Report of the European Medicines Agency Conference on RNA-Based Medicines.

RNA-based medicines have potential to treat a large variety of diseases, and research in the field is very dynamic. Proactively, The European Medicines Agency (EMA) organized a virtual conference on February 2, 2023 to promote the development of RNA-based medicines. The initiative addresses the goal of the EMA Regulatory Science Strategy to 2025 to "catalyse the integration of science and technology in medicines development." The conference focused on RNA technologies (excluding RNA vaccines) and involved different stakeholders, including representatives from academia, industry, regulatory authorities, and patient organizations. The conference comprised presentations and discussion sessions conducted by panels of subject matter experts. In this meeting report, we summarize the presentations and recap the main themes of the panel discussions.

Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair-HPLC-MS.

The relatively large molecular size, diastereoisomeric nature, and complex impurity profiles of therapeutic phosphorothioate oligonucleotides create significant analytical challenges for the quality control laboratory. To overcome the lack of selectivity inherent to traditional chromatographic approaches, an ion pair liquid chromatography-mass spectrometry (LCMS) method combining ultraviolet and mass spectrometry quantification was developed and validated for >35 different oligonucleotide drug substances and products, including several commercialized drugs. The selection of chromatographic and spectrometric conditions, data acquisition and processing, critical aspects of sample and buffer preparation and instrument maintenance, and results from method validation experiments are discussed.

Strategies for Identity Testing of Therapeutic Oligonucleotide Drug Substances and Drug Products.

A risk-based approach for routine identity testing of therapeutic oligonucleotide drug substances and drug products is described. Risk analysis of solid-phase oligonucleotide synthesis indicates that intact mass measurement is a powerful technique for confirming synthesis of the intended oligonucleotide. Further risk assessment suggests that the addition of a second, sequence-sensitive identity test, which relies on a comparison of some property of the sample to a reference standard of proven identity, results in a sufficient test of identity for most oligonucleotide drug substances and products. Alternative strategies for drug product identity testing are presented. The analysis creates a common way to communicate risk and should result in a harmonized approach to identity testing that avoids the unnecessary analytical burden associated with routine de novo sequencing, without compromising quality or patient safety.

Impurity Qualification Toxicology Study for a 2'-O-Methoxyethyl-Modified Antisense Inhibitor in Mice.

Safety assessment of drug impurities is a routine part of the drug development process. For oligonucleotide-based drugs, impurities can arise from impurities in starting materials, as by-products of the manufacturing process or from degradation, and are generally structurally similar to the parent oligonucleotide. To study the potential impact of impurities, a representative batch of a 2'-O-methoxyethyl (MOE) antisense oligonucleotide (ASO) was compared to batches of drug that were enriched with nine of the common impurities encountered with the chemical class. Mice were treated for 3 months with weekly subcutaneous injection of 10 or 30 mg/kg. The impurity content of the parent batch was 0.25%-2.5% of total drug substance. The enriched impurity mixtures contained from 3% to 10% of the various impurities. The expected common class effects were observed at the 30 mg/kg/week dose level in hematology, serum chemistry, and histopathology. However, there were no differences between the representative batch of material and those enriched with impurities. Based on these data, common oligonucleotide impurity studies do not appear to contribute to the overall toxicology profile.

Assay determination by mass spectrometry for oligonucleotide therapeutics.

Phosphorothioate oligonucleotide drugs typically contain product-related impurities that are difficult to resolve chromatographically from the parent oligonucleotide due to the size of these compounds and the large number of stereoisomers that comprise the parent. The presence of co-eluting impurities hinders the process of determining assay based on chromatographic separation alone. A mass spectrometry-based purity assessment of the main chromatography peak can be used to quantify co-eluting impurities and enable the accurate determination of assay, but a more direct measure of assay was desired due to the complexity of measuring all co-eluting impurities by mass spectrometry. Therefore, we developed an assay method that utilizes the specificity of mass spectrometry to measure the amount of active pharmaceutical ingredient in a sample, which eliminates the need for chromatographic separation of impurities from the product. This procedure uses a single quadrupole mass spectrometer and incorporates an internal standard that is co-sprayed with the analyte to compensate for the drift commonly associated with mass spectrometry-based quantitation. Using the mass spectrometry response ratio for sample to internal standard enables the method to achieve excellent linearity (R2 = 0.998), repeatability (relative standard deviation = 0.5%), intermediate precision (0.6%), and accuracy, with measured assay values consistently within 2.0% of expected. The results indicate the method possesses the accuracy and precision required for measuring assay in clinical and commercial stage pharmaceutical products. Since the method is based on the specificity of the mass spectrometer, and does not rely on chromatographic separation of impurities, the procedure should be applicable to a wide variety of oligonucleotide therapeutics regardless of sequence or chemical modifications.

Determination of oligonucleotide deamination by high resolution mass spectrometry.

A novel analytical approach capable of measuring deaminated degradation products of oligonucleotide therapeutics is described. The method employs high-resolution mass spectrometry to assess the shift in isotopic distribution that accompanies deamination. Isotopic Distribution Factors (IDF), derived directly from the peak heights of the isotopic pattern, are employed to measure deamination levels of as little as 0.5%. Results obtained from application of the method to a phosphorothioate diester oligonucleotide exposed to various temperatures were used to determine deamination rates.

Rapid Oligonucleotide Drug Impurity Determination by Direct Spectral Comparison of Ion Pair-Reversed Phase HPLC ESI MS Data.

RATIONALE: Quantitative Ion-Pair (IP)-HPLC MS methods are employed to determine the complex impurity profiles of oligonucleotide therapeutics. While impurities that co-elute with the main product are routinely monitored, the large number of early and late eluting impurities makes their individual measurements tedious and time-consuming. An improved method is needed for routine analyses. METHODS: A Combined Ranking (CR) index is developed to provide a composite value for both qualitative and quantitative impurity changes. Positive and Negative Impurity Change (PIC/NIC) indices are developed to determine the degree and direction of change. Optimized experimental conditions are determined for the characterization of trace levels of impurities. Replicate analysis, blank subtraction, and signal processing approaches are used to enhance the S/N. Dot-product and Euclidean distance equations monitor spectral changes. RESULTS: Spiking experiments with individual and complex impurity mixtures show the method can distinguish among samples differing in impurity content by 0.2% wt. The method has been applied to monitor changes in impurity profile among different batches of the same oligonucleotide and changes in the same batch over time (stability analysis). The method permits rapid determination of changes in the types and amounts (increases and decreases) of impurities present. CONCLUSIONS: A novel approach for the rapid determination of changes in the impurity profile of oligonucleotide therapeutics has been developed. The straightforward data treatment and the speed and simplicity of the approach make the method easy to implement and use. Possible quality control applications include drug substance and drug product stability studies, and the assessment of batch-to-batch variability.

Impurities in Oligonucleotide Drug Substances and Drug Products.

This white paper, which is the 10th in a series intended to address issues associated with the development of therapeutic oligonucleotides, examines the subject of product-related impurities. The authors consider chemistry and safety aspects and advance arguments in favor of platform approaches to impurity identification and qualification. Reporting, identification, and qualification thresholds suitable for product-related impurities of therapeutic oligonucleotides are proposed.

Industrial-scale manufacturing of a possible oligonucleotide cargo CPP-based drug.

This chapter describes the manufacturing process to a certain level for a possible oligonucleotide cargo and a peptide API in a multi-kilogram scale from a manufacture's point of view. In the concluding remarks, possible conjugation methods will be discussed from an industrial-scale perspective.

Characterization of high molecular weight impurities in synthetic phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides manufactured by standard phosphoramidite techniques using 2'-deoxyadenosine- or 2'-O-(2-methoxyethyl)-5-methylcytosine-loaded solid supports contain branched impurities consisting of two chains linked through the exocyclic amino group of the 3'-terminal nucleoside of one chain and the 3'-terminal hydroxyl group of another via a P(O)SH group. These impurities are not produced when a universal, non-nucleoside derivatized support is used.

Formation of modified cytosine residues in the presence of depurinated DNA.

[Chemical reaction: See text] Depurination is an important degradation pathway for antisense phosphorothioate oligonucleotides under conditions of thermal stress. We present evidence showing that depurinated oligonucleotides react with cytosine-containing sequences giving products containing a 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residue. Further, we demonstrate that the same product is formed upon treatment of 2'-deoxycytidine with 4-oxo-2-pentenal, the latter being an expected byproduct of serial elimination reactions at apurinic sites. In addition to being important for synthetic oligonucleotides, apurinic site formation in cellular DNA is a common occurrence. Because repair of these sites can result in the production of 4-oxo-2-pentenal, it is interesting to speculate whether 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residues can form in vivo.

Trichloroacetaldehyde modified oligonucleotides.

Some commercial batches of dichloroacetic acid (DCA) contain traces of chloral (trichloroacetaldehyde). Using such DCA to effect detritylation during solid-phase oligonucleotide synthesis results in the formation of a family of process impurities in which the atoms of chloral (Cl3CCHO) are incorporated between the 5'-oxygen and phosphorus atoms of an internucleotide linkage. The structure was elucidated by HPLC with UV and MS detection, digestion of the oligonucleotide, synthesis of model compounds, and 1H and 31P NMR spectroscopy. By understanding the chemistry behind its formation, we are now able to limit levels of this impurity in synthetic oligonucleotides by limiting chloral in DCA.

Formation of 4,4'-dimethoxytrityl-C-phosphonate oligonucleotides.

Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr-C-phosphonate (DMTr=4,4'-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr-C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr-C-phosphonate moiety located internally or at the 5'terminal hydroxy group. DMTr-C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.

Antisense phosphorothioate oligodeoxyribonucleotide targeted against ICAM-1: synthetic and biological characterization of a process-related impurity formed during oligonucleotide synthesis.

A phosphorothioate-linked oligonucleotide bearing a 3'-terminal phosphorothioate monoester has been synthesized and characterized. This oligonucleotide has been identified as a process-related impurity formed during synthesis of ISIS 2302. Biological properties of the compound have been determined. Based on these data, it can be concluded that this species (3'-TPT) has biological properties similar to parent drug.

Synthesis of high quality phosphorothioate oligonucleotides as antisense drugs. Use of I-linker in the elimination of 3'-terminal phosphorothioate monoesters.

Detritylation of a 5'-O-DMT-2'-deoxyadenosine moiety attached to solid support under acidic condition leads to depurination during oligonucleotide synthesis. Deprotection followed by reversed phase HPLC purification leads to desired oligonucleotide contaminated with significant levels of 3'-terminal phosphorothiaote (3'-TPT) monoester (n-1)-mer. However, it is demonstrated that attachment of dA nucleoside through its exocyclic amino group to solid support leads to substantial reduction of 3'-TPT formation thereby improving the quality of oligonucleotide synthesized.