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

Abstract

IntroductionMonoclonal 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. MethodsTwo 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). ResultsRadiometric 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. ConclusionsThe 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.