PEGylated Protein Analysis by Size-Exclusion and Reversed-Phase UPLC

Importance of the Topic

PEGylation has become a vital modification to improve the pharmacokinetics, solubility, and stability of protein therapeutics. Monitoring the degree of PEG attachment and detecting free polymer are critical quality attributes that impact both efficacy and safety, especially in light of emerging anti‐PEG antibodies in patients.

Study Objectives and Overview

This study compared two UPLC approaches—size‐exclusion (SE‐UPLC) and reversed‐phase (RP‐UPLC)—for simultaneous analysis of non‐PEGylated protein, free activated PEG (aPEG), and the PEG‐protein conjugate. The goal was to determine which method provides sufficient resolution and sensitivity for routine quality control of bioconjugates.

Methodology and Instrumentation

Samples of a 50 kDa protein, 40 kDa aPEG, and their conjugate were analyzed using a Waters ACQUITY UPLC H‐Class Bio System with TUV and ELSD detectors. SE‐UPLC employed dual BEH columns (200 Å and 450 Å) at 40 °C, isocratic mobile phase (200 mM ammonium formate/5% ACN), and UV detection at 280 nm. RP‐UPLC used a BEH300 C4 column at 90 °C with a water/ACN gradient containing 0.1% TFA, detecting both UV (280 nm) and ELSD signals.

Key Results and Discussion

SE‐UPLC successfully separated non‐PEGylated protein from the conjugate but failed to resolve the conjugate from the free PEG due to similar hydrodynamic radii (Rh ratio ≈1.07). Theoretical Rh predictions confirmed this limitation for many protein/PEG combinations. In contrast, RP‐UPLC achieved complete baseline resolution of all three species, aided by elevated column temperature and dual detection. ELSD allowed quantitation of low‐level free PEG that was undetectable by UV alone.

Benefits and Practical Applications

• RP‐UPLC provides robust separation of protein, aPEG, and conjugate in a single run.
• ELSD detection enhances sensitivity for free PEG quantitation.
• SE‐UPLC remains useful for distinguishing unmodified protein versus PEGylated species when large MW PEGs are used.

Future Trends and Potential Applications

Advances in stationary phases and detector technologies may further improve selectivity and sensitivity. Integration of mass spectrometry, UHPLC columns with tailored pore sizes, and computational Rh prediction tools will streamline method development. These approaches will support high‐throughput screening of novel bioconjugates and assessment of anti‐PEG antibody impacts.

Conclusion

While SE‐UPLC can separate unmodified and PEGylated proteins under favorable conditions, it often fails to resolve free polymer from conjugate. RP‐UPLC with a C4 column and dual UV/ELSD detection provides a superior, comprehensive assay for quality control of PEGylated therapeutics.

References

• Banerjee SS, et al. Poly(ethylene glycol)–Prodrug Conjugates: Concept, Design, and Applications. J Drug Deliv. 2012.
• Armstrong JK, et al. Antibody against PEG adversely affects PEG‐asparaginase therapy. Cancer. 2007;110:103–11.
• Garay R, Labaune J. Immunogenicity of polyethylene glycol (PEG). Open Conf Proc J. 2011.
• Fee CJ, Van Alstine JM. Purification of PEGylated proteins. Protein Purif. 2011;149:339–.
• Li N, et al. Quantitation of free PEG in PEGylated protein by SEC‐HPLC with RI detection. J Pharm Biomed Anal. 2008;48:1338.
• Koza S, Lauber M, Fountain KJ. Analysis of Multimeric Monoclonal Antibody Aggregates. Waters App Note. 2013.
• Fee CJ, Van Alstine JM. Prediction of viscosity radius and SEC behavior of PEGylated proteins. Bioconjug Chem. 2004;15:1304–13.
• Fee CJ, Van Alstine JM. PEG‐proteins: Reaction engineering and separation issues. Chem Eng Sci. 2006;61:924–39.