Quantitative analysis of free polyethylene glycols in PEGylated peptides using reverse-phase high-performance liquid chromatography and charged aerosol detection
- 1National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, Beijing, China
- 2Beijing Institute of Petrochemical Technology, Beijing, China
- 3Taizhou Institute for Food and Drug Control, Taizhou, Zhejiang, China
Abstract
PEGylation is a well-established strategy used to enhance the pharmacokinetic and pharmacodynamic properties of peptide drugs. However, free polyethylene glycol (PEG) remains a critical process-related impurity. Conventional detection methods for nonchromophoric compounds such as PEG, which include the use of evaporative light scattering (ELS) and refractive index (RI) detectors, are limited by their low sensitivity and poor compatibility with the gradient elution. Herein, systematically optimized reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with charged aerosol detection (CAD) was developed and validated for the quantification of free PEG in three representative PEGylated peptide therapeutics: PEG-loxenatide, pegmolesatide, and visepegenatide. The proposed method implements a co-optimization strategy for CAD parameters, with the power function value (PFV) applied during CAD signal acquisition and the power law(PL) setting adjusted during data processing.This dual optimization effectively linearizes the inherently nonlinear CAD response across a broad concentration range, enabling a linear relationship between PEG concentration and detector response. Method validation demonstrated satisfactory specificity, precision, linearity, accuracy, robustness, and solution stability in accordance with the applicable guidelines and pharmacopoeial requirements. Practical applicability was confirmed by analyzing multiple drug batches and accelerated stability samples.A Wilcoxon signed-rank test showed no statistically significant difference between the results obtained under the default and co-optimized settings (P > 0.05), indicating that the two settings produced comparable results under the conditions evaluated. The proposed HPLC-CAD method provides a practical analytical approach in the analysis of PEGylated peptide pharmaceuticals by achieving a linearized CAD response through PFV and PL co-optimization, providing an effective approach for free PEG quantification in routine quality control.
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BibTeX
@article{sun2026quantitative,
title = {{Quantitative analysis of free polyethylene glycols in PEGylated peptides using reverse-phase high-performance liquid chromatography and charged aerosol detection}},
author = {Yue Sun and Xinyue Hu and Yue Huang and Yuqin Yang and Xiaoming Zhang and Yi Li and Ping Lyu and Jing Li},
journal = {PLOS One},
year = {2026},
month = sep,
doi = {10.1371/journal.pone.0357292},
url = {https://doi.org/10.1371/journal.pone.0357292},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Quantitative analysis of free polyethylene glycols in PEGylated peptides using reverse-phase high-performance liquid chromatography and charged aerosol detection
AU - Yue Sun
AU - Xinyue Hu
AU - Yue Huang
AU - Yuqin Yang
AU - Xiaoming Zhang
AU - Yi Li
AU - Ping Lyu
AU - Jing Li
PY - 2026
DA - 2026/09/03
JO - PLOS One
DO - 10.1371/journal.pone.0357292
UR - https://doi.org/10.1371/journal.pone.0357292
AB - PEGylation is a well-established strategy used to enhance the pharmacokinetic and pharmacodynamic properties of peptide drugs. However, free polyethylene glycol (PEG) remains a critical process-related impurity. Conventional detection methods for nonchromophoric compounds such as PEG, which include the use of evaporative light scattering (ELS) and refractive index (RI) detectors, are limited by their low sensitivity and poor compatibility with the gradient elution. Herein, systematically optimized reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with charged aerosol detection (CAD) was developed and validated for the quantification of free PEG in three representative PEGylated peptide therapeutics: PEG-loxenatide, pegmolesatide, and visepegenatide. The proposed method implements a co-optimization strategy for CAD parameters, with the power function value (PFV) applied during CAD signal acquisition and the power law(PL) setting adjusted during data processing.This dual optimization effectively linearizes the inherently nonlinear CAD response across a broad concentration range, enabling a linear relationship between PEG concentration and detector response. Method validation demonstrated satisfactory specificity, precision, linearity, accuracy, robustness, and solution stability in accordance with the applicable guidelines and pharmacopoeial requirements. Practical applicability was confirmed by analyzing multiple drug batches and accelerated stability samples.A Wilcoxon signed-rank test showed no statistically significant difference between the results obtained under the default and co-optimized settings (P > 0.05), indicating that the two settings produced comparable results under the conditions evaluated. The proposed HPLC-CAD method provides a practical analytical approach in the analysis of PEGylated peptide pharmaceuticals by achieving a linearized CAD response through PFV and PL co-optimization, providing an effective approach for free PEG quantification in routine quality control.
SN - 1932-6203
ER -