Microscale Native Size Exclusion Chromatography High Resolution Mass Spectrometry for the Determination of the Chelator-To-Antibody Ratio of Immunoconjugates

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Abstract
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. Radiometric titration, although widely used, is labor-intensive 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. Thanks to the microscale flow applied, high concentrations of volatile salts, up to 1000 mM ammonium acetate, could be used to minimize secondary interactions. 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 to radionuclide titration for the analysis of the same immunoconjugate model, reporting similar results. Finally, we demonstrated the broad applicability of the method by testing diverse immunoconjugates (ipilimumab), including cysteine and lysine-linked chelator samples, as well as other smaller protein models, such as nanobodies. With a 20-min run time, the averageCAR, dispersity index, glycosylation profile, and impurities could all be determined. This approach offers a broadly applicable alternative for radiometric titration for the characterization of immunoconjugates.

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