LC-MS Method Development for Antibody Drug Conjugates Using a Non-Toxic ADC Mimic

By: Brian Gau, Ben Cutak, Pegah Jalili, Kevin Ray, MilliporeSigma, St. Louis, MO


ADCs conjugated at Cys residues of inter-chain disulfide bonds give mAbs bearing 0-4 pairs of drug-linkers. Characterizing this distribution is paramount to understanding ADC efficacy and process control. Here we show how LC-MS and immunopurification methods may be optimized using a non-toxic surrogate of the ADC, an “ADC-mimic”, that behaves very similarly to approved Cys-linked ADC therapeutics. Its dansyl-cadaverine-SMCC mimic-linker imparts a 668 Da mass shift upon conjugation to Cys residues.

ADC Mimic




Native SEC-MS
    Column / T:     Tosoh TSK Gel SW3000XL, 300 x 2.0 mm, 4 μm / 25 °C
    Isocratic flow:  100 mM ammonium acetate, pH 7, 70 μL/min
    Handling:         PNGase F 37 °C 18 h

Reduced SEC-MS
    Column / T:     Tosoh TSK Gel SW3000XL, 300 x 2.0 mm, 4 μm / 25 °C
    Isocratic flow:  30% ACN 0.1% TFA, 70 μL/min
    Handling:         PNGase F 37 °C 18 h; 50 mM DTT 37 °C 1 h

Antibody subunit RPLC-MS
    Column / T:     BIOshell A400 Protein C4, 300 μm x 15 cm, 3.4 μm / 65 °C
    Phases:          Water, 0.1% TFA; ACN, 0.1% TFA
    Gradient flow: Desalt 5% organic; 31%-32.5% organic / 4 min; 35%-38% organic /10 min, 40 μL/min
    Handling:        IdeS 37 °C 3 h; 7.4 M GndCl / 100 mM DTT 50 °C 1 h

Immunopurification from serum
    Affinity:           Goat Anti-Human Fc, biotin conjugate
    Serum:           Cynomolgus monkey
    Beads:            Streptavidin magnetic beads
    Buffers:          TBST wash; pH 2.8 IgG elute

   General purification scheme:

General purification scheme


Native SEC-MS

Native SEC-MS

Reduced SEC-MS

Reduced SEC-MS


Antibody subunit RPLC-MS

Antibody subunit RPLC-MS

Figure 1. ADC-mimic characterization by native, reduced, and IdeSreduced (antibody subunit) LC-MS methods. Each annotation counts the number of mimic-linkers conjugated to the protein or subunit.


50 °C Mimic Serum Stability

Mimic Serum Stability


Figure 2. ADC-mimic serum stability. 400 μL of monkey serum was spiked with 8 μg ADC-mimic and incubated at 37 or 50 °C for 0.5 – 18 h. The mimic was recovered by immunopurification and concentrated by 10000 MWCO filtration. DAR was analyzed by reduced SEC-MS. Loss of mimic-linker was only observed with serum samples, not buffered samples (data not shown).



We have developed a non-toxic ADC-mimic, with Cys-conjugation to dansyl-cadaverine-SMCC. By native SEC-MS, reduced SEC-MS, and antibody subunit RPLC-MS, its DAR and physiochemical properties are comparable to approved Cys-linked ADC therapeutics. The ADC-mimic was used to develop an in vitro stability assay. We find that the mimiclinker experiences deconjugation in cynomolgus monkey serum similar to Cys-linked ADCs (Wei C. et al, Anal Chem, 2016, 88, 4979-4986).


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