CEI-MS-11 — Determination of aggregate content by size-exclusion chromatography
Determination of aggregate content by size-exclusion chromatography
§1Scope
§1.1Scope
Applies to peptides and proteins prone to self-association, and mandatorily to the amylin analogue class, to Fc-fusion and antibody-conjugate products, and to cationic amphipathic peptides. Reverse-phase purity does not detect aggregates and for these classes a reverse-phase result alone is recorded by the Institute as an incomplete characterisation.
§2Principle
Molecules are separated by hydrodynamic volume as they traverse a porous stationary phase under isocratic aqueous conditions. Larger species access less pore volume and elute earlier. Because separation is non-denaturing, non-covalent aggregates present in solution are preserved and detected — which is precisely what a denaturing reverse-phase separation destroys.[1,2]
§3Apparatus and reagents
§3.1Apparatus
- HPLC or UHPLC with an aqueous-compatible pump and a low-dispersion flow path
- Size-exclusion column of pore size appropriate to the molecular-mass range
- Ultraviolet detector at 280 nm where aromatic content permits, otherwise 214 nm
- Multi-angle light-scattering and refractive-index detectors where absolute molar mass is required
§3.2Reagents and reference materials
- Isotonic aqueous mobile phase at physiological pH, typically phosphate-buffered saline or a phosphate–arginine buffer
- Molecular-mass calibration standards spanning the range
- A control sample of known monomer content
References cited on this page
References are numbered in order of first citation in this document. Each superscript in the text links to its entry below.
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. ICH Harmonised Tripartite Guideline 1999;Step 4 version. identifier not held by the Institute
- United States Pharmacopeial Convention. General Chapter ⟨788⟩ Particulate Matter in Injections. United States Pharmacopeia — National Formulary (USP–NF) 2024;USP 2024 Issue 1. identifier not held by the Institute
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