Automation of Sample Preparation
for Accurate and Scalable
Quantification and Characterizationof Biotherapeutic Proteins Using theAgilent AssayMAP Bravo Platform
Application Note
Automated Protein Sample Preparation for LC/MS

Authors

Abstract

Michael Bovee, Jason Russell,

The Agilent AssayMAP Bravo platform automates a wide variety of LC/MS

Steve Murphy

sample prep workflows on a single instrument that is easy to use, gives highly

Agilent Technologies, Inc.

reproducible results, reduces hands-on time, and can simultaneously process

Madison, WI

8–96 samples. We report results from two workflows that are routinely used toUSAquantify and characterize biotherapeutic proteins. Peptide quantification was
performed using the AssayMAP Bravo to purify 96 antibody samples in parallelwith PG-W (immobilized Protein G) cartridges, digest the antibodies, and clean
up the peptides with C18 cartridges. LC/MS analysis indicated that the workflow
was highly reproducible with CVs < 10 %. Sequence coverage characterization
was performed using the AssayMAP Bravo to purify antibody samples, digest theantibodies with three different proteases in parallel, and clean up the peptides.
LC/MS analysis indicated that the combined sequence coverage from all three
enzymes was 99 % and 100 % for the heavy and light chains, respectively. Finally,
we report analytical figures of merit for antibody purification using PG-W and
PA-W (immobilized Protein A) cartridges and the AssayMAP Affinity Purification
application. Antibodies were purified with CVs less than 5 %, recovery greaterthan 90 %, and elution volumes of less than 10 µL.

Introduction

Experimental

An ever-increasing number of potential
biotherapeutic proteins must be
rapidly and accurately characterized sodata‑driven decisions can be made tomove the best candidates efficiently
through the drug discovery anddevelopment process. This has resulted
in a sample processing bottleneck thatdemands more rapid, scalable, andreproducible methods to supplant current
low-throughput and laborious manual
sample prep methods for common
workflows such as protein purificationand quantification, whole protein mass
determination, sequence coverage
analysis, and characterization of posttranslational modifications (PTMs).

AssayMAP applications

The Agilent AssayMAP Bravo platform
addresses these challenges by enabling
the parallel processing of samples
through a variety of LC/MS sample
prep workflows, with low variability andminimal hands on time. The AssayMAP
Bravo platform consists of easy-to‑use
yet flexible software optimized for LC/MS
sample prep workflows, the Bravo
liquid handler fitted with a 96-channel
AssayMAP syringe head, and disposable
5 µL packed-bed cartridges that mate withthe syringes. The liquid handling of the
AssayMAP Bravo provides precise flow
control through the cartridges, thereby
allowing chromatography to be performed
in a highly parallel format that enableshigh reproducibility, excellent sample
recovery, efficient washing, and very low
elution volumes. This Application Notedemonstrates the utility of the platform
for widely-used bioprocess workflows andbenchmarks analytical figures-of-merit for
automated antibody purification.

Antibody purification

The AssayMAP Bravo is controlled bystand-alone automated operations called
applications. Every application can beused independently or combined withother applications to execute workflows.
The common workflows are delivered
with the component applications bundled
together. Each application is optimized
to give maximum flexibility with theminimum number of inputs to ensureease-of-use. The workflows characterized
in this Application Note use threedifferent AssayMAP applications; Affinity
Purification, In-Solution Digestion1, andPeptide Cleanup1. While the applications
have different user-selectable features,reagents, and labware options, they
maintain a consistent organization withthree sections: Application Settings,
Deck Layout, and Labware Table(Figure 1). Before a run is executed, theapplication evaluates all entry fields
and alerts the user of any incompatible
settings that could lead to costly andtime-consuming mistakes.

The Affinity Purification application uses
Agilent PA-W and PG-W cartridges, which
are packed with immobilized protein A
and protein G resin, respectively. The
buffers and solutions used for thisapplication note are listed in Table 1, but
their composition may be varied to meetdifferent experimental design and purity
requirements. The main steps conductedby this application are:•Priming the cartridges to wet themand remove entrapped air•

Equilibrating the cartridge toestablish optimal conditions fortarget binding•

Loading samples on cartridges•

Washing the cartridges with up to
two wash solutions•

Eluting the purified antibody

Figure 1. Affinity Purification application user interface. The user modifies default Application Settingsand Labware selections as appropriate for the run, places consumables at the indicated deck locations,and clicks Run Affinity Purification. The settings shown were used for the experiment in Figure 3. User
Guides provide detailed explanations of all steps.

2

Cartridge performance was benchmarked
using antibody isotypes that suit therespective binding selectivity of protein Gor protein A; human IgG (hIgG) for PG-W
(protein G strongly binds all four subtypes
present in the polyclonal antibody
preparation) and subtype hIgG1 for PA-W
(protein A does not bind hIgG3).
Quantitative recovery was examinedby serially diluting hIgG1 (1–100 μg)
into 25 μL samples of particulate-free
Chinese hamster ovary (CHO) cell culture
supernatant (CCS), loading the sample
onto PA-W and PG-W cartridges in
triplicate, and washing the cartridges.
Bound antibody was eluted with 10 μL of
5 % acetic acid into an existing collection
volume of 10 μL of 5 % acetic acid for a
total of 20 μL (Figure 2). Eluted sample
from this type of experiment is routinely
quantified using a UV/Vis plate reader,but for this experiment quantification
was done using an Agilent 1260 Bio-inert
LC System with an Agilent Poroshell
300SB-C18 column (2.1 × 75 mm, 5 µm)
for greater sensitivity. The relationshipbetween loaded and eluted mass wasplotted.
Reproducibility was examined by seriallydiluting hIgG (12.5, 25, 50, and 100 μg)
into 100 μL of CCS, loading the samples
onto PG-W cartridges, and washing the
cartridges. A full plate of 96 samples(24 at each load mass) was purified andeluted with 50 μL of 5 % acetic acid into ahalf-area UV plate for quantification usinga plate reader. A relatively large elution
volume was used in this experiment tominimize variability introduced by theplate reader. The mass recovered ineach sample was plotted, and CVs were
calculated for each set of 24 replicates.

Table 1. Samples, reagents, and analytical instrumentation.
On-deck samples and reagents
Wash station

Deionized water, for all protocols

Affinity Purification v1.0
Cartridges

PA-W (G5496-60000), PG-W (G5496-60008)

Prime and equilibrate buffer

Phosphate-buffered saline (PBS)

SampleshIgG , hIgG1, or sequenced monoclonal antibody

Cartridge Wash 1

1 M NaCl in PBS

Cartridge Wash 2

PBS

Elution and syringe wash buffer

5 % acetic acid or 12 mM HCl plus 0.1 M NaCl

Existing collection solution

Nothing, 5 % Acetic acid, 12 mM HCl plus 0.1 M NaCl, or
550 mM Tris base, 9 M urea, 15 mM TCEP

In-Solution Digestion v1.0
Wash plate

50 mM NaOH

Alkylant

120 mM iodoacetamide (20 mM final)

Diluent mixture

3 mm TCEP (4 mM final)

Protease

trypsin, 0.075 μg/μL in 50 mM acetic acid (1:20 final)
chymotrypsin, 0.075 μg/μL in 1 mM HCl (1:20 final)
Glu-C, 0.075 μg/μL in purified water (1:20 final)

Peptide Cleanup v2.0
Cartridges

C18 Cartridges (5190-6532)

Priming and syringe wash

0.1 % TFA, 50 % acetonitrile

Equilibration and cartridge wash buffer

0.1 % TFA

Elution buffer

0.1 % TFA, 50 % acetonitrile

Eluate collection plate
Analytical instrumentation

0.1 % TFA

Automation
Core automation platform

Agilent AssayMAP Bravo (G5542A)

Agilent AssayMAP Bravo accessories

Risers, 146 mm (G5498B#055),
Peltier Thermal Station with STC controller (G5498B#035),
Custom Plate Nest (G5498B#017),
PCR Plate Insert (G5498B#013),
Orbital Shaking Station w/control Unit (G5498B#033)

Additional accessories

Agilent PlateLoc Thermal Microplate sealer (G5402A)

LC/MS
Mass spectrometer

Agilent 6550 iFunnel Q-TOF LC/MS,
Dual Agilent Jet Stream ESI

LC systems and columns

Agilent 1290 Infinity LC System
Agilent 1260 Infinity Bio-Inert LC System
Agilent AdvanceBio Peptide Mapping columns, C18:
Analytical - 2.1 × 250 mm, 2.7 µm (p/n 651750-902)
Guard - 2.1 mm Fast Guard (p/n 851725-911)
Agilent Poroshell 300SB-C18:
Analytical - 2.1 × 75 mm, 5 µm (p/n 660750-902)

3

The peptide quantification workflow
used the Affinity Purification, In‑Solution
Digestion, and Peptide Cleanup
applications. Ninety-six samples
containing 15 μg of a sequenced
monoclonal antibody (mAb) in 100 μL
CCS were purified using the Affinity
Purification application with PG-W
cartridges. The antibody was eluted
with 10 μL of 5 % acetic acid directly
into 20 μL of existing collection volumecontaining concentrated denaturant
and reductant in Tris buffer (Table 1)
for a final composition of 365 mM Tris
pH 8.1, 6.0 M urea, 10 mM TECP. The
eluates were incubated at 60 °C for
30 minutes. The AssayMAP Bravo deck
was reconfigured and the samples werealkylated, diluted, and Agilent proteomics
grade trypsin was added at 1:20
(enzyme:substrate) using the In-Solution
Digestion application. Reactions (180 μLeach) were incubated overnight at 37 °C,
followed by acidification with 20 μL of
10 % trifluoroacetic acid (TFA). The deck
was rearranged for the Peptide Cleanup
application and the samples were loaded
on C18 reversed-phase cartridges andwashed with 0.1 % TFA. Peptides were
eluted off the C18 cartridge with 15 μL of
0.1 % TFA in 50 % acetonitrile and directly

Affinity purification
characterization

The quantitative recovery range for PA-W
and PG-W cartridges was examinedusing the Affinity Purification application.
A serial dilution of 1–100 μg hIgG
or hIgG1 was loaded onto PG-W or PA-W
cartridges, respectively. The cartridges
were washed, and bound antibodies
were eluted and quantified. Recovery
exceeded 90 % of the loaded mass foreach cartridge type across the entire
range. Figure 2 illustrates the relationshipbetween loaded and eluted antibody
mass with the identity line shown inblack. The left panel shows the entire
range, while the right panel shows the
0 to 10 μg region.

An antibody sequence coverage
workflow used the Affinity Purification,
In‑Solution Digestion, and Peptide
Cleanup applications. The workflow
was run essentially as described above
for 15 μg samples of a sequenced mAb
in 100 μL CCS, except three different
proteases were used in parallel in the
In-Solution Digestion application. The
proteases used were trypsin (Agilent),
chymotrypsin (Promega Corp.), and Glu-C(Promega Corp.). Ten microliters (1 μg) of
each sample was analyzed using the 1290
HPLC and 6550 iFunnel Q-TOF LC/MS,
Dual Agilent Jet Stream ESI. The resulting
data were analyzed with SpectrumMill
software.
A

B

100

PG-W

80

PA-W

10

60
40
20
0

PG-W
PA-W

8
Eluate (µg)

Protein characterization workflows

Results and Discussion

mixed into 135 μL of 0.1 % TFA. A 10 μL(1 μg) amount of each sample wasanalyzed using an Agilent 1290 HPLC andan Agilent 6550 iFunnel Q-TOF LC/MS,
Dual Agilent Jet Stream ESI. The resulting
data were analyzed with MassHunter
BioConfirm software.

Eluate (µg)

Minimal elution volume was examined fortwo eluents with different stringencies
by loading 50 μg of antibody in duplicate
onto PA-W and PG-W cartridges andwashing the cartridges using the Affinity
Purification application. To examine
differences in the elution profiles,
antibodies were eluted from eachcartridge type with 5 % acetic acid or
12 mM HCl, 0.1 M NaCl (pH 2), using
a custom protocol that collected theeluate in 2 or 3 μL fractions. These small
volume fractions were eluted into wells
containing the same eluent for a total
volume of 50 µL. Quantification was
performed using an Agilent 1260 HPLC
as described above. Duplicates wereaveraged, and eluted mass per fraction
was plotted with respect to cumulative
elution volume.

6
4
2

0

20
40
60
80
Human IgG load (µg)

100

0

0

2

8
4
6
Human IgG load (µg)

Figure 2. Affinity Purification quantitative recovery using PG-W and PA-W cartridges.

4

10

100
75
Eluate (µg)

50
25
0

Minimum elution volume

Figure 4A shows maximum recovery inless than 10 uL of 5 % acetic acid for both
PG-W and PA-W cartridges. This eluent
exhibits high stringency and is a good
choice for keeping the sample volume low
at the beginning of a multistep workflowthat culminates in LC/MS analysis.
Figure 4B shows maximum recovery in
21 µL or less for PA-W, or 30 μL or less for
PG-W, when eluting with 12 mM HCl,
0.1 M NaCl. This eluent is a good choice
when a gentler elution solution is required.
The first fraction (void volume) contained
no protein for either eluent, which
increased the volume and lowered thepurified antibody concentration. The
Affinity Purification application allows theuser to specify the start and end points
for eluate collection, and automaticallydiscard the void volume, if desired.

24

48
Sample number

72

96

Figure 3. Affinity Purification reproducibility. The results from the purification of 96 hIgG samples using
the Affinity Purification application.

Table 2. Calculated recoveries and % CV.
Sample number

Load (µg)% Recovery

% CV

1–24

12.5

95.4

1.3

25–48

25

93.5

1.8

49–72

50

92.3

1.8

73–96

100

91.0

1.1

30
µg eluted per fraction

Precise flow rate control combined withsmall cartridge bed volumes enables
quantitative elution in very small volumes,
but eluent formulation and stringency
must also be evaluated to ensure
success. The elution profiles of PG-W
and PA-W cartridges with two eluentsof different stringencies were examined.
Fifty micrograms of hIgG or hIgG1 wasloaded onto PG-W or PA-W cartridges,
respectively. The cartridges were washed,
and bound antibodies were eluted witheither 5 % acetic acid (Figure 4A) or
12 mM HCl and 0.1 M NaCl (Figure 4B),
using 2 µL or 3 µL fraction collection,
respectively. Eluted antibody mass foreach fraction was plotted with respect tocumulative elution volume.

0

A

30

PG-W
PA-W
µg eluted per fraction

Reproducibility across an entire plateof 96 samples was tested by parallel
purification of hIgG in CCS, using PG-W
cartridges. Twenty-four cartridges wereused at each of four antibody massloads. The cartridges were washed, andeluted antibody was quantified (Figure 3).
Percent recovery and CVs were calculated(Table 2).

20

10

0

2

4
6
8
10
12
Cumulative elution volume (µL)

B
PG-W
PA-W

20

10

0

3

6

9

12 15 18 21 24 27 30 33 36
Cumulative elution volume (µL)

Figure 4. Minimum elution volume for antibody purification. Fifty micrograms of bound antibody was
eluted in 2- or 3‑μL fractions and mixed into a total volume of 50 μL for quantification. The eluted massfor each fraction was plotted with respect to cumulative elution volume.

5

Workflow for mAb peptide
quantification by LC/MS

Automated bottom-up proteomics workflows

A sample preparation workflow,
illustrated in Figure 5, was performed on
the AssayMAP Bravo in which a full plateof 96 samples each containing 15 μg of
mAb in CCS was processed using theAffinity Purification, In-Solution Digestion,
and Peptide Cleanup applications
described above. LC/MS analysis wasdone and extracted ion chromatograms(EIC) were generated for six proteotypic
peptides, from which relative protein
abundance could be inferred. CVs of
5–8 % were calculated from EIC peakareas for all 96 samples, which indicatedthat reproducibility of the entire workflow
was very high. An overlay of the EICs
from the first row of 12 samples is shownin Figure 6.
×106
3

Purify653CountsClean up

4

1

1

0

2

3

4

LC/MS

Figure 5. Workflow diagram for biotherapeutic antibody sample preparation. For this study, replicatesof mAb in particulate-free CCS were purified in parallel using the Affinity Purification application,
digested using the In-Solution Digestion application, and then the resulting peptides were purified andconcentrated on C18 reversed-phase cartridges using the Peptide Cleanup application, resulting insamples ready for LC/MS analysis.

22Digest5
6Acquisition time (min)78Peptide sequence% CV

1. SLSHSPG

7.8

2. TSTSPIVK

5.6

3. SQVFLK

8.0

4. FTGSGSGTQFSLK

6.6

5. VNSAAFPAPIEK

5.0

6. DVLTITLTPK

7.2

9

Figure 6. Peptide Quantitation Workflow reproducibility indicated by the overlay of EIC for six mAb peptides from 12 samples, and the CV calculated from thepeak area of the same six peptides from 96 samples.

6

Workflow for mAb sequence
coverage

A sample preparation workflow was
performed on the AssayMAP Bravo inwhich samples containing 15 μg mAb
in CCS were processed using Affinity
Purification, In-Solution Digestion, andPeptide Cleanup applications as describedabove (Figure 5), with the exception thatthree proteases (trypsin, chymotrypsin,or Glu-C), were tested in parallel for the
In-Solution Digestion step, to determine
sequence coverage for a mAb in asingle experiment. LC/MS analysis was
performed on the desalted peptides todetermine sequence coverage.

Distinct % AA
peptides coverage
99
26
79
33
69
19
49

Heavy chain
Trypsin
Chymotrypsin
Glu-C
Distinct
peptidesLight chain
Trypsin
Chymotrypsin
Glu-C

16
25
10

% AA
coverage
100
97
88
52

Figure 7. mAb sequence coverage. Green indicates combined amino acid coverage from all threeproteases, blue for individual proteases, and white shows lack of coverage. Combined coverage was 433
of 439 heavy chain amino acids, and all 214 light chain amino acids. By contrast, trypsin-only reactions
covered only 349 (79 %) for the heavy chain and 209 (97 %) for the light chain. The number of distinct
peptides and the percent coverage were determined using SpectrumMill software.

In Figure 7, sequence coverage isillustrated using colored bars to represent
detected peptides from N- to C-terminus(left to right). Upper and lower green bars
represent aggregate coverage for theheavy and light chains, respectively, andvarying shades of blue represent peptides
detected in digests by the individual
proteases. White space indicates a lackof sequence coverage for that region.
No single enzyme achieved complete
coverage of either polypeptide chain.
Most notably, a large gap in the dark blue
bars is seen for a 70 amino acid region
in the heavy chain that lacks trypsin
cleavage sites, possibly because theinstrumentation was tuned for sensitive
detection of much smaller peptides.
Digests with chymotrypsin and Glu-C
each produced detectable peptides within
this and other regions where tryptic
digestion left gaps, resulting in coverageof all but six amino acid positions forthe heavy chain. Light chain coveragefor this antibody was nearly complete
using trypsin alone, but digestion withchymotrypsin completed the sequence
coverage by filling in the remaining fiveamino acids.

7

Conclusions

Reference

The Agilent AssayMAP Bravo is a flexible
and easy-to-use automation platform thatallows the execution of workflows forbiotherapeutic protein sample preparation
prior to LC/MS. The automation oftedious, but critical, liquid handling steps
reduces hands-on time and maximizes
throughput, with high reproducibility.
Characterization of the Affinity
Purification cartridges showed a linear
range of quantitative binding and recovery
from 1–100 μg of loaded antibody,which could be eluted in 10 μL or less,with CVs below 5 %. More importantly,
the AssayMAP platform permits users
to combine the automation of Affinity
Purification, In‑Solution Digestion, andPeptide Cleanup into a single workflowthat maintains high reproducibility
throughout, as demonstrated by CVs
less than 10 % for the entire Peptide
Quantification workflow presented in thisApplication Note.

1. Russell, J.D.; et al., Automation for
LC/MS Sample Preparation: HighThroughput In-Solution Digestion
and Peptide Cleanup Enabled by the
Agilent AssayMAP Bravo Platform,
Agilent Technologies Application Note,publication number 5991-2957EN,
2013.

www.agilent.com/lifesciences/
assaymap
For Research Use Only. Not for use in diagnostic
procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2014, 2016
Published in the USA, March 16, 2016
5991-4872EN