ESI-MS sprays antibody-drug-conjugate (ADC) sample in liquid phase through a capillary at high voltage into the MS instrument. This measures the mass over charge ratio (m/z). For ADCs the electrospray process results in multiply protonated molecules with a distribution of ion species at m/z range from 1-3000. A deconvolution algorithm calculates the mass of the intact non-protonated antibody to determine the mass of the ADC.
The ESI process requires a quete pure preparation of the sample, without interfering salts or detergents. Reversed phase HPLC (Agilent 1200 system) using a short C8 column purifies the sample. Then a Q-TOF mass spectrometer (Bruker Maxis Impact system) performs the analysis. We determine the mass of the sample 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 ADCs we analyze the sample in the intact form and after reduction with DTT. The drug-to-antibody ratio (DAR) distributions is then determined from the deconvoluted data.
A specific protease cleaves the ADC into smaller peptides. These are then analyzed by mass spectrometry and the observed peptides correlated to the antibody amino acid sequence.
The identified peptides in the peptide map thus confirm 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 typically gives a sequence coverage of 30-90%. Two or more proteases increases the coverage to 95-100%.
Detailed characterization of ADCs requires careful investigation of the antibody sequence. This is done to select the best proteases, and choose between nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides. The difference between the two techniques relates 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 includes a UV-trace of the chromatogram.
Peptide mapping by nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with C18 capilary column. It connects to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument runs either with a short or long gradient. The Q-TOF is in data dependent analysis mode (DDA), switching between MS and MS/MS mode.
The analysis provides a Base peak chromatogram. In addition, we collect all MS/MS data for peptides with mass/charge ratio from 200-1600 dalton.
High-flow LC-MS/MS with UV
We perform peptide mapping using high-flow LC-MS/MS on a Agilent 1200 system. It runs at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument runs 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 collects all MS/MS data for peptides with mass/charge ratio from 200-1600 dalton.
Sample Preparation Guidelines
Submit Antibody-Drug-Conjugate (ADC) samples 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 most often works on samples containing small amounts of salts, urea or detergent, but you obtain the best result 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 ADCs by mass spectrometry CANNOT be conducted on samples from gels or PVDF membranes!