The chemical synthesis of oligonucleotides provides an attractive and versatile way of producing precisely designed drugs with specific structural (and therefore functional) features. These products require detailed characterization, but just as importantly, the nature of the impurities in the sample must also be investigated.
This requirement is clearly defined in regulatory documents from the EMA (1) and FDA (2), both of which invoke ICH Q3 guidelines (3-6). These documents recognize two major classes of impurities that must be considered:
It is important to recognize that impurity investigations are not just a regulatory requirement but should also be performed during manufacturing process development to assess the process itself and make any necessary adjustments based on the impurity profiles obtained.
Product-related impurities are associated with the actual drug product itself and arise due to the nature of the chemical synthesis process. The process inevitably suffers from incomplete reaction cycles and by-product formation, resulting in the presence of minor levels of truncated and elongated species of the product, with larger products naturally more likely to have a larger and wider array of impurities due to the greater number of cycles.
This can lead to a complex and challenging impurity profile with a number of minor components present in the sample that are structurally similar to one another, due to the limited number of different nucleotides used in the product synthesis. Given the structural similarities among these impurities, mass spectrometry (MS) combined with LC separation technologies is a powerful tool for identifying and characterizing them. MS provides high mass accuracy and resolving power, allowing for the detection of mass differences and fragmentation patterns that reveal specific nucleotide loss or additions.
According to the EMA guidelines (1), chemical reaction by-products and cross-reactions can also generate modified chemical structures, such as:
Again, these chemical modifications give rise to mass differences that are readily detectable by mass spectrometry.
One other area of impurity that must be considered (and is recognized in the guidelines) is that of enantiomer formation. For products containing a phosphorothioate (PS) linkage between bases, two isomeric species may be formed. This can occur at each PS linkage, resulting in a mixture of stereoisomers.
Since different stereoisomer species can have different biological properties in terms of binding activity and susceptibility to enzymic degradation, it is important to assess the diastereomeric profile of the sample. This can also be performed using state-of-the-art mass spectrometers fitted with ion mobility systems. These instruments are capable of separating molecular species with the same mass based on the different collisional cross-sectional profiles of the molecules. This is a function of shape and varies for different stereoisomers even though they possess the same mass. The application of this type of mass spectrometry provides a second dimension of chromatographic separation of potential stereoisomers e.g., during an on-line LC-MS analysis.
Aggregates and free single chains (the latter specifically in double-stranded products) also need to be investigated. This can be performed by SEC-QELS-MALS analysis, where the relative abundance of each species can be determined.
Assessment of impurities must also be considered beyond the actual process of manufacture. It is required that degradants and degradation pathways are also investigated (1). As such, the types of mass spectrometry and aggregation studies detailed above need to be repeated during product storage to assess for breakdown products or aggregation over time and develop as full an understanding as possible of the product’s characteristics and behavior.
Process-related impurities are derived from the chemical process itself or from the reagents used in the process. These are also required to be investigated (1, 2).
Areas that are particularly, but not exclusively, noted are residual solvents and heavy metals, for which specific ICH guidelines exist and are referenced by both regulatory agency documents (5, 6). Both types of impurity are amenable to the relevant form of mass spectrometry.
For solvents, headspace GC-MS can be used to detect and quantify any residual organic solvents left in the product.
For heavy metals, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is the method of choice. ICP-MS allows for the identification of a very wide range of metals at very low abundances.
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
Investigations into the impurity profiles of chemically synthesized oligonucleotides provide meaningful data for process development and optimization of manufacturing. Furthermore, clear expectations for the investigations into impurities have been put forward by regulatory authorities.
Meaningful information to fulfil both areas can be obtained using state-of-the-art mass spectrometry techniques that allow both product- and process-related impurities to be studied. Aggregation assessment and investigations into complexing of oligonucleotide chains can be performed by SEC-QELS-MALS. The application of these techniques allows for the generation of detailed impurity profiles from this new and exciting class of drug product.
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