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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 (click the link to view lysine-drug conjugate peptides from ADC standard)
  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 (see our 2019 ASMS poster).

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 calculated DAR shown below are that of MSQC4-AF488, and MSQC8, antibody-drug conjugate mimics, consisting of the MSQC4 standard mAb with dansyl fluorophores attached to lysine or cysteine residues, respectively. Analysis examples include intact and pre-treated samples under either denaturing or native LC conditions*.

*Denatured LC conditions expose samples to acidic (pH~2) and organic solvents during separation. Native conditions expose samples to aqueous salt buffers (pH~6-7) to favor maintaining stable protein fold.

Denatured Intact ADC analysis

The heterogenous addition of AF488, to produce the ADC lysine mimic (bottom), yields a broad later-eluting peak compared to the MSQC4 mAb standard chromatogram (top).

The baseline subtracted ESI spectrum shown below consists of all drug addition states of lysine conjugated ADC mimic under the 3.870 minute elution peak.  

The deconvoluted mass spectrum (below) demonstrates the ability of ProMass to accurately calculate intact drug-conjugate states in the heterogeneous MSQC4-AF488 sample, with no observed mass artifacts.

The expanded deconvoluted spectrum (below) clearly resolves the individual glycoform clusters, which are separated by a single AF488 molecule addition per cluster. The DAR for the MSQC4-AF488 shown is 5.7.

Analysis of reduced and PNGase F treated ADC

The heavy and light drug-conjugated protein chains elute in two distinct peaks, following reduction of cysteine disulfide bridges and removal of N-linked glycans.

Below are the baseline subtracted ESI and deconvoluted spectra under the 5.480 minute peak. The deconvoluted mass spectrum demonstrates the ability of ProMass to accurately calculate drug-conjugate states of the heterogeneous MSQC4-AF488 heavy chain, with no observed mass artifacts. The DAR for the heavy chain of the MSQC4-AF488 is 3.7.

The baseline subtracted ESI and deconvoluted spectra under the 5.180 minute peak are shown below. The DAR for the light chain of the MSQC4-AF488 is 1.7.

The overall DAR for the reduced and PNGase F treated MSQC4-AF488 sample shown is 5.4.

Native intact ADC analysis

Cysteine conjugated ADCs sacrifice structural stability by losing disulfide bridges to bind drug molecules. The loss of disulfide bridges reduces the energy required to separate and unfold the protein chains. Native LC conditions favor the ADC maintaining its most stable fold compared to the denatured conditions described in the previous examples. Below are the ESI and deconvoluted spectra MSQC8 cysteine conjugated ADC mimic analyzed under native LC conditions:

The folded state of the protein reduces the number of sites capable of picking up a positive charge, this results in the higher m/z peaks observed in the spectrum above.

The deconvoluted mass spectrum (above) demonstrates the ability of ProMass to accurately calculate intact drug-conjugate states in the heterogeneous MSQC8 sample, with no observed mass artifacts. The DAR value of MSQC8, shown above, is 4.9.

Native analysis of PNGase F treated ADC

Removal of glycans from cysteine conjugated ADC reduces the heterogeneity of the ADC sample. Despite the loss of structural stability as a consequence of sacrificing disulfide bridges, native LC conditions favor the protein chains remaining folded and bound together. Below are the ESI and deconvoluted spectra of deglycosylated MSQC8 cysteine conjugated ADC mimic analyzed under native LC conditions:

The fold of the protein reduces the number of sites available to carry a positive charge, this results in higher m/z charge states shown above.

The DAR value of PNGase F treated MSQC8, shown above, is 4.9.