Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 253–258

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Development of simple isocratic HPLC methods for siRNA

quantitation in lipid-based nanoparticles
Zongyun Huang ∗ , William P. Fish
Bristol-Myers Squibb, Drug Product Science & Technology, 1 Squibb Drive, New Brunswick, NJ 08901, USA

a r t i c l e i n f o a b s t r a c t

Article history: This paper describes the development of simple and user-friendly HPLC methods that can quantitate the
Received 8 February 2019 amount of small interfering RNA (siRNA) in lipid-based nanoparticle (LNP) formulations. The methods
Accepted 12 April 2019 have been used as alternative chromatographic approaches to the size exclusion chromatography in order
Available online 27 April 2019
to perform “fit for purpose” analysis such as determining the amount of released siRNA from LNP formu-
lations as a part of in-vitro release testing. Two HPLC conditions were optimized using reversed phase (a
Keywords:
250 × 4.6 mm Waters XSelect CSH column) and cation exchange columns (a 250 × 4.6 mm Zorbax SCX-
Oligonucleotide
300 column) maintained at 30 ◦ C with a mobile phase of 0.1 M ammonium bicarbonate aqueous solution
siRNA
Lipid nanoparticle
containing 20–30% acetonitrile. All the siRNA variants were excluded from pores in stationary phase and
Liquid chromatography led to a single peak eluted earlier than the solvent front. The impact of chromatographic conditions and
Size exclusion chromatography column configurations on method specificity, accuracy, and sensitivity have been investigated. Validation
Analytical method data for both methods in terms of precision, linearity, accuracy and sensitivity are also presented.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction siRNA (free plus encapsulated) in the sample matrix. Tradition-

Small interfering ribonucleic acids (siRNAs) are short dou-
ble stranded RNAs which can silence the expression of specific
post-transcriptional genes by the direct sequence-specific cleavage
leading to translation repression and consequent degradation of
messenger RNA [1,2]. SiRNAs have become a promising therapeu-
tic tool for several pathological areas such as viral infections, cancer,
genetic disorders, fibrosis, and autoimmune disorders [3]. In order
for siRNA to reach the targets to show efficacy, effective drug deliv-
ery systems are required to facilitate transfection because siRNA is
susceptible to degradation by endogenous enzymes, and is too large
(approximately 13 kDa) and too negatively charged to cross cellular
membranes. Lipid-based nanoparticles (LNP) are commonly used
for siRNA delivery due to their effective protection from plasmatic
nucleases, desirable particle size and non-immunogenicity [4–6].
Various analytical tests are required to characterize many in-vitro
features of siRNA drug products made of nanoparticles in order to
assure the desired in-vivo performance. Typically, Encapsulation
Efficiency, a measure of how much siRNA is inside the LNP, and in-
vitro release, a measure of how much siRNA will leak from the LNP
over time, are two critical quality attributes used to monitor LNP
quality. Monitoring these attributes is challenging as there is a need
to measure “free siRNA” (RNA outside of the LNP), as well as the total
ally, size exclusion chromatographic (SEC) methods are used for
siRNA determination with both advantages and drawbacks. Dou-
ble and single stranded siRNA can be separated adequately using
SEC columns with appropriate pore size, however, the susceptibil-
ity of SEC columns to back pressure requires low flow rate resulting
in broadened peak shape and long run time. Band broadening of
siRNA peaks often posts challenges in quantifying siRNA at low
concentration (i.e. < 1 ␮g/mL) In addition, SEC columns are usually
costly and have shorter column life times when unfriendly sample
matrixes are used such as the ones containing substantial amount of
lipids. Nevertheless, the methodologies for determining total siRNA
amount are not restricted to SEC, provided that the alternative
technique demonstrates specificity, accuracy and sensitivity.
The objective of this study is to develop a fast, user-friendly
and robust isocratic HPLC method with adequate sensitivity for the
analysis of large numbers of samples in timely matter. This paper
discusses the course of development for such an HPLC method
for the determination of total siRNA content in various sample
matrixes and media.
2. Experimental
2.1. Materials and reagents

∗ Corresponding author. ACS reagent grade chemicals from MilliporeSigma (Burlington,

E-mail address: zongyun.huang@bms.com (Z. Huang). MA, USA) were used unless otherwise indicated. Milli-Q purified

https://doi.org/10.1016/j.jpba.2019.04.026
0731-7085/© 2019 Elsevier B.V. All rights reserved.
254 Z. Huang, W.P. Fish / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 253–258

Fig. 1. Chromatograms of a solution containing 0.1 mg/mL siRNA using a Waters CSH C18 (3.5 ␮m particle size) 15 cm × 4.6 mm column operated at different column
temperatures of (A) 60 ◦ C and (B) 25 ◦ C; flow rate: 1.0 ml min−1 ; injection volume: 10 ␮l; UV detection: 260 nm; mobile phase A: 0.1 M ammonium bicarbonate buffer, B:
acetonitrile with initial composition of 97.5:2.5 (%A:%B) followed by a 15-minute gradient from 97.5:2.5 to 80:20 (%A:%B) with 5 min hold at 20%B. Run time: 20 min. Sample
diluent: TE buffer. Peak identity: 1 = system peak attributed to TE buffer, 2 = double strand siRNA, 3 = single strand siRNA.

water (MilliporeSigma), sodium dodocyl sulfate (SDS), ammonium
bicarbonate, monobasic sodium phosphate, dibasic sodium phos-
phate, phosphoric acid, HPLC grade acetonitrile and isopropyl
alcohol were used to prepare the mobile phases and sample
solutions. siRNA drug substances and formulated LNP drug prod-
uct were provided by Bristol-Myers Squibb Drug Product Science
and Technology Department (New Brunswick, NJ, USA). Tris-EDTA
buffer solution (TE), phosphate buffered saline solution (PBS) and
10% TritonTM X-100 solution were used as received.
2.2. Chromatographic conditions for the determination of siRNA
A Waters Alliance HPLC system equipped with an ultraviolet
detector (Waters Corporation, Milford, MA) was used for all the
studies. Chromatographic data were recorded and processed using
Waters Empower software.
Several chromatographic conditions for siRNA quantitation
were evaluated during the course of method development and
a number of analytical columns were used including Ascen-
tis Express C18 (2.7 ␮m, 15 cm × 4.6 mm i.d., Sigma-Aldrich, St.
Louis, MO), Waters Xselect CSH C18 (3 ␮m, 15 cm × 4.6 mm i.d.;
Waters Corporation, Milford, MA, USA), and Zorbax SCX-300
(5 ␮m, 15 cm × 4.6 mm i.d.; Agilent Technologies, Santa Clara,
CA). The details of these method conditions are discussed in
detail in the Results and Discussion section. The HPLC conditions
were optimized using a Waters Xselect CSH C18 column (5 ␮m,
25 cm × 4.6 mm i.d.) maintained at 30 ◦ C with a mobile phase of
0.1 M ammonium bicarbonate aqueous solution and acetonitrile
(80:20 v/v), a flow rate of 1.0 mL/min, UV detection at 260 nm, an
injection volume of 10 ␮L, and a run time of 5 min.
2.3. Sample preparation
A stock standard solution of siRNA was prepared at a con-
centration of approximately 5 mg/mL in purified water. Working
standards were prepared in TE, PBS and HEPES buffer solu-
tions by diluting the stock siRNA solution to a concentration of
10 ␮g/mL. The LNP sample solutions were prepared by recon-
stituting lyophilized LNP with Milli-Q water and diluting the
reconstituted LNP drug product samples in different buffer dilu-
ents to a target siRNA concentration ranging from 1 to 8 ␮g/mL.
The diluent for the determination of total siRNA content in LNP
drug products is TE buffer with 1% TritonTM X-100 or SDS, in order
to solubilize the lipid components.
2.4. Method validation
The method for siRNA quantitation were validated for speci-
ficity, linearity, accuracy, precision, sensitivity and solution
stability. Details are described in the Results and Discussion section.
 Z. Huang, W.P. Fish / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 253–258 255

Fig. 2. Example chromatograms of a solution containing 0.1 mg/mL siRNA using a Waters CSH C18 (3.5 ␮m particle size) 15 cm × 4.6 mm with different mobile phase
composition between 0.1 M ammonium bicarbonate and acetonitrile: (A) 95:5 v/v, (B) 90:10 v/v and (C) 80:20 v/v. Column temperature: 30 ◦ C; flow rate: 1.0 ml min−1 ;
injection volume: 10 ␮l; UV detection: 260 nm. Sample diluent: TE buffer. Peak identity: 1 = siRNA, 2 = system peak attributed to TE buffer.

3. Results and discussion
3.1. Evaluation of gradient RPLC method
The analyte used for evaluation is an siRNA substance with
approximate molecular masses of 13,000 and 6500 g/mole for
double and single strand, respectively. It also contains numer-
ous RNA variants with similar molecular masses. The retention
behavior of oligonucleotide analytes in RPLC is different from that
of small-molecule analytes with relatively low molecular mass
(MW < 2000 g/mole). Partitioning of the analyte between station-
ary and mobile phases is believed to play an important role in
retaining small-molecule analytes [7,8]. Oligonucleotides behave
more like polymeric analytes due to their high molecular mass
and their “retention” in gradient RPLC is governed through the
contributions of multiple effects. In RPLC, polymeric analytes,
such as oligonucleotides, tend to be adsorbed and precipitated on
the immobilized hydrophobic ligands on the stationary phase. As
the organic strength (e.g. acetonitrile level) in the mobile phase
increases, they are desorbed and re-dissolved. The threshold of
mobile phase strength to desorb/re-dissolve polymers primarily
depends on the hydrophobicity of analytes. Once desorbed/re-
dissolved, the polymer homologues with different molecular mass
are excluded from the column at different elution times in a gra-
dient elution [9–11]. Fig. 1 shows chromatograms of a solution
containing 0.1 mg/mL siRNA using gradient elution varying the ace-
tonitrile fraction in mobile phase from 5 to 20% for 15 min. siRNA
eluted as double and single strand peaks at 5.0 and 7.3 min, respec-
tively. Using a column temperature at 60 ◦ C, significant changes of
the peak area ratio between the double and single strand siRNA
peaks was observed, indicating denaturing of the double strand
siRNA (Fig. 1a). This method can quantitate both double and single
strand siRNA with adequate precision and accuracy, however, its
ability to accurately quantitate total siRNA content, which consists
of double and single stranded siRNA and all the variants, has several
drawbacks especially when very diluted samples are analyzed: 1).
since siRNA and its variants are exhibited as multiple peaks in the
chromatogram, the sensitivity of each individual peak is reduced;
2). the sum of area responses of all the siRNA and variants peaks
needs to be calculated and any changes to the peak area ratio may
impact the accuracy of total siRNA determination because peak area
increases with increasing retention times; 3). The run time of the
gradient RPLC method is relatively lengthy (>15 min) for single ana-
lyte quantitation. Therefore, the analytical approach using gradient
elution was not preferred for our particular task to quantitate total
siRNA content.
3.2. Development of isocratic HPLC methods
The method development strategy for isocratic elution is to
exclude all siRNA peaks prior to the solvent front as a single peak
using the polymeric characteristics of oligonucleotides. A similar
analytical approach has been used successfully for quantitation of
a polymeric impurity in asunaprevir drug product by our research
team [12]. The isocratic conditions were evaluated with varying
acetonitrile level in the mobile phase on several reversed phase
columns. As shown in Fig. 2, a significant amount of siRNA was
re-dissolved and excluded from the C18 column when the ace-
tonitrile level in the mobile phase reached 10% v/v. Fig. 3 shows
a plot of siRNA peak area responses versus the percent acetoni-
trile levels in the mobile phase under isocratic conditions. For
RPLC, the total area response of siRNA increased with increasing
percent acetonitrile in the mobile phase and reached a plateau
when mobile phase with 15% acetonitrile is used, indicating that
all the variants of siRNA were re-dissolved and excluded from
the column. This analytical approach enables the separation of
256 Z. Huang, W.P. Fish / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 253–258

As shown in Fig. 4, separation between siRNA and the solvent front

Fig. 3. Plot of peak area response of siRNA versus acetonitrile percent of mobile
phase on a Waters C18 (3.5 ␮m particle size) 15 cm × 4.6 mm column.
siRNA from all other non-polymeric components by leveraging the
size-exclusion effect of porous stationary phase particles. The res-
olutions between the siRNA peak and solvent front peaks were
examined using C18 columns packed with particles of different
types and pore sizes including partially porous (Ascentis Express
C18, fused-core, 2.7 ␮m particle size and 90 Å pore size) and fully
porous (XSelect CSH C18, 3.5 ␮m particle size and 130 Å pore size;
Aquity CSH C18, 1.7 ␮m particle size and 90 Å pore size) substracts.
peak depends on the pore size, not the particle size, of the substrate
stationary phase particle. Therefore, Xselect CSH C18 column was
selected for siRNA quantitation due to the favorable porous prop-
erty of its packing materials. The optimized RPLC conditions for the
determination of total siRNA are described in the Experimental sec-
tion above. Mobile phase containing 20% acetonitrile was chosen
based on the data shown in Fig. 3 to ensure all the siRNA variants
are eluted prior to the solvent front and a longer length Xselect CSH
column (25 cm) was selected to increase the separation.
The elution behavior of siRNA was also studied on a strong cation
exchange column to examine the exclusion effect from both ion
and size. All the siRNA was eluted before solvent front using a Zor-
bax SCX column with a 100% aqueous buffer because the negatively
charged ligands on the stationary phase excluded the siRNA (a weak
acid) in addition to the size exclusion effect. When analyzing siRNA
in samples containing non-ionic surfactants such as TritonTM X100,
RPLC method is less advantageous due to potential interference
resulting from the strong retention and UV absorption of Triton. The
conditions using Zorbax SCX columns is a viable alternative for ana-
lyzing such samples because Triton is less retained on ion exchange
stationary phases. An example chromatogram for LNP sample solu-
tions with Triton using SCX column were shown in Fig. 5a. The
Triton peak eluted at 4 min and the baseline was re-equilibrated to
the initial level at approximately 6 min due to a late eluting system
peak attributed to the TE buffer resulting in a final run was time

Fig. 4. Example chromatograms of siRNA sample solutions in TE buffer using 15 cm × 4.6 mm columns packed with different particles: (A) Ascentis Express C18 fused-core
particles (2.7 ␮m particle size, 90 Å pore size, partially porous), (B) Xselect CSH C18 fully porous particles (3.5 ␮m particle size, 130 Å pore size) and (C) Acquity UPLC CSH C18
fully porous particles (1.7 ␮m particle size, 130 Å pore size) in isocratic elution. Column temperature 30 ◦ C; flow rate: 1.0 ml min−1 ; injection volume: 10 ␮l; UV detection:
260 nm; mobile phase: 0.1 M ammonium bicarbonate/acetonitrile (80:20 v/v). Peak identity: 1 = siRNA, 2 = system peak attributed to TE buffer.
 Z. Huang, W.P. Fish / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 253–258 257

Fig. 5. (A) An example chromatogram of an LNP sample solution in TE buffer containing 1.2 ␮g/mL siRNA with 1% Triton using the IEC condition. (B) An example chromatogram
of an LNP sample solution containing 4 ␮g/mL siRNA with 1% SDS using the RPLC condition. RPLC conditions are described in the Experimental Section. Conditions using 15 cm
x 4.6 mm Zorbax SCX-300 column are column temperature 30 ◦ C; flow rate: 1.0 ml min−1 ; injection volume: 10 ␮l; UV detection: 260 nm; mobile phase: 0.1 M ammonium
bicarbonate/acetonitrile (70:30 v/v). Peak identity: 1 = siRNA, 2 = system peak attributed to TE buffer, 3 = Triton.

Table 1
Results for Validation and Sample Analysis.

Linear Regression

Column Type Range Correlation Coefficient

Reversed Phase 0.05 – 15 ␮g/mL 0.9995

SCX Phase 0.05 – 15 ␮g/mL 0.9998

Accuracy

% Recovery vs. siRNA Amount Expected in Sample Solution

Concentration of siRNA in Spiked Sample Solution (␮g/mL)
Reversed Phase SCX Phase
1.0 97.2 98.4
5.0 99.3 100.4
10.0 99.2 99.3

Repeatability of 6 Sample Preps for Total siRNA Determination of LNP Drug Product

%RSD (6) 1.1

Sensitivity - Injection Repeatability of 0.025 ␮g/mL siRNA in the Presence of LNP

Column Type Area (6) % RSD (6)

Reversed Phase 487 2.8

SCX Phase 543 1.9

Results for siRNA Amount in LNP Drug Product Sample Solutions in Different Diluents

Sample Type Sample Diluent Theoretical Conc. (mg/mL) Conc. Found (mg/mL) Column Used

Reconstituted Solution TE buffer/1% Triton 3.0 2.982 SCX

Frozen Solution TE buffer/1% Triton 2.0 1.984 SCX
Reconstituted Solution TE buffer/1% SDS 3.0 2.976 Reversed Phase
Frozen Solution TE buffer/1% SDS 2.0 2.014 Reversed Phase
258 Z. Huang, W.P. Fish / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 253–258
of 7 min. Fig. 5b shows an example chromatogram of LNP sample
solution without TritonTM X100 using the optimized RPLC condi-
tions and all the system peaks were eluted within 3.5 min, reducing
the run time to 4 min. The resolution between siRNA and the clos-
est peaks is larger than 2.0 in both chromatograms shown in Fig. 5
which is sufficient for accurate quantitation.
3.3. Method validation and applications
Several key parameters including specificity, linear, accuracy,
precision, sensitivity and solution stability were evaluated for both
method conditions. Specificity was demonstrated by the absence of
a siRNA peak in the sample chromatograms of a placebo LNP solu-
tion. Moreover, apart from the siRNA peak, no other peaks in the
sample chromatograms exhibited absorbance at 260 nm (typical
maximum absorbance of oligonucleotides) which further verified
method specificity. The method sensitivity of 0.025 ␮g/mL was
obtained in the presence of LNP for both methods. The results of the
total siRNA content determination of various samples in different
sample diluents demonstrated adequate accuracy and robustness
for these two techniques. The validation results for all parameters
are consistent with ICH validation criteria [13] as shown in Table 1,
indicating the methods have acceptable analytical performance.
With the short run time, chromatographic simplicity and ability
to handle different sample diluents, these methods offer increased
laboratory efficiency when supporting LNP process development
and scale up efforts compared to traditional fluorescent based
assays.
4. Conclusions
Analytical approaches to determine siRNA content in LNP drug
product samples have been studied. Two user-friendly and iso-
cratic HPLC conditions with short run times were developed using
reversed phase and cation exchange columns with a mobile phase
containing 20–30% acetonitrile. The siRNA variants were excluded
from pores in stationary phase and eluted as a single peak before
solvent front. It was observed that the separation of siRNA peak
from other components was impacted by the pore size of substract
particles rather than the type and particle size of stationary phases.
These two HPLC methods can quantitate siRNA amount in LNP sam-
ples with adequate accuracy, sensitivity and precision, and reduce
the testing time significantly.
Acknowledgement
The authors gratefully acknowledge the valuable insights pro-
vided by Sherif Badawy and Rao Mantri of Bristol Myers Squibb Co.
in their review of this article.
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