Structural Characterization of Antibody Drug Conjugates(ADCs) by a Combination of Intact, Middle-up and Bottom-up
Approaches using CESI-MS
Stephen Lock,1 Nassur Said,2 Rabah Gahoual,2 Alain Beck,3
Yannis-Nicolas François,2 and Emmanuelle Leize-Wagner2
1 SCIEX,UK
2 Laboratoire de Spectrométrie de Masses des Interactions et des Systèmes (LSMIS),
Université de Strasbourg, Strasbourg, France
3 Centre d’Immunologie Pierre Fabre, Saint-Julien-en-Genevois, France

Introduction
Antibody drug conjugates (ADCs) represent a rapidly growingclass of biopharmaceuticals. ADCs are formed by the conjugationof an active drug species to a monoclonal antibody,1 and theyoften result in a distribution of products containing varying
numbers of active drugs bound at different locations aroundthe antibody.
Capillary electrospray ionization (CESI) is the integration ofcapillary electrophoresis (CE) and electrospray ionization (ESI)
into a single process in a single device (Figure 1). CESI-MS

Figure 1. OptiMS - Ultra low flow ESI Interface.

2operates at low nL/min flow rates and offers several advantages,Materials and methods

including increased ionization efficiency and a reduction inChemicals: All chemicals were reagent grade and wereion suppression. CESI-MS separates analytes by their chargeand size, and it is therefore a separation mechanism thatis complementary to more traditional techniques, such as
reversed-phase LC.
This document summarizes the work recently published by theresearch group at LSMIS at the Université de Strasbourg.3 In
this application note, we will show how CESI-MS can be used
to characterize a gold standard ADC, brentuximab vedotin(BV). BV is a cysteine-linked mono-methyl auristatin E (MMAE)
monoclonal antibody (mAb), which was attached without
disrupting the heavy and light chain linkages of the mAb. We will
3

purchased from Sigma Aldrich or alternative suppliers.3
Brentuximab vedotin was produced by Millennium
Pharmaceuticals/Takeda.
Sample Preparation: BV was buffer exchanged with 200 mM
ammonium acetate buffer (pH 7.0) using Amicon filters before
intact analysis.3
For middle-up analysis, BV was cleaved at the hinge region
by proteolysis using IdeS (FabriCATOR, Genovis) to obtain
Fc/2 fragments and an F(ab’)2 fragment. After digestion wascompleted, the sample was again buffer exchanged into 200 mM

show how CESI-MS is used as a nano-spray infusion device toammonium acetate buffer (pH 7.0) before CESI-MS analysis.

identify the drug antibody ratio of the sample. We will also show

For bottom-up analysis, a sample of desalted IdeS cleaved

how CESI-MS can be used to study the “middle-up” structure forthis protein, for example, by analyzing the light chain, Fab andF(ab’)2 subunits. Finally, a bottom-up analysis of a tryptic digest
will be performed by CESI-MS to fully sequence the ADC.

ADC, which had undergone buffer exchange and was diluted
with RapiGest (Waters) and reduced with dithiothreitol (DTT).
Before enzymatic digestion, 10% of acetonitrile was added tothe sample. The reduced protein was then digested with trypsin
overnight, isopropanol (40%) and formic acid (1% v/v) wereadded and the final solution diluted using ammonium acetate(50 mM, pH 4.0) to produce a final concentration of 2.2 μM.3
p1

CESI-MS method: For the analysis of intact and digested
samples, a bare fused silica OptiMS cartridge (30 μm ID x 91 cm)
from SCIEX was used and was thermostatted using recirculating
liquid coolant regulated at 20°C used. For the analysis of tryptic
digests, the sample was injected hydrodynamically (10 psi,60 s) and peptides were separated using conditions shown inTable 1 with a background electrolyte of 10% acetic acid. For MS

Important:• A separation current above 5 μA might cause permanent
damage to the separation capillary.
• Generally, please do not apply >2000V to generateelectrospray as it may result in capillary damage.

analysis of tryptic digests, a SCIEX TripleTOF® 5600+ LC-MS/Results

MS System was fitted with the NanoSpray III Ion Source. Gas

When CESI is used as a nano-spray infusion device, the sample®

1 and 2 were not used, the curtain gas was set to 5 psi andthe temperature of the interface was set at 150 °C. Ionization
at these very low-flow rates occurred by simply applying theionspray voltage (1,450 V). MS data was acquired using a TOF
survey scan (m/z 100–2,000 amu), which triggered MS/MSacquisition (m/z 100–2,000 amu).
PressureDirection(psi)is simply pushed to the MS detector at low nL/min flow rates.4
Desalted intact BV was analyzed in this mode to confirm themolecular weight of this ADC and measure the drug to antibody
ratio (DAR), which is the drug loading on the antibody andtypically ranges from 0 to 8. An example of the data achieved
is shown in Figure 2, which gives an overview of the analysisof BV by CESI-MS.

ActionTime(min)Voltage(kV)

SolutionRinse3.5

100Forward00.1 M NaOH

Rinse

1.5100Forward0Water

Rinse

3100

Forward00.1 M HClRinse

5100

Forward010% Acetic acid

Rinse

275Reverse

010% Acetic acid

Rinse

60s

10Forward

0Sample VialInjection

10s

5Forward

010% Acetic acid

Separation

350.1

Forward20

10% Acetic acid

Voltage

210Forward

1

10% Acetic acid

Table 1. CESI separation conditions used for the analysis of tryptic digests.

For nano-spray infusion, the CESI capillary was flushed with
10% acetic acid (5 min, 50 psi) and then with sample (5 min,50 psi), and MS data was acquired at 5 psi. After each analysis,
the capillary was flushed with 10% acetic acid (10 min, 50 psi) toprevent carryover. For these nano-spray infusion experiments,
MS data was acquired using a maXis 4G system (Bruker).
The maXis system was optimized for each analysis using theactual sample and ion funnels with values that ranged from
300–400 Vpp. The electrospray voltage ranged from 1,200–
1,800 V, drying gas was set at 1.5 L/min and the source
temperature was set at 150 °C.

Figure 2. Overview of brentuximab vedotin structural characterization
using sheathless CE-MS. (a) Native MS infusion for average DAR
determination and drug loading distribution assessment. (b) Middle-up
and bottom-up analysis.

Based on the charge state of the deconvoluted mass spectrumof the intact BV shown in Figure 2, the average DAR value wasbetween 3.8 and 3.9. A distribution of baseline resolved species
of BV was observed with masses that corresponded to theintact mAb linked with 0 to 8 payloads of the drug. The massaccuracies of intact BV were in total agreement with the results
reported in the literature.3

p2

Using the same experimental conditions as the intact analysis, aCESI-MS has been previously used for the analysis of tryptic

middle-up sample was analyzed to elucidate the location of thedigests of mAbs.5 The next set of experiments performed on

drug loading on the mAb, which had been cleaved at the hinge

BV were the bottom-up analysis of a tryptic digest to determine

region (Figure 2). The raw data highlighted multiply charged

the location of post-translational modification (PTM) sites,

protein peaks in 3 separate regions across the mass range ofthe location of the drug linked to the mAb and the amino acid

2,000–6,000 amu (Figure 3). When deconvoluted, the multiplysequence. The tryptic digestion protocol had been adapted fromcharged protein envelopes depicted in Figure 3 showed that thethe classical approach to improve the overall digestion, which

free light chain (LC) protein subunit (MW = 25040.1 ± 0.1 Da) had

was affected by the presence of the drug molecules bound to

1 drug molecule linked to it as well as 2 glycation modifications.

the mAb.3 CESI-MS analysis took less than 35 min. Due toThe smaller Fab subunit (approximately 48–55 kDa) had 0–4

the sensitivity and efficiency of the CESI-MS analysis, 100%molecules of drug linked to the protein and was present as asequence coverage could be obtained for BV in a single injection

dimer. The F(ab’)2 subunit (approximately 97–108 kDa) had 0–8

with the identification of the peptides based on their accurate

drug molecules attached to the protein subunit. As the drug units

molecular weight as well as sequence data from MS/MS analysis.

were spread across multiple regions of the mAb, the IdeS middle-The presence of the organic solvent in the sample preparation

up sample could not be used to calculate the DAR for ADCs, but

did not seem to have an effect on the separation of the tryptic

does give information about the location of the drug molecules

peptides demonstrated by the detection of small (3 amino acids)

within BV.

to large (63 amino acid) peptides.
Modified peptides (including N-glycopeptides) were detected in
the analysis, for example, TKYPREEQYN297STYR was observed
to have 11 glycoforms. Regarding drug-loaded-peptides, 4 weredetected (Figure 4), which was aided by the presence of organic
solvent in the sample preparation to prevent the loss of thesehydrophobic modified peptides.

Figure 3. (a) MS spectra corresponding to native MS nano ESI infusionof middle-up BV. (b) Charge state deconvoluted mass spectra of(1) LC- drug conjugated subunit, (2) Fab subunits with the incorporationof 0 to 4 drug molecules and Fc/2 homodimers and (3) F(ab’)2 subunits
with the incorporation of 0 to 8 drug molecules.

Figure 3. MS and MS/MS spectra of drug-loaded peptides. (a) [GEC]- 1 drug, (b) [SCDK] -1 drug, (c) [THTCPPCPAPELLG] - 1 drug and(d) [THTCPPCPAPELLG] - 2 drug moleculesp3

Identification of these drug containing peptides was confirmed
by MS/MS data analysis, which highlighted the presence ofseveral diagnostic drug fragment ions. A single drug loaded
peptide was located on the light chain of the mAb (GEC),
and the other peptides were present on the heavy chain. TheTHTCPPCPAPELLG peptide actually had the potential ofcontaining 2 drug molecules, and these 2 different peptides havebeen separated by CESI-MS.

Conclusions
A CESI-MS protocol for structural characterization of ADC
molecules has been developed. By using CESI-MS in 2 different
modes, several properties of an ADC could be confirmed,including:• DAR ratio calculation using native conditions• Drug distribution on the F(ab’)2 and Fc/2 using nano-spray
infusion CESI-MS analysis of an IdeS digested middle-up
sample using native conditions• 100% sequence coverage of the ADC and identification of
the drug location as well as the location of other PTMs
• Characterization of drug loaded peptides by analysis ofMS/MS spectra

References
1. Chari, R.V.J. Targeted cancer therapy: conferring specificity
to cytotoxic drugs. Accounts Chem. Res. (2008), 41: 98–107.
2. Busnel, J.-M. et al. High Capacity Capillary ElectrophoresisElectrospray Ionization Mass Spectrometry: Coupling aPorous Sheath-less Interface with Transient-Isotachophoresis.
Anal. Chem. (2010), 82: 9476–9483.
3. Said, N., Gahoual, R., Kuhn, L., Beck, A., François, Y.-N.,
Leize-Wagner, E. Structural characterization of antibody
drug conjugate by a combination of intact, middle-up andbottom-up techniques using sheathless capillary electrosprayTandem mass spectrometry as nanoESI infusion platform
and separation method. Analytica Chimica, Acta (2016).
http://dx.doi.org/10.1016/j.aca.2016.03.006.
4. Gahoual, R., Busnel, J.M., Wolff, P., Francois, Y.-N.,
Leize-Wagner, E. Novel sheathless CE-MS interface as an
original and powerful infusion platform for nanoESI study:
from intact proteins to high molecular mass noncovalent
complexes. Anal. Bioanal. Chem. (2014), 406: 1029–1038.
5. Gahoual, R., Busnel, J.M., Beck, A., Francois, Y.-N.,
Leize-Wagner, E. Full antibody primary structure andmicrovariant characterization in a single injection usingtransient isotachophoresis and sheathless capillaryelectrophoresis-tandem mass spectrometry. Anal. Chem.
(2014), 86: 9074–9081.

For further information on this topic we would like to referreaders to the full scientific publication on which this applicationnote is based.3

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