As previously discussed in this blog series, peptides can be produced through both recombinant and chemical means. The choice depends on several factors, including manufacturing capabilities and cost. If the structural features of a proposed peptide drug cannot be achieved through biological systems, chemical synthesis must be employed. In some instances, chemical modification of a biologically manufactured peptide may also be necessary to generate the final product.
Regardless of the production route, product-related impurities are likely to be present due to the nature of the manufacturing process. The EMA (1) and FDA (2) guidelines on synthetic peptides are clear on the requirement for detailed analysis of product-related impurities. While ICH Q6A (3) was not written specifically for peptides, it is recognized to have applicability to synthetic peptides since it refers to chemical drug substances. Similarly, recombinant peptides must also be investigated for impurities arising from biosynthesis mechanisms.
For synthetic peptides, product-related impurities can be formed in several ways. These relate to the nature of the chemistry used, reaction yields, potential by-product formation, and in-solution effects. Consequently, areas requiring investigation include, but are not limited to:
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Techniques used to assess product-related impurities must be highly sensitive and capable of clearly discriminating between impurities from the product and other impurities. Mass spectrometry (MS) is ideally suited to this, with modern instruments being highly sensitive and equipped with state-of-the-art detection and fragmentation mechanisms. When combined with highly resolving chromatography systems, impurity profiles can be readily generated for low-abundance species. MS can provide both intact and fragmentation data to allow characterization / identification of these impurities.
A large amount of information on product-related impurities is generated during structural characterization of the product itself. For example, intact mass analysis and peptide mapping (for larger peptides) both provide information on the total population of species present in the sample, often down to very low levels. However, analyses specifically dedicated to impurity profiling are still necessary. This can include:
Chromatographic buffer systems are sometimes incompatible with mass spectrometry. In such cases, peaks may be collected and buffer-exchanged before MS analysis or alternatively analyzed using two-dimensional liquid chromatography-mass spectrometry (2D LC-MS).
Despite their common molecular mass, even isomeric impurities can be investigated by mass spectrometry. These may arise, for example, from the misincorporation of a specific stereoisomer at a particular site, such as the presence of an L-amino acid in place of a required D-amino acid intended to achieve a specific pharmacokinetic profile.
Such stereoisomers may be resolved chromatographically on standard LC-MS systems. However, a more refined analysis can be achieved using ion mobility separation in appropriately equipped mass spectrometers, such as BioPharmaSpec’s Waters Cyclic IMS system.
A general approach to screen a peptide chain for D- and L-isomers involves:
It must be noted, however, that the limitations of amino acid hydrolysis (e.g., susceptibility to acid) apply here as well.
Peptides can also be produced by recombinant means, in which case the impurity profile is governed by biological, rather than chemical, processes. Many of the same factors require consideration as with synthetic peptides, including truncated and extended species and side chain chemical modifications. However, stereoisomer-related issues are generally not relevant due to the stereospecific nature of biological production systems. Amino acid misincorporations remain a possible impurity and may require investigation.
Although recombinant peptides do not fall under the remit of ICH Q6B (4), which considers recombinant proteins, the principles concerning product-related impurities should be considered in the same manner. The risks and critical areas in the production process are comparable and should be evaluated accordingly.
Product-related impurities must be thoroughly investigated to achieve a comprehensive understanding of the peptide product’s composition. While some of this analysis naturally occurs during product characterization, specific investigations focused on identifying product-related impurities are essential when these impurities are suspected. Mass spectrometry is a key tool for this, offering high sensitivity and the ability to generate detailed structural information even from low-abundance components.
Determining the relative abundance of impurities is important and, depending on their levels, detailed characterization may be necessary to fully define the product. Additionally, employing orthogonal analytical methods is critical to the analysis of a particular structural aspect. For example, Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) can serve as an orthogonal technique to Size Exclusion Chromatography with Multi-Angle Laser Light Scattering (SEC-MALS), where it is applicable, for aggregation analysis. Similarly, multiple chromatographic techniques may be used to comprehensively investigate product-related impurity profiles.
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