Antibody-drug conjugates

  • Detailed characterization of ADC
  • Drug-to-antibody ratio (DAR) profiling
  • With or without deglycosylation

Do you want to know the exact number and position of drug molecules attached to the antibody in your ADC?

Antibody-drug conjugates (ADCs) are normally site-directed cancer drugs that consist of an antibody with one or more cytotoxic drug molecules attached (conjugated) to it.

The antibody has a very specific binding to cancer cells, thus directing the drug to the exact target of the treatment. Most often, an antibody-drug conjugate is a mixtures of antibodies with 1, 2, 3 or more attached drug molecules in various positions.

Alphalyse offers analytical services in order to determine the exact distribution of drug-to-antibody ratio (DAR) in your ADC.

Our service for determination of DAR distribution includes:

  • Exact masses are determined by UV LC-ESI-MS
  • Distribution of intact ADC is determined
  • Exact mass distribution of de-glycosylated ADC
  • Calculation of DAR distribution in percentage based on MS peak intensity

Innovation collaboration supported by Eurostars.

Alphalyse collaborates with ChromaCon in Switzerland. Together, we develop purification technologies and analytical services for separation and detailed characterization of antibody-drug conjugate (ADC) with various drug-to-antibody ratio (DAR) distributions. This innovation collaboration is supported by Eurostars.

MoreLess details

The process:

  • 1 Order the standard analysis or contact us for a custom project proposal
  • 2 We find the number and position of drug molecules attached to the antibody
  • 3 You receive a report with calculated DAR distribution in percentage
  • 4 Follow-up by phone or email

More information:

Find out how the analysis was developed

Case: Collaboration with Chromacon Improving cancer drugs based on ADCs

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Knowledge center

More information

We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

Technical details

DAR Distribution

ESI-MS sprays the 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 multiple protonated molecules with a distribution of ion species at m/z range from 1 to 3000 Da. A deconvolution algorithm calculates the mass of the intact non-protonated antibody to determine the mass of the ADC.

The ESI process requires a 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.

Peptide mapping

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.

Nano-flow LC-MS/MS

Peptide mapping by nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with a C18 capillary 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 m/z ratio of 200-1600 Da.

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

Sample requirements for ADC analysis

Submit Antibody-Drug-Conjugate (ADC) samples in liquid form 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 and urea, 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 high-quality 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.

Meet the experts

For this type of analysis our experts include:

Do you need help?
Jakob Bunkenborg

PhD in Analytical Chemistry

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