By ESI-MS, antibody-drug-conjugate (ADC) sample in liquid phase are sprayed through a capillary at high voltage into the MS instrument where the mass over charge ratio (m/z) is measured. For ADC’s the electrospray process results in multiply protonated molecules with a distribution of ion species at m/z range from 1-3000. The mass of the ADC is determined using a deconvolution algorithm that calculates the mass of the intact non-protonated antibody. The ESI process requires that the sample preparation is quite pure without interfering salts or detergents.
The sample is purified by reversed phase HPLC (Agilent 1200 system) using a short C8 column before being analyzed on a Q-TOF mass spectrometer (Bruker Maxis Impact system). The mass of the sample is determined by deconvolution of the obtained raw spectra using the MaxEnt algorithm. The high resolution and accuracy of the Q-Tof instrument result in very accurate mass determination within 0-3 Da of the theoretical mass.
For ADC’s the sample is analyzed in the intact form and after reduction with DTT.
From the deconvoluted data the drug-to-antibody ratio (DAR) distributions can be determined.
In Mass spectrometric Peptide Mapping the ADC is cleaved into smaller peptides using a specific protease. The peptides are analyzed by mass spectrometry and the observed peptides correlated to the antibody amino acid sequence.
The identified peptides in the peptide map thus confirms the specific amino acid sequences covered by the peptide map, as well as the attachment sites for Drug-conjugates. Peptide mapping using a single protease will typically give a sequence coverage of 30-90%. By using two or more proteases this coverage can be increased to 95-100%.
Detailed characterization of an ADC’s requires careful investigation of the antibody sequence to select the best proteases, and choice of nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides. The difference between the two techniques is related to the amount of material needed for the analysis – nano-flow can be conducted on low microgram antibody whereas the high-flow analysis requires at least 20 microgram per analysis. The high-flow analysis include a UV-trace of the chromatogram.
The peptide mapping using nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with C18 capilary column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.
The analysis provide a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge ratio from 200-1600 dalton.
high-flow LC-MS/MS with UV
The peptide mapping using high-flow LC-MS/MS is performed on a Agilent 1200 system running at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.
The analysis provides both a UV- and a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge ratio from 200-1600 dalton.
Sample Preparation Guidelines
Antibody-Drug-Conjugate (ADC) samples can be submitted in liquid or as lyophilized material.
The chromatographic purity should be >90%
Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS analysis can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents.
Minimum amount ~ 100 micrograms of ADC
Put the ADC into microcentrifuge tube
Use Eppendorf Safe Lock tubes, or similar tubes from your lab
Lyophilize or speed vac the ADC to a solid sample to ensure stability during shipment
Alternatively, freeze the liquid sample for cold shipment
Please note that MW determination of intact ADC’s by mass spectrometry CANNOT be conducted on samples from gels or PVDF membranes!