Charge Detection Mass Spectrometry and a Glu-C/Lys-C Digestion-Based Data-Dependent Approach Suggest Mono-PEGylation of a Heterogenous Therapeutic Protein

There have been various strategies to increase protein's half-life in blood, such as glycosylation or protein fusion, but one of the most widely used approaches is the addition of PEG (polyethylene-glycol). Attaching a large, polydisperse PEG moiety to the therapeutics makes the analytical characterization challenging due to conjugation chemistry and increased heterogeneity. In this study, we aimed to develop a mass-spectrometry-based workflow to mitigate challenges. Using Orbitrap-based charge detection mass spectrometry, we determined the intact mass of a heavily glycosylated protein modality which is likely to carry a 30 kDa PEG. It has been demonstrated that the technique is suitable for impurity analysis, such as the remaining underivatized PEG in the formulated drug. Our data suggest mono-PEGylation of glycoprotein. Analyzing the digests of the protein generated by Lys-C and Glu-C and the combination of both enzymes in data-dependent acquisition allowed us to identify PEGylation sites on K45 and K52 residues and the <i>N</i>-terminus. Software-assisted data processing of PEGylation from digests yielded by the three digestion conditions generated results complementary to each other and led to a highly confident assignment of PEGylation sites. The methodology introduced here overcomes bottlenecks caused by PEGylation and can be routinely used for the comprehensive characterization of PEGylated therapeutics.