Application Note

A streamlined drug-to-antibody ratio
determination workflow for intact
and deglycosylated antibody-drug
conjugates
Using the Agilent AssayMAP Bravo andAgilent 6545XT AdvanceBio accurate-mass
quadrupole time-of-flight LC/MS system
Authors
Jerry Han, Zach Van Den
Heuvel, and Steve Murphy
Agilent Technologies, Inc.
Santa Clara, CA 95052

Introduction
Antibody drug conjugates (ADCs), a fast-growing class of biomolecule, comprisea monoclonal antibody (mAb) conjugated to a small molecule drug throughsynthetic linkers. The ratio of the conjugated drug to mAb (drug-to-antibody ratioor DAR) is one of the critical quality attributes for ADC development because it canaffect efficacy and safety1,2. Monitoring the changes in DAR distribution in vivo canprovide important insights into the biotransformation of ADCs (for example, drug
deconjugation)3,4. However, measurement of average DAR and DAR distribution
in circulation by LC/MS requires tedious and error-prone sample preparation
procedures such as affinity capture of ADCs from complex matrices (for example,
serum/plasma) and deglycosylation of the purified antibodies. To address thechallenge of this complex sample preparation workflow (Figure 1), this Application
Note automates affinity purification and deglycosylation on the Agilent AssayMAP
Bravo platform5,6. To enable isolation of ADCs with all DAR species (0 to N) inserum, affinity purification was performed using the target antigen. Deglycosylation
was used to simplify the mass spectrum of the purified antibodies by eliminating
the various glycoforms, and to improve the detection limit. The whole automatedprocess, including antigen immobilization, affinity purification, and on-cartridge
deglycosylation, can be completed in approximately five hours, with minimalhands‑on time.
This Application Note demonstrates a streamlined ADC DAR workflow using
the AssayMAP Bravo, with an Agilent 6545XT AdvanceBio LC/Q-TOF, andAgilent BioConfirm DAR Calculator software. The workflow is simple to implement,
highly reproducible, scalable, and minimizes hands-on time.

Experimental
Materials
Recombinant human HER2 extracellulardomain (ECD) was purchased from
ACRO Biosystems (Newark, DE;
HE2-H5225). Trastuzumab emtansine(T‑DM1) was manufactured by
Genentech. EZ-Link Sulfo-NHS-LC‑Biotin,
Pierce Biotin Quantitation Kit, and Zeba
Spin Desalting Columns, 7K MWCO,
0.5 mL were from Thermo Fisher
Scientific (Grand Island, NY; 21327,
28005 and 89882). Rat serum wasfrom Sigma-Aldrich (St. Louis, MO;
R9759). Rapid PNGase F was from New
England BioLabs (Ipswich, MA; P0711).
AssayMAP Streptavidin cartridges werefrom Agilent Technologies (Santa Clara,
CA; SA-W, G5496-60010). 10X HEPES
buffer, 100 mM, 1.5 M NaCl, pH 7.4(HBS-N) was from GE Healthcare Life
Sciences (Marlborough, MA; BR100670).
5 M NaCl was from Promega (PAV4221).
1 M Tris-HCl, pH 8.0 was from Quality
Biological (351-007-101). All otherchemicals were from Sigma-Aldrich(St. Louis, MO).

Biotinylation of HER2 ECD
To obtain a final concentrationof 2 mg/mL, 1 mg of lyophilizedrecombinant HER2 ECD wasreconstituted in 500 μL of Milli-Q
water. Half (0.5 mg in 250 µL) of thereconstituted HER2 ECD was biotinylatedusing EZ-Link Sulfo-NHS-LC-Biotin per
the manufacturer’s instructions. The
molar fold excess of biotin was 250, andthe incubation was one hour at room
temperature. After labeling, the extra
Sulfo-NHS-LC-Biotin and Sulfo‑NHS(by‑product) were removed by gel
filtration using Zeba Spin Desalting
Columns. The biotinylation levels in thelabeled HER2 ECD were estimated usinga Pierce Biotin Quantitation Kit, per the
manufacturer’s instructions.

2

Sample preparation

LC/MS

DAR calculation

Figure 1. ADC DAR characterization workflow.

Automated antigen immobilization,affinity capture, and on-cartridge
deglycosylation
Automation setup: The biotinylated
antigen immobilization, ADC purification,
and ADC deglycosylation were carriedout using the Immobilization, Affinity
Purification, and On-Cartridge Reaction
Applications on the AssayMAP Bravo
platform. Figures 2–4 show the decklayouts and labware used for theseprocedures. Table 1 summarizes the
samples and reagents. The solution
volumes required for each application
were determined using their respective
Reagent Volume Calculator. To prepare
the platform for the Immobilization
Application, 48 (six columns) ofstreptavidin (SA-W) cartridges weretransferred from the cartridge rack
(deck 6 on the AssayMAP Bravo) to theseating station (on deck 2) using the
Cartridge Transfer v1.0 utility (Table 2B).
SA-W cartridge conditioning: SA-W
cartridges were primed with 100 µL of
1 % formic acid (FA) at a flow rate of
300 µL/min to clear any air entrappedin the resin. This was followed by anequilibration step with 100 µL of 1 % FA
at a flow rate of 5 μL/min. This step
removes any streptavidin that is weakly

bound to the cartridge resin and would
come off in the elution step, where
it could interfere with downstream
analysis. Figure 2A shows theapplication settings. The SA-W cartridge
conditioning and the biotinylated antigen
immobilization runs can be combined
into a single run (see Wu; et al.7), butwere separated here for simplicity.
Biotinylated HER2 ECD immobilization:
To generate the affinity (bait)
cartridges for affinity purification, 3 µgof the biotinylated HER2 ECD were
immobilized on each of the 48 individual
streptavidin cartridges (SA-W) using
the Immobilization v1.0 application.
Figure 2B shows the application settings.
The cartridges primed in the SA-W
condition step were equilibrated with
10 bed volumes of binding buffer (50 µL,
10 mM HEPES, 150 mM NaCl, pH 7.4) at
10 µL/min. Then, 3 μg of the biotinylated
HER2 ECD in 100 µL of binding buffer
were loaded into each equilibrated
cartridge at a flow rate of 5 µL/min. Afterthe biotinylated HER2 ECD was loaded,
the cartridges were washed with 10 bed
volumes (50 µL) of binding buffer at
10 µL/min. The cartridges were thenready for the affinity purification step.

A

B

Figure 2. Immobilization application settings. A) Conditioning SA-W cartridges; (B) immobilization of
biotinylated HER2 ECD on SA-W cartridges.

3

Figure 3. Affinity purification application settings.

Figure 4. On-cartridge reaction application settings.

4

ADC affinity purification: To obtain
different concentrations of T-DM1 insera from 20 to 0.3125 μg/mL, plus a
no-T‑DM1 control (Table 2A), various
amounts of T-DM1 (Trastuzumab
emtansine, an ADC that targets HER2
ECD) were spiked into clarified rat sera.
The dilution set was also preparedin water for direct injection onto the
LC/MS. For affinity capture, the serum
samples were further diluted 1:1 withbinding buffer (10 mM HEPES, 150
mM NaCl, pH 7.4) immediately before
loading them onto the biotinylated HER2
ECD‑SA-W cartridges. Affinity purification
was carried out using the Affinity
Purification v1.0 application. Figure 3
shows the application settings. Briefly,
100 µL of diluted rat serum containing
T-DM1 was passed through the affinity
cartridge at a flow rate of 2 µL/min.
Each row of cartridges (n = 6) was
loaded with a different concentration of
T-DM1 (see Table 2B for plate layout).
After the samples were loaded, thecartridges were sequentially washed with
10 bed volumes (50 µL) of the high-salt
HEPES buffer (50 µL, 10 mM HEPES,
1 M NaCl, pH 7.4), then 10 bed volumes(50 µL) of binding buffer at a flow rateof 10 µL/min. The cartridges were thenready for deglycosylation.
Deglycosylation: To compare DAR
analysis with and without glycans
present, half of the cartridges withpurified T-DM1 were incubatedwith PNGase F (columns 1–3),
and the other half of the cartridges(columns 4–6) were incubated with
deglycosylation buffer without PNGase F(no deglycosylation controls) (Table 2B).

Table 1. Samples and reagents.
Samples and reagents
Wash station

Milli-Q water, for all protocols

Immobilization v1.0
SA-W cartridge conditioning
Cartridges

SA-W (G5496-60010)

Prime

1 % FA

Equilibrate

1 % FA

Biotinylated HER2 ECD immobilization
Cartridges

Conditioned SA-W

Equilibrate and cartridge wash buffer

10 mM HEPES, 150 mM NaCl, pH 7.4

SampleBiotinylated HER2 ECD

Affinity purification v1.0
Cartridges

Biotinylated HER2 ECD-SA-W

SampleRat sera spiked with different concentrations of ADC

Cartridge wash 1

10 mM HEPES, 1 M NaCl, pH 7.4

Cartridge wash 2

10 mM HEPES, 150 mM NaCl, pH 7.4

On-cartridge reaction v1.0
Cartridges

ADC-bound biotinylated HER2 ECD-SA-W

Equilibrate and chase buffer

20 mM Tris, pH 8.0

Deglycosylation enzyme

PNGase F

Cartridge wash 1

10 mM HEPES, 1 M NaCl, pH 7.4

Cartridge wash 2

0.003 % FA

Elution buffer

1 % FA

Neutralization solution

0.5 % ammonium hydroxide

Table 2. Experimental design. (A) Serial dilutions and loading amounts and (B) plate layout for affinity
purification and deglycosylation.

A

B

Serial dilutions and loading amounts
ADC concentration in rat
serum (µg/mL)

Serum volume used(µL)

Mass loaded on
cartridge (ng)Mass loaded on LC
column (ng)

20

50

1000

200

10

50

500

100

5

50

250

50

2.5

50

125

25

1.25

50

62.5

12.5

0.625

50

31.25

6.25

0.312550

15.625

3.125

0

50

0

0

Plate layout for affinity purification and deglycosylation (ADC concentration in rat serum (µg/mL))
1

2

3

4

5

6

A

20

20

20

20

20

20

B

10

10

10

10

10

10

C

5

5

5

5

5

5

D

2.5

2.5

2.5

2.5

2.5

2.5

E

1.25

1.25

1.25

1.25

1.25

1.25

F

0.625

0.625

0.625

0.625

0.625

0.625

G

0.31250.31250.31250.31250.31250.3125

H

0

0

0

0

0

0

Deglycosylation

No deglycosylation5

On-cartridge deglycosylation was
carried out using the On-Cartridge
Reaction v1.0 application. Figure 4
shows the application settings. Briefly,
the T-DM1‑bound cartridges were
equilibrated with 10 bed volumes(50 µL) of the deglycosylation buffer(20 mM Tris, pH 8.0) at a flow rate of
10 µL/min. For the reaction step, 4 µL of
the heated PNGase F (columns 1–3) ordeglycosylation buffer (columns 4–6)
was aspirated from deck position 4(a Peltier heater/cooler) onto each
T-DM1‑bound cartridge. This step was
followed by a 2-µL slow aspiration over
30 minutes. The software always loads
the first 4 µL of the reaction volume at
10 µL/min, then loads the remaining
volume (2 µL in this example) overthe duration time set on the form.
During the reaction, the cartridgetips were in contact with the solution
in the wells to maintain an elevatedtemperature inside the cartridge byconductance. To give a temperatureof approximately 37 °C inside thecartridges (reaction temperature), thePeltier temperature was set at 45 °C.
Although only 6 µL of solution was
aspirated through the cartridges, 12 µL
of 1:12 diluted PNGase F (columns 1–3)
and deglycosylation buffer alone(columns 4–6) was aliquoted into the
PCR plate at position 4 at the beginningof the run. Volume more than theamount aspirated through the cartridgeis necessary due to volume loss from
evaporation, and the need to haveat least 3 µL per well throughout thecourse of the run to maintain contact
between the bottom of the well and thetip of the cartridge for heat transfer.
The amount of excess volume required
depends on the temperature setting
and the reaction time. To collect thereleased glycans remaining in the
cartridges after the reaction, 25 µL of
deglycosylation buffer (reaction chase)
was aspirated through each cartridge
into the syringe probes at a flow rate of
5 μL/min. The combined flow-throughs(reaction chase, released N-glycans,6and enzyme solution) collected in thesyringes were then dispensed into theempty flow‑through collection plate. The
cartridges were washed with 10 bed
volumes of the high-salt HEPES buffer(50 µL, 10 mM HEPES, 1 M NaCl, pH
7.4) at a flow rate of 10 μL/min. Thiswas followed by a low stringency washwith 10 bed volumes of 0.003 % FA at
the same flow rate to remove high‑salt
HEPES buffer (not compatible with MS)
and some nonspecifically bound entities.
Deglycosylated (columns 1–3) or intact(columns 4–6) T-DM1 immobilized on
the cartridges were eluted with 15 µL
of 1 % FA at a flow rate of 5 µL/min
into the elution plate that contained
15 µL of 0.5 % ammonium hydroxide
per well (existing collection volume) toimmediately neutralize the samples. The
final volume of each sample was then
30 µL. Finally, the diluted unprocessed
T-DM1 samples in water were added tothe empty wells along with the processed
samples, and the plate was sealed using
an Agilent PlateLoc. The samples werethen ready for LC/MS analysis.

LC/MS analysis
Prepared samples were analyzed
on an Agilent 6545XT AdvanceBio
accurate‑mass quadrupole time‑of‑flight
LC/MS system coupled with an
Agilent 1290 Infinity II UHPLC system.
The LC/MS parameters were set asshown in Tables 3A and 3B. For eachsample, 6 µL out of 30 µL (20 %) were
injected for analysis.

Table 3. LC and MS parameters. A) LC parameters used to separate ADCs. B) MS
parameters used to acquire MS data for intact ADCs.

A

B

Parameter

Value
Agilent 1290 Infinity II UHPLC system

ColumnAgilent PLRP-S 1000Å 8 µm 150 × 2.1 mm (PL1912-3802)

Sample thermostat

5 °C

Mobile phase A

0.1 % FA in water

Mobile phase B

0.1 % FA in acetonitrile

Gradient (segmented)

Time (min)
0–1
1–2
2–4
4–4.5
4.5–5.5
5.5–6
6–8.5

Stop time

8.5 minutes

Column temperature

60 °C

Flow rate

0.4 mL/min

%B
25–25
25–37
37–37
37–50
50–50
50–25
25–25

Agilent 6545XT AdvanceBio accurate-mass quadrupole time-of-flight MS system
Ion mode

Positive ion mode

SourceAgilent Dual Jet Stream

Drying gas temperature

350 °C

Drying gas flow

12 L/minSheath gas temperature

400 °C

Sheath gas flow

11 L/min

Nebulizer

60 psiCapillary voltage

5,500 V

Nozzle2,000 V

Fragmentor voltage

380 V

Skimmer

140 V

Oct RF Vpp

750 V

Acquisition parameters
MS modeHigh (30,000 m/z) mass range
Extended mass range (2 GHz)MS only mode
Mass Range 1,000–5,000 m/z

MS data analysis
Raw data files were analyzed by
Agilent MassHunter BioConfirm
software. Average spectra were extracted
and deconvoluted from the total ion
chromatograms (TICs) at a retention
time between 3.05 and 3.85 minutes.
Table 4 shows the deconvolution
parameters. For DAR analysis, thedeconvoluted spectra were exported andanalyzed by Agilent MassHunter DAR
Calculator. The default parameters in
DAR Calculator were used to determine
the DAR value for intact (nonreduced),lysine-linked ADCs, except the max
DAR peak number was set at 7. To
assess reproducibility and performanceof quantitative analysis, extracted
ion chromatograms (EICs) in the m/z
range 2,000–4,000 were extracted andintegrated using Agilent MassHunter
Qualitative Analysis.

Results and discussion
ADC DAR characterization
To address the challenges of complex
sample preparation for ADC DAR
characterization in serum using theAgilent AssayMAP Bravo platform, acomplete workflow was developed. The
automated sample preparation workflow
includes:•Immobilization of biotinylated target
antigens on AssayMAP streptavidin
cartridges (SA-W)•

Purification of ADCs from complex
matrices•

Deglycosylation of the purified ADCs
on-cartridge

Two result plates were generated:•

A flow-through plate containing thesoluble reaction products, in thiscase N-glycans, which were notanalyzed in this study•

The elution plate, containing
purified and desalted intact anddeglycosylated ADCs, which wereanalyzed by LC/MS

Table 4. Agilent MassHunter BioConfirm parameters for deconvolution.
Parameter

Agilent MassHunter BioConfirm Deconvolute (MS): Protein

Deconvolution algorithm

Maximum entropy

Deconvolution settings

Mass Range: 140–160 KDa
Mass Step: 1.0 Da

Use limited m/z range

2,400–3,600 m/z

Baseline

Subtract baseline
Baseline factor: 7.00

AdductProtonIsotope width

AutomaticThree application protocols in theProtein Sample Prep WorkBench wereused consecutively: Immobilization
v1.0, Affinity Purification v1.0, and
On-Cartridge Reaction v1.0. Using
the application settings in this study(Figures 2–4), the approximate
instrument time for immobilization
was one hour, affinity purification
1.2 hours, and on-cartridge
deglycosylation 1.3 hours. The entire
automated sample process can becompleted in approximately 4.5 hours.
AssayMAP makes it possible tocomplete the preparation of up to
96 samples simultaneously, and begin
LC/MS analysis in one day with minimalhands‑on time.

Preparation of biotinylated antigen
and antigen-coupled cartridges
Recombinant human HER2 ECD(Accession number AAA75493,
extracellular domain: amino acids
Thr23–Thr652), containing the target
binding site for T-DM1, was used as thebait for affinity purification. To avoid
nonspecific endogenous antibody
capture, a biotinylated antigen affinity
purification approach was used withstreptavidin rather than using a protein
A or G cartridge. All DAR species(0 to N) can be isolated by antigen
purification. As the first step to couple
the antigen to AssayMAP streptavidin
cartridges, HER2 ECD was labeledwith Sulfo‑NHS‑LC‑Biotin throughprimary amines. There are 16 potential
primary amine labeling sites in HER2
ECD: 15 lysine residues and the protein

N-terminal end. It was determinedthat 250-fold molar excess resulted inlabeling approximately nine biotin groups
per HER2 ECD molecule in one hour atroom temperature. In this study, 3 µgof bait antigens (~6-fold molar excess
compared to the maximum amountof ADCs to be captured) was used forimmobilization on each cartridge. This
mass of affinity purification ligand
was far below the binding capacity of
the SA-W cartridge but was chosen tominimize the consumption of affinity
ligand while still enabling high levels oftarget recovery.

Affinity purification of ADCs
from serum
To mimic the kinds of in vivo ADC
samples that are often analyzed during
ADC development, serially diluted
T-DM1 was spiked into rat serum to final
concentrations of 20, 10, 5, 2.5, 1.25,
0.625, 0.3125, and 0 µg/mL (Table 2A).
The key parameters for efficient binding
during the loading step are the molar
excess of affinity ligand to target andresidence time for binding. The residence
time is controlled by the loading flow
rate. We optimized both parameters to
use the lowest molar excess of affinity
ligand to minimize cost, while using thehighest flow rate possible to minimize
run time. The compromise used was tohave a molar excess of approximately
6-fold and a loading flow rate of
2 µL/min. These variables may need to
be optimized for other binding partners.

7

On-cartridge deglycosylation
For ADC DAR characterization, multiple
glycoforms complicate the MS spectrum
and reduce the accuracy of the DAR
calculation. Therefore, a deglycosylation
step is commonly applied to remove
glycans before LC/MS analysis. In thisstudy, a newly developed on-cartridge
reaction approach was carried out
to deglycosylate ADCs after affinity
purification on the cartridge. Comparedwith the traditional deglycosylation
reaction in solution, on-cartridge
deglycosylation streamlines theentire process of protein purification,
deglycosylation, and buffer exchange.
PNGase F is separated from ADCs
during this process, thereby avoiding ion

suppression in the LC/MS analysis8. The
deglycosylation was performed at 37 °C
to preserve ADC integrity and minimize
evaporation while achieving complete
deglycosylation in 30 minutes or less.
The N-glycans are available for labeling,
if desired.

Evaluation of sample preparation
performance by LC/MS
T-DM1 samples prepared using an
Agilent AssayMAP Bravo platform, as
described above, and unprocessed
T-DM1 controls were separated on a
PLRP-S column with an Agilent 1290
Infinity II UHPLC system, and analyzed
on an Agilent 6545XT AdvanceBio
accurate‑mass quadrupole time-of‑flight

MS system. TICs, EICs, extracted
spectra, deconvoluted spectra, and DAR
values were generated from the raw
data using the MassHunter software
suite (see MS data analysis for detail).
Figure 5 shows LC/MS data from 200 ng
of control and purified T-DM1 (on-column
assuming 100 % recovery during sample
preparation). The TICs showed a highdegree of purity for the samples spiked
into serum and good recovery comparedto the control T-DM1 (Figures 5A–C).
The extracted spectra and deconvoluted
spectra of the control, affinity purified,and affinity purified plus deglycosylated
T-DM1 samples indicate that the affinity
purification of ADCs did not introduce
bias. It also showed that on‑cartridge

Processed T-DM1
Affinity purified T-DM1

Unprocessed T-DM1

Affinity purified and deglycosylated T-DM1

A

B

C

D

E

F

TIC

Extracted
spectrumG

DAR = 3.5

H

DAR = 3.5

I

DAR = 3.5

Deconvoluted
spectrum

Figure 5. Representative TICs, extracted spectra, and deconvoluted spectra from 200 ng of T-DM1 before and after sample preparation. TIC of unprocessed
T-DM1 (A), affinity purified intact T-DM1 (B), affinity purified and deglycosylated T-DM1 (C); extracted spectrum of unprocessed T-DM1 (D), affinity purified intact
T-DM1 (E), affinity purified and deglycosylated T-DM1 (F); deconvoluted spectrum of unprocessed T-DM1 (G), affinity purified intact T-DM1 (H), and affinity
purified and deglycosylated T-DM1 (I).
8

deglycosylation simplified thedeconvoluted spectrum and increased
signal intensity, as expected, by removing
most glycoforms (Figure 5D–I).

3.0
2.8

A

200 ng; %CV = 1.01

Affinity purified T-DM1

100 ng; %CV = 3.67

2.6

50 ng; %CV = 2.63

2.4

25 ng; %CV = 1.60
12.5 ng; %CV = 2.26

2.2

6.25 ng; %CV = 4.73

Counts2.0

3.125 ng; %CV = 14.50

1.8

0 ng

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
×10
3.2

2.7

2.8

2.9

3.0

3.1

8

3.0
2.8

3.2
3.3
3.4
3.5
Acquisition time (min)

3.6

3.7

B

3.8

3.9

4.0

200 ng; %CV = 1.07

Affinity purified/deglycosylated T-DM1

100 ng; %CV = 4.94
50 ng; %CV = 6.74

2.6

25 ng; %CV = 5.79

2.4

12.5 ng; %CV = 5.04

2.2

6.25 ng; %CV = 5.47

2.0

3.125 ng; %CV = 9.01

1.8CountsTo evaluate the reproducibility of thisworkflow, EIC-based quantification
was applied to both purified andpurified/deglycosylated T-DM1
from rat serum spiked with various
concentrations of T-DM1, as describedabove. The EICs were extracted
and integrated over the range of
2,000–4,000 m/z. The area under
the curve (AUC) was used for relative
quantification. Figure 6 shows theoverlaid EICs and %CV between test
replicates (n = 3). All %CV values
were below 10, except the 3.125 ngof the glycosylated T-DM1 samples(CV % = 14.50) (Figure 6). The overall
low CVs and the tightly overlapped EICs
demonstrate the excellent reproducibilityof this workflow.

×108

0 ng

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
2.7

2.8

2.9

3.0

3.1

3.2
3.3
3.4
3.5
Acquisition time (min)

3.6

3.7

3.8

3.9

4.0

Figure 6. The workflow reproducibility evaluated by the EICs of the affinity purified T-DM1. The ADC T-DM1
was spiked in rat serum at concentrations of 20 to 0.3125 µg/mL and recovered by affinity purification
with or without deglycosylation, followed by intact LC/MS analysis. The column loading masses of T-DM1,
based on the initial sample inputs, are indicated (see Table 2A). The replicate traces are overlaid and shownin the same color. The %CV between replicates was calculated based on the integrated AUCs (n = 3).

9

To determine DAR values, thedeconvoluted spectra from control,
purified, and purified/deglycosylated
T-DM1 samples, described above(Table 2A), were analyzed using
Agilent DAR Calculator. DAR0-7 wereused to determine the average DAR. The
average DAR values from three replicateswere calculated to be approximately
3.5 ±0.1 across all samples (Figure 8),
which are consistent with the reported
DAR value9. Based on the results ofthe control T-DM1, the limit of DAR
measurement is 3.125 ng on-column
T-DM1. The lowest serum concentrationof T-DM1 that could reliably be measured
after affinity purification on the
AssayMAP platform was 0.3125 µg/mL,
although the spectrum was noisy at thislevel. Deglycosylation of the purified
T-DM1 samples simplified the mass
spectrum, allowing a more confident
measurement of DAR at 0.3125 µg/mL
serum concentration (Figure 8).

10

×108
40

A

35

Affinity purified T-DM1
y = 3E+07x + 2E+08
R² = 0.9987

AUC

30
25
20
15
10
5
0

0

×108
40

20

40

60
80
Load on column (ng)

100

120

100

120

B
Affinity purified/deglycosylated T-DM1

35

y = 3E+07x + 9E+07

30
AUC

The performance of this workflow for thequantification of affinity purified T-DM1
at the intact protein level was further
evaluated based on the linear dynamic
range of the assay. The AUCs for the
purified and purified/deglycosylated
T-DM1 were plotted (Figure 7).
Regression analysis indicated excellent
linear responses for both purified(R2 = 0.9987) and purified/deglycosylated(R2 = 0.9996) T-DM1 from serum
samples with 0.3125–10 µg/mL T-DM1(3.125 to 100 ng of column loading
amounts based on initial spiked amount).
Note that only 20 % of the eluate wasinjected for this analysis. These results
demonstrated the feasibility of using
the AssayMAP Bravo platform toisolate ADCs from complex matrix forquantification analysis.

R² = 0.9996

25
20
15
10
5
0

0

20

40

60
80
Load on column (ng)Figure 7. Linear dynamic range analysis of affinity purified and affinity purified/deglycosylated T-DM1 from serum samples. The T-DM1 was spiked into
rat serum to a final concentration of 0.3125 to 10 ng/µL. T-DM1 from thesesamples were affinity purified with or without deglycosylation, followed byintact LC/MS analysis. The EICs in the 2,000-4,000 m/z range were extracted
and integrated. The AUCs, after removing background, were plotted against the
on-column T-DM1 loading amounts from these samples (see Table 2A). Linear
regression analysis was carried out to assess the linearity.

Unprocessed T-DM1

Affinity purified/deglycosylated T-DM1

Affinity purified T-DM1

3.5

3.5

3.5

3.5

3.4

3.5

3.5

3.4

3.4

3.5

3.4

3.4

3.5

3.4

3.4

3.5

3.6

3.5

3.5

3.6

3.6

200 ng

100 ng

50 ng

25 ng

12.5 ng

6.25 ng

3.125 ng

Figure 8. Representative deconvoluted spectra and average DAR values for unprocessed, affinity purified and affinity purified/deglycosylated T-DM1. T-DM1 was
spiked into rat serum at different concentrations over the range 0.3125–20 µg/mL and recovered by affinity purification with or without deglycosylation, followed
by intact LC/MS analysis. DAR values were determined from deconvoluted spectra using Agilent DAR Calculator. The on-column loading masses based on initial
inputs are shown on the left (see Table 2A). The average DAR value averaged from replicates is shown in the upper-right corner in each spectrum (n = 3).

11

Conclusion

References

A complete solution was provided toaddress the challenges of in vivo ADC
DAR analysis. A typical workflow torecover ADCs from serum followed bydeglycosylation before LC/MS analysis
was demonstrated with automatedprocedures using the Agilent AssayMAP
Bravo platform. The automated antigen
immobilization, affinity purification, andon-cartridge deglycosylation could becompleted in approximately 4.5 hours
with excellent reproducibility, minimalhands‑on time, and the ability to scale
as needed. With the high performanceof the Agilent 6545XT AdvanceBio
accurate-mass quadrupole time-of-flight
LC/MS system, the prepared samples
can be analyzed seamlessly to yieldhigh-quality data for both qualitative andquantitative studies.

1.

Acknowledgement
We would like to thank Jing Chenfor extensive work developing andoptimizing the workflow presented
here, and Bill Rust, Maryann Shen, andShuai Wu for advice and critical readingof this Application Note.

www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2018
Printed in the USA, February 21, 20185991-9010EN

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