High throughput and high confidence sequence variant analysis in therapeutic antibodies using Evosep One liquid chromatography tandem mass spectrometry with synthetic heavy peptides

Sequence variants are anomalous misincorporations of amino acids into the primary structure of therapeutic antibodies during DNA replication and protein biosynthesis. As these low abundance variants contribute to molecular heterogeneity and could negatively impact the safety and efficacy of a protein therapeutic, analytical methods like liquid chromatography tandem mass spectrometry (LC-MS2) are used to monitor them with the goal of establishing control strategies that limit their occurrence. Current LC-MS2 strategies depend on relatively long gradients that minimize coelution between abundant non-variant peptide peaks and trace-level variants to limit ion suppression that can potentially conceal the latter. However, lengthy LC gradients reduce the number of samples that can be analyzed per day, limiting the practicality of LC-MS2 when analyzing large sample sets. Furthermore, confident variant identification partly depends on capturing rich MS2 spectra that localize any amino acid misincorporations, which can be challenging due to the low abundance of this class of analyte. This work drastically reduces the cycle time to run each therapeutic antibody sample with roughly the same or even more variant identifications, compared to traditional LC-MS2 analysis, by integrating an Evosep One LC platform with an Orbitrap Fusion Lumos mass spectrometer. It also introduces a novel strategy using synthetic peptides that contain heavy isotopes placed near both termini to validate lower confidence variants in one targeted LC-MS2 run according to retention time, precursor mass signal, and MS2 fragment patterns shared with the heavy peptide variant. Taken together, this approach enables high-throughput sequence variant analysis at 30 samples per day as well as validation for lower confidence variants that can be integrated into therapeutic antibody process development and characterization.