Oligonucleotide drugs, as a new class of biomolecular therapeutics, demonstrate significant potential in gene expression regulation through precise targeting of mRNA. However, the complexity of these drugs presents a series of challenges for preclinical bioanalysis, including demands for detection sensitivity, specificity, and stability. Among these, LC-MS/MS technology is highly favored for its outstanding performance in quantitative analysis and metabolite identification. With continuous advancements in technology, various innovative analytical techniques have emerged, providing richer options for the research and development of oligonucleotide drugs.
Q1. Has the improvement of the HILIC method made it a viable option for the bioanalysis of oligonucleotides? Is the use of ion-pairing agents still the preferred method?
Mimi Wan: Compared to ion-pair reverse phase liquid chromatography (IP RP HPLC), the HILIC method shows somewhat weaker performance in terms of separation selectivity and sensitivity. While there have been numerous attempts to replace ion-pair systems with HILIC methods, there have been few instances of achieving comparable analytical performance. The HILIC method has certain application potential in the bioanalysis of oligonucleotides, particularly in situations where ion-pair reagents are not used. However, ion-pair reverse phase liquid chromatography remains the preferred method due to its superior performance in terms of separation efficiency and sensitivity.
Q2. In this session of the Cloud Lecture Hall, you discussed the impact of pH on the adsorption loss of oligonucleotide drugs. So, why is a high pH mobile phase used in bioanalysis? What are the advantages and limitations of using this high pH mobile phase for analysis?
Mimi Wan: In bioanalysis, using a high pH mobile phase offers several advantages. Firstly, a high pH environment can significantly enhance the stability of oligonucleotides. DNA and RNA are more stable under neutral to alkaline conditions, so high pH conditions help protect the integrity and stability of oligonucleotides. From the perspective of LC-MS analysis, the increased retention of oligonucleotides under high pH conditions contributes to improved separation and resolution. Additionally, high pH mobile phases can reduce non-specific adsorption, thereby enhancing the recovery, accuracy, and reproducibility of the method. However, high pH mobile phases also have some limitations. Specifically, they can affect the stability of column materials, particularly silica-based columns, which may impact the lifespan and maintenance costs of the chromatography columns.
Q3. What are the options for selecting bioanalysis platforms for oligonucleotide drugs?
Mimi Wan: With the increasing structural diversity of oligonucleotides and advancements in sample preparation and analytical techniques, their bioanalysis is continuously evolving. The choice of bioanalytical methods for oligonucleotides is still based on the principle of suitability for the intended purpose and should be evaluated according to specific circumstances. For example, when analyzing small sample volumes, if LC-MS cannot meet the analytical requirements, researchers may consider using HELISA or qPCR as alternative methods. However, if it is known that HELISA or qPCR may be influenced by certain factors, then, conditions permitting, LC-MS may become the preferred choice due to its higher accuracy.
Q4. Is the administration of intrathecal injection in cynomolgus monkeys performed through lumbar puncture? How is cerebrospinal fluid collected, and at what location is it collected? How much experience is there in research involving intrathecal injection of small nucleic acids?
Mimi Wan: In cynomolgus monkeys, intrathecal injection is performed at the L3/L4 or L4/L5 intervertebral spaces through lumbar puncture, with continuous cerebrospinal fluid collection from the cisterna magna. The Medicilon research team has extensive project experience and can perform intrathecal injections in monkeys and rats, providing customized solutions to meet client needs.
If you have any questions or specific topics you would like to explore regarding preclinical research in new drug development, please feel free to leave your comments and suggestions in the comment section. Medicilon hopes to collaborate with you to explore the fascinating world of new drug development.
