EMA Guideline on Synthetic Peptides: Impurity Thresholds, Characterisation and Orthogonal Analysis

1 Key facts

2 Impurities: types and thresholds

• Thresholds: peptide-related impurities are reported above 0.1%, identified above 0.5% and qualified above 1.0%, citing Ph. Eur. 2034; ICH Q3A/B do not apply.
• Co-eluting and unresolved peaks: where several impurities form one peak, the thresholds apply to the combined peak unless otherwise justified.
• Peptide-related impurity types listed: stereoisomers (epimerisation), deletion sequences, truncated (acetylated) sequences, insertion sequences; degradation products such as oxidation, hydrolysis, isomerisation, deamidation, aspartimide, diketopiperazine and pyroglutamic acid; and HMW impurities such as aggregates, dimers and oligomers.
• Non-peptide impurities: process reagents, residual solvents, elemental impurities, mutagenic impurities and nitrosamines.

3 Specifications and characterisation

• HMW impurities: must be specified by type (e.g. dimers, aggregates); a single parameter "high molecular weight impurities" is not acceptable. The method named here is SEC.
• Identification: at least two orthogonal methods are recommended; impurity methods must be studied for stability-indicating properties and mass balance and must reach the 0.1% reporting threshold.
• Characterisation: primary structure by MS, LC-MS/MS, amino acid analysis, NMR and Edman degradation; secondary structure by far-UV CD, FTIR and NMR; tertiary structure by near-UV CD and NMR; aggregation by ThT dye assay and intrinsic tryptophan fluorescence.
• Other: counter-ion content needs a justified upper limit; assay is normally expressed on a counter-ion-free, anhydrous basis; biological assays are generally not required for release and stability.

4 Synthetic peptides referencing a biological product (Section 6)

This is the section with the largest impact on generic peptides. A chemically synthesised peptide cannot follow the biosimilar route; instead it is compared with a reference product sourced from the EU market:

1. Structure: a full analytical panel covering primary and higher-order structure; higher-order methods include CD, FTIR, 2D NMR, fluorescence and DSC.
2. Purity: orthogonal size-, charge- and hydrophobicity-based methods; ThT assay where relevant.
3. Impurities: each peptide-related impurity at 0.1–0.5% is compared with the reference; impurities absent from the reference must not raise immunogenicity concerns and must be qualified above 1.0%.
4. Immunogenicity: in silico T-cell receptor binding prediction or in vitro T-cell activation assays may help.
5. Forced degradation: comparative studies, including shear/mechanical stress.
6. Function: cell-based assays should be used.
7. Stability: shelf life cannot be derived from the reference product.

5 Investigational medicinal products (Section 7)

• Monitor individual impurities in amino acid building blocks from early development; limits based on criticality are expected in Phase II/III.
• Phase I may rely on a prior-knowledge risk assessment for HMW impurities; Phase II/III needs experimental data.
• Identify peptide-related impurities above 1.0% and qualify them in non-clinical studies; use orthogonal and stability-indicating methods early.

6 What it means for analytical work

1. HMW impurities controlled by type

   Section 4.4

   Suggested data package

   Aggregate type and level

   Methods

   SEC quantitation, with a column-free method to confirm aggregate type (AUC is not named in the guideline; this is our recommended orthogonal check)

   Quickgene provides

   SV-AUC aggregation analysis

2. Higher-order structure comparison

   Sections 4.3, 6

   Suggested data package

   Quantitative similarity of secondary and tertiary structure (correlation, RMSD)

   Methods

   Far-/near-UV CD

   Quickgene provides

   Circular dichroism

3. Orthogonal charge-based purity

   Section 6

   Suggested data package

   Charge-variant distribution and apparent pI comparison

   Methods

   icIEF

   Quickgene provides

   icIEF analysis

4. Cell-based functional assays

   Section 6

   Suggested data package

   Receptor activation dose–response and relative potency

   Methods

   Reporter-gene cell assays

   Quickgene provides

   Cell-based potency testing; CHO-GLP-1 reporter cell line

5. Impurity-by-impurity comparison at 0.1–0.5%

   Section 6

   Suggested data package

   Location of known impurities and method specificity

   Methods

   RP-HPLC, LC-MS with impurity reference standards

   Quickgene provides

   Semaglutide, tirzepatide and retatrutide impurity standards

6. Immunogenicity of impurities absent from the reference

   Section 6

   Suggested data package

   Impurity immunogenicity data

   Methods

   In vitro T-cell activation assays with high-purity impurities

   Quickgene provides

   Custom synthesis of high-purity impurities for immunogenicity testing

7 Frequently asked questions

• How does the guideline relate to Ph. Eur. 2034? It adopts the 0.1% / 0.5% / 1.0% thresholds of 2034 and adds peptide-specific expectations on co-eluting peaks, HMW impurities and comparison with biological reference products.
• How does it differ from the FDA? The 2021 FDA guidance covers only five generics referencing rDNA-origin products and focuses on impurity-by-impurity comparison with a 0.5% cap on new impurities; the EMA guideline covers new actives, generics and clinical-trial material. See the regulatory requirements overview for a three-region comparison.

References