{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T18:15:20Z","timestamp":1788459320378,"version":"build-2803163510"},"reference-count":30,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Youth Development Research Foundation","award":["2024A2"],"award-info":[{"award-number":["2024A2"]}]},{"DOI":"10.13039\/501100011198","name":"State Key Laboratory of Drug Research","doi-asserted-by":"crossref","award":["2025SKLDRS0312"],"award-info":[{"award-number":["2025SKLDRS0312"]}],"id":[{"id":"10.13039\/501100011198","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>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\u2009&gt;\u20090.05), 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.<\/jats:p>","DOI":"10.1371\/journal.pone.0357292","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T17:32:41Z","timestamp":1788456761000},"page":"e0357292","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Quantitative analysis of free polyethylene glycols in PEGylated peptides using reverse-phase high-performance liquid chromatography and charged aerosol detection"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-0901-022X","authenticated-orcid":true,"given":"Yue","family":"Sun","sequence":"first","affiliation":[{"name":"National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyue","family":"Hu","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Huang","sequence":"additional","affiliation":[{"name":"Beijing Institute of Petrochemical Technology","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuqin","family":"Yang","sequence":"additional","affiliation":[{"name":"Taizhou Institute for Food and Drug Control","place":["Taizhou, Zhejiang, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoming","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Li","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Lyu","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9521-9028","authenticated-orcid":true,"given":"Jing","family":"Li","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, State Key Laboratory of Drug Regulatory Science, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"key":"pone.0357292.ref001","doi-asserted-by":"crossref","first-page":"1978","DOI":"10.1002\/jssc.201901340","article-title":"Recent advances in the bioanalytical methods of polyethylene glycols and PEGylated pharmaceuticals","volume":"43","author":"Z Zhang","year":"2020","journal-title":"J Sep Sci"},{"issue":"2","key":"pone.0357292.ref002","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1080\/09205063.2020.1825304","article-title":"PEGYLATION: an important approach for novel drug delivery system","volume":"32","author":"D Yadav","year":"2021","journal-title":"J Biomater Sci Polym Ed"},{"issue":"9","key":"pone.0357292.ref003","doi-asserted-by":"crossref","first-page":"1257","DOI":"10.1080\/17425247.2016.1182485","article-title":"Polyethylene glycol (PEG): a versatile polymer for pharmaceutical applications","volume":"13","author":"AA D\u2019souza","year":"2016","journal-title":"Expert Opin Drug Deliv"},{"issue":"5","key":"pone.0357292.ref004","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1615\/CritRevTherDrugCarrierSyst.v25.i5.10","article-title":"PEGylation: an approach for drug delivery. A review","volume":"25","author":"A Jain","year":"2008","journal-title":"Crit Rev Ther Drug Carrier Syst"},{"issue":"1","key":"pone.0357292.ref005","article-title":"PEGylated therapeutics in the clinic","volume":"9","author":"Y Gao","year":"2023","journal-title":"Bioeng Transl Med"},{"issue":"3","key":"pone.0357292.ref006","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1038\/nrd1033","article-title":"Effect of pegylation on pharmaceuticals","volume":"2","author":"JM Harris","year":"2003","journal-title":"Nat Rev Drug Discov"},{"issue":"4","key":"pone.0357292.ref007","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1517\/14728214.2015.1113254","article-title":"What is the future of PEGylated therapies?","volume":"20","author":"M Swierczewska","year":"2015","journal-title":"Expert Opin Emerg Drugs"},{"issue":"5","key":"pone.0357292.ref008","doi-asserted-by":"crossref","first-page":"315","DOI":"10.2165\/00063030-200822050-00004","article-title":"The impact of PEGylation on biological therapies","volume":"22","author":"FM Veronese","year":"2008","journal-title":"BioDrugs"},{"key":"pone.0357292.ref009","author":"European Medicines Agency","year":"2025"},{"key":"pone.0357292.ref010","volume-title":"General monograph for pegylated recombinant protein and polypeptide products for human use. 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