Review highlights state-of-the-art RPLC methods for therapeutic oligonucleotide and peptide-conjugate analysis
Background
Therapeutic oligonucleotides (ONs) are a rapidly growing drug class, with over 25 approvals in the last decade. Accurate characterization, especially for impurity profiling and novel conjugates like peptide-ONs, is critical for drug development and quality control. Traditional methods like Ion-Pair Reversed-Phase Liquid Chromatography coupled with Mass Spectrometry (IP-RPLC-MS) are widely used but face challenges with complex structures and
Study Design
This review critically examined the state-of-the-art developments in Ion-Pair Reversed-Phase Liquid Chromatography coupled with Mass Spectrometry (IP-RPLC-MS) for therapeutic oligonucleotide (ON) analysis. It focused on selecting optimal chromatographic conditions for three key applications: impurity profiling of single-stranded ONs, characterization of novel conjugates (e.g., lipid- and peptide-conjugated ONs), and double-stranded siRNA analysis under non-denaturing conditions. Finally, recent advancements in ion-pair-free RPLC were discussed for pharmaceutical and bioanalytical applications.
Results
The review highlights significant advancements in IP-RPLC-MS for therapeutic oligonucleotide (ON) characterization, emphasizing optimized conditions for impurity profiling of single-stranded ONs. It details strategies for effectively characterizing novel conjugates, including lipid- and peptide-conjugated ONs, which present unique analytical challenges due to their increased complexity. These methods are crucial for ensuring the purity and structural integrity of these advanced therapeutics. Furthermore, the review discusses specialized approaches for double-stranded siRNA analysis in non-denaturing conditions, crucial for maintaining their structural integrity and biological activity. It also explores the emergence of ion-pair-free RPLC methods, offering greener and potentially more MS-compatible alternatives for pharmaceutical and bioanalytical applications, addressing limitations of traditional IP-RPLC. These developments are vital for the continued progress and quality control of ON-based therapies. > The review underscores the critical role of appropriate ion-pair reagent selection and mobile phase optimization in achieving high-resolution separations for diverse ON structures.
Key Findings
- Advances in IP-RPLC-MS enable improved impurity profiling for single-stranded oligonucleotides.
- Optimized RPLC conditions are critical for characterizing complex lipid- and peptide-conjugated oligonucleotides.
- Specialized RPLC methods facilitate non-denaturing analysis of double-stranded siRNA.
- Ion-pair-free RPLC approaches are emerging as greener alternatives for oligonucleotide analysis.
Why It Matters
Optimized RPLC methods are crucial for advancing peptide-oligonucleotide conjugate (POC) therapeutics, ensuring their purity and structural integrity. For researchers and developers, this review provides a roadmap for selecting appropriate analytical techniques, directly impacting the quality control and regulatory approval of novel ON drugs. Understanding these chromatographic nuances can accelerate the development of more stable and effective peptide-conjugated ONs, potentially leading to safer and more targeted therapies. This directly informs analytical protocols, helping to refine characterization strategies for complex bioconjugates and ensuring the reliability of preclinical and clinical data.
oligonucleotides
rplc
mass-spectrometry
peptide-conjugates
analytical-chemistry
drug-development
