PEGylation,the controlled covalent conjugation of polyethylene glycol to therapeutics,enhances therapeutic efficacy through optimized pharmacokinetics.However,to date no high-molecular-weight PEGylated small-molecule ...PEGylation,the controlled covalent conjugation of polyethylene glycol to therapeutics,enhances therapeutic efficacy through optimized pharmacokinetics.However,to date no high-molecular-weight PEGylated small-molecule prodrugs have received regulatory approval.This technological gap can be partially attributed to the exponential proliferation of metabolic intermediates resulting from multi-payload conjugation strategies,which introduces unprecedented analytical complexities in metabolite profiling and pharmacokinetic characterization.To address this challenge,we developed a liquid chromatography-triple-quadrupole/time-of-flight mass spectrometry platform for PEG20k-(irinotecan)3,a Phase III clinical candidate.Our methodology employs payload stoichiometry-based chromatographic resolution for clustering isomeric PEG species.Complementarily,diagnostic product ions at m/z 699.83,569.27,and 587.28 enable systematic differentiation between double-loaded,single-loaded,and released irinotecan payload.This approach successfully identifies eight metabolic clusters spanning from PEG-conjugates,cleaved PEG segments,and released small-molecule species.Its demonstrated capacity to deconvolute complex metabolic profiles—through payload-stoichiometry based chromatographic resolution coupled with diagnostic ion analysis—positions this workflow as an attractive tool for accelerating the development of PEGylated small-molecule therapeutics.展开更多
基金support from the National Natural Science Foundation of China(Nos.82030107,82304443,82373944)the Hainan Provincial Natural Science Foundation of China(No.825QN288).
文摘PEGylation,the controlled covalent conjugation of polyethylene glycol to therapeutics,enhances therapeutic efficacy through optimized pharmacokinetics.However,to date no high-molecular-weight PEGylated small-molecule prodrugs have received regulatory approval.This technological gap can be partially attributed to the exponential proliferation of metabolic intermediates resulting from multi-payload conjugation strategies,which introduces unprecedented analytical complexities in metabolite profiling and pharmacokinetic characterization.To address this challenge,we developed a liquid chromatography-triple-quadrupole/time-of-flight mass spectrometry platform for PEG20k-(irinotecan)3,a Phase III clinical candidate.Our methodology employs payload stoichiometry-based chromatographic resolution for clustering isomeric PEG species.Complementarily,diagnostic product ions at m/z 699.83,569.27,and 587.28 enable systematic differentiation between double-loaded,single-loaded,and released irinotecan payload.This approach successfully identifies eight metabolic clusters spanning from PEG-conjugates,cleaved PEG segments,and released small-molecule species.Its demonstrated capacity to deconvolute complex metabolic profiles—through payload-stoichiometry based chromatographic resolution coupled with diagnostic ion analysis—positions this workflow as an attractive tool for accelerating the development of PEGylated small-molecule therapeutics.
