An Integrated Workflow for
Automated Calculation of
Antibody‑Drug Conjugate (ADC)
Drug‑to-Antibody Ratio (DAR)

Using Automated Sample Preparation,
Agilent MassHunter Walkup, and Agilent MassHunter
BioConfirm DAR Calculator Software

Application Note
Biotherapeutics and Biosimilars

Authors

Abstract

David L. Wong, Tanner Stevenson, andAn easy-to-use and fully integrated method for the determination of

Jing Chendrug‑to‑antibody ratios (DARs) of antibody-drug conjugates (ADCs) has been

Agilent Technologies, Inc.

developed using the Agilent AssayMAP Bravo, Agilent MassHunter Walkup

Santa Clara, CA, USA

software, and the Agilent BioConfirm DAR calculator.

Introduction
Antibody-drug conjugates (ADCs)
represent a new generation of targetedbiotherapeutics that make up a rapidly
growing segment of the drug discovery
pipeline. They are created by attachingpotent cytotoxic drugs through a linker
to monoclonal antibodies (mAbs)
that target specific cells. They deliver
those drugs to the desired tissue
while limiting toxicity to nontargetedtissues1. Different types of conjugation
chemistries are used to create ADCs,including cysteine-linked, lysine-linked,
and site-specific conjugation. Dependingon the type of conjugation chemistry,different methodologies are used for thecharacterization of ADCs2-3.
The production and characterization ofpotent, highly selective, and low‑toxicity
ADCs are more complicated than theprocesses used for traditional mAbs.
The number of drug molecules attachedto each antibody can vary considerably
depending on the conjugation chemistry
and reaction conditions. Achieving theproper average drug-to-antibody ratio(DAR) is crucial to optimizing the efficacy,
and minimizing the toxicity of the ADC.
Rigorous quality control is required toensure that ADCs have a consistent DAR,
which is frequently determined using
liquid chromatography coupled to mass
spectrometry (LC/MS).

Reduction and deglycosylation are often
done offline before analysis to reduce thecomplexity of the ADC LC/MS results
used for the DAR calculation. Suchsample preparation prior to LC/MS andsubsequent calculation of the DAR values
are often manually intensive, limiting
throughput and introducing variability. Inaddition, the scientists developing andanalyzing ADCs are often not experts inLC/MS analysis. This Application Notedescribes an integrated workflow for
ADC DAR determination that automates
sample preparation and DAR calculation,
while enabling operators who are notexperts in LC/MS to obtain high quality
results.
The Agilent AssayMAP Bravo isan automated sample preparation
platform whose simple user interface
gives non-automation experts theability to efficiently purify, reduce,and deglycosylate ADCs prior toLC/MS analysis. Automated sample
preparation enables robust ADC DAR
analysis by minimizing variability insample preparation, which is a major
source of data variability in the ADC
DAR workflows. Agilent MassHunter
Walkup Software allows users to walkup to the LC/MS instrument with theirprocessed samples, input minimal sample
information, choose the appropriate ADC
analysis method, place the samples inposition as directed by the system, andwalk away. The users are not required
to know how to set up or optimize theLC/MS system for their application. Thisis done by the system administrator, alsousing the MassHunter Walkup Software.

2Following the LC/MS run, AgilentMassHunter BioConfirm DAR Calculator
software provides an easy-to-use solution
for the calculation of DAR, which is thensent to the analyst as part of an email
report on the analysis results.

Experimental
Material

Rapid PNGase F was obtained fromNew England Biolabs. Eppendorf 96-well
PCR plates were from Eppendorf, andTris chloride and Tris(hydroxymethyl)aminomethane were obtained from
EMD Millipore. All other chemicals
were sourced from Sigma-Aldrich. The
formulated ADC was from Genentech.

In-solution reductionand deglycosylation

Lyophilized ADC was reconstitutedin deionized (DI) water to 5 mg/mL,
aliquoted and stored at –80 °C until
used. Reduction and deglycosylation
were performed manually using theAgilent AssayMAP Bravo Platformand Protein Sample Prep Workbench
software. For the automated workflow,
5 μL of ADC were dispensed into eachwell of the first four columns of a 96-well
Eppendorf PCR plate (position A1-D4).
Subsequently, 10 µL of 10 mM Tris buffer(pH = 7.5) (position A1-D2), or 35 mM
DTT in 10 mM Tris buffer (pH = 7.5)
(position A3-D4) were added to thesamples. Finally, 20 µL of 10 mM Trisbuffer (pH = 7.5) (positions A1-D1 andA3-D3) or rapid PNGase F (diluted 1:40)
in 10 mM Tris buffer (pH = 7.5) (A2-D2and A4-D4) were added to the samples.
The sample plate was then sealed using
an Agilent PlateLoc Thermal Plate Sealer,and incubated at 50 °C for 10 minutes.

LC/MS analysis

LC/MS analyses were conducted
on an Agilent 1290 Infinity II UHPLC
system coupled with an Agilent 6230
Accurate‑Mass TOF LC/MS systemequipped with an Agilent Dual Jet StreamESI source. Tables 1 and 2 list the LC/MSparameters used. Approximately 1.4 µg ofsample was injected for each run.

Table 1. Liquid chromatography parameters.
Agilent 1290 Infinity II LC System
ColumnAgilent PLRP-S, 300 Å, 2.1 × 50 mm, 5 µm (p/n PL 1912-1501)

Thermostat

5 °C

Solvent A

0.1 % Formic acid in water

Solvent B

0.1 % Formic acid in acetonitrile

Gradient

Intact ADC
Time (min)
0–2
2–10
10–13
13–15

Column temperature

80 °C

Flow rate

0.5 mL/min

Injection volume2 µL

Data Analysis

Data analysis was performedautomatically by MassHunter
BioConfirm B.08.00 and DAR Calculator
B.01.01 software following sample
submission and data acquisition
through the MassHunter Walkup.
BioConfirm automation was enabledin the acquisition method by linking a
BioConfirm method containing DAR
Calculator parameters.

%B
3–10
10–45
45–70
70

Reduced ADC
Time (min) %B
0–5
3–20
5–6
20–95
6–1095

Table 2. Mass spectrometer parameters.
Agilent 6230 Accurate-Mass TOF LC/MS system
Intact ADC

Reduced ADC

SourceDual Agilent Jet Stream

Dual Agilent Jet Stream

Raw data obtained from LC/MS were
processed automatically by the Intact
Protein Deconvolution with the DAR
Calculation custom feature in BioConfirm.
This process deconvoluted the data usingthe Maximum Entropy deconvolution
algorithm (Table 3), exported thedeconvoluted spectra to a csv file, andcalculated the average DAR for the ADC
sample (Table 4).

Gas temperature

325 °C

290 °CGas flow

13 L/min13 L/min

Nebulizer

60 psig

35 psig

Sheath gas temperature

350 °C

350 °C

Sheath gas flow

12 L/min12 L/min

VCap

4,000 V

4,000 VNozzle voltage

2,000V

2,000 VFragmentor

400 V

175 V

Skimmer

65 V

45 V

The drug-to-antibody ratio was calculatedusing the following formula:

Mass range

800–8,000 m/z

300–3,200 m/z

Scan rate

1.0 spectra/sec

1.0 spectra/sec

Acquisition mode

Positive,
High (10,000 m/z) mass range

Positive,
Standard (3200 m/z) mass range

DAR =ni=0i×Ai

AtotalEquation 1.

Where i denotes the number of drugs
attached to the antibody (DAR species),and Ai denotes the area under each DAR
species peak cluster, which includes all
glycan peaks.
If necessary, peak limits can be manually
adjusted in the DAR Calculator software
after reviewing the automated data
analysis results.

Table 3. BioConfirm deconvolution parameters.
Intact ADC

Reduced ADC

Deconvolution algorithm

Maximum entropy

Maximum entropy

Mass range (Da)

140,000–160,000

20,000–60,000

Mass step

11Use limited m/z range

1,800–5,000

1,000–3,200

Subtract baseline

77Adduct

ProtonProton

Isotope width

Automatic/20 Da

Automatic/20 Da

Peak signal-to-noise (S/N)

¡30

¡30

Maximum number of peaks

100100Calculate average mass using 25 %top % of peak height

25 %Deconvolution tab

Maximum entropy tab3

Results and Discussion

Table 4. DAR Calculator parameters.

Integrated workflow using
AssayMAP Bravo, Walkup
Software, and the DAR Calculator

Parameters tab

The AssayMAP Bravo Platform
enables the automated reduction anddeglycosylation of antibodies and ADCs,
thus reducing human error, assuring
reproducibility, and allowing the analyst
to walk away and perform other tasks(Figure 1). This open access automation
solution is specifically designed forbiomolecule sample preparation using
miniaturized, packed-bed chromatography
cartridges, simple and reliable automatedprocesses, and an application-based userinterface.
MassHunter Walkup software allows
scientists not familiar with LC/MS toaccess instruments with ease. Even
novice users can take full advantage ofpowerful LC/MS capabilities, without
assistance from expert staff, by simply
providing some basic information,choosing a method, and placing
samples as directed. The results arrivein the user’s email inbox automatically(Figure 1). Administrators of the LC/MS
systems can take advantage of easy
method setup and management, as wellas management of the user queue.
The BioConfirm B.08.00 ADC DAR
Calculator requires minimal input fromthe user, primarily the molecular weightsof the unconjugated antibody and thedrug/linker mass. It instantaneously
calculates the average DAR from thedeconvoluted ADC peaks produced by
BioConfirm software, using auto peak
integration. Walkup software can thensend DAR calculator results to the user inan email report (Figure 1).

Intact ADC

Reduced ADC

Processing method

IntactReducedLinker type

Lysine-linked

N/AIntact DAR 0 mass (Da)

145170 (Degly)/148059 (Native)

N/A

Heavy chain DAR 0 mass (Da)N/A

49,149 (Degly)/50,756 (Native)

Light chain DAR 0 mass (Da)N/A

23,439Drug/linker mass (Da)

957.5

957.5

Selected peaks

DAR 0–7

DAR 0–4

Peak area

Use total area

Use total area

Peak baseline

Use y = 0

Use y = 0

Maximum DAR peak number

84

PDF

PDF

Properties tab

Report tab
Report type

Intactglyco

IntactdeglycoReducedglyco

ReduceddeglycoAgilent AssayMap Bravo Platform
automated sample prep

LC/TOF MS

Agilent MassHunter
Walkup Software

Figure 1. Integrated Agilent MassHunter Walkup ADC calculation workflow, from automated sample
preparation on the Agilent AssayMAP Bravo Platform to LC/TOF MS analysis, automated DAR
calculation, and delivery of email report.

4Intact, glycosylated ADC•Intact, deglycosylated ADC•

Reduced, glycosylated ADC•

Reduced, deglycosylated ADC

987654321Intact ADCsATIC

×10 7
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.20.10Intact deglycosylated ADCsCTICCounts

×10 6

glycoforms G0F/G0F, G0F/G1F, G1F/G1F
or G0F/G2F, and G1F/G2F. For intact
deglycosylated ADC, nine peak groups
were also observed with mass matching
D0–D8 conjugation states. All glycoforms
observed in glycosylated ADC samples
were eliminated, and the peak mass
was reduced correspondingly. The peakintensities were increased several foldcompared to the intact ADCs.

5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2
Acquisition time (min)×10 31.0B
Deconvoluted
151,084.86
0.9MS spectrum
0.8150,128.06 152,044.90
0.7
0.6
153,002.97
0.5
149,172.70
0.4
153,963.17
0.3
148,213.91
0.2
0.1
0
148,000 150,000 152,000 154,000
Deconvoluted mass (amu)×10 3Counts•

Figure 2 shows the total ion
chromatographs (TICs) and deconvoluted
spectra of intact glycosylated (Figures 2Aand 2B), and intact deglycosylated(Figures 2C and 2D) ADCs. For intact
glycosylated ADCs, nine peak groups
were observed with masses matching
D0–D8. The four major peaks observedin each peak group correspond withCounts

The performance of the integratedworkflow with automated sample
preparation was compared to asimilar workflow using manual
sample preparation (reduction anddeglycosylation), with a lysine-conjugated
ADC sample. Four different sample
preparation conditions were used foreach workflow type. In the automatedintegrated workflow, four technicalreplicates were prepared on theAssayMAP Bravo for each of the four
sample conditions (Figure 1). Each sample
was then run three times on the LC/MS.
In the manual integrated workflow, asingle technical replicate was preparedmanually for each of the four sample
conditions, and each sample was run six
times on the LC/MS. The four sample
conditions for each workflow were as
follows:

Automated integrated
workflow results

CountsDrug-to-antibody ratio
determinations

5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2
Acquisition time (min)D

Deconvoluted

4.5
148,033.84
MS spectrum
148,991.19
4.0
147,075.50
3.5
3.0
149,947.53
2.5
146,611.69
2.0
150,905.21
1.5
152,824.00
1.0 145,162.43
151,864.00
0.50146,000 148,000 150,000 152,000
Deconvoluted mass (amu)

Figure 2. Total ion chromatographs (TICs) and deconvoluted spectra of intact glycosylated (A,B) andintact deglycosylated (C,D) ADC prepared using the Agilent Bravo AssayMAP Platform.

5The deconvoluted spectra were thenanalyzed in the BioConfirm DAR
calculator. Figure 3 shows representativegraphs of the DAR calculations ofintact glycosylated (left) and intact
deglycosylated ADC (right). The
average DAR value calculated fromfour technical replicates was 3.5 for theintact glycosylated ADC. The DAR value
calculated for the intact deglycosylated
ADC was also 3.5.
Figure 4 shows the TICs anddeconvoluted spectra of reduced
glycosylated (Figure 4A–4C), and reduced
deglycosylated ADC (Figure 2D–2F).
For light chains, four peak groups wereobserved (D0–D3) for both glycosylated
and deglycosylated reduced ADC.

Figure 3. Representative DAR calculation of intact glycosylated and deglycosylated ADC prepared using
the Agilent Bravo AssayMAP Platform.
×10 8
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0Reduced ADCsATIC

TIC

0.6
0.2

55.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2
Acquisition time (min)B

23,439.65

Deconvoluted
MS spectrum
light chain

0×10
1.0

6

5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2
Acquisition time (min)E

23,439.72

Deconvoluted
MS spectrum
light chain

0.8
0.63

CountsCountsD

0.4

424,397.39

2

0.4

25,354.48

10

Reduced deglycosylated ADCs

0.8

22,00024,00026,000Deconvoluted mass (amu)

×10 4
C 50,755.79
9Deconvoluted
51,713.89
MS spectrum
8heavy chain
7652,671.51543253,629.63
10
50,000 51,000 52,000 53,000 54,000
Deconvoluted mass (amu)

24,397.40

0.2

26,311.79

0×10 51.0

25,354.57
26,310.55

22,00024,00026,000Deconvoluted mass (amu)

F 49,149.99 50,107.78

0.8
0.6

Counts51.2
CountsCounts×10×10 8

1.0Counts

For heavy chains, four peak groups
were observed for glycosylated andreduced ADC, while five peaks wereobserved for deglycosylated and reduced
ADCs (D0–D4). For each peak group,
the four major peaks observed in theglycosylated and reduced ADCs represent
G0, G0F, G1F, and G2F glycoforms. After
deglycosylation, only one major peak wasobserved in each peak group representing
fully deglycosylated ADCs. The minor
peak observed for deglycosylated heavy
chains is due to ADC with an extra linker.
The peak intensities for the reduced anddeglycosylated chains were increased
approximately 2 to 4-fold compared to thereduced and glycosylated ADC chains.

DeconvolutedMS spectrum
heavy chain

51,065.59

0.40.2052,023.02
52,980.24

49,000 50,000 51,000 52,000 53,000
Deconvoluted mass (amu)

Figure 4. TIC and deconvoluted light and heavy chain spectra of reduced glycosylated (panels A–C) andreduced deglycosylated (panels D–F) ADC prepared using the Agilent Bravo AssayMAP Platform.

6Figures 5 and 6 show representative
reports from the automated DAR
calculator in BioConfirm for both reduced
glycosylated and reduced deglycosylated
ADC. The DARs calculated for thefour technical replicates of reduced
glycosylated and deglycosylated ADC
were 3.2, and 3.0, respectively. The DARvalue calculated from the reduced ADC
was slightly different from that of theintact ADC reported previously1.

Comparison to manual
sample preparation

The same set of experiments wasperformed with the same ADC sample,
using manual sample preparation insteadof the AssayMAP Bravo Platform. As
expected, the DAR values obtainedusing manual sample preparation andautomated sample preparation were
nearly identical (Table 5). Automation
would not be expected to give a different
DAR value, but automation does offer
several advantages, including reduced
labor and eliminating human error. The
preconfigured software protocols allow
users to run sample preparation withthe click of a few buttons. With the96-well format, the same amount of timeis required to set up automated sample
preparation for 1 or 96 samples.

Figure 5. Representative report from the Agilent DAR calculator for reduced glycosylated ADC.

Figure 6. Representative report from the Agilent DAR calculator for reduced deglycosylated ADC.
Table 5. Comparison of results, using either automated (Bravo AssayMAP) or manual sample preparation.

Intact ADCIntact
deglycosylated
ADC

Reduced
ADC

Reduceddeglycosylated
ADC

Automated sample prep
(Total number of injections)

12121212

Average DAR

3.5

3.5

3.2

3.0

Manual sample prep
(Total number of injections)

66

6

6

Average DAR

3.6

3.5

3.4

3.0

7

Conclusions

References

Scientists developing antibody‑conjugatedbiotherapeutics need to obtainaccurate and reliable DAR information
without having to become experts insample preparation, operating mass
spectrometers, interpreting the data,or manually calculating the DAR.
Agilent provides an integrated workflowthat delivers reliable DAR values right tothe user’s email, while requiring very little
hands-on input. The Agilent AssayMAP
Bravo Platform automates sample
preparation, and Agilent MassHunter
Walkup software ensures optimal MS
data with little or no knowledge of
operation of the instrument. Finally,
Agilent BioConfirm software provides
automatic calculation of the average DAR
for all forms of the ADC, including intact
and deglycosylated configurations, aswell as reduced heavy and light chains.
For reliable DAR results obtained witha minimum of effort or expertise, thisintegrated workflow using the AssayMAP
Bravo Platform, MS instruments withWalkup software, and the Agilent
MassHunter BioConfirm DAR calculator
is ideal.

1. Perez, H. L., et al. Antibody-drug
conjugates: current status and future
directions. Drug Discovery Today 2014,19(7), 869-81.
2. Xu, K., et al. Characterization of thedrug-to-antibody ratio distribution
for antibody–drug conjugates inplasma/serum. Bioanalysis 2013, 5(9),1057‑1071.
3. Beck, A, et al. Cutting-edge mass
spectrometry methods for themulti‑level structural characterizationof antibody-drug conjugates. Expert
Reviews of Proteomics 2016, 13:2,157-183.

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This information is subject to change without notice.
© Agilent Technologies, Inc., 2016
Published in the USA, October 4, 2016
5991-7366EN