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Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation

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Abstract

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography–mass spectrometry (LC–MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Correlation between log PEG MW and retention time observed by reversed-phase LC-MS with in-source fragmentation

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Acknowledgements

The authors would like to thank the Pharmaceutical Candidate Optimization—Metabolism and Pharmacokinetics group at Bristol–Myers Squibb for supplying samples. We thank Dr Petia A. Shipkova, Dr Dieter M. Drexler, Dr Purnima Khandelwal, Dr David K. Lloyd, and Dr Adrienne A. Tymiak for helpful discussions and critical review of the manuscript.

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Authors and Affiliations

  1. Pharmaceutical Candidate Optimization – Bioanalytical and Discovery Analytical Sciences, Route 206 and Province Line Road, Princeton, NJ, 08543-4000, USA

    Bethanne M. Warrack & Guodong Chen

  2. Spectrix Analytical Services, Route 206 and Province Line Road, Princeton, NJ, 08543-4000, USA

    Brian P. Redding

  3. Analytical and Bioanalytical Development, 1 Squibb Drive, New Brunswick, NJ, 08903-0191, USA

    Mark S. Bolgar

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  1. Bethanne M. Warrack
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  2. Brian P. Redding
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Correspondence to Bethanne M. Warrack.

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Warrack, B.M., Redding, B.P., Chen, G. et al. Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation. Anal Bioanal Chem 405, 4283–4287 (2013). https://doi.org/10.1007/s00216-013-6795-3

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