Disulfide mapping and the scrambling problem
A correct mass proves nothing about how the cysteines are paired. What does, and how rarely it is done.
Alternative techniques
Size-exclusion chromatography for aggregates and conjugates
The only routine method that sees an aggregate, and the only sensible way to characterise a polydisperse conjugate.
Reverse-phase chromatography runs in an organic-rich, acidic mobile phase that dissociates most non-covalent aggregates before they reach the detector. It therefore reports the monomer content of a sample that may have been substantially aggregated in the vial. Size-exclusion chromatography, run in a near-physiological aqueous buffer, does not dissociate them and is the method that sees them.
Conditions
| Parameter | Typical setting |
|---|---|
| Column | Silica or polymeric SEC, 3–5 µm, 150–300 Å pore, 300 × 7.8 mm |
| Mobile phase | 50–100 mM phosphate or acetate, pH 6.8–7.4, 150–300 mM NaCl |
| Flow | 0.4–0.8 mL/min, isocratic |
| Detection | UV 280 nm (protein) or 214 nm (peptide); MALS and refractive index where absolute mass is required |
| Calibration | Molecular-weight standards spanning the range; relative rather than absolute unless MALS is used |
The salt is not optional. Without it, charged analytes interact electrostatically with the stationary phase and elute at the wrong volume — usually early, which looks like an aggregate.
What it is for, in this index
- Recombinant compounds. IGF-1 LR3, IGF-1 DES and follistatin-344 are proteins with disulfides. Their monomer fraction, dimer content and higher-order aggregate content are measurable only by SEC, and no reverse-phase purity figure bears on them.
- Conjugates. PEG-MGF's 5 kDa mPEG is polydisperse, so its reverse-phase "main peak" is a 1–2 minute hump. SEC separates the conjugate from free polymer and from free peptide and gives the mono/di/unreacted distribution, which is the meaningful characterisation.
- Aggregation-prone peptides. Glucagon fibrillates within hours; cagrilintide and pramlintide form disulfide-linked dimers; LL-37 self-associates. For these the SEC monomer fraction after reconstitution is more informative than the RP purity of the dry powder.
Normative references
Cited because they are the documents the acceptance criteria above are taken from. The index applies them as written and states every deviation.
- USP <621> — Chromatography, size-exclusion
- Ph. Eur. 2.2.30 — Size-exclusion chromatography
- USP <1052> — Biotechnology-derived articles
- ICH Q6B — Specifications for biotechnological products
Related methods
Peptide content versus purity — the distinction most buyers get wrong
Purity is a ratio of areas. Content is a fraction of mass. A 99 % pure vial can contain 78 % peptide, and the difference is the whole ball game.
Content and mass
Forced degradation and stability-indicating capability
Deliberately damaging a sample to prove the method can see damage — and what the resulting degradant profile says about storage.
Physical tests