Aggregate Analysis by SEC
Size-Exclusion HPLC for Peptide Aggregates
Principle
Size-exclusion chromatography (SEC) separates molecules by hydrodynamic volume. Larger aggregates elute first; monomers elute later. SEC is the primary method for aggregate quantification in peptide products.
Method Parameters
| Parameter | Recommended Value |
| Column | Silica-based diol (200–300 Å pore) |
| Mobile phase | 100 mM phosphate + 200 mM NaCl, pH 7.0 |
| Flow rate | 0.5 mL/min |
| Temperature | 25 °C (ambient) |
| Detection | UV 214, 280 nm |
| Injection | 10–50 µg |
| Run time | 30–40 min |
| Calibration | Molecular weight standards |
Aggregate Classification
| Species | Apparent MW | Elution Order | Concern Level |
| Soluble oligomers | Dimer–hexamer | Before monomer | Medium |
| Sub-visible particles | 0.1–25 µm | Filtered | High |
| Visible particles | >25 µm | Filtered | Critical |
| Monomer | Expected MW | Main peak | — |
| Fragments | <Expected MW | After monomer | Low |
Acceptance Criteria
| Application | Monomer | Aggregate | Fragment |
| Research peptide | ≥95% | ≤3% | ≤2% |
| GMP peptide (parenteral) | ≥98% | ≤2% | ≤1% |
| Peptide therapeutic | ≥99% | ≤1% | ≤0.5% |
🔗 Related: HPLC Analysis | Stability Testing | Impurity Profiling | Peptide Quality Control Guide