High-molecular weight impurity screening by size-exclusion chromatography on a reversed-phase column

Area of Science:

• Analytical Chemistry
• Pharmaceutical Analysis
• Polymer Science

Background:

• Monitoring high-molecular weight (MW) impurities during active pharmaceutical ingredient (API) synthesis is critical but challenging.
• Existing methods lack generalized approaches for trace polymeric impurities with diverse physicochemical properties.
• Reversed-phase chromatography (RPC) stationary phases (SPs) in size-exclusion chromatography (SEC) mode offer potential due to efficiency and solvent compatibility.

Purpose of the Study:

• To develop a generalized SEC-based screening method for detecting high-MW impurities in API synthesis.
• To establish screening conditions effective for a wide range of polymer types and physicochemical properties.

Main Methods:

• Developed a size-exclusion chromatography (SEC) approach utilizing a C18 stationary phase (SP).
• Employed seven polymer standards with varied functional groups (hydrophobic, heterocyclic, ionic, hydrophilic) as model impurities.
• Evaluated nine mobile phases, including tetrahydrofuran-based, buffered methanol, and buffered acetonitrile systems.

Main Results:

• The developed SEC system demonstrated excellent linearity (R² > 0.99) from 0.05-1.0% w/w for model impurities.
• Successfully identified two real high-MW impurities: BMT-041910 (polymeric degradation) and poly(phenyl thiirane) (by-product polymerization).
• Achieved a quantitative limit better than 0.1% w/w for both identified real-world impurities.

Conclusions:

• The developed SEC platform provides a robust and generalized method for screening unknown high-MW impurities in API synthesis.
• This approach is applicable to low-abundance polymeric impurities with diverse chemical characteristics.
• The method enhances the safety and quality control of drug substance development.