Previous BioPharmaSpec blogs discussed the product and process-related impurities in oligonucleotides and the necessity of their analysis for obtaining a comprehensive understanding of product composition and as well as to obtain feedback for optimizing production processes. These impurities are recognized by both the EMA (1) and FDA in both general and product-specific guidelines (2 and e.g. 3) as requiring assessment, quantitation (if necessary), and control.
While many general concepts related to process-related impurities, as described in ICH Q3A(R2) (4) may be applicable to oligonucleotides, this document explicitly excludes this drug due to the necessity for higher reporting thresholds. Nevertheless, it acknowledges that impurities can arise at any stage of the manufacturing process, whether through the process itself, the chemicals used, or the raw materials. Typical impurity classes include reagents, ligands, catalysts, and elemental impurities, including heavy metals. While this provides a general awareness of likely contaminants, each specific manufacturing process must be carefully evaluated to identify potential impurities. Investigations of these will invoke several analytical techniques.
ICP-MS for Elemental Impurity Detection
Elemental impurities are of particular concern, as highlighted by both the EMA and FDA. Both agencies refer to ICH Q3D (5) for this impurity class. The most sensitive and broadly applicable analytical technique for detecting elemental impurities is ICP-MS (Inductively Coupled Plasma Mass Spectrometry). This method works by atomizing the sample through interaction with plasma in the instrument’s source, with the resulting ions being analyzed mass spectrometrically. ICP-MS can detect a wide range of components in a single analysis. Depending on the matrix, detection levels can be as low as parts per billion (ppb) or lower.
It is also important to note that the EMA guidelines require measuring the counter-ion level in the sample, which can also be performed using ICP-MS (1).
Sources of Residual Solvents
Residual solvents are another category of impurities identified by both the EMA and FDA, and in each case, ICH Q3C is noted as the document to refer to for this area of investigation (6). This class of impurity originates from the production process itself and may be residue from processes such as column washing. Solvents used during production and manufacturing must be assessed in the drug substance and, if necessary, the drug product.
Headspace GC-MS for Residual Solvent Detection
Headspace gas chromatography-mass spectrometry (GC-MS) can be used to test for the presence of these volatile solvents. This procedure involves sampling volatile material from the gas space (headspace) above the heated sample and injecting the sampled gas into a GC-MS system for analysis.
Risk Assessment and Impurity Identification
The range and quantity of impurities present in a product depend on the nature of the manufacturing process. Risk assessment methodologies can be employed to evaluate the potential presence of specific impurities, including genotoxins and the formation of particulate matter. Careful consideration should be given to assessing the process and identifying potential sources of impurities, including raw materials and other components associated with the manufacturing process.
Analytical Strategies for Impurity Detection
Focused analytical methods, such as chromatographic purification coupled with mass spectrometry (e.g., on-line LC-MS or GC-MS), can be employed to investigate these process-related impurities. If impurities have a low ionization potential, making mass spectrometry less feasible as a detection method, other techniques such as evaporative light scattering can be employed.
Methods must be sensitive enough to detect low levels of impurities, and the matrix in which the impurity is being assessed must be carefully considered, as it may adversely affect the level of sensitivity. Multiple reaction monitoring (MRM) can be used to reduce background interference and improve specificity, thereby significantly increasing the level of quantitation and detection. The use of internal standards appropriately matched to the sample allows for accurate quantitation where quantitation is required and possible.
Method Validation for Impurity Assessment
Depending on the nature of impurity and/or whether impurity assessment is part of release testing, the method of impurity evaluation may need to be either qualified or fully validated. In both instances, this can be carried out according to ICH Q2(R2) guidelines (7). A detailed discussion on method qualification and validation strategies for impurity analysis is available in reference (8).
Design effective structural characterization studies for oligonucleotide therapeutics.
Example data from the analysis of unmodified Inotersen and Inotersen.
Process-related impurities are a key consideration in oligonucleotide product development. They can potentially be derived from all sources that contribute to the production process, and steps must be taken to identify, quantitate (if necessary), and ultimately ensure they are reduced to either at or below the maximum permitted or acceptable level. A range of high-sensitivity mass spectrometric techniques can be applied to these investigations, with appropriate method qualification being performed if needed.
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