Core Contents and Technical Points for Quality Research of Peptide Active Pharmaceutical Ingredients

Quality research on peptide active pharmaceutical ingredients (APIs) shall follow the principles of comprehensive characterization, precise control, and full-process traceability. Its core contents include impurity control, structural elucidation, stability studies, and assay and purity determination. Meanwhile, it must comply with the latest requirements of regulatory authorities such as NMPA, EMA, and FDA to ensure product quality is controllable, safe, and effective.

1. Impurity Control of Peptide APIs

Peptide APIs have complex impurity sources, mainly including process impurities, degradation impurities, and foreign impurities. Among them, process impurities (racemates, deletion peptides, insertion peptides, and aggregates) and degradation impurities (oxidation products, deamidation products, and hydrolysis products) are the key control targets. Targeted detection and control strategies shall be established based on impurity profiling.

1.1 Main Impurity Types and Control Methods

Racemization impurities (D-amino acid isomers) are the most common chiral impurities in peptide synthesis, caused by the formation of oxazolone intermediates during the activation and coupling of protected amino acids. Amino acids such as His, Cys, and Ser are high-risk components, with conventional racemization rates ranging from 1% to 15%. Control methods include the use of low-racemization coupling reagents (e.g., HATU, ynamide reagents), optimization of base reagents and reaction temperature, and application of novel protecting groups (e.g., DNPBS). Detection is performed by chiral HPLC-MS (Pirkle columns, crown ether columns), with a limit of detection (LOD) of 0.01% to meet ICH Q6A requirements.

Deletion peptides and insertion peptides are impurities caused by incomplete coupling or over-dosing in cyclic SPPS reactions. Control focuses on optimizing coupling conditions, prolonging coupling time, and adopting online end-point monitoring (ninhydrin method, HPLC method). During purification, the elution range is precisely controlled by preparative HPLC to limit such impurities below 0.5%.Aggregate impurities are formed by cross-linking between amino and carboxyl groups of peptide chains, which tend to increase immunogenicity. Control methods include optimizing the purification process, reducing peptide concentration, and adding anti-aggregation reagents (e.g., Arg, Tween-80). Detection is performed by size-exclusion chromatography (SEC-HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Degradation impurities mainly result from oxidation, deamidation, and hydrolysis during production or storage. Among them, Met and Trp are prone to oxidation, while Asn and Gln are prone to deamidation. Control methods include optimizing storage conditions (low temperature, light protection, dry environment), using antioxidants (e.g., vitamin C, EDTA), and controlling pH and temperature during production. Detection is performed by RP-HPLC-MS to accurately identify degradation product structures and set reasonable limits (generally ≤1.0%).

Foreign impurities include residual solvents, heavy metals, and residual reagents (coupling reagents, protecting group reagents), which shall comply with ICH Q3C (residual solvents) and ICH Q3D (elemental impurities). Residual solvents are detected by GC, heavy metals by ICP-MS, and residual reagents by HPLC-MS, with limits complying with relevant pharmacopoeial standards.

1.2 Impurity Profiling and Limit Setting

Impurity profiling shall employ a combination of multiple analytical methods (RP-HPLC, SEC-HPLC, chiral HPLC, LC-MS/MS) to comprehensively identify potential impurities and clarify their sources and structures. Limit setting shall be based on toxicological data and clinical application risks, following the principle of risk control. Generally, the limit for known impurities is ≤0.5%, and for unknown impurities: ≤0.1% (reporting threshold), ≤0.5% (identification threshold), ≤1.0% (qualification threshold), in compliance with the EMA Guideline on Synthetic Peptides issued in 2025.

2. Structural Elucidation: Ensuring Product Identity and Activity

Structural elucidation of peptide APIs shall clarify the primary structure (amino acid sequence, modification sites) and higher-order structure (secondary and tertiary structures) to ensure consistency with the target peptide, which is the foundation of efficacy and safety.

2.1 Primary Structure Elucidation

The core is to confirm the amino acid sequence, peptide bond linkage, and side-chain modification sites (e.g., disulfide bonds, acylation, phosphorylation). Amino acid sequencing uses a combination of Edman degradation and LC-MS/MS. Edman degradation determines the sequence stepwise, while LC-MS/MS resolves the peptide structure via fragment ion peaks; the two methods complement each other to ensure accuracy. Disulfide linkage sites are identified by reduction-alkylation combined with LC-MS/MS, especially for cyclic peptides to confirm cyclization sites and linkage patterns. Side-chain modification sites are characterized by high-resolution mass spectrometry (HRMS) to precisely identify modifying groups and positions.

2.2 Higher-Order Structure Elucidation

The activity of peptides depends on specific spatial conformations (e.g., α-helix, β-sheet, cyclic structures). For innovative peptides and generic drugs, consistency in higher-order structures must be verified. Common methods include circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), and fluorescence spectroscopy. CD rapidly analyzes secondary structure content; FTIR confirms peptide bond configuration; NMR elucidates detailed tertiary structures. For generic drugs, higher-order structure consistency with the reference listed drug must be demonstrated; for innovative drugs, the correlation between higher-order structure and biological activity shall be defined.

3 Assay and Purity Determination: Quantifying Product Quality

3.1 Purity Determination

Purity of peptide APIs is divided into chemical purity and chiral purity. Chemical purity is determined by RP-HPLC (detection wavelength 214 nm), with routine requirements of ≥98% for marketed products and ≥95% for clinical products. Chiral purity is determined by chiral HPLC, requiring D-amino acid isomers ≤0.1%. To ensure accuracy, multiple chromatographic methods (RP-HPLC, SEC-HPLC, IEX-HPLC) are used to cover all types of impurities.

3.2 Assay

Assay refers to the actual mass percentage of the target peptide in the peptide API, excluding non-peptide components (moisture, residual salts, counterions). Common methods include amino acid composition analysis, nitrogen determination, and HPLC external standard method. Amino acid composition analysis hydrolyzes peptides into single amino acids under acidic conditions, followed by derivatization and HPLC detection, serving as the core basis for net peptide content. The HPLC external standard method is widely used in industry and shall undergo full method validation (linearity, precision, accuracy) to ensure R² ≥ 0.99 and RSD ≤ 2.0%.

4.Stability Studies: Ensuring Product Shelf Life

Peptide APIs are structurally sensitive and prone to degradation under temperature, humidity, light, pH, and other factors. Stability studies shall be conducted in accordance with ICH Q1A, including stress testing, accelerated testing, and long-term testing, to clarify stability characteristics and shelf life.

Stress testing (high temperature, high humidity, light) identifies critical factors and determines storage conditions. Accelerated testing (40℃±2℃, RH 75%±5%) is performed for 6 months. Long-term testing (25℃±2℃, RH 60%±5%) is performed for 24 months. Changes in purity, assay, and impurities are monitored, and shelf life is predicted based on accelerated data.Typical storage conditions for peptide APIs are refrigeration at 2–8℃, protected from light, sealed, and freezing is avoided to prevent peptide aggregation. Some products require nitrogen filling to prevent oxidation.

In stability studies, special attention shall be paid to the formation and changes of degradation products. If degradation products exceed limits, production processes and storage conditions shall be optimized. Compatibility stability studies shall also be conducted to evaluate compatibility with excipients and solvents, supporting formulation development.

5. Quality Control System and Compliance Requirements

The quality control system for peptide APIs shall cover the whole process of R&D, production, storage, and transportation, comply with GMP, and establish a complete system of quality standards, test methods, and record traceability.Quality standards shall include description, identification, tests (impurities, moisture, residual solvents, heavy metals, microbial limits), assay, and purity determination. Test methods shall undergo full method validation (precision, accuracy, linearity, LOD, LOQ) to ensure reliable results.

Regulatory requirements:

NMPA: Peptide APIs must obtain GMP certification and submit complete process validation reports, impurity profiling reports, and stability data；FDA: Generic drugs must meet quality standards consistent with the reference listed drug and submit DMF files in eCTD format；EMA: Peptide APIs must comply with the EMA Synthetic Peptide Guideline effective in 2026, specifying impurity classification thresholds and structural elucidation requirements to meet Ph. Eur. standards.