Application Note
Biopharma

Analysis of mRNA-Lipid Nanoparticles
in Human Plasma using SEC-Static
Dual-Angle Light Scattering andRefractive Index Detection
Authors

Introduction

Brian Liau, Suresh Babu C.V.,
and Ravindra Gudihal
Agilent Technologies, Inc.

In recent years, lipid nanoparticles have been used effectively for clinical deliveryof siRNA, mRNA, and other nucleic acid payloads. As one of the breakthroughtechnologies to emerge from the COVID-19 pandemic, mRNA-loaded lipid
nanoparticles (mRNA-LNPs) have been proven to be a safe and efficacious meansof expressing proteins in human tissues without the potential for inducing genetic
mutations. They are now being investigated for a variety of therapeutic applications
beyond vaccination against infectious diseases, including oncology, personalized
medicine, and rare genetic diseases.1

Li Zhang, Melgious Jin YanAng, and Yi Yan Yang
Bioprocessing Technology
Institute

The stability of mRNA-LNPs in their storage buffers was of widespread interest
during the pandemic because it was one of the primary determinants of vaccine
shipping conditions, and therefore, the degree of logistical challenge faced during
distribution. However, the stability of mRNA-LNPs in physiological fluids such as
blood plasma is an equally important consideration when developing new drugs
because it strongly influences the drug’s efficacy, pharmacokinetics, and organ
selectivity. The FDA guidance to industry on Biological Products that Contain
Nanomaterials2 reflects this, stating that "the impact of human plasma and blood on
the stability of nanomaterials... should be examined".
In this application note, size exclusion chromatography (SEC) with online
dual‑angle light scattering and differential refractive index (RI) detection was used
to characterize the reaction kinetics of mRNA-LNPs in human plasma. It was
shown that the degradation kinetics of a widely used mRNA-LNP composition canbe measured in real time by changes in hydrodynamic radius (Rh ) and molecularweight (Mw).

The study shows that having an online, high-resolution SEC
step greatly increases the power of light scattering analysis,
as it eliminates interference from plasma componentsthat typically confound bulk dynamic light scattering (DLS)
analyses. In addition, as the technique profiles all particles
in the sample without making implicit assumptions about
the size of mRNA-LNPs, it is an ideal tool for the unbiased
examination of the fate of mRNA-LNPs in complex media.

mRNA-LNP plasma incubation

Experimental

Human plasma powder was reconstituted in Milli-Q wateraccording to the manufacturer’s instructions, then centrifuged
at 10,000 g for 10 minutes and filtered through a filter with acore size of 0.2 μm to remove particulates. Serum albumin
was dissolved in 50 mM sodium phosphate buffer (pH 7.2) at
a concentration of 40 mg/mL. mRNA-LNPs were diluted 1:10(v:v) into either filtered plasma or serum albumin solution,then immediately injected at 15-minute intervals for SEC
multidetector analysis.

Human plasma powder (P9523-5ML) and bovine serum
albumin (A3782-5G) were purchased from Sigma-Aldrich.

Size exclusion chromatography and the Bio-SEC
multidetector system

Production of mRNA-LNPs

As shown in Figure 1, the following equipment stack
was used:

mRNA-LNPs were produced using the same composition
as Comirnaty1, the COVID-19 vaccine pioneered by Pfizer
and BioNTech. mRNA was in vitro transcribed from a
PCR‑amplified dsDNA template, purified using spin columns(Monarch RNA cleanup kit, New England Biolabs), thendissolved in 1 mM sodium acetate buffer (pH 4.7) to form theaqueous phase. ALC-0315, ALC-0159, DSPC, and cholesterol
were purchased from MedChemExpress and dissolved inethanol at a molar ratio of 46.3 : 1.6 : 9.4 : 42.7 to form theorganic phase. The lipid and mRNA solutions were allowed toundergo controlled mixing in a benchtop microfluidic device(NanoAssemblr Platform, Precision NanoSystems) withthe organic phase and aqueous phase, at a volume ratio of
3:1. The total flow rate was 12 mL/minute and the N:P ratio
was 6:1. The resulting mRNA-LNPs were buffer-exchanged
into physiological saline solution and concentrated inultracentrifuge tubes with a molecular weight cut-off (MWCO)of 30 K at 4 °C × 2,500 g × 60 minutes for a total lipid
concentration of approximately 2.2 mg/mL. The formed LNPs
were stored at 4 °C before being characterized by bulk DLS(Zetasizer, Malvern, UK) or the Agilent 1260 Infinity II Bio-SEC
multidetector system.

Agilent 1260 Infinity II
Bio-SEC Multidetector
System (G7805AA)

Agilent Aquagel-OH Mixed-M
4.6 × 250 mm, 8 µm

Figure 1. Instrumentation and consumables.

2

Agilent 1290 Infinity II
Bio LC System– Agilent 1290 Infinity II Bio Flexible Pump (G7131A)– Agilent 1290 Infinity II Bio Multisampler (G7137A)– Agilent 1290 Infinity II Multicolumn Thermostat (G7116B)
with Agilent InfinityLab Quick Connect Bio heat exchanger,
standard flow (G7116-60071)– Agilent 1290 Infinity II Diode Array Detector (G7117B)
equipped with an Agilent InfinityLab Max-Light Cartridge
Cell, 10 mm (G4212-60008)– Agilent 1290 Infinity II Refractive Index Detector (G7162B)– Agilent 1260 Infinity II Bio-SEC Multidetector System(G7805AA) featuring Bio-inert Static Dual-Angle andDynamic Light Scattering Detector

Agilent 1290 Infinity II
Diode Array (G7117B)
and Refractive Index(G7162B) Detectors

Table 1. Agilent 1260 Infinity II Bio-SEC multidetector system parameters.
Chromatographic Conditions
Mobile Phase

50 mM sodium phosphate, pH 7.2

Flow Rate

0.6 mL/min

Injection Volume

15 µL

Run Time

15 minThermostat Autosampler

5 °C

Temperature TCC

30 °C

LS Detector

30 °C, 5 Hz

Results and discussion
Analysis of mRNA-LNPs in phosphate buffer

System Calibration
Concentration

10 mg/mL

dn/dc

0.186

Extinction Coefficient

light scattering. Two 4.6 × 250 mm PL aquagel‑OH
SEC columns with different molecular weight cutoffs,
500,000 Da (part number PL1549‑5801) and 10,000,000 Da(part number PL1549-5800) were used for this study. Dataacquisition and analysis was performed using Agilent Bio-SEC
software (A.02.01).

0.66 mL/mg/cm

DLS Operational Parameters
Correlator Run Time

5s

Correlator Function Clip Time

10 µs

R²

0.80

Eluent Viscosity

0.0079 p (viscosity of water at 30 °C)

Eluent Refractive Index

1.333 (refractive index of water)

The diode array detector was used primarily for systemcalibration and was not required for light scatteringexperiments. The quaternary pump was used in isocraticmode with only a single solvent line active to reducebaseline fluctuations in the RI detector. The Bio-SEC
multidetector system was equipped to perform dual‑angle
light scattering at 15 and 90°, as well as dynamic

mRNA-LNPs were fabricated using microfluidic technology
and buffer-exchanged into 154 mM NaCl solution. They werediluted into 50 mM sodium phosphate buffer (pH 7.2) to a
final lipid concentration of 0.22 mg/mL prior to analysis witheither bulk DLS or the Bio-SEC multidetector system.
Figure 2 shows a representative SEC chromatogram.
Baseline regions (blue shading) were carefully chosen,bracketing the region of interest (green shading) before eachtrace was individually checked for accurate baselining andintegration. As the lipid-formed LNPs do not have strong UVchromophores, the RI detector was necessary for estimating
the concentration of eluting analytes. Positive and negative
deflections in the RI tracing away from the region of interest
were attributed to dissolved air and salts in the injected
sample. In the region of interest, the overlaid red dots indicate
Rh measurements from the online DLS detector, and thethick blue line indicates Mw measurements from dual-angle
light scattering.

Green highlight:
Peak of interest
Blue highlights:
Baseline region
RI: salts

RI: dissolved air

Figure 2. Representative SEC chromatogram.

3

As shown in Table 2, SEC multidetector Rh and polydispersity
measurements showed excellent agreement with bulk DLS.
As expected, both techniques showed the sample to be close
to monodisperse. In addition, the Bio-SEC multidetector
provided estimates of the sample Mw and radius of gyration(Rg ) using static light scattering. These measurements,
which are orthogonal to DLS, can be useful because they
may indicate differences in particle composition that may
not affect Rh, and may also yield insights into particle
shape. In this case, Rg/Rh is approximately 0.9, which is
close to the ideal value of 0.77 expected of monodisperse,
compact spheres.

Chromatographic resolution of mRNA-LNPs andplasma components
As separation in SEC depends on the transient inclusion ofanalytes into porous chromatography resin, the resolutionof analytes depends on both column length and pore size.
In cases where a single column proves insufficient, twocolumns in series may provide superior resolution. In addition,columns with different pore sizes may be used to separate
analytes over a wider range of molecular weights than can beaddressed with a single type of column.
Figure 3 shows overlaid light scattering (LS90°) and refractive
index chromatograms for mRNA-LNPs and human plasma

Table 2. Physical characteristics of mRNA-LNPs.
Rh (nm)Polydispersity*

Zeta Potential (mV)

Rg (nm)Mw (MDa)

Bulk DLS

37.75

0.079

–0.47

–

–

Agilent 1260 Infinity II Bio-SEC
multidetector system

31.94

1.017

–

28.87

43.1

* Polydispersity is calculated differently for DLS and the Agilent 1260 Infinity II Bio-SEC multidetector system.
In DLS, the polydispersity index is scaled such that values < 0.05 are rarely seen other than with highly
monodisperse standards, and values > 0.7 indicate a very broad size distribution. In dual‑angle light scattering,
polydispersity is defined as Mw/Mn.

A

Single column
LS90°, mRNA-LNP in PBS
LS90°, Human plasma

B

C

D

Single column
RI, mRNA-LNP in PBS
RI, Human plasma

Dual column
RI, mRNA-LNP in PBS
RI, Human plasma

Dual column
LS90°, mRNA-LNP in PBS
LS90°, Human plasma

Figure 3. LS90° and refractive index chromatograms for mRNA-LNPs and human plasma injected separately onto single or dual-column SEC configurations.

4injected separately in single or dual-column configurations.
The single-column configuration comprised a wide pore
4.6 × 250 mm PL aquagel-OH column (10 MDa MWCO),
and the dual-column configuration comprised the wide porecolumn followed by a second narrow pore 4.6 × 250 mm
column (0.5 MDa MWCO).
Figure 3A clearly shows that the single-column configuration
was unable to adequately resolve plasma components from
mRNA-LNPs, whereas the dual-column configuration in
Figure 3B provides acceptable resolution for much of themRNA-LNPs’ peak. In addition, the dual-column configuration
resolves plasma components into two clear peaks, with thetaller peak eluting at approximately 8.5 minutes, dominatedby serum albumin (supporting data not shown). Figures 3C
and 3D show that the RI signal attributable to mRNA‑LNPs
is much weaker than the LS90° signal and may easily bedominated by the much larger RI of the plasma components.
Hence, good chromatographic separation (Figure 3D) isimperative for accurate determination of mRNA-LNPs Mw,
since even minor coelution with plasma components canlead to large errors in mRNA‑LNPs concentration estimates,
thereby causing inaccurate Mw calculations.

Kinetics of mRNA-LNPs reaction with human plasma
Figure 4 shows mRNA-LNPs incubated in human plasma
injected at 15-minute intervals and separated in adual‑column configuration. The LS90° chromatograms clearlyshow the mRNA-LNPs peak at 6.7 minutes decreasing in
intensity over time, while the plasma component peak at
7.7 minutes increases correspondingly in intensity. Evidently,
mRNA-LNPs are reacting with plasma components anddecreasing in size, leading to an increase in retention timeand coelution with plasma components at 7.7 minutes. Online
DLS measurements (left inset) show that the Rh of plasmacomponents in the 7.7-minute peak range from 12 to 16 nm;
thus, the final size range of mRNA-LNPs after degradation
can be inferred.
The inset figures on the right of Figure 4 show the futility ofattempting to track this reaction using either the RI detector(Figure 4B) or via UV absorbance of the encapsulated mRNAat 260 nm using a diode array detector (Figure 4C). Dueto its limited sensitivity, the RI detector shows only verysmall changes in the intensity of the peak attributable tomRNA-LNPs. These changes are more evident in the UV
absorbance chromatogram. However, in both cases, the fate

B

A

Refractive index

Online DLS

mRNA-LNPs
Degraded mRNA-LNPs+ plasma components

C

UV Abs260
mRNA-LNPs
mRNA-LNPs

Figure 4. LS90° chromatograms of mRNA-LNPs incubated in human plasma over 120 minutes. (A) a representative trace showing online DLS measurements (reddots) across the three peaks. Refractive index (B) and UV absorbance (C) traces corresponding to the LS90° chromatograms.
5

of the mRNA‑LNPs is unclear because the overwhelming
signal from plasma components obscures where degraded
mRNA‑LNPs elute. Only the light scattering detector, with its
higher sensitivity to particulate matter, is able to clearly makethis determination.

more than Mw =A

Σ N iM i4
Σ N iM i3

over time, which weights heavier particles muchB

16,00025

10,000Rh (nm)

LS90 Area (mV‧s)

12,000
8,000
6,000
2,000
20

40

60

80

100

120

140

0

D

Intact mRNA-LNP peak Mw

40
35
25

Mz+1 (MDa)

Mw (MDa)

30
20
15
10
5
0

15

5
0

Time (min)

C

20

10

4,000
0

Intact mRNA-LNP average Rh

3530

y = –90.128x + 16,711
R² = 0.9764

14,000. As shown, Mz+1 remains approximately

In contrast, Figure 6 shows that mRNA-LNPs react withserum albumin, the most abundant component in bloodplasma, very differently than with whole plasma. Figure 6A
shows that the decrease in mRNA-LNP peak area is far more
limited than in plasma and is not associated with any shift
in retention time. Unlike in plasma, changes in peak area
(Figure 6B) and Mw (Figure 6C) indicate that the reaction
was completed within 30 minutes. There was no significant
change in Rh, which averaged 29.8 ± 3.3 nm over the courseof the experiment, whereas Mw increased from approximately
40 to 50 MDa after 30 minutes. Taken together, these data areconsistent with a protein-coating process.

Intact mRNA-LNP peak area

18,000Σ N iM i

constant over time at approximately 40 MDa, which canbe attributed to the signal from intact mRNA-LNPs. These
data suggest that there is little to no increase in the size ofmRNA‑LNPs due to, e.g., protein coating, and that degradationis the dominant process.

Figure 5 shows the changes in the physical characteristicsof the mRNA-LNPs peak over time. Figure 5A shows that theintensity of the peak decreases linearly with time, indicatingthat the reaction follows zero-order kinetics and is expected
to go to completion in approximately 180 minutes. Figure 5Bshows the average Rh of particles in the mRNA‑LNPs
peak, measured by online DLS. The average Rh falls fromapproximately 32 nm in sodium phosphate buffer toapproximately 25 nm within 30 minutes of plasma incubation,
providing direct evidence that the mRNA-LNPs are decreasing
in size. Figure 5C corroborates this, as the apparent Mw ofmRNA-LNPs decreases linearly with time. Figure 5D shows
Mz+1 =

Σ N iM i2

0

20

40

60

80

Time (min)

100

120

140

50
45
40
35
30
25
20
15
10
5
0

PBS

Plasma, 0 min

Plasma, 15 min

Intact mRNA-LNP peak Mz+1

0

20

40

60

80

100

Time (min)Figure 5. Physical characteristics of mRNA-LNPs incubated in human plasma as measured by online static dual-angle light scattering and DLS.

6

Plasma, 30 min

120

140

mRNA-LNPs

LS90 area (mV‧S)

Serum albumin
18,00017,00016,00015,00014,00013,00012,00011,00010,000Intact mRNA-LNP peak area

0

20

40

60
80
Time (min)

100

120

140

Intact mRNA-LNP Mw

60

Mw (MDa)

50
40
30
20
10
0

0

20

40

60

80

100

120

140

Time (min)Figure 6. LS90° chromatogram and Mw of mRNA-LNPs incubated in serum albumin.

Conclusion

References

This study demonstrates the power of SEC with amultidetector system for the study of LNPs as: (i) it profiles
all particles in a sample regardless of changes in size, (ii) it
is far more sensitive to particulates than refractive index
or UV absorbance detection, allowing nanoparticles to be
detected even when obscured by sample matrix, (iii) SEC
can eliminate interference from complex matrices such asplasma, facilitating accurate light scattering measurements,and (iv) it enables orthogonal determination of size andmolecular weight through retention time, static, anddynamic light scattering in a single experiment, increasing
experimental certainty.

1. Tenchov, R.; Bird, R.; Curtze, A. E.; Zhou, Q. Lipid
Nanoparticles–From Liposomes to mRNA Vaccine
Delivery, a Landscape of Research Diversity andAdvancement. ACS Nano 2021.

www.agilent.com
For Research Use Only. Not for use in diagnostic procedures.
RA45250.5916550926
This information is subject to change without notice.
© Agilent Technologies, Inc. 2024
Printed in the USA, February 14, 2024
5994-6854EN

2. Drug Products, Including Biological Products, that Contain
Nanomaterials – Guidance for Industry. U.S. Food andDrug Administration, April 2022.