Comparison of analytical methods for antibody conjugates with application in nuclear imaging – Report from the trenches

Irene V. J. Feiner, Beatrice Longo, Vanessa Gómez-Vallejo, Javier Calvo, Marion Chomet, Danielle J. Vugts, Albert D. Windhorst, Daniel Padro, Matteo Zanda, Luka Rejc, Jordi Llop*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


Introduction: Monoclonal antibodies (mAbs) are widely used in nuclear imaging. Radiolabelling with positron emitting radionuclides, typically radiometals, requires the incorporation of a bifunctional chelator for the formation of the radiometal-mAb complex. Additionally, mAbs can be conjugated with small molecules capable to undergo bioorthogonal click reactions in vivo, enabling pre-targeting strategies. The determination of the number of functionalities attached to the mAb is critically important to ensure a good labelling yield or to guarantee pre-targeting efficacy. In this work, we compare three different analytical methods for the assessment of average functionalisation and heterogeneity of the conjugated mAbs. Methods: Two selected mAbs (Trastuzumab and Bevacizumab) were randomly conjugated through lysine residues with 3–10 equivalents p-isothiocyanatobenzyl-desferrioxamine (p-NCS-Bz-DFO) or 20–200 equivalents trans-cyclooctene-N-hydroxysuccinimide ester (TCO-NHS). The DFO- or TCO-to-mAb ratio were determined using three different methods: direct titration (radiometric for DFO-conjugated mAbs, photometric for TCO-conjugated mAbs), MALDI/TOF MS mass analysis (Matrix-Assisted Laser Desorption-Ionization/Time of Flight Mass Spectrometry), and UPLC/ESI-TOF MS mass analysis (Ultra High Performance Liquid Chromatography/Electrospray Ionization-Time of Flight Mass Spectrometry). Results: Radiometric and photometric titrations provided information on the average number of DFO and TCO functionalities per mAb respectively. MALDI/TOF MS provided equivalent results to those obtained by titration, although investigation of the heterogeneity of the resulting mixture was challenging and inaccurate. UPLC/ESI-TOF MS resulted in good peak resolution in the case of DFO-conjugated mAbs, where an accurate discrimination of the contribution of mono-, di- and tri-substituted mAbs could be achieved by mathematical fitting of the spectra. However, UPLC/ESI-TOF MS was unable to discriminate between different conjugates when the smaller TCO moiety was attached to the mAbs. Conclusions: The three techniques offered comparable results in terms of determining the average number of conjugates per mAb. Additionally, UPLC/ESI-TOF MS was able to shed a light on the heterogeneity of the resulting functionalised mAbs, especially in the case of DFO-conjugated mAbs. Finally, while using a single analytical method might not be a reliable way to determine the average functionalisation and assess the heterogeneity of the sample, a combination of these methods could substantially improve the characterization of mAb conjugates.
Original languageEnglish
Pages (from-to)24-33
Number of pages10
JournalNuclear Medicine and Biology
Publication statusPublished - 1 Nov 2021

Cite this