LAUSR

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 product quality, monitor stability, and ultimately ensure drug efficacy and patient safety. However, due to the heterogeneity in size, multiplicity of functional groups, and highly convoluted MS charge distribution profile, 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 discernable mass spectra of key impurities and degradants. Second, the CRMS method was applied to an investigation of 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 sensitive characterization of the entire impurity profile of the high molecular weight multi-functionalized PEG-maleimide conjugation intermediate.