Analysis of PEGylated Proteins with Agilent AdvanceBio SEC Columns

Importance of the Topic

PEGylation has become a cornerstone in modern biopharmaceutical development due to its ability to enhance solubility, reduce immunogenicity, extend circulating half-life, and protect proteins from proteolytic degradation. Reliable analytical methods are essential to ensure the purity and safety of PEGylated therapeutics. Size exclusion chromatography (SEC) remains the standard approach for detecting high-molecular-weight impurities and evaluating aggregation in PEG-protein conjugates.

Objectives and Study Overview

This study aimed to develop and validate a simple, sensitive SEC method for assessing the purity and aggregate content of PEGylated granulocyte colony stimulating factor (PEG-GCSF). Using an Agilent AdvanceBio SEC column with a bio-inert LC system, the method was evaluated for linearity, sensitivity (LOD/LOQ), precision, and its capacity to separate monomeric drug from stress-induced aggregates.

Methodology

Calibration standards of PEG GCSF spanning 12.5–2,000 µg/mL were prepared in 150 mM sodium phosphate buffer (pH 6.8). Linearity, limit of detection (LOD), and limit of quantitation (LOQ) were determined from signal-to-noise ratios. Forced-stress samples were generated by incubating 2 mg/mL PEG GCSF at 55 °C for up to 180 min to induce aggregation. Method precision and system suitability were assessed via replicate injections of the monomeric standard.

Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary LC System (pump, autosampler, column compartment, diode array detector)
• Agilent AdvanceBio SEC column, 130 Å, 7.8 × 300 mm, 2.7 µm particles
• Mobile phase: 150 mM sodium phosphate buffer, pH 6.8
• Flow rate: 0.8 mL/min; injection volume: 10 µL; detection wavelengths: 214 nm and 280 nm

Main Results and Discussion

The method demonstrated excellent linearity (R2 = 0.9994) over the tested range. LOD and LOQ were determined as 3.125 µg/mL and 12.5 µg/mL, respectively. Precision studies yielded retention time RSD of 0.023% and peak area RSD of 0.081% for the monomer peak. Forced-stress experiments revealed a time-dependent increase in aggregate levels, with the monomer fraction decreasing from ~96% in unstressed samples to ~70% and ~6% after 120 min and 180 min at 55 °C.

Benefits and Practical Applications

This bio-inert SEC method eliminates the need for organic modifiers, simplifying mobile phase preparation and reducing potential protein denaturation. High precision and sensitivity make it ideal for routine quality control of PEGylated proteins in research and manufacturing settings. The robust separation of monomer and aggregates ensures reliable purity estimates aligned with regulatory guidelines.

Future Trends and Opportunities

Potential advancements include integrating multi-angle light scattering or mass spectrometry detectors for absolute molecular mass determination. High-throughput, automated SEC platforms could accelerate screening of novel PEGylated constructs. Continued development of ultra-wide pore and hydrophilic bonded stationary phases may further improve aggregate resolution and expand applications to other bioconjugates.

Conclusion

A straightforward, reproducible SEC method using the Agilent AdvanceBio SEC column and bio-inert LC system was developed for quantitative purity and aggregate analysis of PEG GCSF. The approach meets system suitability criteria, offers excellent sensitivity and precision, and supports critical QC testing for PEGylated therapeutics.

Reference

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