Author: Dr. Richard Easton. Date: 29th July 2026
Peptides, defined by the FDA as amino acid polymers of up to 40 units in length, represent a highly significant class of drug products (1,2). Investigations into the biological activities of peptides have led to the development of a variety of production processes and modifications of the basic peptide chain to produce enhanced therapeutics. A common example is GLP-1 products, although many other peptide-based medicines exist (3,4).
As a result, currently peptides can be produced either recombinantly or, more commonly, by synthetic methods, an approach that is less frequently used for proteins. The synthetic approach provides a relatively straightforward means of introducing chemical modifications, including the incorporation of entities such as acyl chains. In addition, synthetic production allows for the inclusion of non-canonical amino acids or D-forms of naturally occurring amino acids at defined locations, rather than the L-forms of amino acids typically found in proteins (4).
These varied peptide species require comprehensive characterization to demonstrate a clear understanding of both the final product structure and the manufacturing process used to produce it. This includes assessment of the impurity profile. Regulatory authorities recognize these requirements, and the guidelines developed for the analysis of peptide products encompass these critical aspects.
Recently, the EMA’s guidance document on the development and manufacture of synthetic peptides came into effect (5), and the details of this guidance have been described in various summary articles (6,7). The document recognizes the wide range of different procedures currently applied to produce modern peptide drugs, including the use of non-canonical amino acids, specific chiral forms of amino acids, and peptide conjugates, whether intended to enhance activity or to serve as true conjugation strategies in which the peptide acts as the carrier. Therefore, peptides are developed to fulfil two distinct roles: as the active component itself or as a delivery mechanism for another active component.
Regardless of their intended role, the EMA expects comprehensive structural characterization to be performed from the primary sequence level through higher order structure (HOS) assessment, where relevant, alongside aggregation analysis and detailed impurity investigations. This includes assessment of the levels of detected impurities within the product.
The expectations are clearly outlined within the guidelines, and orthogonal methods are emphasized to be used for the assessment of both the peptide product and the purity of the product. This requirement is repeatedly highlighted throughout the guideline, covering both the product and product-related impurities.
For conjugated peptides, a thorough assessment of the products of the conjugation reaction is expected, alongside demonstration that no detrimental effects have occurred elsewhere on the molecule. Additionally, forced degradation studies are described, and the ability of analytical procedures to clearly detect degradation components is discussed.
Analytical methods are therefore expected to be stability-indicating and capable of assessing products of forced degradation. This applies not only to characterization of the peptide product itself but also to additional assessments, including higher order structure and aggregation studies.
BioPharmaSpec offers independent, EMA- and FDA-aligned structural characterization of synthetic and conjugated peptides, using state-of-the-art instrumentation and orthogonal methods to assess primary sequence, higher-order structure, chirality, aggregation, forced degradation, and impurity profiles, building the analytical package that supports a robust regulatory submission.
Synthetic peptide production introduces the possibility of both intended and unintended stereochemical effects, particularly through the incorporation of D-amino acids into the peptide chain. These need to be assessed to confirm that amino acid chirality in the final product is as expected; that is, present only at the specific amino acid locations where they have been deliberately incorporated and are not introduced elsewhere.
The FDA’s current regulatory guidance for peptides, which predates the EMA guidance by few years, naturally shares several of the same expectations for structural evaluation. These include the requirement for detailed structural characterization from the primary sequence level through higher structural levels, and aggregation assessment. The need to use orthogonal methods to analyze the product is also strongly encouraged.
There is no clear consideration regarding forced degradation studies for peptides beyond stress for aggregates. However, impurity profiling remains a key consideration for the FDA, and analytical methods employed must be capable of detecting, identifying, and characterizing impurities, while also providing quantitative assessment of their levels present relative to the drug substance.
Although chirality is not explicitly discussed, for synthetic peptides where this is of a known risk either due to the synthetic chemistry process itself or from specific inclusion of D-amino acids, studies should be performed to assess that any incorporated D-amino acids are correct both in terms of the acid itself and the location in the chain. Additionally, it should be demonstrated that unintended chirality has not been introduced elsewhere in the peptide.
Although peptides are not generally expected to demonstrate significant higher order structure (HOS), both the FDA and EMA require HOS to be assessed and characterized where necessary. This relies on orthogonality to be effective, an expectation highlighted by both regulatory agencies. The EMA explicitly states the requirement to assess tertiary structure as well as secondary structure. While only secondary structure assessment is explicitly mentioned in the FDA guidance, avoiding tertiary structure assessment would be unwise when generating data to submit to the agency.
In summary, the EMA and FDA guidance documents for expected peptide analytical considerations demonstrate both similarities and differences in their approach to handling this product class. The EMA document incorporates some aspects of structural investigation including expectations for forced degradation studies that are not specifically detailed within the FDA guidance, apart from a stress assessment of aggregation. However, the precise attributes defining a molecule to fall into a peptide category are less clearly established within the EMA document. The EMA acknowledges peptides as being at the “interface of small molecules and proteins”, whereas the FDA provides a more useful polymer length definition (2).
Both regulatory authorities place strong emphasis on impurity profiling, with the EMA providing a fuller and clearer definition of impurities to assess. In addition, both agencies invoke ICH guidelines Q3C and Q3D for solvent and elemental impurity considerations, respectively. While both documents provide frameworks for peptide characterization, the EMA guidance appears to provide a more comprehensive approach in terms of analytical consideration, clarity of expectation, and positioning within the broader compliant landscape.
The FDA guidance remains serviceable in terms of its general principles; however, considering its publication date (2021), it may benefit from an update that reflects the current landscape of peptide products, including the current incorporation of all the chemical diversity, rather than its current focus on synthetic versions of peptides previously produced through recombinant approaches.
