Overcoming the Challenges of Metabolite Identification and Profiling for Developing Oligonucleotides

The use of oligonucleotides (i.e., “oligos”) was first proposed in the late 1970s, but has not achieved widespread popularity until recently. Developments in delivery method and target specificity for oligos have led to oligos’ resurgence in popularity. These therapies are particularly of interest because their mechanism of action allows for targeting virtually any nucleic acid sequence including previously “undruggable” disease targets. Therefore, the development of oligo drugs can help to address unmet medical needs.A global data search revealed that 17 oligonucleotide drugs were approved between 1998 and 2023.Additionally, more than 100 Phase 2 or Phase 3 clinical trials are currently underway. However, many unique challenges remain for oligo drugs. The novelty, complexity and evolving regulatory guidance of these therapies can pose greater technical challenges than conventional drugs. In this article, we will focus on how to overcome the challenges of metabolite identification (MetID) and profiling.

What are oligos?

Oligonucleotides are synthetic single or double strands of modified RNA (ribonucleic acid) or DNA (deoxyribonucleic acid). They are less than 100 nucleotides (Nt) and can be used to modulate gene expression by binding to RNA or DNA through the Watson-Crick base pairing principle.

In theory, oligos can target any gene of interest because only the nucleotide sequence of interest along the targeted DNA or RNA needs to be selected. This characteristic makes oligos an exciting therapeutic option within precision medicine.

There are four types of oligos, but all share the properties of being negatively charged and hydrophilic, although they range in molecular weight:

• Antisense oligonucleotides (ASOs)

• Single strand DNA or RNA: 5k-10kDa (~18-30 nucleotides)

• Small interfering RNAs (siRNA)

• Double strand RNA: 14k~17kDa (~20-25 nucleotides for each strand)

• Aptamers

• Single strand DNA or RNA: ~6k-15kDa (~20-100 nucleotides)

• Micro RNAs (miRNA):

• Single strand RNA: 5K-10kDa (~20-25 nucleotides)

With the opportunity for novel and targeted treatment comes unique challenges in delivery, stability and specificity. The action sites for oligos lay in the intracellular space, so they must overcome several biological barriers to reach their pharmacological targets in vivo. These barriers include the following:

• Structural instability

• Easy degradation by nuclease in vivo

• Negatively charged molecular structures

• Difficulty in penetrating cell membranes

• Low escape efficiency from endocytosomes to cytoplasm

• Immunogenicity with the human immune system

Overcome the sample analysis challenges of oligos during MetID

Recent advancements in chemical modification and delivery systems have allowed drug developers to overcome many of the aforementioned challenges of oligos with novel and targeted treatments. Since oligo therapies are intended to alter a biological target, drug developers must be able to accurately determine oligo concentration in biological samples. However, challenges remain for the bioanalysis of oligos.

These characteristics of oligos also present significant challenges for sample analysis. Due to their vulnerable nature, oligos require special assays and handling methods to overcome those challenges in the processes of sample treatment and LC-MS analysis, including the following:

• Readily degraded by nucleases

• Protein and nonspecific binding

• Low recovery of extraction

• Poor chromatographic retention

• Multiple charge states

• Ion suppression

Specific experimental materials, reagents and methods were developed for sample treatment, e.g., liquid-liquid extraction and solid-liquid extraction, which could result in acceptable recovery and stability.

As oligos are highly polar compounds, they are difficult to obtain retention and good peak shape by conventional reversed-phase chromatography. Due to this challenge, ion-pair reversed-phase liquid chromatography (IP-RPLC), hydrophilic interaction chromatography (HILIC), and ion exchange chromatography (IEC) are typically used instead. The most popular method is ion-pair reversed-phase chromatography, which mainly uses triethylamine (TEA), N, N-diisopropylethylamine (DIPEA), triethylamine acetate (TEAA) and hexafluoroisopropanol (HFIP) to form ion-pair reagents.

The study systems of oligos during MetID

It is essential to understand oligo drug metabolism when identifying metabolites that are formed during in vitro and in vivo studies. Metabolites have the potential to be pharmacologically active and could cause off‐target toxicity. Furthermore, the mechanisms of oligos are quite different from conventional metabolic pathways that are described for small-molecule drugs. Identifying the metabolites in early studies may help researchers determine if there is potential for off‐target toxicity, which is important when interpreting the results of preclinical and clinical studies. The tissue distribution of these compounds is also of interest.

Cross-species MetID can help compare metabolic profiles to find if there are unique metabolites to humans in vitro and to support animal species selection for toxicology testing. By comparing the in vitro metabolic data with that from in vivo animals, the correlation of in vitro and in vivo metabolism can be speculated.

Unlike small-molecule drugs, which are mainly metabolized by cytochrome P450-mediated mechanisms, oligos are metabolized mainly by nuclease-mediated cleavage of phosphodiester bonds. Cleavage at the 3-terminus by exonucleases is the major metabolic fate for oligonucleotides, followed by 5-exonuclease and endonuclease cleavage events. Commonly, plasma, S9, and tissue homogenate are used as the in vitro metabolism models to evaluate the metabolism of oligos. Preclinical in vivo MetID studies involved plasma, urine, bile, feces and tissues from animals to evaluate metabolism and clearance pathways in animals, which will facilitate quantitative bioanalysis of any relevant metabolites, including chain-shortened metabolites.

Application of high-resolution mass spectrometry for oligo MetID and profiling

Developments in high-resolution mass spectrometry (HRMS) have contributed to the resurgence of oligo therapy. Oligo metabolites produced by exo- and endo-nuclease activity have been identified using mass spectrometry with accurate mass measurement; however, data interpretation is difficult and time-intensive. This is mainly due to the large number of fragment ions generated from oligos, which can largely be mitigated through improvements and advances in software such as automated spectrum deconvolution and data analysis.

Negative ion ESI-MS has been used for oligo detection and analysis due to the charged phosphodiester backbone. Ion-pairing (IP) reversed-phase LC has been used for the analysis of oligonucleotides, which could overcome the challenge of the high polarity and hydrophilicity that lead to poor retention on conventional reversed-phase chromatography and could be compatible with MS-based techniques. Although ion-pairing reagents lead to ion suppression on mass spectrometry, oligo-dedicated HRMS enables highly sensitive and accurate detection that distinguishes mass difference within 10 ppm, making it possible to identify metabolites of oligos.

HRMS is well-suited to profile and identify oligo metabolites, however, many drug developers do not have the resources and experience to do so in-house. Drug developers should seek out a high-quality laboratory testing partner to assist them with bioanalysis for oligo metabolites.

Figure 1. Platform for whole process of oligonucleotide MetID (siRNA and ASO as example)

A final word on oligonucleotides

Inherent properties related to oligos, such as instability and negative charge, can pose significant technical challenges to develop novel and targeted oligo therapies; those challenges remain for oligo MetID and profiling studies. HRMS enables accurate detection of ion fragments, thereby making it possible to identify oligo metabolites. Drug developers should seek out a laboratory testing partner that has extensive experience with metabolite testing for oligo drugs. The ability to do so could greatly accelerate oligo therapy development.

If you want to learn more details about overcoming the challenges of metabolite identification and profiling for developing oligonucleotides, please read the article now.

Authors：Gengyao Qin,Liqi Shi,Guoxiang Huang,Weiqun Cao

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