Microscale native size exclusion chromatography high resolution mass spectrometry for the determination of the chelator-to-antibody ratio of immunoconjugates.

BACKGROUND: Positron emission tomography (PET) using radiolabeled antibodies (radioimmunoconjugates) is a powerful imaging technique to track the distribution of therapeutic antibodies. Accurate determination of the average number of chelators per antibody (CAR) is essential for (radio)immunoconjugate characterization, which is important for maximizing tumor uptake and image quality. Determination of the CAR is considered a critical quality attribute. Radiometric titration, although widely used, is labor-intensive, material- and time-consuming, and requires approximately half a day per sample. To overcome this analytical bottleneck, we developed a microscale size exclusion chromatography - mass spectrometry (SEC-MS) analysis.

RESULTS: By applying microscale flow, high concentrations of volatile salts, up to 1000 mM ammonium acetate, could be used to minimize secondary interactions. Moreover, high isCID energy of 140 eV was applied to reduce adduct formation and enhance the MS sensitivity. The method was optimized using model radioimmunoconjugates based on intact trastuzumab, with a particular focus on the impact of buffer concentration on both chromatographic effects and CAR determination. A final buffer concentration of 600 mM ammonium acetate was selected. The method was compared with radionuclide titration for analysis of the same immunoconjugate model and yielded similar results. Finally, we demonstrated the broad applicability of the method by testing diverse immunoconjugates (e.g., ipilimumab, rituximab), including cysteine- and lysine-linked chelators (e.g., DFO, DOTA, RESCA), as well as other smaller protein models, such as nanobodies.

SIGNIFICANCE: The optimized microflow native SEC-MS method, with a rapid 20-min run time, provides comprehensive characterization, including determination of the average CAR, dispersity index, glycosylation profile, and mAb impurities. Crucially, this native approach is applicable to all tested immunoconjugates, including chemically sensitive species such as those with maleimide and thiourea linkages, which often challenge denaturing methods like RPLC-MS. This establishes SEC-MS as a broadly applicable alternative to radiometric titration for the comprehensive characterization of immunoconjugates.