Characterization of singly and multiply PEGylated insulin isomers by reversed-phase ultra-performance liquid chromatography interfaced with ion mobility mass spectrometry

Highlights: • LC-MS is coupled with ion mobility (IM) to separate bioconjugate mixtures. • Separation is achieved in two dimensions by polarity and shape/charge. • Conjugation sites are revealed by adding in-source dissociation (ISD). • The multidimensional LC-ISD-IM-MS approach is validated with PEGylated insulin. • PEGylation sites are confirmed by LC-ISD-IM-MS analysis after disulfide reduction. - Abstract: Conjugation of poly(ethylene glycol) (PEG) to protein drugs (PEGylation) is increasingly utilized in the biotherapeutics field because it improves significantly the drugs' circulatory half-life, solubility, and shelf-life. The activity of a PEGylated drug depends on the number, size, and location of the attached PEG chain(s). This study introduces a 2D separation approach, including reversed-phase ultra-performance liquid chromatography (RP-UPLC) and ion mobility mass spectrometry (IM-MS), in order to determine the structural properties of the conjugates, as demonstrated for a PEGylated insulin sample that was prepared by random amine PEGylation. The UPLC dimension allowed separation based on polarity. Electrospray ionization (ESI) of the eluates followed by in-source dissociation (ISD) truncated the PEG chains and created insulin fragments that provided site-specific information based on whether they contained a marker at the potential conjugation sites. Separation of the latter fragments by size and charge in the orthogonal IM dimension (pseudo-4D UPLC-ISD-IM-MS approach) enabled clear detection and identification of the positional isomers formed upon PEGylation. The results showed a highly heterogeneous mixture of singly and multiply conjugated isomers plus unconjugated material. PEGylation was observed on all three possible attachment sites (ε-NH₂ of LysB29, A- and B-chain N-termini). Each PEGylation site was validated by analysis of the same product after disulfide bond cleavage, so that the PEGylated A- and B- chain could be individually characterized with the same pseudo-4D UPLC-ISD-IM-MS method.