Methods & Standards
Methods & Standards
HPLC Identity & Purity
| Parameter | Specification |
|---|---|
| Column type | Reverse-phase C18, 150 × 4.6 mm, 3.5 µm |
| Mobile phase A | 0.1% trifluoroacetic acid in water (v/v) |
| Mobile phase B | 0.1% trifluoroacetic acid in acetonitrile (v/v) |
| Gradient | 5–95% B over 20 min; linear |
| Flow rate | 1.0 mL/min |
| Detection wavelength | 220 nm (primary); 280 nm (aromatic confirmation) |
| Injection volume | 10 µL |
| Column temperature | 40 °C |
| Run time | 30 min |
| Purity threshold | ≥98.0% area (main peak, UV 220 nm) |
| Integration method | Peak area ratio; Empower 3 software |
| System suitability | Tailing factor ≤2.0; theoretical plates ≥2000 |
Mass Spectrometry Confirmation
| Parameter | Specification |
|---|---|
| Instrument type | ESI-MS (electrospray ionization, single quadrupole) |
| Ionization mode | Positive ion; ESI+ |
| Mass range | m/z 100–3000 (full scan); targeted MRM for confirmation |
| Capillary voltage | 3.5 kV |
| Desolvation temperature | 350 °C |
| Cone voltage | 25–40 V (compound-dependent) |
| Molecular ion threshold | Observed [M+H]⁺ or [M+nH]ⁿ⁺ within ±0.5 Da of theoretical MW |
| Charge states evaluated | +1, +2, +3 (peptide-dependent) |
| Testing frequency | Every lot; no exceptions |
| Pass criterion | Observed MW matches theoretical within tolerance; no unlabeled fragments >5% |
Endotoxin Testing (LAL)
| Parameter | Specification |
|---|---|
| Method | Kinetic-chromogenic LAL (limulus amebocyte lysate) |
| Regulatory standard | USP <85> — Bacterial Endotoxins Test |
| Reagent | Lonza PyroGene™ rFC Assay (recombinant Factor C) |
| Standard curve | 0.005–50 EU/mL; R² ≥0.98 required |
| Product threshold | ≤0.50 EU/mg (standard); ≤0.25 EU/mg (high-sensitivity lots); ≤0.10 EU/mg (select compounds) |
| Spike recovery | 50–200% (within-run control per USP <85>) |
| Testing frequency | Every lot release; independent of HPLC result |
| Laboratory | Third-party ISO 17025-accredited laboratory |
| Hold time | Sample tested within 14 days of synthesis completion |
Lot Release Criteria
| Test | Threshold | Pass/Fail Rule |
|---|---|---|
| HPLC purity | ≥98.0% area (UV 220 nm) | Fail if any lot <98.0% |
| MS identity | Observed MW within ±0.5 Da | Fail if MW deviation exceeds tolerance |
| Endotoxin | ≤0.50 EU/mg | Fail if any replicate exceeds limit |
| Water content (Karl Fischer) | ≤6.0% w/w | Fail if moisture >6.0% |
| Appearance | White to off-white lyophilized powder; no visible particles | Fail if colored, clumped, or particulate |
| Net weight (fill verification) | Labeled quantity ±5% | Fail if fill outside tolerance |
| COA issuance | All tests passed | COA not issued until all criteria met; lot quarantined pending release |
All methods documented per ICH Q2(R1) validation guidelines.