# Therapeutic siRNA quantification in lipid nanoparticles using ion-pair reversed-phase chromatography

Ludivine Ferey, Sandy Al Bardawil, Emilie Dols, Aurore Guédin, Viet-Ha Phan, Philippe Barthélémy, Jeanne Leblond Chain

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Ludivine Ferey, Sandy Al Bardawil, Emilie Dols, Aurore Guédin, Viet-Ha Phan, et al.. Therapeutic siRNA quantification in lipid nanoparticles using ion-pair reversed-phase chromatography. Journal of Pharmaceutical and Biomedical Analysis Open, 2025, 5, pp.100072. 10.1016/j.jpbao.2025.100072. hal-05048019

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# Therapeutic siRNA quantification in lipid nanoparticles using ion-pair reversed-phase chromatography

Ludivine Ferey*, Sandy Al Bardawil, Emilie Dols, Aurore Guédin, Viet-Ha Phan, Philippe Barthélémy, Jeanne Leblond Chain

University of Bordeaux, ARNA Laboratory, INSERM U1212, UMR CNRS 5320, Bordeaux F-33076, France

## ARTICLE INFO

**Keywords:**

- Ion-pair reversed-phase liquid chromatography
- Small interfering RNA
- Lipid nanoparticle
- Melting temperature
- UV-Vis spectroscopy

## ABSTRACT

The clinical development of lipid nanoparticles encapsulating small interfering RNA (siRNA-LNP) requires robust and efficient analytical methods for quality assessment. In this study, we investigated the chromatographic profiles of siRNAs using Ion-Pair Reversed-Phase (IP-RP) HPLC at six different temperatures, including the melting temperature of duplexes, on two RP columns. The column temperature was identified as a critical parameter influencing chromatographic profiles, with characteristic profiles evolving in a temperature-dependent manner, corresponding to the melting temperatures ($T_m$) of the two siRNA duplexes analyzed, across both tested columns. Moreover, the siRNA dissociation observed on the columns was consistent with the melting temperatures of the duplexes measured by UV-Vis spectroscopy under HPLC mobile phase conditions. An IP-RPHPLC method using 100 mM TEAA with ACN and a column temperature of 80 °C, above the $T_m$ of the analyzed duplexes, was successfully applied for the quantitative determination of siRNAs in LNP formulations. A simple, one-step sample preparation was employed by directly adding a neutral surfactant to the LNP samples before injection. This method proved highly accurate, as shown by a 100 % recovery of siRNA immediately following microfluidic formulation. Additionally, the impact of the dialysis purification process on siRNA recovery was assessed, revealing a 30 % loss of siRNA. Importantly, the results obtained from this method were consistent with those from the fluorescence reference method. This method helps improve the quality control of siRNA-LNP therapeutics, thereby enhancing their clinical translation potential and supporting the development of next-generation RNA-based therapies.

## 1. Introduction

The current emergence of therapeutic oligonucleotides, as demonstrated by their growing presence in the market and advanced stages of clinical trials, along with forthcoming regulatory guidelines, necessitates the development of efficient analytical methods for quality control of these drugs [1-4]. Among them, five small interfering RNA (siRNA)-based therapeutics have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA): Patisiran, Givosiran, Lumasiran, Inclisiran, Vutrisiran [5,6].

siRNA are double stranded compounds composed of an antisense strand pharmacologically active, and a sense strand to stabilize the duplex. Once into the cytoplasm of target cells, the duplex is recognized by the RNA-induced silencing complex (RISC) [2]. AGO2 (Argonaute RISC Catalytic Component 2) is a key component of the RISC [7]. It first selects the guide strand of the siRNA duplex, which is complementary to

the target mRNA, while the passenger strand is degraded. Once loaded with the guide strand, AGO2 then directs the RISC to bind to the complementary mRNA. Finally, due to its endonuclease activity, AGO2 enables to cleave the target mRNA at the site of complementarity, resulting in mRNA degradation and subsequent gene silencing.

The recent approvals brought needs of regulatory guidelines, which are continuously published and corrected. EMA recently updated the Guideline on the development and Manufacture of Oligonucleotides [8]. In particular, the identification of the duplex and the single strands should be demonstrated, for instance using denaturing as well as non-denaturing conditions. The determination of the melting temperature is also recommended.

Lipid nanoparticles (LNP) are very broadly described systems for RNA delivery [9,10]. They have moved from the simple goal to improve cell internalization of therapeutic nucleic acids to highly modular delivery platform through specific targeting, controlled and sustained drug

<footnote>* Corresponding author.
E-mail address: ludivine.ferey@u-bordeaux.fr (L. Ferey).</footnote>
release. Indeed, LNPs offer flexibility of composition, ease of surface functionalization, biocompatibility, and biodegradability. Improved analytical methods are highly needed for siRNA quantification in LNP formulations under development [11–16]. Although several methods have been developed for lipid quantification in lipid-based nanoformulations [14,17], the encapsulation of siRNA in LNP raised the need for adapted methods for RNA quantification. Among the most reported methods, fluorescence assays based on specific probes, such as Ribogreen® or SYBR™ Gold dye-binding assay [18], offer the advantage to be simple and cheap, but come with high variability [15]. Indeed, the complex nature of the matrix containing many lipids of different types and detergent affects the fluorescence measurement [15]. That is why, more accurate and robust orthogonal separation techniques should be encouraged for siRNA quantification in LNPs, such as HPLC hyphenated with UV or MS.

As opposed to MS-based analysis that requires costly instrumentation and qualified personnel [19–22], HPLC-UV offers a rapid and accessible technique to analyze RNA from lipid nanoparticles [2,11–13]. Especially, the ion-pair reversed-phase HPLC mode (IP-RPHPLC) appears to be the method of choice for siRNA [4, 11–16, 23–26] but also for larger mRNA analysis [27]. In IP-RPHPLC chromatographic mode, the retention of highly polar nucleic acids is achieved through ion pairing with hydrophobic counterions, such as alkylammoniums. The retention and resolution of oligonucleotides are influenced by several factors, including the type of ion-pairing agent, the nature of the stationary and mobile phases, and the structural characteristics of the oligonucleotides. The primary driving force for oligonucleotide separation is the difference in their length/charge. However, additional factors, such as sequence composition, nucleotide chemical modifications, and the presence of hydrophobic labels, also play crucial roles in the separation process [25].

Today, the complexity of siRNA-LNP formulations involving multiple lipid components remains a challenge to be overcome [15,16]. Indeed, LNP are typically composed of cationic lipids, cholesterol, phospholipids, and PEG-conjugated lipids, in decreasing order of quantity. We reported previously the development and the validation of a HPLC-DAD/ELSD method for the simultaneous quantification of 7 lipids involved in LNPs [17].

Although siRNA quantification studies in LNPs have become more common, many still lack quantification data or fail to include rigorous accuracy assessments, such as evaluating siRNA recovery at various stages of LNP formulation for quality control, or comparing results with those obtained using orthogonal techniques [11,15,16]. Worryingly, several studies proceed to *in-vitro* and *in-vivo* experiments without providing any quantification data, further highlighting the lack of reliable analytical methods in the field [15,28–31]. Furthermore, the extraction step is typically labor-intensive and time-consuming, and it may introduce biases in quantification, as the efficiency of siRNA recovery can vary based on the extraction conditions [12,15,16]. Such variability can lead to inconsistent results, making it difficult to achieve accurate and reproducible measurements of siRNA content in LNP formulations.

These challenges underscore the need for simpler and more reliable analytical methods that are essential to facilitate and accelerate the clinical translation process of new advanced formulations [16].

We propose herein, a simple and accurate method for analysing siRNA content in LNPs using IP-RPHPLC. Total siRNA content in lipid nanoparticle formulations was measured just after the microfluidic formulation and after the purification process by dialysis and results were compared to those obtained using the orthogonal fluorescence reference method.

A quick and easy-to-implement sample preparation was used, involving a single-step disruption of the nanoparticles through the addition of a neutral surfactant. This work aims to enhance the bank of analytical methods for RNA quality control, thereby supporting the development of reliable and effective RNA-based therapeutics.

## 2. Experimental

### 2.1. Chemicals

All chemical and solvents used were of analytical grade or of HPLC quality. Triethylamine (TEA) ≥ 99.5 % was purchased for Sigma-Aldrich (Saint-Quentin Fallavier, France). Acetonitrile (ACN) for HPLC was provided by VWR (Fontenay-sous-Bois, France). A MilliQ RG water purification system (Millipore, Bedford, MA, USA) was used to provide ultrapure water. SYBR™ Gold 4X was purchased from Invitrogen (ThermoFisher, Bordeaux, France).

### 2.2. siRNA structures, synthesis, and purification

Two types of siRNAs were determined in lipid nanoparticles. siRNA against green fluorescent protein (siRNA-GFP) was used as a siRNA model widely reported for the development of lipid nanoparticle formulations, as well as a control siRNA sequence (siRNA-CTR), targeting no mRNA from the whole genome [32]. siRNA-GFP duplex was made of 23 base pairs with the sense strand (5'-(AGC AGC ACG ACU UCU UCA AGU CC)3') and the antisense strand (5'-(ACU UGA AGA AGU CGU GCU GCU UC)3'). siRNA-control duplex (siRNA-CTR,) 21 nucleotides in length, was composed of the sense strand (5'-(UAG CGA CUA AAC ACA UCA AUU)3') and the antisense strand (5'-(AAU UGA UGU GUU UAG UCG CUA)3') [33].

siRNAs, sense and antisense strands were synthesized using the conventional phosphoramidite chemistry using an automated H8 synthesizer (K&A Laborgeraete, Germany) at a 1 µmol scale. After synthesis, each sequence was cleaved from support by incubation for 4 h at 55 °C in 1 mL EtOH/NH₄OH 1:3 v/v, then centrifuged and rinsed three times with EtOH/CH₃CN/H₂O 3:1:1 v/v. Deprotection was carried out with 1h30 incubation at 65 °C with a solution of N-methylpyrrolidinone/triethylamine/Triethylamine trihydrofluoride (TEA.3HF) 15.0/7.5/10.0 v/v. After cooling, butanol was added to precipitate the oligonucleotide, washed and concentrated to dryness. RNA samples were purified using polyacrylamide gel (PAGE purification, 20 %, 18 Watts, 2 h) and recovered from the gel slice by electroelution (10 Watts, 25 min). After precipitation in NaOAc pH5.3/EtOH overnight, the RNA sequences were dried, resuspended in water and quantified by DeNovix DS-11 spectrophotometer (DeNovix, USA). For siRNA duplexes, once each individual strand synthesized and purified, the two strands of a duplex were mixed in equimolar ratio, hybridized at 90 °C during 5 min, and allowed to cool to room temperature.

For identity confirmation, all ON samples were characterized by MALDI-ToF mass spectrometry.

### 2.3. Microfluidic formulation of siRNA-LNPs and recovery assessment using the SYBR™ Gold assay

Ionizable switchable LNPs encapsulating siRNAs were prepared by microfluidic mixing. The ionizable lipid CSL3 [32], a helper lipid (1, 2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and the lipopoly(ethyl oxazoline) (PolyEtOx), as an alternative polymer to address allergic reactions attributed to PEGylated lipids [17] were mixed in molar ratio of 50.0:10.0:37.5:2.5, respectively in ethanol. Lipid nanoparticles were prepared by microfluidic mixing using a Dolomite system (Mitos P-pumps basic 30 mL, Mitos Flow Rate Sensor, connected to a 3D Flow Focusing 100 µm hydrophilic, Dolomite, Serlabo, France). Lipids in ethanol and siRNA in 1X PBS were injected into the chip under specific flow and concentration conditions. The total flow rate was 100 µL/min and a flow rate ratio of 3:1 PBS/ethanol was applied. The final concentration of siRNA was 0.52 µM and total lipids was 0.175 mM.

The lipid/nucleotide (N/P) ratio was 4, corresponding to the molar ratio of the amine group within the ionizable lipid to the phosphate groups of the siRNA backbone. At this ratio, ionizable lipids are in excess as compared to negative charges of siRNA.
The resulting suspension was dialyzed (D-Tube Dialyzer, Midi, MWCO 12–14 kDa, Sigma Aldrich, France) against PBS1X buffer pH 7.4 overnight at RT and stored at 4 °C. The dialysis step removes ethanol and free lipids from the formulations as well as siRNAs not involved in liposomes. After dialysis, the sample volume increased slightly within the dialysis cassette. This increase was recorded and taken into account in the calculation to determine siRNA recovery.

siRNA recoveries were assessed by the SYBR™ Gold assay. Briefly, this dye emits fluorescence when in close contact to nucleic acids, allowing for the quantification of accessible siRNA in the solution against a calibration curve. To ensure the detection of all siRNA in the sample, LNP were broken down using Triton 10%. Briefly, 50 µL SYBR™ Gold 4X was added to 100 µL of LNP (diluted 15/100 in PBS 1X) or siRNA (5–125 nM in PBS, for the calibration curve). Fluorescence (exc/em 495/537 nm) was read 1 h after the addition of 10 µL Triton 10% v/v in water. The fluorescence intensity was measured using TECAN infinite M1000 PRO (Tecan, Switzerland). 4 measurements were performed per well and samples were prepared in triplicate. The concentration of siRNA was determined against the calibration curve and the recovery (%) was determined by the ratio of the siRNA against the initial feeding amount (0.52 µM).

## 2.4. LC instrumentation and IP-RPHPLC conditions

siRNA-LNP analyses in IP-RPHPLC were performed using an HPLC Ultimate 3000 system from Dionex-Thermo Scientific (USA), composed of a quaternary pump (LPG-3400 SD), a thermostated autosampler (WPS-3000), a thermostated column compartment (TCC-300SD) and a Diode Array Detector (DAD-3000).

siRNA chromatographic analysis was performed on two different columns: a sub-2 µm Acquity BEH C18 column (50 \* 2.1 mm, 1.7 µm) from Waters and a DNAPac RP column (50 \* 2.1 mm, 4 µm) from Thermo Electron SAS. The Acquity BEH C18 column is a silica-based particle column grafted with C18, while the DNAPac column consists of a large-pore, intrinsically hydrophobic polymer resin with 4 µm polymer particles, specifically designed for the separation of single- and double-stranded oligonucleotides. Both columns are stable over a wide range of pH (1–12) and temperature conditions (up to 90 °C). The HPLC method makes use of a gradient elution. Mobile phase A consisted of a 100 mM triethylammonium acetate (TEAA) buffer adjusted to pH 7.0 with acetic acid and mobile phase B was composed of 100 mM TEAA in 50/50 (v/v) water/ACN with the same amount of acetic acid as mobile phase A to prevent an ionic strength gradient. The gradient elution started at 90/10 (v/v) A/B to 100% B in 7 min. The column temperature was varied between 20 °C and 80 °C and set to 80 °C for siRNA determination in LNP samples. The flow rate was 0.25 mL/min and the injection volume was 2 µL. The detection wavelength was 260 nm. The siRNA samples were prepared at a concentration of 5 µM in water.

For siRNA quantitative determination in LNPs, 1 % (v/v) Triton-X-100 was added to the formulations.

Chromeleon 7.2 software allowed HPLC instrument control and data acquisition and processing.

## 2.5. Thermal melting experiments

Experiments to determine the melting temperature ($T_m$) of siRNA-GFP and siRNA-CTR were performed by UV-spectrophotometry (Cary 100, Agilent Technologies) using 10 mm quartz cuvettes (Hellma Analytics, Paris, France). siRNA samples were diluted at 3 µM in various salt conditions: 100 mM TEAA (pH 7), 100 mM TEAA with 25% acetonitrile, supplemented with acetic acid to match the acetic acid concentration of the 100 mM TEAA solution (pH 7); and 20 mM sodium cadmium (NaCaco) buffer pH 6.6, with or without 80 mM NaCl to reach a total salt concentration of 100 mM, similar to the mobile phase. Heating was programmed at 0.2 °C/min from 10 °C to 80 °C then from 80 °C to 10 °C and again from 10 °C to 80 °C (3 ramps) (waiting time 5 min at 10 °C and

0 min at 80 °C). The absorbance was recorded at 260 nm and 345 nm (blank). The normalized absorbance ($Abs_{260}-Abs_{345}$) was plotted as a function of the temperature to obtain the UV absorbance spectra. To determine the $T_m$ values, the first derivative of the melting curve was plotted against temperature and the maximum was determined graphically. The mean of the three ramps was considered as the $T_m \pm SD$ ($n = 3$).

# 3. Results and discussion

## 3.1. Development of an IP-RPHPLC method for siRNA determination in LNPs

Dealing with siRNA analysis using IP-RPHPLC, the temperature of column appears as one of the most critical parameter on peak shape. Indeed, each siRNA duplex has its own melting temperature ($T_m$) depending on its sequence and chemistry. If the column temperature is below $T_m$, then the siRNA is analysed under its native conformation, i.e. a duplex adopting a double-helix structure, strongly associated through hydrogen bonds interaction. In that case, the siRNA duplex elutes as a single peak [13]. Otherwise, if the column temperature exceeds $T_m$, siRNA duplexes are submitted to a fast melting during injection and consequently, each strand elutes as a sharp single-peak [13]. At a column temperature close to siRNA $T_m$, the sample may be a complex mixture of several conformations in equilibrium including the denatured single strands and the intact duplex. For these reasons, chromatographic analysis close to the melting temperature of siRNAs should be avoided. In other words, siRNA analysis should be performed either under native conditions at a temperature sufficiently below $T_m$ or under denaturing conditions at a temperature sufficiently above the $T_m$.

$T_m$ values of these two duplexes were determined by UV-Vis spectroscopy and $T_m$ were determined graphically at the maximum of the first derivatives of these UV melting curves (Fig. 1, Table S1, Fig. S1). To mimic chromatographic conditions, experiments were conducted in 100 mM TEAA and 25% acetonitrile was added to reproduce the increasing percentage of acetonitrile during gradient elution. As illustrated on Fig. 1, the siRNA-GFP duplex was destabilized around 62.6 °C ± 0.3 in 100 mM TEAA and siRNA-CTR at 53.4 °C ± 0.3. The higher $T_m$ for siRNA-GFP is consistent with its longer sequence (23 vs 21 nucleotides). The $T_m$ values decreased in the presence of 25% acetonitrile, reaching 53.0 °C ± 1.9 for siRNA-GFP and 42.1 °C ± 0.5 for siRNA-CTR, demonstrating the destabilization induced by organic solvent, as previously reported [34]. Noteworthy, the values obtained in 100 mM TEAA are lower than $T_m$ values typically reported in literature for siRNA duplexes [34]. Therefore, $T_m$ were measured in 20 mM sodium cadmium, a thermostable buffer with or without 80 mM NaCl to match the salt concentration of mobile phase (100 mM). The $T_m$ of siRNA-GFP was 69.9 °C ± 0.3 and increased to 77.3 °C ± 0.4 in the presence of 100 mM total salt concentration (Fig. S1). Similarly, the $T_m$ of siRNA-CTR was 60.5 °C ± 0.9 and increased to 68.7 °C ± 0.3 with higher salt concentration (Fig. S1), showing that salts stabilized the duplex, as expected [34]. Despite being at the same concentration, the nature of the salt (TEAA vs NaCl) drastically impacted the $T_m$ value of the siRNA duplexes (Table S1). Altogether, these results indicate that $T_m$ values are affected by the siRNA sequences, salt composition, concentration, and the presence of organic solvent.

In our study, we first used an Acquity BEH C18 column with a mobile phase consisting of 100 mM TEAA at pH 7 and gradient elution with ACN, conditions classically described in the literature for siRNA analysis in IP-RPHPLC [12,13].

Fig. 2 and S2 display the overlay of siRNA-GFP and siRNA-CTR duplexes, respectively, at column temperatures of 20 °C, 30 °C, 40 °C, 50 °C, 65 °C, and 80 °C using the same mobile phase conditions. The chromatographic profile of siRNA duplexes, compared to their single-stranded counterparts, allowed for the study of siRNA melting in LC. On-column melting of both siRNA duplexes was observed starting at 40
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Thermal stability of siRNA. UV absorbance of siRNA-GFP (A) or siRNA-CTR (C) normalized by their respective absorbance at 345 nm. First derivatives of normalized absorbances for T<sub>m</sub> quantification of siRNA-GFP (B) and siRNA-CTR (D). [siRNA] = 3 µM, 100 mM triethylammonium acetate with or without 25 % acetonitrile, 10–80 °C, 0.2 °C/min, 3 ramps. T<sub>m</sub> values were determined graphically from each ramp. T<sub>m</sub> value are reported as mean ± SD (n = 3).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Effect of column temperature on the IP-RPHPLC analysis of siRNA-GFP duplex between 20 °C and 80 °C using an Acquity BEH C18 column (50 × 2.1 mm, 1.7 µm). Mobile phase A: 100 mM TEAA in water adjusted to pH 7.0 with acetic acid. Mobile phase B: 100 mM TEAA in 50/50 water/ACN (v/v) with the same concentration of acetic acid as eluent A. Gradient: 90/10 A/B (v/v) to 100 %B in 7 min. Flow rate: 0.25 mL/min. Sample: siRNA at 5 µM in milli-Q water. Injected volume: 2 µL. UV detection at 260 nm. siRNA, sense strand, and antisense strand are represented by solid, dotted, and dash-dotted lines, respectively.</figcaption></figure>

°C, as indicated by the appearance of additional peaks corresponding to the single-stranded forms of siRNA-GFP (Fig. 2) and the distortion of the siRNA-CTR duplex peak, which showed a shoulder at the retention time of the single-stranded RNAs (Fig. S2). These values are consistent with the T<sub>m</sub> values observed with 25 % acetonitrile (Fig. 1), which corresponds to the proportion of acetonitrile in the mobile phase at t<sub>R</sub> = 3 min. The melting of siRNA duplexes persisted up to 65 °C. Indeed, at 50 °C, for siRNA-GFP, two distinct peaks with different areas were observed (Fig. 2), confirming the coexistence of the duplex and single-stranded forms, while for siRNA-CTR, the peak remained distorted, suggesting incomplete melting (Fig. S2). A temperature of 65 °C or higher was required for total melting of both siRNA duplexes upon injection onto the column under studied IP-RPHPLC conditions. This was evidenced by the clear separation of the two single-stranded forms in the siRNA-GFP duplex sample (Fig. 2), along with narrower and more Gaussian peaks for siRNA-CTR (Fig. S2) at 65 °C (Gaussian peak with w<sub>1/2</sub> = 0.086 min), compared to 40 °C (distorted peak with w<sub>1/2</sub> = 0.203 min) and 50 °C (distorted peak with w<sub>1/2</sub> = 0.097 min). It is important to note that the conditions used did not allow for the separation of the sense and antisense strands of siRNA-CTR (Fig. S2). However, complete dissociation of the siRNA-CTR duplex was observed by the emergence of a thin and Gaussian peak during column heating

(Fig. S2).

At 20 °C and 30 °C, below the T<sub>m</sub>, injections of siRNA duplexes produced single peaks, distinct from the antisense and sense single-strand peaks for both siRNAs (Fig. 2 & S2). Below the T<sub>m</sub>, the siRNAs are analyzed under native conditions, maintaining their duplex forms. Notably, in both siRNA samples, a smaller peak was detected at the same retention times as the single strands, indicating the presence of an excess of single strands in the duplex samples (Fig. 2 & S2).

The same study of the chromatographic profile of siRNA duplexes in IP-RPHPLC as a function of column temperature was also performed using the DNAPac column, which is specifically designed for the analysis of large nucleic acid duplexes due to its larger pore size resin. Similar observations were made on this column, with duplex melting observed at 40 °C for both siRNAs (Fig. 3 & S3). For siRNA-GFP, a transient coexistence of the duplex and single strands persisted at 50 °C (Fig. 3). A minimum temperature of 65 °C for siRNA-GFP (Fig. 3) and 50 °C for

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Effect of column temperature on the IP-RPHPLC analysis of siRNA-GFP duplex between 20 °C and 80 °C using a DNAPac RP column (50 × 2.1 mm, 4 µm). Mobile phase A: 100 mM TEAA in water adjusted to pH 7.0 with acetic acid. Mobile phase B: 100 mM TEAA in 50/50 water/ACN (v/v) with the same concentration of acetic acid as eluent A. Gradient: 90/10 A/B (v/v) to 100 %B in 7 min. Flow rate: 0.25 mL/min. Sample: siRNA at 5 µM in milli-Q water. Injected volume: 2 µL. UV detection at 260 nm. siRNA, sense strand, and antisense strand are represented by solid, dotted, and dash-dotted lines, respectively.</figcaption></figure>
siRNA-CTR (Fig. S3) was required to achieve complete fusion of the duplexes into sense and antisense strands, explained by the lower T<sub>m</sub> of siRNA-CTR compared to siRNA-GFP (Fig. 1). In contrast to siRNA-GFP, no separation of the single-stranded species was observed for siRNA-CTR (Fig. S3), as previously noted with the Acquity BEH C18 column. Below the melting temperature, at 20 °C and 30 °C, the duplex peaks appeared again distinct from the single-stranded peaks for both siRNAs (Fig. 3 & S3). Consequently, the siRNA chromatographic profiles appeared comparable between the columns, highlighting the robustness of our method across different column types.

Main differences observed between the two columns were broader peaks and lower resolution on the DNAPac RP column compared to the Acquity BEH C18. Specifically, at 65 °C, the widths at half-peak height (w<sub>1/2</sub>) for siRNA-GFP were around 0.09 min on the Acquity BEH C18 column (Fig. 2), compared to 0.17 min on the DNAPac RP column (Fig. 3). This resulted in less effective separation of the single strands at temperatures above the melting temperature for siRNA-GFP on the DNAPac RP column (Fig. 3). Moreover, at temperatures below the duplex melting temperature, siRNA-GFP peaks were distorted, with significant tailing appearing on DNAPac RP column (Fig. 3).

For these reasons, the Acquity BEH C18 column was selected for siRNA quantification in LNP formulations. Moreover, a column temperature of 80 °C was chosen for quantitative analyses, as it ensured complete dissociation of the duplex strands, resulting in the best chromatographic profiles. In contrast, temperatures below the T<sub>m</sub> showed additional peaks due to the presence of excess single strands and peak distortions, particularly evident for siRNA-GFP.

## 3.2. Application to siRNA assay in LNPs

This method was then applied to the quantitative determination of siRNAs encapsulated in LNP. Such application is of high interest because the literature mainly focus on the determination of the relative quantity of siRNAs in LNP through the determination of encapsulation yield rather than the determination of absolute quantity of siRNAs in LNP [15,18].

For purposes of siRNA quantification, calibration curves of siRNAs were established using the developed IP-RPHPLC method on the range 0.075–0.75 µM, including the theoretical concentration of siRNAs (0.52 µM) in LNP. For both siRNAs, a good linearity of the method was proved by determination coefficients above 0.995.

LNP composed of 50 mol% cationic lipid (CSL3), 37.5 mol% cholesterol, 10 mol% DSPC, 2.5 mol% lipopoly(ethyloxazoline) were then prepared by microfluidic mixing. The targeted concentration of siRNAs in LNP was 0.52 µM. A siRNA-GFP-LNP sample was also analyzed before dialysis to assess the impact of the dialysis process on siRNA recovery in LNP samples. Before HPLC analysis, LNP samples were prepared by adding the neutral detergent Triton™ X-100 at 1 % (v/v) in order to solubilize the lipid components [11,12].

The quantification results obtained from IP-RPHPLC and SYBR™ Gold methods are presented in Table 1.

For siRNA-GFP formulation, the mean recovery of siRNA was 101.7 % before dialysis, indicating complete extraction of siRNA from siRNA-LNP and confirming the accuracy of the developed method for siRNA quantification in LNP.

After dialysis, the recovery decreased to 72.9 % for siRNA-GFP LNP, while siRNA recovery was 71.9 % for siRNA-CTR LNP. These results were consistent with those obtained using the SYBR™ Gold assay. Furthermore, the observed loss during the dialysis step aligned with the 40 % loss of lipids in LNP formulations reported by our group [17] and other studies [35].

The optimized IP-RPHPLC method was thus proven to be reliable for: i) determining the amount of active substance encapsulated in finished LNP products, and ii) assessing RNA losses during the formulation process.

## 4. Conclusion

IP-RPHPLC methods were investigated to assess the impact of column type and temperature on the chromatographic profiles of siRNA duplexes. Column temperature was identified as a critical parameter that governs the strand melting of duplexes on the column, with the effect being dependent on each duplex's specific melting temperature (T<sub>m</sub>). Notably, the impact of column temperature on chromatographic profiles was consistent across the two columns tested. Before conducting quantitative analysis of siRNAs in lipid nanoparticle (LNP) formulations using IP-RPHPLC, it is therefore recommended to perform a temperature study to evaluate the effect of column temperature (both above and below the T<sub>m</sub>) on the chromatographic profiles of siRNA peaks. Additionally, the melting temperature (T<sub>m</sub>) of the duplex under analysis can be determined using an orthogonal method, such as UV spectroscopy, under mobile phase conditions for predictive analysis.

A fast and user-friendly IP-RPHPLC method is proposed for determining siRNA content in LNP samples. The method demonstrated good recovery of siRNA from LNP samples using a simple addition of a non-ionic surfactant to the LNP samples before HPLC injection. The impact of the dialysis process was assessed, revealing a loss of approximately 30 % of siRNA content in the samples. These results were consistent with those obtained using the SYBR™ Gold fluorescence method.

In summary, this study presents an analytical method that can be easily implemented in routine LNP preparation. We believe that the standardized quantification of siRNA during the siRNA-LNP preparation process will significantly enhance their clinical translation.

### CRediT authorship contribution statement

**Sandy Al Bardawil:** Investigation. **Ludivine Ferey:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization. **Jeanne Leblond-Chain:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition. **Philippe Barthélémy:** Writing – review & editing, Supervision, Resources, Project administration. **Viet-Ha Phan:** Investigation. **Aurore Guédin:** Investigation, Formal analysis, Data curation. **Emilie Dols:** Visualization, Validation, Investigation, Formal analysis, Data curation.

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence

<caption>**Table 1**</caption>

Quantitative determination of siRNA concentrations and recoveries in LNP using the developed IP-RPHPLC method, with comparison to the SYBR™ Gold method.

<table><thead><tr><th>LNP sample</th><th>siRNA calculated concentration by HPLC (µM)</th><th>Recovery HPLC (%)</th><th>siRNA calculated concentration by SYBR™ Gold (µM)</th><th>Recovery SYBR™ Gold (%)</th></tr></thead><tbody><tr><td>siRNA-GFP before dialysis</td><td>0.529 ± 0.018</td><td>101.7 ± 3.4</td><td>nd</td><td>nd</td></tr><tr><td>siRNA-GFP after dialysis</td><td>0.379 ± 0.020</td><td>72.9 ± 4.0</td><td>0.375 ± 0.008</td><td>72.1 ± 1.60</td></tr><tr><td>siRNA-CTR after dialysis</td><td>0.374 ± 0.019</td><td>71.9 ± 3.9</td><td>0.375 ± 0.006</td><td>72.1 ± 1.20</td></tr></tbody></table>

nd: not determined.
the work reported in this paper.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jpbao.2025.100072.

## Data availability

No data was used for the research described in the article.

## References

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