Pros and Cons of Different Chromatographic Technologies in High-Throughput Bioanalysis of Oligonucleotide Therapeutics

Oligonucleotide therapeutics (ONs), which consist of short synthetic strands of DNA or RNA, possess the ability to modulate gene expression and have emerged as promising medical therapeutics. ONs can be categorized into three distinct types based on their therapeutic mechanism: anti-sense Oligonucleotides (ASOs), small interference RNA (siRNA), and aptamer RNAs. However, the early generations of ONs faced significant challenges, such as issues related to in vivo instability and cellular uptake, which require innovative solutions through chemical modifications and the evolution of delivery systems. The precision and efficiency of bioanalysis are crucial in the development of ON-based therapeutics. However, the incorporation of chemical modifications and complex delivery systems has introduced new challenges in the bioanalysis of ONs. To address these challenges, various chromatographic technologies have been deployed, each possessing unique strengths and limitations. These technologies include reverse-phased liquid chromatography (RP-LC), ion-pairing LC (IP-LC), ion exchange LC (IE-LC), and hydrophilic interaction LC (HILIC). In our laboratory, we have accumulated extensive experience in the development and validation of various bioanalytical methods for different types of ONs. These methods encompass RP-LC-MS/MS, IP-LC-MS/MS, HILIC-LC-MS/MS, IP-LC-High-Resolution MS (IP-LC-HRMS), and IE-LC-Fluorescence Detection (IE-LC-FLD). In this presentation, we systematically evaluate and compare the pros and cons of these techniques for the high-throughput bioanalysis of various ONs including ASO and SiRNA of varying lengths. Our goal is to provide insights into the practical implications of utilizing these technologies, which we will illustrate their utility through multiple real-case studies. These case studies will not only exemplify the strengths and limitations of each chromatographic technology but also offer valuable insights for the high-throughput bioanalysis of ONs.