Monitoring Peptide PEGylation by HPLC with Charged Aerosol Detection

Purpose: The goal of this work was to develop a fast and accurate method to monitor PEGylation reactions by using HPLC with complementary UV/Vis and charged aerosol detection. It is an increasingly common practice to modify biotherapeutic peptides, proteins and small molecules by attaching chemical groups designed to enhance properties such as bioavailability, potency or stability. One example of this is the PEGylation reaction that attaches hydrophilic polyethylene glycol (PEG) moieties tailored to increase the drug’s aqueous solubility and circulating half life. Chemists developing extended-life biotherapeutics need to measure free PEG, partially PEGylated intermediates, and final PEGylated peptide/protein in order to optimize reaction conditions, assess the quality of final product, and study drug product stability. Methods: Peptides and proteins were modified by reaction with MS(PEG)n reagents. These are methyl-terminated, polyethylene glycol compounds with defined PEG chain lengths of discrete molecular weight (n equals 4 to 24 PEG units). They are activated as NHS esters for covalent PEGylation of primary amines on proteins through the formation of stable, irreversible amide bonds. A high resolution separation on the MAbPac RP resolves PEGylation reagents, Tris-quenched PEG, unreacted insulin, and several PEGylated insulins that differ in the number and sites of attached PEG. The UV/Vis detector provides information on the quantity and molar ratio of insulin and PEGylated insulin, but can’t measure PEGylation reagents or hydrolyzed PEG. Charged aerosol detection (Figure 1) easily quantifies the PEG related species that are missed by UV, and provides information on the number of PEGs attached to each peptide. Results: The method was used to characterize the reaction products from MS(PEG)8 PEGylation of insulin and human immunoglobulin G (IgG). Quantitative performance including precision, detection limits and dynamic range is presented. Figures of merit include sensitivity at the low-nanogram on-column level, dynamic range over two orders of magnitude, and peak area precision averaging less than two percent RSD. Dave Thomas1, Ian Acworth1, Scott Meier2, Barbara Kaboord2, Hua Yang3, and Carl Fisher3; 1Thermo Fisher Scientific, Chelmsford, MA, 2Thermo Fisher Scientific, Rockford, IL, 3Thermo Fisher Scientific, Sunnyvale, CA Monitoring Peptide PEGylation by HPLC with Charged Aerosol Detection Future Steps This preliminary work shows that UHPLC with charged aerosol detection is well suited to monitor the reagents, intermediates and products of protein and peptide PEGylation reactions. Future work will include:  Applying this UHPLC-CAD method to monitor the stability and forced degradation behavior of PEGylated peptide and proteins.  Improving the separation of IgG and PEGylated IgG.  The development of optimized workflows for PEGylation reaction and PEGylated product stability monitoring that are simple and robust enough for routine quality control purposes.  Further investigations into applying UHPLC-CAD to monitor the reagents, intermediates and products of other protein conjugation reactions such as XTENylation, HESylation, PASylation or conjugation with glycopolymers.5