Application of triple quadrupole mass spectrometry for the characterization of antibody–drug conjugates

Abstract

Antibody–drug conjugates (ADCs) are an inherently heterogeneous class of biotherapeutics, the development of which requires extensive characterization throughout. During the earliest phases of preclinical development, when synthetic routes towards the desired conjugate are being assessed, the main interest lies in the determination of the average drug-to-antibody ratio (DAR) of a given batch as well as information about different conjugation species. There has been a trend in mass spectrometry (MS)–based characterization of ADCs towards the use of high-resolving mass spectrometry for many of these analyses. Considering the high cost for such an instrument, the evaluation of cheaper and more accessible alternatives is highly motivated. We have therefore tested the applicability of a quadrupole mass analyzer for the aforementioned characterizations. Eight ADCs consisting of trastuzumab and varying stoichiometries of Mc-Val-Cit-PABC-monomethyl auristatin E conjugated to native cysteines were synthesized and served as test analytes. The average DAR value and molecular weights (Mw) of all detected chains from the quadrupole mass analyzer showed surprisingly high agreement with results obtained from a time-of-flight (TOF) mass analyzer and hydrophobic interaction chromatography (HIC)–derived values for all investigated ADC batches. Acquired Mw were within 80 ppm of TOF-derived values, and DAR was on average within 0.32 DAR units of HIC-derived values. Quadrupole mass spectrometers therefore represent a viable alternative for the characterization of ADC in early-stage development. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-01699-0) contains supplementary material, which is available to authorized users.

The Metabolomics Approaches Based on LC-MS/MS for Analysis of Non-Halal Meats in Food Products: A Review

“…It has been widely used in targeted MS analysis. Quadrupole cannot be utilized for untargeted metabolomics analysis due to its low resolving power, which is less than 2000 FWHM (full width at half maximum) [48]. Time of flight (TOF) is an example of a medium-resolution mass analyzer with a resolving power capacity that ranges from 12,000 to 50,000 FWHM.…”

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

“…In 2020, our research group successfully conducted native IEX-QTOF/MS analysis of a site-specific ADC that resulted in both DAR determination and charge variant characterization in a single analysis. 49) Furthermore, subunit analysis after enzymatic digestion of the ADC was also performed. These results suggest that the native IEX-QTOF/MS for ADC characterization has great potential.…”

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody–Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry

“…16,17,19−21 Analysis of ADCs/AOCs of a low-to-medium sample complexity could be efficiently conducted with Orbitrap Fourier transform MS (FTMS), 8,9,20−23 time-of-flight (TOF) MS, 4,18,20,24 and triple quadrupole MS platforms. 25 The increased complexity of ADC samples has paved the way to a broader application of highresolution MS instruments, 22,26 inadequate at resolving highly congested mass spectra derived from complex ADC/AOC samples.…”

“…High levels of selectivity and specificity of mass spectrometry (MS), typically coupled to liquid chromatography (LC), render it an analytical technique of choice to provide the required accuracy needed in deducing the DAR values. ,,, The currently employed MS methods differ by the resolution performance (low vs high resolution) and the degree of protein unfolding (denaturing vs native MS conditions). ,,− The native MS approach has been shown to have advantages for the analysis of both noncovalently and covalently bound ADC samples compared to analysis performed under denaturing conditions. ,,, Lower-resolution levels, ∼5000–15,000 at the target m / z peak, are common for mass measurements of proteins in the native MS approach. ,,− Analysis of ADCs/AOCs of a low-to-medium sample complexity could be efficiently conducted with Orbitrap Fourier transform MS (FTMS), ,,− time-of-flight (TOF) MS, ,,, and triple quadrupole MS platforms . The increased complexity of ADC samples has paved the way to a broader application of high-resolution MS instruments, , which may still prove to be inadequate at resolving highly congested mass spectra derived from complex ADC/AOC samples.…”