Drug-to-Antibody Ratio (DAR)
Calculation of Antibody-Drug
Conjugates (ADCs)
Using Automated Sample Preparation and Novel DAR
Calculator Software

Application NoteAuthors

Introduction

Jing Chen and Steve Murphy

Antibody-drug conjugates (ADCs) are a new class of biotherapeutics that represent a
rapidly growing portion of the drug discovery pipeline in pharmaceutical companies.
ADCs are monoclonal antibodies chemically linked to biologically active small moleculedrugs. By combining potent cytotoxic drugs with target-speciﬁc antibodies, ADCs deliver
cytotoxic drugs to the diseased tissue while limiting the toxicity in the nontargetedtissues.

Agilent Technologies, Inc.

Drug-to-antibody ratio (DAR) is the average number of drugs conjugated to theantibodies, which is an important attribute of ADCs. The DAR value affects the efﬁcacyof the drug, as low drug loading reduces the potency, while high drug loading cannegatively affect pharmacokinetics (PK)1 and toxicity. With the current conjugation
chemistry, that is lysine side-chain amidation or cysteine interchain disulﬁde bond
reduction, a drug load of 0 ~8 drugs (D0 ~ D8) per antibody is commonly observed.
LC/MS is a popular analytical method for measuring the DAR and the drug load
distribution of ADCs. It is an essential method used in the identiﬁcation of various
drug-loaded ADCs species. In most cases, the intact ADC can be analyzed directly using
LC/MS to determine the DAR value. The ADCs may be reduced before LC/MS analysis
when speciﬁc DAR information on the light and heavy chains is needed. Additionally,the ADCs may be deglycosylated prior to LC/MS analysis to further reduce spectrum
complexity.
ADC sample preparation prior to LC/MS analysis is typically done manually which
can introduce variability and limit throughput. The Agilent AssayMAP Bravo is aneasy-to-use automated sample preparation system that gives great reproducibility,
labor savings in the form of reduced hands-on time, scalability (8–96 samples can be
run simultaneously), simple person-to-person and site-to-site method transfer, and it
minimizes the possibility of human error. AssayMAP Bravo is an excellent platform forproviding automated sample preparation of the aforementioned reactions.

The AssayMAP Bravo automatedsample preparation platform together
with the Agilent LC/MS andMassHunter/BioConﬁrm/DAR Calculator
software provides a reproducible andeasy solution for the DAR calculation ofADCs. In this Application Note, intact
and reduced ADCs, with or without
deglycosylation, were processed inparallel using the Agilent AssayMAP
Bravo platform, and analyzed using
the Agilent LC/MS. The DAR wasthen determined with the Agilent DAR
calculator.

Experimental

LC/MS analysis

MaterialRapid PNGase F was purchased fromNew England Biolabs (Ipswich, MA).
Eppendorf 96-well PCR plates were from
Eppendorf (Hauppauge, NY); Tris chlorideand Tris(hydroxymethyl)aminomethane
were purchased from EMD Millipore(Billerica, MA). All other chemicals werefrom Sigma-Aldrich (St. Louis, MO).

In-solution reduction anddeglycosylation using
Agilent AssayMAP Bravo
Lyophilized ADCs were reconstituted
in deionized (DI) water to 5 mg/mL,
aliquoted and stored at –80 °C until used.
All reagent transfers were performed
using the Agilent AssayMAP Bravo
platform (Santa Clara, CA) through Protein
Sample Prep Workbench software. Fivemicroliters of ADCs were dispensed into
each well of the ﬁrst four columns of a96-well Eppendorf PCR plate (positionA1-H4). Subsequently, 10 µL of 10 mM
Tris buffer (pH = 7.5) (position A1-H2),
or 35 mM DTT in 10 mM Tris buffer(pH = 7.5) (position A3-H4) were added
to the samples. Finally, 20 µL of 10 mM
Tris buffer (pH = 7.5) (positions A1-H1 andA3-H3) or rapid PNGase F (diluted 1:40)
in 10 mM Tris buffer (pH = 7.5) (A2-H2and A4-H4) were added to the samples.
The sample plate was sealed using anAgilent PlateLoc Thermal Plate Sealer(Santa Clara, CA) and incubated at 50 °C
for 10 minutes.

2LC/MS analyses were conducted onan Agilent 6550 iFunnel Q-TOF (SantaClara, CA) equipped with a Agilent DualJet Stream ESI source coupled withthe Agilent 1290 Inﬁnity UHPLC system(Waldbronn, Germany). Table 1 andTable 2 list the LC/MS parameters used.
One microgram of sample was injected
for each run.

Data analysis
Raw data obtained from LC/MS weredeconvoluted with Agilent MassHunter
Qualitative Analysis Software(Version B.07.00, Build 7.0.7024.0),
with BioConﬁrm using the Maximum
Entropy deconvolution algorithm. Thedeconvolution parameters were set asfollows:•

For intact ADCs, mass range was
set to 140,000–160,000 Da andmass step at 1 Da•

For reduced ADCs, mass range
was set to 20,000–60,000 Da, mass
step to 0.1 Da•

For both intact and reduced ADCs,
average mass was calculatedusing top peak height at 25 %The rest of the parameters were set
to default. The deconvoluted spectra
were then exported as a .csv ﬁle andimported into an Agilent DAR calculator.
After inputting/selecting D0 mass anddrug/linker mass, the DAR calculator
automatically selects, annotates andintegrates mass peak groups of ADCs
with various drug loadings, and calculates
the average DAR and generates a peaklist table.

Results and Discussion
To determine the DAR of an example
lysine conjugated ADC, ADCs wereprepared in four different conditions(n = 8 for each condition); intact
glycosylated, intact deglycosylated,reduced glycosylated, and reduced
deglycosylated using the AssayMAP
Bravo platform. They were analyzed
with an Agilent 1290 Inﬁnity UHPLC
system equipped with a 2.1 mm ID
1000Å PLRP-S LC column which
was coupled to the 6550 Q-TOF,
followed by data analysis using
Agilent MassHunter/BioConﬁrm/DAR
Calculator software (Figure 1).

Table 1. Liquid chromatography parameters.
Agilent 1290 Inﬁnity LC System
Parameter

IntactReducedColumn

Agilent PLRP-S, 1000 Å
2.1 × 150 mm, 8 µm

Agilent PLRP-S, 1000 Å
2.1 × 150 mm, 8 µm

Sample thermostat

5 °C

5 °C

Mobile phase A

0.1 % Formic acid in water

0.1 % Formic acid in water

Mobile phase B

0.1 % Formic acid in acetonitrile

0.1 % Formic acid in acetonitrile

Gradient

0–5 minutes, 20 %B
5–10 minutes, 20–90 %B

0–5 minutes, 20 %B
5–6 minutes, 20–75 %B
6–10 minutes, 75–90 %B

Post time

None

None

Column temperature

80 °C

80 °C

Flow rate

0.4 mL/min

0.4 mL/min

Table 2. Mass spectrometer parameters.
Parameter

Agilent 6550 Q-TOF LC/MS System

Ion mode

Positive ion mode

SourceAgilent Dual Jet StreamAgilent Dual Jet Stream

Drying gas temperature

290 °C

290 °C

Drying gas ﬂow

14 L/min14 L/minSheath gas temperature

400 °C

400 °C

Sheath gas ﬂow

12 L/min12 L/min

Nebulizer

40 psi40 psiCapillary voltage

4,500 V

4,500 V

Nozzle1,500 V

1,500 V

Positive ion mode

Fragmentor voltage

250 V

250 V

Oct RF Vpp

750 V

750 V

Acquisition parameters
MS modeHigh (10,000 m/z) mass range,
Extended mass range (2 GHz),MS only mode,
Mass Range 1,800–6,000 m/z

High (10,000 m/z) mass range,
Extended mass range (2 GHz),MS only mode,
Mass Range 800–4,000 m/z

IntactGlyco

IntactDeglycoReduced
Glyco

Sample preparation

ReducedDeglycoLC/MS

Figure 1. Workﬂow of the DAR calculation of ADCs.

3

DAR calculation

Figure 2 shows the overlaid total ion
chromatograph (TIC), extracted spectra,
and the deconvoluted spectra of intact
glycosylated (Figure 2 panels A-C),
and intact deglycosylated (Figure 2panels D-F) ADCs of eight replicates.
Both intact glycosylated and intact
deglycosylated ADCs eluted in lessthan 1 minute with a mass envelope
centered around 3,000 Da with charge
states between +35 and +66. For intact
glycosylated ADCs, nine peak groups
were observed with masses matching
D0–D8. The four major peaks observedin each peak group corresponds withglycoforms G0F/G0F, G0F/G1F, G1F/G1F

1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0A
TIC
CountsCounts

×10 8

6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6
Acquisition time (min)
×10 3
3.5

×10 8
1.8 D
1.61.4TIC
1.2
1.0
0.8
0.6
0.4
0.206.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6
Acquisition time (min)
×10 3

B6

Extracted spectraCounts

2.5

E7

3.0
Countsor G0F/G2F, and G1F/G2F. For intact
deglycosylated ADC, 10 peak groups wereobserved with mass matching D0–D9. All
glycoforms observed in glycosylated ADC
samples were eliminated, and the peakmass was reduced correspondingly. Thepeak intensities were increased about2-fold compared to the intact ADCs.

2.0

4

1.5

3

1.0

20.5

10

Extracted spectra

50
2,000 2,200 2,400 2,600 2,800 3,000 3,200 3,400 3,600 3,800 4,000
Mass-to-charge (m/z)

2,000 2,200 2,400 2,600 2,800 3,000 3,200 3,400 3,600 3,800 4,000
Mass-to-charge (m/z)×10 4

F65Counts

Deconvoluted spectraCounts

×10 4
2.4 C
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0148,000

Deconvoluted spectra

432
10150,000
152,000
154,000
Deconvoluted mass (amu)156,000

146,000

148,000
150,000
152,000
Deconvoluted mass (amu)

154,000

Figure 2. Total ion chromatograph, extracted spectra, and deconvoluted spectra of intact glycosylated (panels A-C) and intact deglycosylated (panels D-F) ADCs.

4

The deconvoluted spectra were thenanalyzed in an Agilent MassHunter
DAR calculator. Figure 3 shows a
representative graph of the DAR
calculation of intact glycosylated
ADC. The DAR values calculated fromeight replicates were 3.6 (%CV = 0)
for the intact glycosylated ADCsand 3.88 (%CV = 1.1) for the intact
deglycosylated ADCs.

Figure 3. Representative DAR calculation of intact glycosylated ADCs.

5

Figure 4 shows the overlaid TIC, extracted
spectra, and the deconvoluted spectraof reduced glycosylated (Figure 4panels A-D), and reduced deglycosylated(Figure 4 panels E-H) ADCs of eight

4.5Counts

TIC

×104

×104

2.0

1.5
1.0

0.5

0.5
1,000 1,200 1,400 1,600 1,800 2,000 2,200 2,400 2,600 2,800
Mass-to-charge (m/z)

×105

C3.0
Deconvoluted spectra
light chain

2.0
1.51.5
1.0

0.5

0.5
0

22,500 23,000 23,500 24,000 24,500 25,000 25,500 26,000 26,500
Deconvoluted mass (amu)

×1048

D
3.0

1.5

H
Deconvoluted spectra
heavy chain

6
Counts2.0

22,500 23,000 23,500 24,000 24,500 25,000 25,500 26,000 26,500
Deconvoluted mass (amu)

7

Deconvoluted spectra
heavy chain

2.5

Deconvoluted spectra
light chain

2.0

1.0

×104

G

2.5
CountsCounts01,000 1,200 1,400 1,600 1,800 2,000 2,200 2,400 2,600 2,800
Mass-to-charge (m/z)

2.5Counts2.0

1.00

Extracted spectra

2.51.5×105

F

3.0Counts

Counts3.5
Extracted spectra

2.5

3.0

TIC

6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6
Acquisition time (min)

B

3.00E

4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.50

6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6
Acquisition time (min)3.5

and heavy chain charge states between+13 and +53. For light chain ADCs, fourpeak groups were observed (D0–D3) forboth glycosylated and deglycosylated
ADCs. The signal intensities are similar
between the two conditions.

×108ACounts

×108
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

replicates. Both reduced glycosylated andreduced deglycosylated ADCs eluted inless than 1 minute with a mass envelope
centered around 1,400 Da, with lightchain charge states between +6 and +27

5
4
3

1.0

20.5

100

50,00051,00052,00053,000Deconvoluted mass (amu)54,00048,50049,50050,50051,500Deconvoluted mass (amu)

52,500Figure 4. TIC, extracted spectra, and deconvoluted light and heavy chain spectra of reduced glycosylated (panels A-D) and reduced deglycosylated (panels E-H) ADCs.

6

For heavy chain ADCs, ﬁve peak groups
were observed for both glycosylated
and deglycosylated ADCs (D0–D4).
For each peak group, the four major
peaks observed in the glycosylated
ADCs represent G0, G0F, G1F, and G2F
glycoforms. After deglycosylation,
only one major peak was observedin each peak group representing fully

deglycosylated ADCs; a minor peak
was also observed in each peak grouprepresenting ADCs with an extra
linker. The peak intensities were also
increased about 2-fold compared tothe reduced glycosylated ADCs. The
exported .csv ﬁles were processed by theAgilent DAR Calculator. Figure 5 showsa representative report from the DAR

calculator of reduced glycosylated ADCs.
The DAR calculated eight replicates of
reduced glycosylated and deglycosylated
ADCs were 3.23 (%CV = 2.2), and3.21 (%CV = 2.6), respectively. The DAR
value calculated from the reduced ADC
was slightly different from that of theintact ADC as reported previously2.

Light Chain Results

DAR Report

Deconvoluted Spectrum
DAR Calculation Results
ADC Information
Data File
Processing Method
Report Time

Reduced-001
Reduced9/30/2015 11:50 AM

Property
Heavy Chain DAR 0 Mass
Light Chain DAR 0 Mass
Drug/Linker Mass

Combined DAR
Heavy Chain DAR
Light Chain DAR

Theoretical (Da)Not Given
Not Given
Not Given

3.2
1.1
0.5

Observed (Da)50594.1
23439.3
979.8

Heavy Chain Results
Deconvoluted Spectrum

DAR Peak List
DAR Peak

Theoretical Mass(Da)Not Given
Not GivenObserved Mass (Da) Area

% Area0123439.3
24396.9

1.60E+006
5.32E+005

64.75
21.48

23

Not Given
Not Given

25354.3
26311.5

2.89E+005
5.21E+004

11.67
2.10

DAR Peak List
DAR Peak

Theoretical Mass(Da)Observed Mass (Da) Area

% Area0

Not Given

50594.1

1.18E+006

33.89

123

Not Given
Not Given
Not Given

51552.1
52509.553629.8

1.14E+006
8.16E+005
2.98E+005

32.88
23.50
8.59

4

Not Given

54587.6

3.97E+004

1.14Page 2 of 2

Page 1 of 2

Figure 5. Representative report from the Agilent DAR calculator of reduced ADCs.

7Conclusion

References

Together with the use of rapid PNGase F,
automated sample preparation with
the AssayMAP Bravo allows for rapid
deglycosylation in a high-throughput
manner (less than one hour). The userfriendly control software Protein Sample
Prep Workbench and the precision of
the AssayMAP Bravo platform enableeasy and reproducible reduction anddeglycosylation of ADC samples forDAR calculation. The Agilent 1290
Inﬁnity UHPLC/6550 Q-TOF massspectrometer generates high resolution
accurate mass spectra for accurate andstraightforward DAR calculation with
MassHunter/BioConﬁrm/DAR Calculator
software. The Agilent PLRP-S column
delivers excellent peak shape in theformic acid mobile phase, enhancing
sensitivity. Agilent’s automatedrobotics, LC/MS, software package, andconsumables provide a complete solution
for rapid, easy, accurate and reproducible
DAR calculation of ADCs.

1. Perez, H. L; et al. Antibody-drug
conjugates: current status and future
directions. Drug Discovery Today 2014,19(7), p.869-81.
2. Basa, L. Chapter: Drug-to-Antibody
Ratio (DAR) and Drug Load
Distribution by LC-ESI-MS. Antibody
Drug Conjugates. Methods in
Molecular Biology; L. Dury (Ed.);
Humana Press: New York, 2013; 1045,
pp 285-293.

www.agilent.com/chem/bioconﬁrm
For Research Use Only. Not for use in diagnostic
procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2015
Published in the USA, October 5, 2015
5991-6263EN