Oligonucleotide-based therapeutics are revolutionizing the treatment of a variety of diseases, from genetic disorders to cancers. These cutting-edge drugs harness the power of small strands of nucleic acids, carefully designed to target specific genetic sequences in the body.
There are several types of oligonucleotides, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and aptamers, each with a unique mechanism of action. To date, over 20 oligonucleotide-based drugs have been approved by regulatory agencies such as the FDA and EMA, with many more in development, signaling the potential for widespread therapeutic application.
As the field of oligonucleotide therapeutics expands, a key challenge in the development and manufacturing process is the accurate characterization of stereoisomers. These are variations of molecules that share the same chemical formula but differ in the spatial arrangements of their atoms.
This blog will explore:
Despite the immense potential of oligonucleotide drugs, their synthesis is inherently complex. One critical concern, recognized in both the EMA and FDA guidelines, (2, 3), is the presence of stereoisomers. In particular, phosphorothioate (PS)-linked oligonucleotides are prone to forming diastereomers during synthesis and each PS linkage can produce a distinct diastereomer. As a result, the final product may consist of a heterogeneous mixture of species, each with its own biological activity, half-life, and interaction with its target. This variability poses significant challenges for the development of safe and effective therapies.
Stereoisomers present a significant analytical challenge in oligonucleotide characterization. Specialized techniques are required to investigate their isomeric profile.
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
Cyclic IMS Mass Spectrometry (cIMS-MS) enhances the resolution of ion mobility spectrometry by allowing for ion separation through repeated passes using a cyclic ion mobility region. This technique has proven to be particularly useful for resolving:
The technique separates molecular species based on their collisional cross-section (CCS), which is determined by their shape and size. Despite having the same mass, diastereomers will exhibit different CCS values due to their different three-dimensional structures. These differences enable clear separation and identification, something that cannot be achieved using conventional mass spectrometry methods.
Additionally, cIMS-MS provides enhanced resolution, more precise separation, and a second dimension of analysis. When coupled with initial liquid chromatographic separation, it becomes the analytical methodology of choice for characterizing stereoisomeric profiles.
While other techniques such as LC-MS offer valuable insight into oligonucleotide composition, cyclic IMS-MS delivers a superior capability for stereoisomer analysis. Its ability to distinguish components with identical masses based on shape represents a significant leap forward in oligonucleotide characterization.
As oligonucleotide therapeutics continue to gain traction in the pharmaceutical industry, addressing the challenges posed by stereochemistry is essential for regulatory approval and clinical success. BioPharmaSpec’s adoption of cutting-edge technologies, such as Waters Cyclic IMS mass spectrometer, places state-of-the-art instrumentation at the forefront of stereoisomer investigations and marks a major step in meeting these analytical challenges. By enabling precise separation and characterization of oligonucleotide stereoisomers, cyclic IMS-MS enhances our ability to develop safer and more effective therapeutics.
As a thought leader in the oligonucleotide field, BioPharmaSpec continues to pioneer and drive forward innovative solutions, helping ensure that next-generation oligonucleotide therapies reach patients with the highest standards of quality and efficacy.
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