Radiolabeled antibodies (radioimmunoconjugates) provide significant opportunities for targeted therapy and noninvasive disease monitoring via positron emission tomography (PET). A key factor affecting tumor uptake and imaging quality is the number of chelators attached to each antibody, known as the chelator-to-antibody ratio (CAR). Correctly determining the CAR is essential for quality control. Although radiometric titration is commonly used for this measurement, it is labor-intensive, consumes a lot of material, and takes up to half a day per sample.
In this study, CAST scientists Annika van der Zon et al., in collaboration with Bram Weijers and Danielle Vlugts (Amsterdam UMC), introduce a rapid, microscale aqueous size-exclusion chromatography–mass spectrometry (SEC-MS) method to measure CAR. She tested ammonium acetate buffer concentrations up to 1000 mM to reduce secondary interactions with the SEC stationary phase. To improve MS sensitivity and decrease adduct formation, a high in-source collision-induced dissociation (isCID) energy of 140 eV was used. The method was optimized for various immunoconjugates based on chromatographic performance and MS sensitivity, thereby enabling precise CAR calculation.
The optimized protocol, which uses 600 mM ammonium acetate (pH 6.8), was successfully applied to various immunoconjugates, yielding CAR results comparable to those obtained by traditional radiometric titration. In addition to measuring CAR, the SEC-MS method provides additional data, including dispersity index, glycosylation patterns, and antibody impurities. Most notably, the entire analysis takes only 20 minutes.
The method’s broad applicability was demonstrated across various immunoconjugates, including cysteine- and lysine-linked chelators, smaller proteins like nanobodies, and multiple chelator types (DFO, DOTA, and RESCA). This showcases its versatility as a fast, informative, and efficient analytical tool.
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https://www.sciencedirect.com/science/article/pii/S0003267026001649
Figure 1: Example of MS data obtained by our SEC-MS method. Trastuzumab-mal-DFO measurement with (A) m/z spectrum with the charge states and (B) deconvoluted spectrum with the different CARs (CAR0 (green), CAR1 (dark blue), CAR2 (light blue), CAR3 (red), CAR4 (pink), CAR5 (dark yellow)), CAR6 (light yellow), and glycoforms distribution ((1) G0/G0F, (2) G0F/G0F, (3) G0F/G1F, (4) G1F/G1F, and (5) G1F/G2F).