Utilization of Tandem-Column UHPLC for High-Throughput Peptide Mapping of Therapeutic Proteins

Liquid chromatography-mass spectrometry (LC-MS) peptide mapping is indispensable for site-resolved evaluation of chemical modifications, also known as critical quality attributes (CQAs) when shown to impact molecule quality, safety, and/or efficacy in therapeutic proteins. This approach is often constrained by long gradients due to challenges associated with monitoring isoaspartic acid (isobaric) and asparagine deamidation (near-isobaric) formation. A high-flow, ultrahigh-pressure LC (UHPLC) workflow (700 μL/min; >1000 bar) was developed to retain specificity for these modifications while increasing injection-to-injection throughput by ∼10-fold (123 min vs 12.5 min with a dual binary pump configuration). Using a panel of 51 synthetic peptides corresponding to sequences in therapeutic proteins shown to be susceptible to these modifications, the rapid method delivered markedly tighter retention time control than the reference method (median standard deviation 0.01 vs 0.34 min; range 0.6 vs 42.6 s; % RSD 0.1% vs 0.9%). Coupling two complementary stationary phases in series increased the number of observed peaks in a 10:1 unmodified:modified mixture of all synthetic peptides (42/51 vs 38/51). Artificial on-column degradations were minimized by introducing a low-temperature trap upstream of the analytical column(s). A dual binary pump, multicolumn configuration further doubled the duty cycle by moving column wash/equilibration offline. These results establish a readily implemented, practical route to fast, artifact-aware, site-resolved peptide mapping for the characterization of therapeutic proteins without sacrificing separation specificity or quantitative control. With 1-2 s chromatographic peaks, MS duty cycle limited MS/MS acquisition on the Q Exactive platform. Pairing the chromatographic conditions with higher duty cycle instruments in future studies is expected to enable identification-rich acquisitions when coupled to these rapid separations.