Characterization of High Molecular Weight Multi-Arm Functionalized PEG-Maleimide for Protein Conjugation by Charge-Reduction Mass Spectrometry Coupled to Two-Dimensional Liquid Chromatography.

A current trend in drug development involves the use of high molecular weight, branched, and functionalized polymers for protein conjugation and drug delivery. Accurately characterizing these polymers is critical to control the product quality, to monitor the stability, and ultimately to ensure the drug efficacy and patient safety. However, due to the heterogeneity in size, the multiplicity of functional groups, and the highly convoluted charge-distribution profile in mass spectra, the characterization of these polymers is highly challenging from both chromatography and mass spectrometry perspectives. To overcome these challenges, we developed a strategy utilizing charge-reduction mass spectrometry (CRMS) coupled with two-dimensional HPLC (2D-LC). We then applied the workflow to characterize a 40 kDa 8-arm polyethylene glycol (PEG) functionalized with a maleimide terminal group for protein conjugation. The development was carried out in stages, where first we focused on the development of a CRMS method to simplify the charge profile of the polymers and then coupled it to HPLC to obtain discernible mass spectra of key impurities and degradants. Second, the CRMS method was applied to an investigation of the size-variant impurity resolved by reversed-phase size-exclusion 2D-LC. Finally, a separate size-exclusion reversed-phase 2D-LC- CRMS method was developed to capture a wider range of process-related impurities and reaction intermediates from the PEG- maleimide polymers throughout the conjugation process. The combination of these experiments using the 2D-LC-CRMS strategy enables the sensitive characterization of the entire impurity profile of the high molecular weight multifunctionalized PEG-maleimide conjugation intermediate.