figure7). It is clear that in our example formulation, this combination of TFR, FRR, chip and formulation could beconsidered for larger scale continuous production forpre-clinical and ultimately clinical trials. In addition tooptimizing the variables explored above, one may also
consider the effect of changing the concentration ofinjected reagents, or the N/P ratio of the formulation
selected for further optimization using Sunshine.

Parameter

Description

Aqueous Phase (A)1 x PBS pH 7.4

Organic Phase (O)Phospholipon 90G (1 mg/mL), DBAB (0.1 mg/mL) inethanol

Dilution (D)Flow Rate Ratio(FRR)
Total Flow Rate (TFR)

1 x PBS pH 7.4
2:1:2, 2:2:2, 2:3:2 (A:O:D)
3 mL/minTable 2: Parameters for the synthesis of liposomes utilizingSunshine.

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PROCESS OPTIMIZATION AND PRE-CLINICAL PRODUCTION OF LIPID NANOPARTICLES USING SUNSHINE

Figure 8: Liposome produced continuously at a TFR of 3 mL/min at varying FRR. Nanoparticles ranged from 19 nm to 57 nm, mean n=3.
The error bars represent standard deviation of the mean. PDI remains consistent between runs-by-runs ranging from 0.15 to 0.2 with astandard deviation of 0.01.

Sunshine is ideally suited to optimizing the formation
process of lead candidate formulations, whilst enabling a seamless transition into continuous manufacturing ahead of clinical trials. The data presented
here show the principles of optimizing a selected
formulation to target required key performance indicators such as size, PDI and encapsulation efficiency.

Figure 8 shows that liposomes based on phospholipon 90G were produced using a range of FRRs
(Aqueous:Organic:Dilution) in either protocol mode orcontinuous production mode, using a constant TFRof 3 mL/min. The nanoparticle sizes ranged between
19 to 57 nm and differed by less than 7 nm between
protocol and continuous mode, and the PDI remained
consistent ranging from 0.15 to 0.2, showing an
expected size increase as the proportion of organic toaqueous was increased. We can see from a process optimization perspective that lower PDIs wereachieved at a flow rate ratio of 2:3:2.

The data shown focuses on the impact of tuning total
flow rate and flow rate ratio using Sunshine to rapidly
complete consistent sets of triplicate data for all conditions tested, in a hands-free automated manner. Inaddition to these parameters, users can investigate
reagent concentrations to achieve a higher yield process, tune N/P ratio to deliver more mRNA per particle, as well as test Unchained Labs’ range of Sunnies
to assess the ideal microfluidic mixing geometry.

Summary
In developing any nanoparticle-based drug delivery
product, it is key to identify both the correct formulation and the best process conditions in order tosuccessfully manufacture nanomedicines with thecorrect performance characteristics. Since particle
performance is underpinned by attributes such as
the size, shape and structure of the nanoparticle, thesynthesis process and mixing conditions are critical toensuring highly effective and safe nanomedicines.

Once a suitable particle formation process has been
identified, the same system can be set up to executea continuous production run with identical process
conditions in a matter of minutes, allowing liters ofnanoparticles to be produced for pre-clinical trials
and more extensive downstream characterization.
5PROCESS OPTIMIZATION AND PRE-CLINICAL PRODUCTION OF LIPID NANOPARTICLES USING SUNSHINE

Particles produced this way are consistent with those
produced using the system’s protocol mode, making
the transition to continuous product manufacturingsimpler than ever, with minimal process validation
required.

and dilution phase, phosphate buffer saline (PBS)
x 1 at pH 7.4 was used. All prepared solutions were
filtered with a 0.2 µm filter prior to use. The organic and aqueous were mixed automatically using
the Sunshine protocol.

Materials and methods

The organic and aqueous phase were mixed automatically using the Sunshine protocol on a Sunny
190 XT. The lipid nanoparticle samples were transferred to dialysis kits (Pur-A-lyzer™ Midi Dialysis
Kit, Sigma, UK) and placed in a beaker containing
1 L of PBS 1x pH 7.4. The dialysis was run for 3 h toremove the ethanol and exchange the buffer medium. The post-dialysis nanoparticle samples werestored in the fridge for further investigation.

For the formation of mRNA-LNPs: 1,2-distearoyl-sn-glycero-3-phosphochloline (DSPC)(Avanti, UK) 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG PEG-2000)(Avanti, UK), cholesterol (Sigma Aldrich, UK),
and SM-102 (BroadPharm, San Diego, US) weredissolved in ethanol (reagent grade 99%, SigmaAldrich, UK). The solution of 10mM lipid formulation
contained SM-102, DSPC, cholesterol and DMGPEG-2000 in ethanol with the relative molar ratioof 50:10:38.5:1.5%. For the aqueous phase, 89.44µg/mL of Luciferase mRNA in 50 mM of acetate
buffer at pH 4 (nuclease-free) was employed.

Particle size distribution and polydispersity index(PDI) were determined by DLS. All samples weregenerated in triplicate, the mean and standard
deviation (SD) were reported.
The encapsulation efficiency (EE%) of mRNA-LNPs
was quantified by RiboGreen assay - using Triton
X-100 to break open the LNPs to analyze concentration of total mRNA in the sample and comparing this value to the concentration of free mRNA,
determined in the absence of Triton. The fluorescence intensity of RiboGreen was measured on aMicroplate reader (FLUOROstar, BMG, Germany).

To produce liposomal particles for continuousproduction comparisons, phospholipon and DDAB,phospholipon 90G (Lipoid, Switzerland) anddimethyl-dioctadecyl-ammonium bromide (DDAB)(Fischer Scientific, UK) were dissolved in ethanol(reagent grade 99%, Sigma Aldrich, UK) at 1 mg/mL and 0.1 mg/mL respectively. For the aqueous

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