Analytical Methods to Qualify and Quantify PEG and PEGylated Biopharmaceuticals

Význam tématu

Polyethylene glycol (PEG) and its covalent attachment to proteins (PEGylation) improve solubility, bioavailability and reduce immunogenicity of biopharmaceuticals. Precise characterization of PEGylated products and residual PEG reagents is crucial for quality control and process optimization in pharmaceutical development.

Cíle a přehled studie / článku

The study aimed to develop and assess an online two-dimensional liquid chromatography (2D LC) system coupled with charged aerosol detection (CAD) for simultaneous qualification and quantification of protein PEGylation extents and remaining unreacted PEG reagents. The approach integrates size-exclusion chromatography and reversed-phase separation in an automated workflow for real-time reaction monitoring.

Použitá metodika a instrumentace

• System: Thermo Scientific Dionex UltiMate 3000 x2 Dual RSLC with dual-gradient pumps, WPS-3000TRS autosampler, TCC-3000RS column compartment and high-pressure valves.
• Detectors: DAD-3000RS (UV 214/280 nm) and Corona Ultra RS charged aerosol detector.
• Software: Chromeleon 7.1 with user-defined program functions for online sample preparation and autosampler-based reaction quenching.
• Chromatographic setup:
  • Dimension 1: MAbPac SEC-1 column (4×300 mm, 5 μm, 300 Å), mobile phase 10 mM ammonium acetate (pH 6.7) with 5% acetonitrile, flow 0.4 mL/min.
  • Dimension 2: Acclaim C8 column (3.0×150 mm, 3 μm), gradient elution with ammonium acetate, acetonitrile and water, flow 1.0 mL/min.
• Sample preparation: Manual mixing for rabbit IgG and autosampler-based mixing for BSA using MS(PEG)8 reagents at varying molar ratios, quenched with Tris HCl.
• Loop volume: 500 µL trap between SEC and C8 dimensions for fraction transfer.

Hlavní výsledky a diskuse

• SEC first dimension effectively separated PEGylated proteins, free PEG reagent and buffer salts under volatile conditions.
• On-line transfer to C8 column provided focused peaks for PEG species and removal of salts, improving detection.
• Charged aerosol detection enabled quantification of both hydrolyzed and unreacted PEG derivatives, which lack UV chromophores.
• Standard curves from five PEG reagent concentrations allowed accurate determination of residual PEG at initial and 40 min post-quench time points.
• Linear correlation observed between molar excess of reagent and degree of protein PEGylation; saturation observed above 40× excess.
• Automated BSA experiments validated trends seen with IgG, demonstrating method robustness.

Přínosy a praktické využití metody

• Simultaneous assessment of conjugated and free PEG species accelerates process development and QC workflows.
• Online sample preparation and reaction monitoring reduce manual handling and variability.
• Charged aerosol detection circumvents limitations of UV-only methods for non-chromophoric analytes.
• Workflow adaptability supports different proteins and PEG reagent chemistries for industrial applications.

Budoucí trendy a možnosti využití

• Integration with mass spectrometry via flow splitting for detailed structural identification of PEGylation products.
• Extension to alternative bioconjugation strategies and other polymeric reagents.
• Advances in 2D-LC automation and miniaturization to enhance throughput and reduce solvent consumption.
• Application in high-throughput screening of biopharmaceutical modification reactions.

Závěr

The developed 2D LC method with charged aerosol detection provides a comprehensive and automated platform for the qualification and quantification of PEGylated biopharmaceuticals and residual PEG reagents. It offers reliable reaction monitoring, supports process optimization and ensures product quality in biopharmaceutical development.

Reference

• Morar S.; Schrimsher J.; Chavez M. PEGylation of Proteins: A Structural Approach. BioPharm Int. 2006, 4, 34-36.
• Huang L.; Gough P.; DeFelippis M. Characterization of Poly(ethylene glycol) and PEGylated Products by LC/MS with Postcolumn Addition of Amines. Anal. Chem. 2009, 81, 567-577.
• Kou D.; Manius G.; Zhan S.; Chokshi H. Size Exclusion Chromatography with Corona Charged Aerosol Detector for the Analysis of Polyethylene Glycol Polymer. J. Chrom A 2009, 1216(28), 5424-5428.
• MS(PEG)n Reagents; Instructions 1767.4; Thermo Fisher Scientific, 2011.