Maxis mass spectrometer

Mass spectrometric analyses of AFCs were performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system (Dionex, Germering, Germany). Prior to mass spectrometry (MS) analysis, samples (ca. 5 µg) were desalted on a MassPREP (Waters, Saint-Quentin-en-Yvelines, France) desalting cartridge (2.1 × 10 mm, Waters) heated at 80 °C using 0.1% formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B at 500 µL/min. After 1 min, a linear gradient from 5 to 90% B in 1.5 min was applied; the first 1.5 min were diverted to waste. MS data were acquired in positive mode with an ESI source over the m/z range from 900 up to 5000 at 1 Hz and processed using DataAnalysis 4.4 software (Bruker, Bremen, Germany) and the MaxEnt algorithm for spectral deconvolution.

Mass spectrometric analyses of AFCs were performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system (Dionex, Germering, Germany) by the “Fédération de Recherche” ICOA/CBM (FR2708) platform. Prior to mass spectrometry (MS) analysis, samples (ca. 1 µg) were desalted on a MassPREP desalting cartridge (2.1 × 10 mm) (Waters, Saint-Quentin-en-Yvelines, France) heated at 80 °C using 0.1% formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B at 500 µL/min. After 1 min, a linear gradient from 5 to 90% B in 1.5 min was applied; the first 1.5 min were diverted to waste. MS data were acquired in positive mode with an ESI source over the m/z range from 900 up to 5000 at 1 Hz and processed using DataAnalysis 4.4 SR1 software from Bruker (Bremen, Germany) and the MaxEnt algorithm for spectral deconvolution. Deconvolution was carried out in the range 20–60 kDa. The average drug-to-antibody ratio (DAR_average) was calculated according to a previously reported method. Briefly, the percentage abundance of each DARi species represents the relative distribution of each particular drug-loaded FDC species, as monomer and dimer. The DAR_average was then calculated using the percentage peak areas combined with their respective drug load numbers, according to the corresponding formula: $$DA{R}_{average}=0\times DA{R}_0+1\times DA{R}_1+2\times DA{R}_2$$

Mass spectrometric analyses of ADCs were performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system. Prior to MS analysis, samples (ca. 5 µg) were desalted on a MassPREP desalting cartridge (2.1 × 10 mm, Waters) heated at 80 °C using 0.1% formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B at 500 µL/min. After 1 min, a linear gradient from 5 to 90% B in 1.5 min was applied; the first 1.5 min were diverted to waste. HRMS data were acquired in positive mode with ESI source over the m/z range from 900 up to 5000 at 1 Hz and processed using DataAnalysis 4.4 software (Bruker) and MaxEnt algorithm for spectral deconvolution.