Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates

Abstract

Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies.

Keywords: Antibody-drug conjugate (ADC); ion mobility-mass spectrometry (IM-MS); middle level; native mass spectrometry; site-specific bioconjugation; top level.

Figure 1.

Schematic overview of the bioconjugation strategy. A formylglycine (fGly) amino acid residue is produced through the highly selective oxidation of a cysteine residue found within a specific pentapeptide consensus sequence by formylglycine-generating enzyme (FGE). The fGly-containing protein is then further modified using aldehyde specific chemistries.

Figure 2.

Middle-up analysis of CBW-03–106. (a) UV chromatogram of TCEP-reduced CBW-03–106 (a). LC without drug load and HC fragments with one or 2 RED-106 bound molecules were observed. Theoretical and experimental masses of the G0F glycoform obtained by middle-up analyses of CBW-03–106 (b).

Figure 3.

Native mass spectrometry analysis of deglycosylated CBW-03–106. Full scan ESI mass spectra on the m/z range [4 500 – 7 500] of deglycosylated CBW-03–106 in native conditions obtained either on a Q-TOF (a-c) or an orbitrap (d-f) instrument. Zoom on the most intense charge states showing drug load profiles (b,e). Native MS derived drug load profile and subsequent average DAR (c,f): relative intensities of each drug load as a function of the number of drugs loaded onto the mAb. ^•: non-identified impurity; *: loss of one fucose (−146 Da) and **: glycation (+162 Da).

Figure 4.

Native MS and IM-MS for benchmarking bioconjugation strategies. (a-c) Deconvoluted native mass spectra from the Orbitrap showing drug load profiles (left panels) and native IM-MS plots of m/z vs. drift time from the Q-TOF instrument for brentuximab vedotin (a), trastuzumab emtansine (b) and CBW-03–106 DAR4 site-specific ADC (c).

Figure 5.

Native IM-MS analysis of deglycosylated CBW-03-106 and its parental mAb form. Plots of m/z vs. drift time for CBW-03-106 (a) and mAb (b). Extracted ATDs corresponding to the 24+ charge state of the parental mAb (blue) and CBW-03–106 (red) at a trap collision voltage of 4 V (c). Measured ^TWCCS_N2 (nm²) of the parental mAb (blue diamonds) and CBW-03–106 (red diamonds) as a function of ESI charge state (d).

Figure 6.

CIU experiments performed on deglycosylated CBW-03–106 site-specific ADC and its unconjugated mAb form. CIU fingerprints of the 24+ charge state for the unconjugated mAb (a) and the site-specific ADC, CBW-03–106 (b). CIU difference plot between CBW-03–106 and unconjugated mAb (c). Extracted arrival time distributions (ATD) of the unconjugated mAb (blue) and CBW-03–106 (red) at different trap collision voltages (d).