The field of oligonucleotide therapeutics is rapidly advancing, with a significant focus on modulating gene expression in patients. Since the discovery of gene silencing by naturally occurring oligodeoxynucleotides in 1978 (ref.1), extensive research has been conducted to introduce chemical modifications to backbone linkages, sugar moieties, and base structures, enhancing oligonucleotides' potential as therapeutic agents (ref.2,3). Currently, over 20 oligonucleotide drugs have received regulatory (FDA and/or EMA) approval, with 19 functioning through RNA interference or antisense mechanisms to modulate gene expression in patients.
Phosphorodiamidate morpholino oligomers (PMOs, morpholino antisense oligonucleotides) are gaining recognition as a vital class of oligonucleotide therapeutics. PMOs differ significantly from native oligonucleotides. They feature a morpholine ring in place of the ribose/deoxyribose sugar, and the phosphodiester linkage is replaced with a phosphorodiamidate bond. This unique structure gives PMOs enhanced properties.
PMOs have a range of improved properties compared to native oligonucleotides, including:
PMOs are increasingly recognized as a vital class of oligonucleotide therapeutics with four FDA-approved PMO-based drugs to date, namely Casimersen, Golodirsen, Eteplirsen (all developed by Sarepta Therapeutics Inc.), and Viltolarsen (NS Pharma). Furthermore, several PMO-based drugs are currently undergoing clinical trials.
The development of PMO drugs requires compliance with regulatory guidelines mandating the assessment of critical quality attributes (CQAs), including purity, sequence, drug-related impurity profiling, and other non-drug-related substances (ref.4,5).
In contrast to phosphate and phosphorothioate-based oligonucleotides, PMO backbones lack negative charges due to their neutral morpholino groups, presenting unique analytical challenges for this class of drug product.
Analytical strategies commonly used for phosphate- and phosphorothioate-based nucleotides are often not directly applicable to PMOs, particularly in oligonucleotide drug sequencing and product-related impurity investigations (ref.6). For instance, one of the most common impurities in chemically synthesized oligonucleotides is the ‘n-1’ drug-related impurity, where a single nucleotide is missing. In most cases there would be several possible sequence positions for the missing nucleotide. However, because of the neutral backbone of PMOs, it is particularly challenging to chromatographically separate the full-length product from this ‘n-1’ drug-related impurity.
Furthermore, exonuclease-based sequencing of the full-length drug and drug-related impurities is not applicable to PMOs due to their resistance to most hydrolytic enzymes.
Mass spectrometry (MS) is commonly used for sequencing phosphate- and phosphorothioate-based oligonucleotides, typically utilizing negative ion mode to exploit their anionic nature, thereby improving detection limits. Sequence information is often obtained using collision-induced dissociation (CID), where fragment masses are used to determine the sequence of the drug and its related impurities.
In contrast, PMOs are usually sequenced using positive ion mode CID MS/MS due to their lack of inherent negative charge. Unfortunately, under these conditions, the predominant fragmentation pathways often lead to base losses rather than the desired backbone fragmentation, yielding minimal sequence information about the nucleotide sequence of these products.
Given these analytical complexities, advanced sequencing technologies are required to achieve precise characterization of PMO therapeutics.
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
To overcome the analytical hurdles associated with PMO sequencing and impurity profiling, BioPharmaSpec employs state-of-the-art SCIEX ZenoTOF 7600 (ref.7) and Waters cyclic IMS (ref. 8) mass spectrometers. These instruments employ electron-based fragmentation techniques such as:
These techniques use an electron beam to induce fragmentation of pseudomolecular ions. Analysis is performed in positive ion mode, ensuring good sensitivity whereas fragment ions originate from the cleavage of the phosphorodiamidate morpholino backbone, providing the sequence information.
The ion mobility function in the Waters cyclic IMS instrument enables the separation of oligonucleotides based on their collision-cross section (CCS) in the gaseous ions (ref. 9). This CCS-based ion mobility provides an additional tool for oligonucleotide analysis, aiding in the separation and identification of full-length products, drug-related impurities, and their positional isomers.
Our oligonucleotide characterization services are designed to specifically to meet the challenges of oligonucleotide analytical development, ensuring compliance with regulatory guidelines and the safety of your biopharmaceutical product.
PMOs represent a significant and growing class of oligonucleotide therapeutics featuring structural modifications in their oligonucleotide sugar and backbone linkages. The absence of negative charge results in unique physiochemical properties, requiring distinct analytical strategies that differ from those applied to phosphate- and phosphorothioate-based oligonucleotides.
BioPharmaSpec employs state-of-the-art mass spectrometry techniques, including electron-based fragmentation and cyclic ion mobility separation, to provide comprehensive sequence information of PMO drugs and their related impurities. These advanced methodologies facilitate in-depth structural characterization of this analytically challenging oligonucleotide therapeutics.
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