Application of Liquid Chromatography-Fluorescence (LC-FL) Detection Methods for DMPK Quantitative Bioanalysis of Oligonucleotides

The differentiation of oligonucleotide targets and mechanisms of action and the diversification of chemical modifications and delivery systems of oligonucleotides pose many challenges to bioanalytical approaches. It is often necessary to select the appropriate bioanalytical platform based on the structure, the delivery system, and the sensitivity required for the assay of oligonucleotide analytes. Quantitative polymerase chain reaction (qPCR) and hybridization-based enzyme-linked immunosorbent assay (ELISA) methods have high sensitivity and low specificity. The liquid chromatography-mass spectrometry (LC-MS) method has high specificity, but the sensitivity is generally only 5–10 ng/mL in biological sample analysis. The hybridization-based liquid chromatography-fluorescence (LC-FL) assay has high sensitivity and specificity; thus, it is widely used for the quantitative analysis of oligonucleotides in preclinical and clinical pharmacokinetic biological samples. This article introduces the principles, advantages, challenges, and solutions of LC-FL methods and shares some insights on the strength of our experience and research cases.

Principle of the Hybridization-Based LC-Fluorescence (LC-FL) Assay Method

The hybridization-based LC-FL combines probe hybridization and an LC-FL assay. Quantitative oligonucleotide bioanalysis using LC-FL consists of three main steps:

1. Binding of fluorescently labeled peptide nucleic acid (PNA) probes to the target oligonucleotides through Watson-Crick base pairing to form fluorescently labeled complexes.

2. Separation of fluorescently labeled complexes from excess PNA probes by ion-exchange LC.

3. Detection of fluorescently labeled complexes using a fluorescence detector.

Figure 1. Scheme of liquid chromatography- fluorescence analytical method.

PNA is a DNA analog with a peptide backbone replacing the phosphate backbone (see Figure 2). Due to the absence of charged phosphate groups, PNA exhibits electrical neutrality and displays high affinity and specificity for target DNA or RNA through complementary hybridization. PNA is very stable in vivo and in vitro and is resistant to nucleases and proteases ¹. Unique physicochemical properties allow PNA to be widely used in nucleic acid sequence determination, genetic testing, and disease diagnosis.

Figure 2. Comparison of peptide nucleic acid (PNA) and DNA structures ¹.

ATTO fluorescent dyes are usually derivatives of coumarins, rhodamine, or oxazine, which have high solubility, and their wave spectrum covers the range from ultraviolet to near-infrared, making them the most full-band fluorescent markers.

Before adding the PNA fluorescent probe, biological samples are digested with proteinase K to remove most of the matrix proteins, and the oligonucleotide analytes are combined with the ATTO-labeled PNA probe to form a fluorescently labeled complex. Next, the excess PNA fluorescent probe is separated from the fluorescently labeled oligonucleotide complex sample using exchange chromatography. The resolution of oligonucleotides and their metabolites and the resolution of oligonucleotides with secondary structures are selectively controlled by adjusting the pH, salt, and solvent of the eluent. After absorbing a certain wavelength, fluorescent dyes can emit light of another wavelength larger than the wavelength of the absorbed light. A fluorescence detector can quantify the emitted light by detecting the energy of the emitted wave.

Advantages of the LC-FL Method

1. High detection sensitivity for preclinical and clinical sample analysis

Similar to the hybridization-based ELISA method, the hybridization-based LC-FL method can easily achieve a limit of detection of 1 ng/mL or lower and can quantify oligonucleotides in plasma or tissue homogenates without an extraction step after sample pre-treatment ². The fluorescence detector has a stable response, high accuracy and precision, and a wider linear range than the ELISA method. Currently approved oligonucleotides, such as Patisiran, are used for pharmacokinetic analysis using the LC-FL method to determine the concentration of Patisiran in preclinical and clinical samples ³.

2. Good selectivity for quantitative analysis of metabolites

The hybridization-based LC-FL method avoids interference by other components in biological samples that are not fluorescently labeled and has good selectivity. In addition to relying on high-performance LC, the hybridization-based LC-FL method enables the quantitative analysis of oligonucleotide metabolites. Some truncated oligonucleotide metabolites (e.g., n-1, n-2) can efficiently hybridize with fluorescently labeled PNA probes and be separated from the oligonucleotides by ion-exchange chromatography.

3. Unaffected by oligonucleotide structural modifications and delivery systems

Compared to the qPCR method, chemical modifications do not affect the detection sensitivity and specificity of the hybridization-based LC-FL method. This method can be used for the detection of N-acetylgalactosamine (GalNAc)-conjugated oligonucleotides, lipid-nanoparticle (LNP)-delivered oligonucleotides, and oligonucleotides from other delivery systems in preclinical and clinical studies of biological samples.

The Challenges of Hybridization-Based LC-Fluorescence Detection Methods

1. Probe design and screening

The main challenge in developing hybridization-based LC-FL methods is the design and screening of probes, which is critical to the sensitivity and specificity of the assay, and the probes need to be progressively optimized and screened during method development. The design and screening of PNA probes can be a critical factor in determining the cost and time consumption of LC-FL methods. The PNA probe must be complementarily paired with the target oligonucleotide, with a sequence length of 12–21 bases. Because PNA/PNA interactions are stronger and have a higher affinity than PNA/RNA interactions, they can affect the formation of fluorescently labeled complexes. Therefore, any self-complementary sequences, such as inverted repeats, hairpins, and palindromic sequences, should be avoided in the probe design process. Purine-rich sequences should also be avoided because the proportion of purines affects the solubility of PNA probes in aqueous solutions. Therefore, the proportion of purines should be limited as much as possible, and purine extensions should be avoided. Solubility enhancers, such as an O linker, E linker, X linker, or amino acids, can also be added to improve the solubility of PNA probes.

2. Quantitative analysis of metabolites

Since the LC-FL method isolates the target oligonucleotides and their truncated metabolites (e.g., n-1, n-2) by ion-exchange chromatography, metabolite standards are required to confirm the retention times of the metabolites. For double-stranded small interfering RNAs (siRNAs), the metabolites of the sense and antisense strands in the siRNA can only be detected separately since the PNA probe can only detect one strand at a time. In addition, each P=S modification introduces a chiral center, making the chromatographic separation of phosphorothioate-modified oligonucleotides and their metabolites more difficult ⁴.

Column temperature and mobile phase conditions are also essential for the sensitivity and specificity of the method. Therefore, it is necessary to specifically optimize each specific probe and/or target oligonucleotide. LC-FL methods are often used to support late-stage preclinical or clinical studies, whereas LC-tandem mass spectrometry (MS/MS) or LC-HRAM-MS assays are more commonly used in drug discovery or early development stages.

Case Study of Quantitative Analysis of GalNAc-siRNA Conjugates Using an LC-FL Method

The hybridization-based LC-FL method has high sensitivity, accuracy, and precision in quantitative assays. We developed a hybridization-based LC-FL method to quantify GalNAc-siRNA conjugates and validated the method for specificity, selectivity, sensitivity, linear range, and accuracy. This method used an ATTO425-labeled PNA fluorescent probe to complementarily hybridize to the antisense strand (AS) in the GalNAc-siRNA duplex to detect the concentration of the antisense strand. The concentration of GalNAc-siRNA was reported based on the concentration of the antisense strand.

1. Calibration curve and quantitative range

Figure 3 demonstrates the quantitative performance using spiked rat plasma samples after sample treatment. The lower limit of quantitation (LLOQ) was as low as 1 ng/mL, with a linear range between 1 and 200 ng/mL.

Figure 3. Representative chromatograms of the lower limit of quantitation (LLOQ; 1 ng/mL) and control blank samples.

Figure 4. Calibration curve of GalNAc-siRNA hybridized with a PNA fluorescent probe in rat plasma (1–200 ng/mL; linear 1/x², R² = 0.99).

2. Accuracy and precision

The intra-batch accuracy of the quality control samples ranged from 87.1% to 119.7%, and the inter-batch accuracy ranged from 97.4 % to 103.2%. The intra-batch precision (% relative standard deviation [RSD]) ranged from 2.1% to 20.0%, and the inter-batch precision (% RSD) ranged from 7.7% to 19.5%. These data met the requirements for accuracy (± 20%) and precision (± 20%) dictated by regulatory agencies.

3. Separation of metabolites

Due to the very limited volume of the biological samples, the simultaneous separation of the oligonucleotides and their antisense chain metabolites (3'n-1 and 3'n-2) was conducted using ion-exchange high-performance LC and fluorescence detection. Based on ensuring detection sensitivity, the separation achieved excellent results. Figure 5 shows the separation of the tested oligonucleotides and their antisense chain metabolites, 3'n-1 and 3'n-2, by ion-exchange high-performance LC.

Figure 5. Separation of GalNAc-siRNA and its metabolites using ion-exchange high-performance liquid chromatography.

Conclusions

The hybridization-based LC-FL method has high sensitivity and high specificity for oligonucleotide detection. It is widely used for the quantitative analysis of oligonucleotides in preclinical and clinical PK biological samples.

WuXi AppTec DMPK Non-GLP Bioanalytical team has comprehensive bioanalytical capabilities for oligonucleotide drugs and has developed and established five bioanalytical platforms, including LC-MS/MS, LC high-resolution mass spectrometry (LC-HRMS), hybridization-based LC-FL, ligand-binding assays (LBAs), and qPCR. These platforms can support the bioanalysis of oligonucleotides from the early drug screening phase to investigational new drug applications. The team provides a diverse range of bioanalytical platforms and has extensive experience in the development of oligonucleotide analytical methods, enabling high-quality in vitro data delivery and the acceleration of the drug development process.

Figure 6. WuXi AppTec DMPK oligonucleotide bioanalytical platforms.

Click here to learn more about the strategies for oligonucleotides, or talk to a WuXi AppTec expert today to get the support you need to achieve your drug development goals.

Authors: Nan Zhao, Hongmei Wang, Siyu Liu, Hefeng Zhang, Lili Xing

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,500+ global clients, and have successfully supported 1,200+ IND applications.