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Antibody-Drug Conjugate MS

ADC basics

ADCs combine the targeting ability of monoclonal antibodies (mAbs) with cytotoxic agents to selectively kill cells. The selectivity of ADCs makes them an attractive oncology therapeutic to kill tumor cells while sparing normal cells, thereby increasing efficacy and drug tolerance. An ideal ADC would include a highly selective antibody, a biological system stable linker between the mAb and small molecule, and significantly lower cytotoxicity of conjugated small molecule relative to the released small molecule.

Critically important ADC characteristics:

  1. Amino acid sequence of the mAb
  2. Post-translational modifications of the mAb
  3. Amino acid-specific conjugation of the linker or drug to the mAb
  4. Average drug load on the mAb or DAR (Drug Antibody Ratio)

Mass spectrometry analysis is capable of providing the data to describe the characteristics above during discovery, development and production stages of ADC design.

Why should you consider Novatia and ESI/LC/MS for characterization of your ADCs?

Novatia uses electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) to characterize your ADC. Our expert team has more than 38 years of biological mass spectrometry experience. We offer rapid turnaround of high quality MS data and DAR calculations to meet the needs of your ADC project.

LC/MS services

Features and Benefits of Novatia ADC LC/MS ServicesApplications
Excellent mass accuracy, typically +/- 0.01% (i.e., 1 Da in 10 kDa).Identify or Confirm MW and drug load (DAR) of target ADC, with and free of glycans.
Methods for detailed profiling and high-throughput MW determination are available.Identification of attached glycan and/or small molecule, and site of attachment.
Unparalleled ESI spectral deconvolution technology: ProMassIdentification of sequence mutations, post-translational modifications and small molecule attachment.
mAbs can be analyzed in high concentrations of salts, buffers and other contaminants.
Backed by over 38 years of biological mass spectrometry experience


Sample analysis and DAR calculation

DAR calculation can be determined following mAb analysis under standard antibody treatments such as reduction, Deglycosylated only, Deglycosylated and reduction as well as intact (for more information on sample treatment options, follow the mAb MS link).

The sample data and DAR calculation shown below are that of MSQC8, a commercially available antibody-drug conjugate mimic, consisting of the MSQC4 standard mAb with dansyl fluorophores attached to cysteine residues. The reported average DAR for MSQC8 is 4.2 +/- 0.8, while the average DAR per our methods is 4.17 +/- 0.08. Below are sample LC/MS chromatogram, ESI and deconvoluted spectra from an analysis of MSQC8, following deglycosylation and reduction treatments.

The heterogenous addition of the drug, to produce ADC, MSQC8 (top), yields a more complex chromatogram compared to the MSQC4 mAb standard (bottom).

The most intense peak, consists of the 1X drug addition and the unmodified heavy chain of the MSQC4 mAb.  Below is the ESI and deconvoluted spectra of the peak at 8.737 min.

The next intense peak, consists of the 2X and 1X drug additions to heavy chain of the MSQC4 mAb.  Below is the ESI and deconvoluted spectra of the peak at 9.051 min.

The final heavy chain peak, consists of the 3X, 2X, 1X drug additions and unmodified, in descending order.  Below is the ESI and deconvoluted spectra of the peak at 9.235 min.

The most intense light chain peak, consists of the 1X drug addition and unmodified forms.  Below are the ESI and deconvoluted spectra of the peak at 8.087 min.

The final light chain peak consists largely of the unmodified form.  Below are the ESI and deconvoluted spectra from the peak at 7.819 min.