Rapid Antibody Digestion Enabled by
Automated Reversed-Phase Desalting
on the Agilent AssayMAP Bravo
Platform
Application Note
Authors

Introduction

Michael Bovee, Adam Krahenbuhl, andMonoclonal antibodies are a rapidly growing class of biotherapeutics whose rigorous
characterization by mass spectrometry requires reproducible denaturation and digestion.
One of the major bottlenecks in this workflow is the trypsin digestion step, which
typically requires an overnight incubation. This long incubation significantly delays
decisions required to move projects forward. The slow digestion is mainly due to thepresence of denaturant in the digestion reaction. The time required for digestion canbe significantly decreased by removing, rather than simply diluting, the denaturant withsize exclusion chromatography (SEC)1. Although effective, SEC is labor-intensive anddifficult to automate. An alternative approach for removing denaturant is reversed-phase
chromatography, which is more amenable to automation. This Application Note presents
the development of an automated reversed-phase whole protein desalting solution on
the AssayMAP Bravo that decreases the time required for trypsin digestion of denatured
antibodies to as little as 1 hour. When this application is combined with the Affinity
Purification and In Solution Digestion applications on the AssayMAP Bravo, one can
automate the purification, denaturation, reduction, alkylation, cleanup, and digestion of
antibodies. The AssayMAP platform can thereby reduce the time required for a workflowthat begins with affinity purification of antibodies from complex matrices and ends withdigested peptides ready for LC/MS analysis to less than 8 hours.

Steve Murphy
Agilent Technologies, Inc.
Madison, WI

AssayMAP Bravo Platform

The AssayMAP Bravo platform consistsof easy-to-use yet flexible software,
the Bravo liquid handler with an
AssayMAP head, and 5-µL packed‑bed
microchromatography cartridges.
The AssayMAP software contains
pre‑optimized protocols for LC/MS
sample preparation workflows designed
to give bench scientists access to thepower of automation without requiringthem to become automation experts.
The AssayMAP head contains 96 probesyringes, which allows eight to 96
samples to be processed in parallel.
Each syringe probe forms a liquid/liquidcontact with the top of the cartridge
resin bed, allowing positive displacement
control of fluid movement across theresin bed which, combined with theprecise liquid handling capabilities of
the Bravo, enables chromatographic
performance, such as quantitative
binding, efficient washing, and low
elution volumes, in a highly parallel
format. The final component of the
AssayMAP platform is the cartridge,
which can be packed with a variety ofresins to automate sample preparation
for a wide range of LC/MS workflows.
The cartridges are packed to exacting
specifications that ensure packed-bed
consistency and high reproducibility.
The AssayMAP platform supports protein
quantification and characterization
workflows including affinity purification,
enzymatic digestion, protein and peptide
cleanup, peptide mapping, peptide
fractionation, N-Glycan analysis, andphosphopeptide enrichment. The software
for running each of these workflows
comes with default settings to allow
the workflow to be rapidly implemented,
with flexibility that accommodates
optimization if required. This Application
Note demonstrates how to optimize thecritical parameters for denatured antibody
desalting using AssayMAP RP-W
cartridges (reversed‑phase, wide pore).

Experimental Methods
The Protein Cleanup application, like most
AssayMAP applications, has five major
steps: cartridge priming, equilibration,
sample loading, cartridge washing, andelution of the target analyte. Parameters
for each of these steps were optimized
using RP-W cartridges and human
IgG1 (hIgG1) as a model antibody. This
Application Note may be used as a
guide for the optimization of conditions
for other proteins of interest in casethe conditions shown here do not givesatisfactory results.

Materials

Antibodies were purchased from
Sigma-Aldrich (SILuLite) and Athens
Research and Technology (hIgG1 andhIgG polyclonal). Guanidine, TCEP,
iodoacetamide, trifluoroacetic acid, andammonium bicarbonate were purchasedfrom Sigma-Aldrich. Tris base andTris HCl were purchased from EMD
Millipore. Zeba Spin desalting plates werepurchased from Thermo Scientific, andused according to the manufacturer’s
instructions. Cell-free conditioned
medium from a Chinese Hamster Ovary(CHO) cell culture was purchased from
Aldevron and stored in frozen aliquots
at –20 °C. Sequencing grade trypsin waspurchased from Agilent Technologies.

Table 1. Instrumentation and consumables.
Automation
Platform

Agilent AssayMAP Bravo (G5542BA)

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)

Cartridges

PG-W (G5496-60008)
RP-W (G5496-60086)

Analytical instrumentation
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 Poroshell 300SB-C18, 2.1 × 75 mm, 5 µm (660750-902)
Agilent AdvanceBio Peptide Mapping, 2.1 × 250 mm, 2.7 µm (651750-902)
Agilent AdvanceBio Peptide Mapping Fast Guard, 2.1 × 5 mm (851725-911)2

Antibody denaturation, reduction,
alkylation, and dilution

Denaturation and reduction was
performed by adding three volumesof 8 M guanidine, 13.3 mM TECP, and
500 mM Tris pH 8.0 to the antibody
sample to give a final concentrationof 6 M guanidine, 10 mM TCEP, and375 mM Tris pH 8.0. The samples wereincubated for 60 minutes at 60 °C.
Following denaturation and reduction,
samples were alkylated by adding theappropriate amount of iodoacetamide
to the denatured and reduced antibody
sample to give a final concentration of
20 mM. This solution was incubatedfor 30 minutes at ambient temperature
protected from light. The sample wasthen acidified and diluted to 2.5 M
guanidine with 1.75 % TFA (1 % TFA final),
unless otherwise noted, in preparation
for binding to RP-W cartridges. Reactions
were prepared in bulk for Figures 2–13,
which focus on cartridge performanceand comparison to other methods for
reducing denaturant concentration.

Individual reactions were prepared for
Figure 14, which focuses on a complete
workflow using three AssayMAP
applications: Affinity Purification, Protein
Cleanup, and In‑Solution Digestion: Single
Plate.

Running the Protein
Cleanup application

Unless otherwise indicated, the Protein
Cleanup application was run in two
sequential runs to minimize the timethat the eluent was on the deck andsusceptible to evaporation, as changes in
the organic concentration can negatively
impact recovery (Figure 8). The majorityof the total run time for the Protein
Cleanup takes place during the first ofthese two sequential runs. During thefirst run, the default settings (Figure 1)
were used for Initial Syringe Wash, Prime(100 µL of 60 % acetonitrile, 0.1 % TFA
at 300 µL/min), Equilibrate (50 µL of
0.1% TFA at 10 µL/minute), Load Sample(variable volume depending on theexperiment at 5 µL/min), Cup Wash

Figure 1. Protein Cleanup application user interface with default settings.

3(50 µL of 0.1 % TFA), and Internal
Cartridge Wash (50 µL of 0.1 % TFA at
10 µL/min), but the Stringent Syringe
Wash, Elution, and Final Syringe Wash
steps were turned off. At the end of thisrun, the deck was not altered except to
add the eluent plate. The second runof the Protein Cleanup protocol, which
only takes approximately 5 minutes andimmediately follows the first run, had theInitial Syringe Wash, Prime, Equilibrate,
Load Sample, Cup Wash, and Internal
Cartridge Wash steps turned off. The
second run used default settings for theStringent Syringe Wash (50 µL of 60 %acetonitrile, 0.1% TFA unless indicatedotherwise) and Final Syringe Wash.
The Elution settings were 15 µL (60 %acetonitrile, 0.1 % TFA at 5 µL/min unlessindicated otherwise) for Volume, and 2 µL
(dispensed to waste) for Eluate Discard,
which resulted in a total eluent volumeof 13 µL. The Existing Volume was set
to 143 µL (0.1 % TFA for Figures 2–11,
or 50 mM ammonium bicarbonate for
Figures 12–14).

Prime optimization

The concentration of acetonitrile
required to prime the RP-W cartridges
was determined. The Protein Cleanup
application was used as described above
except that RP-W cartridges were primed
with 0–100 % acetonitrile in triplicate.
A 30 μg amount of denatured, reduced,
alkylated, and diluted hIgG1 was cleaned
up per cartridge. Antibody in the eluate
and flowthrough was quantified as
described above. Figure 3 shows theresults.

EluateFlowthrough150
125
Recovery (µg)

Dynamic binding capacity was determinedusing hIgG1 that was serially diluted inwater, then denatured, reduced, alkylated,and diluted. The RP-W cartridges wereprimed, equilibrated, loaded with a serial
dilution of sample (N = 3 per hIgG1 massload), washed, and eluted using theProtein Cleanup application. Antibody
recovered from RP-W cartridges wasquantified using an Agilent 1260 Bio-Inert
HPLC fitted with an Agilent Poroshell
300 SB-C18 column, on the basis ofabsorbance at 214 nm relative to a
standard curve. Figure 2 shows theresults.

175

100
75
50
25
0

0

25

50

75
100
Load (µg)

125

150

175

Figure 2. Quantitative recovery of denatured antibodies
using RP-W cartridges under optimized conditions. An hIgG1
antibody was serially diluted, denatured, reduced, alkylated,
and adjusted to 2.5 M guanidine and 1 % TFA. The samples
were loaded onto RP-W cartridges (n = 3 per data point). The
cartridges were washed, and the bound antibody was eluted.
Flowthroughs and eluates were collected and quantified. Error
bars represent ±1 S.D. The identity line is shown in gray forreference.

100

80

Recovery (%)

Binding capacity under
optimized conditions

EluateFlowthrough

60

40

20

0

0

20

40
60
% Acetonitrile in prime

80

100

Figure 3. Optimization of the priming solution. RP-W cartridges
were primed with increasing concentrations of acetonitrile, andequilibrated in 0.1 % TFA. Denatured, reduced, alkylated, and diluted
hIgG1 was loaded onto RP-W cartridges (n = 3 per data point).
The cartridges were washed, and the bound antibody was eluted.
Flowthroughs and eluates were collected and quantified. Error bars
represent ±1 S.D.

4

Load sample optimization

Human IgG1 was serially diluted in water,then denatured, reduced, alkylated, anddiluted. The Protein Cleanup application
was run three times (n = 3 per sample
per run), once for each guanidine
concentration of 5.0, 2.5, or 1.25 M withrespective load volumes of 155, 310,
or 620 µL. Antibody in the eluate andflowthrough was quantified as describedabove. Figure 4 shows the results.

The effect of sample alkylation
SILuLite antibody was denatured,
reduced, then incubated in the presenceor absence of iodoacetamide as describedabove. A 40 μg amount of these samples
was diluted as described and loaded onto
RP-W cartridges. The cartridges were
washed, and the bound antibody was
eluted using 50, 60, or 70 % acetonitrile in
0.1 % TFA. The eluate was quantified by
HPLC. Figure 5 shows the results.

Eluate, 5 M
Flowthrough, 1.25 M

125
Recovery (µg)

The effect of guanidine concentrationin the sample

Eluate, 2.5 M

150

Flowthrough, 2.5 M
Flowthrough, 5 M

100
75
50
25
0

0

25

50

75
100
Load (µg)

125

150

175

Figure 4. Effect of guanidine concentration on recovery. A serial
dilution of hIgG1 was denatured, reduced, alkylated, and diluted to
1.25, 2.5, or 5.0 M guanidine. The samples were loaded onto RP-W
cartridges (n = 3 per mass load). The cartridges were washed, andbound antibody was eluted. Eluates (closed symbols, solid lines)
and flowthroughs (open symbols, dashed lines) were collected andquantified. Error bars represent ±1 S.D. The identity line is shown ingray for reference.

100

Recovery (% of load)

The effects of guanidine concentration,
alkylation, sample volume, eluent
evaporation, and flow rate were
examined.

Eluate, 1.25 M

175

Alkylated
Not alkylated

80
60
40
20
0

50

60
% Acetonitrile in eluent

70

Figure 5. Effect of alkylation on recovery. SILuLite antibody was
denatured, reduced, and either alkylated or not alkylated before
loading 40 µg onto duplicate RP-W cartridges. Bound antibody
was eluted with varying acetonitrile concentrations in 0.1 % TFA.
Eluates were collected and quantified.

5

A

Eluate (µg)

5075200 µL load
300 µL load
1,000 µL load

0

B

50

25

200 µL load
300 µL load
1,000 µL load

25

0

75

25

C

50

Eluate (µg)

Human IgG1 was denatured, reduced,
alkylated, and diluted as described above.
The samples were further diluted with
2.5 M guanidine to volumes that would
allow 25, 50, and 75 µg to be loaded in
total sample volumes of 200, 300, or
1,000 µL. In this way, the 25, 50, and75 µg mass loads could be applied to
the RP-W cartridges in different total
sample volumes while maintaining the
guanidine concentration at 2.5 M in all
samples. These volumes correspond tothe maximum capacities of the labware
for AssayMAP applications. The Protein
Cleanup application was run once foreach load volume as described above
except that two elution conditions andtwo sample load flow rates were tested.
The eluent was placed on the AssayMAP
deck immediately before the elution
step (Figure 6A) or at the start of the run(Figures 6B and 6C). The sample load flowrate was 5 µL/min for Figures 6A and B,
whereas it was 20 µL/min in Figure 6C.
Antibody in the eluate and flowthroughwas quantified as described above.

75

Eluate (µg)

The impact of sample load volume andeluent evaporation

50
Load (µg)

75

0

0

25

Load (µg)

50200 µL load
300 µL load
1,000 µL load

25

0

0

25

50
Load (µg)

75

Figure 6. Effect of sample volume and eluent evaporation on recovery. Human IgG1 was denatured,
reduced, alkylated, and diluted to 200, 300, or 1,000 µL. The samples were loaded onto RP-W cartridges(n = 3 per data point) at 5 µL/min (Panels A and B) or 20 µL/min (Panel C). The cartridges were washed,
and the bound antibody was eluted. The eluates were collected and quantified. The panels compare
recoveries when the eluent was placed on the deck immediately before the elution step (A), or placedon the deck for the entire run (Prime, Equilibrate, Load, and Wash steps) (B and C), and was therefore
susceptible to evaporation. Error bars represent ±1 S.D.

The effect of sample loading flow rate150

2 µL/min EL
5 µL/min EL
10 µL/min EL

125

20 µL/min EL
2 µL/min FT

100
Recovery (µg)

Human IgG1 was serially diluted,
denatured, reduced, alkylated, anddiluted. The samples were loaded onto
RP-W cartridges at 2, 5, 10, or 20 µL/minin separate runs using the Protein
Cleanup application. For each run, thecartridges were washed and eluted as
decribed. Antibody in the eluate andflowthrough was quantified. Figure 7
shows the results.

75

5 µL/min FT
10 µL/min FT

75

20 µL/min FT

50
25
0

0

25

50

75
Load (µg)

100

125

150

Figure 7. Effect of the sample loading flow rate on recovery. A serial dilution ofhIgG1 was denatured, reduced, and alkylated. The samples were loaded onto
RP-W cartridges (n = 3 per condition) at sample load flow rates of 2 µL/min(red symbols), 5 µL/min (yellow), 10 µL/min (green), and 20 µL/min (blue).
Cartridges were washed, and the bound antibody was eluted. The identity line
is shown in gray for reference. Eluates (closed symbols, EL) and flowthroughs(open symbols, FT) were collected and quantified.

6

The minimum volume required for elution
from RP-W cartridges was examined.
Human IgG1 was denatured, reduced,
alkylated, and diluted as described.
A 50 μg amount of this sample was
loaded onto eight RP-W cartridges using
the Protein Cleanup application. The
cartridges were washed, and elution
was performed at 5 µL/min with 60 %acetonitrile in 0.1 % TFA, using a
special protocol that dispensed eluent
through cartridges in 12 sequential 2-µL
fractions into 48 µL of 0.1 % TFA in theelution plate wells. Eluted antibody wasquantified as described above. Figure 9shows the results.

100

A

50

25

0

B

75
Recovery (%)

75
Recovery (%)

The effect of eluent pH and acetonitrile
concentration was evaluated. Human
IgG1 was denatured, reduced, andalkylated. A 50 μg amount of this sample
was loaded onto RP-W cartridges
using the Protein Cleanup application
as described above except that the
cartridges (n = 3 per elution condition)
were eluted with 40, 50, 60, or 70 %acetonitrile in either 0.1 % TFA (pH 3)
or 50 mM ammonium bicarbonate(pH 8). Eluted antibody was quantified
as described above. Figure 8 shows theresults.

100

50

25

40
50
60
70
% Acetonitrile in eluent

0

40
50
60
70
% Acetonitrile in eluent

Figure 8. Effect of eluent composition on recovery. Human IgG1 wasdenatured, reduced, and alkylated. The samples were loaded onto RPW
cartridges (n = 3 per condition). The cartridges were washed, and thebound antibody was eluted with various acetonitrile concentrationsin (A) 0.1 % TFA, pH 3, or (B) ammonium bicarbonate, pH 8. Error bars
represent ±1 S.D.
4030
µg per fraction

Elution optimization

20

10

0

2

4

6
8
10
Cumulative elution volume (µL)

12

14Figure 9. Minimum required elution volume. Human IgG1 wasdenatured, reduced, and alkylated. Samples were loaded onto RP-W
cartridges (n = 8 per data point). The cartridges were washed, andbound antibody was eluted in 2-µL fractions using a custom elution
protocol with 48 µL of diluent added in advance to each well of theelution plate. Eluted antibody in the 50-µL fractions was quantified.
Error bars represent ±1 S.D.

7

Various concentrations of hIgG1 weredenatured, reduced, alkylated, anddiluted. The Protein Cleanup application
was used in combination with RP-W
cartridges to clean up 10, 25, 50, and75 µg of these samples (n = 12 for eachloaded mass). Eluted mass was quantified
and plotted with respect to loaded
mass. CVs and recoveries for each setof 12 replicates are reported in the table
insert (Figure 10).

100

75
Eluate (µg)

Protein cleanup reproducibility
and recovery

50

25

0

0

Protein cleanup recovery of various
hIgG antibodies

24
Sample number

Samples

Load (µg) % Recovery % CV

1–12

10

104

2.6

13–24

25

103

1.8

25–36

50

103

1.3

37–48

75

99

1.4

36

48

Figure 10. Reproducibility and recovery. Various concentrations of hIgG1 were denatured, reduced, andalkylated. Twelve samples at each concentration were loaded onto RP-W cartridges, the cartridges were
washed, and the bound antibodies were eluted and quantified. CVs and recovery were determined foreach set of 12 replicates (summarized in the inset table).
75

hIgG1
hIgG1 polyclonal
SILuLite mAB

50
Eluate (µg)

Various concentrations of threecommercially available hIgG antibodies
were denatured, reduced, alkylated, anddiluted. Amounts of 25, 50, or 75 µg ofthese samples were desalted on RP-W
cartridges (n = 3) using the Protein
Cleanup application. Eluted antibodies
were quantified as described. Figure 11
shows the results.

12

25

0

0

25

Load (µg)

5075Figure 11. Recovery of three different hIgG antibodies. Various concentrationsof the hIgG1, hIgG polyclonal, and SILuLite antibodies were denatured,
reduced, and alkylated. Samples containing 25, 50, or 75 µg were loaded onto
RP-W cartridges (n = 3 per condition). Eluted antibody was quantified. Error
bars represent ±1 S.D. The identity line is shown in gray for reference.

8

Trypsin digestion time course

Human IgG1 was denatured, reduced,
alkylated, then either diluted to 0.9 M
guanidine, desalted using RP-W
cartridges, or desalted using an SEC
96-well spin plate. SEC samples were
processed according to the instructions
provided by the manufacturer, without
further optimization. A 25 μg amount ofthese diluted or desalted samples were
×107
4.5

digested with trypsin in quadruplicate
for 0.5, 1, 2, 4, or 18 hours at 37 °C. The
reactions were stopped at each time pointby the addition of 10 % TFA to a final
concentration of 1 %, then refrigerated.
Peptides in digestion reactions that
contained 0.9 M guanidine were desaltedusing the AssayMAP Peptide Cleanup
application and C18 cartridges. All
samples were adjusted to the same
protein concentration of approximately

150 µg/mL, and 0.5 µg injections wereanalyzed on an Agilent 1290 HPLC and an
Agilent 6550 Q-TOF mass spectrometeras described below. Figure 12 shows
Total Ion Chromatograms (TICs) for
each desalting method. Twelve peptides
were quantified at each digestion time
point based on MS1 peak area for each
desalting method. Figure 13 shows theresults.
0.5 hours
1 hour2 hours
4 hours
18 hours

A

4

Diluted to 0.9 M guanidine

Counts

3.5
3
2.5
2
1.5
1
0.5
0

0

×107
4.5

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0
5.5
6.0
Acquisition time (min)

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0
5.5
6.0
Acquisition time (min)

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0
5.5
6.0
Acquisition time (min)

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

B

4

Desalted on RP-W

3.5
3
2.5
2
1.5
1
0.5
0

0

×10
4.5

0.5

1.0

7

C

4

Desalted by SEC

3.5
3
2.5
2
1.5
1
0.5
0

0

0.5

1.0

Figure 12. Qualitative analysis of an antibody digest time course. Human IgG1 was denatured, reduced, and alkylated, and samples were: A) diluted to 0.9 M
guanidine; B) desalted using RP-W cartridges and eluted into 50 mM ammonium bicarbonate (ABC) and 5 % acetonitrile final; C) desalted into 50 mM ABC using
size exclusion chromatography. Following these various treatments, the samples were trypsin digested for 0.5 hours (red trace), 1 hour (purple), 2 hours (green),
4 hours (blue), or 18 hours (black). TICs of replicates (n = 4) at each time point were overlaid using Agilent MassHunter, to illustrate the digest progression foreach guanidine concentration reduction method.

9

40

A

Diluted to 0.9 M guanidine

0.5 hours
1 hour2 hours
4 hours
18 hours

Peak area

30
20
10

0

VVSV

×10

6

40

THTC

TPEV

GPSV

NQVS

ALPA

TTPP

FNWY STSG

DTLM WQQG GFYPB

Desalted on RP-W

0.5 hours
1 hour2 hours
4 hours
18 hours

30
Peak area

A workflow beginning with antibody
purification and ending with LC/MS
quantification of digested peptides was
executed using AssayMAP automatedsample preparation. Thirty two samplesof serum-free cell culture supernatantfrom a nonantibody-expressing CHO
cell culture were spiked with hIgG1(n = 16 samples) or Sigma SILuLite(n = 16). The antibodies were purified
from the matrix using the AssayMAP
Bravo Affinity Purification applicationand protein G (PG-W) cartridges using
optimal binding conditions previously
described (Bovee, et al.)2. Briefly, the
PG-W cartridges were loaded with thesamples and washed with PBS, thenthe purified antibodies were eluted with
10 µL of 5 % acetic acid directly into a
96-well PCR plate containing 30 µL of
denaturation solution (8 M guanidine,
13.3 mM TCEP, 0.5 M Tris pH 8) in eachwell for a final concentration of 6 M
guanidine, 10 mM TCEP, and 0.375 M
Tris pH 8. The eluate was automatically
mixed by the AssayMAP Bravo at theend of the run. The eluate plate wasremoved from the deck, sealed, andincubated for 60 minutes at 60 °C
to denature and reduce the purified
antibodies. The plate was allowed to coolat ambient temperature for 5 minutes
before centrifugation for 1 minute at
500xg to collect condensation. The
sample plate and a plate containing
iodoacetamide were placed on the
AssayMAP Bravo deck with opaque lids
along with a nonlidded plate containing
post-alkylation diluent. The In-Solution
Digestion: Single Plate application wasused to alkylate then dilute the denatured
antibodies by removing the lids from thesample plate and iodacetamide platewith the gripper arm, aspirating 5 µL
of 180 mM iodoacetamide per syringe
from the iodacetamide plate, adding theiodoacetamide to the samples (20 mM
final concentration), mixing, relidding bothplates, and incubating for 30 minutes atambient temperature. After alkylation wascomplete, the gripper arm removed thelid from the sample plate, 51 µL of diluent(1.9 % TFA) was aspirated from thediluent plate and added to the samples
to give a final concentration of 2.5 M

×106

20
10

0
×106
20

VVSV

THTC

TPEV

GPSV

NQVS

ALPA

TTPP

FNWY STSG

DTLM WQQG GFYPC

Desalted by SEC

0.5 hours
1 hour2 hours
4 hours
18 hours

15
Peak area

Workflow reproducibility

10

5

0

VVSV

THTC

TPEV

GPSV

NQVS

ALPA

TTPP

FNWY STSG

DTLM WQQG GFYP

Figure 13. Quantitative analysis of an antibody digest time course. Human IgG1 was denatured, reduced,
alkylated, then treated in one of three ways: A) The sample was diluted to 0.9 M guanidine; B) the
sample adjusted to 2.5 M guanidine and 1 % TFA, desalted using RP-W cartridges, and diluted to 5 %acetonitrile; C) the sample was desalted using size exclusion chromatography. (The Y-axis for C wasscaled for easier visual comparison with A and B.) Following these various treatments, the samples
were trypsin digested for 0.5 hours (red trace), 1 hour (yellow), 2 hours (green), 4 hours (blue), or
18 hours (black). All 12 of the conserved peptides were quantified based on MS1 peak area. Error bars
represent ±1 S.D. Peptides ranged from 7–26 amino acids in length, but are abbreviated to the first fouramino acids for simplicity. Complete peptide sequences are shown in Figure 14D.

10

LC/MS data acquisition
and analysis

Peptides were separated with an
AdvanceBio Peptide Mapping C18
column (2.1 × 150 mm, 2.7 μm) at
a flow rate of 0.4 μL/min with a
20‑minute method (from 5 to 40 %B
over 10 minutes; A = 0.1 % formic
acid in water, B = 0.1 % formic acid in
ACN). MS scans were acquired overthe mass range 100–1,700 m/z at ascan rate of 8 spectra/sec. MS/MS
scans were acquired over the mass
range of 100–1,700 m/z at a scan rateof 3.5 spectra/sec. The three mostintense precursor peaks were selected
for auto MS/MS fragmentation. Dynamic
exclusion was set for 12 seconds.
For the hIgG1 and SILuLite antibodies
analyzed by LC/MS in this study, theamino acid sequence was only available
for the SILuLite antibody. Since hIgG1
antibodies have highly conserved
sequences, MS/MS spectra from both
antibodies were searched against the
SILuLite sequence using SpectrumMill(Agilent Technologies). For peptide
identification, a mass tolerance of
10 ppm was permitted for intact peptide
masses, and 50 ppm for CID-fragmented
ions, with allowance for two missedcleavages in the trypsin digests, oxidized
methionine, and deamidated asparagine
as potential variable modifications.
Carboxyamidomethylation (C) was set asa fixed modification.

A

PurifyDenature

DesaltDigestB

Peptide peak area

×106
40

Human IgG1 peptides

30
20
10
0

0

4

12

8
Sample number

16

C
×106
40
Peptide peak area

guanidine and 1 % TFA, the samples
were mixed, and the lid was placedback on the sample. This sample plate
became the load plate for the Protein
Desalting application, which was run as
described above. Finally, the In-Solution
Digestion application was used to initiate
the digestion step through the additionof trypsin (1:25 final by mass) to eachsample. The protocol was automaticallypaused to allow the plate to be sealed, a1-hour on-deck incubation at 37 °C was
performed, the seal was removed, and theprotocol was completed by adding 20 µL
of 10 % TFA to the samples to inactivate
the trypsin (1 % TFA final). Peptides wereanalyzed as described below. Figure 14
shows the results.

SILuLite peptides

30
20
10
0

0

4

8
Sample number

D

12

16

LC/MS

VVSV
TPEV
GPSV
ALPA
NQVS
TTPP
FNWY
STSG
DTLM
WQQG
GFYP
THTC

VVSV
TPEV
GPSV
ALPA
NQVS
TTPP
FNWY
STSG
DTLM
WQQG
GFYP
THTC

% CV
PeptidehIgG1

SILuLiteVVSVLTVLHQDWLNGK

5.5

5.4

TPEVTCVVVDVSHEDPEVK

5.2

4.1

GPSVFPLAPSSK

6.3

4.2

ALPAPIEK

6.3

4.6

NQVSLTCLVK

6.4

4.3

TTPPVLD

7.0

4.3

FNWYVDGVEVHNAK

6.3

5.0

STSGGTAALGCLVK

8.4

6.1

DTLMISR

7.2

5.0

WQQGNVFSCSVMHEALHNHYTQK

11.2

5.7

GFYPSDIAVEWESNGQPENNYK

12.5

6.5

THTCPPCPAPELLGGPSVFLFPPKPK

18.2

17.1

Figure 14. Reproducibility of an antibody sample prep workflow using the Agilent AssayMAP Bravo.
A) Icons illustrate the main steps of the biotherapeutic antibody sample prep workflow that was run
to generate the data in this figure using the AssayMAP Bravo. Human IgG1 or SILuLite (n = 16 foreach antibody) were processed in parallel. Each antibody type was purified from a CHO cell-free matrix
using PG-W cartridges, denatured, reduced, alkylated, desalted on RP-W cartridges, and digested for
60 minutes using the AssayMAP Bravo. The resulting peptides were analyzed by LC/MS. Peak areas
for the 12 fully tryptic peptides are shown in graphs B and C. CVs for individual tryptic peptides are
summarized in the table (D). Peptide abbreviations are underlined in the table.

11Twelve peptides were quantified using
MS1 peak integration. M, M+H, and
M+2H precursor mass for each peptide
were extracted and integrated using
skyline.

Results and Discussion
Trypsin digestion is a key component of
the sample preparation for many protein
quantification and characterization
workflows, yet it remains a majorbottleneck, often requiring overnightincubation for complete digestion. It has
been reported that even small amountsof guanidine hydrochloride greatly inhibit
trypsin activity, and removal of guanidine
results in nearly complete digestionin as little as 30 minutes (Ren, et al.,Analytical Biochemistry 392:12, 20091).
Desalting proteins to hasten trypsin
digestion typically involves SEC, which
is difficult to perform in an automatedfashion, especially for large numbersof samples or large volumes. Desalting
by reversed-phase can address theselimitations because analytes are retainedon the solid phase while salts and matrix
components flow through. This approach
uses much smaller resin bed volumes,
simplifying automation and sample
volume control.

Optimized RP-W
cartridge performance

Reversed-phase desalting of denatured,
reduced, alkylated, and diluted
antibodies using the Protein Cleanup
application, RP-W cartridges, and ahIgG1 under optimal conditions is shownin Figure 2. Briefly, a serial dilution ofhuman IgG1 antibody was denatured
and reduced with guanidine and TCEP,
respectively, followed by alkylation withiodoacetamide. Samples were thendiluted and acidified before loading
onto AssayMAP RP-W cartridges. The
cartridges were washed, and boundantibody was eluted and diluted to reduce
the acetonitrile concentration prior toquantification by HPLC. Figure 1 shows
the Protein Cleanup application withdefault settings. These settings can beused as shown for protein cleanup, but

the highest yield and reproducibility forlarge volume samples will be obtained
if the protocol is run using a load flowrate up to 20 µL/min or by placing theeluate on the deck immediately before theelution step (see Figure 6) to minimize
evaporation altering the concentrationof the eluent. Figure 2 shows that RP-W
cartridges can quantitatively bind andelute up to 75 µg of denatured, reduced,
and alkylated antibody per RP-W cartridgewith less than 10 % breakthrough.

Prime optimization

AssayMAP cartridges are shipped withdry resin, and require a priming step toprepare them for use. The priming step
uses a rapid flow rate to purge entrained
air from the resin bed. RP-W cartridges
require priming with a concentrationof organic solvent sufficient to remove
all air, and condition the surface ofthe resin. This step is essential for
maximum capacity and reproducibility.
To determine the minimum effectiveacetonitrile concentration for priming,
RP-W cartridges were primed with arange of acetonitrile concentrations,equilibrated in 0.1 % TFA, loaded withdenatured, reduced, and alkylated
hIgG1, washed, and eluted directly
into 11 volumes of diluent. The final
dilution step was included to reduce
the organic concentration to 5 % priorto quantification by HPLC. As shownin Figure 3, RP-W cartridges must be
primed with at least 20 % acetonitrile toquantitatively recover antibody during theelution step. Since elution also requires
greater than 20 % acetonitrile (seebelow), reagent preparation is simplified
when the same solution is used for
priming and elution.

Load sample optimization

Sample loading parameters thatwere investigated include guanidine
concentration, alkylation, sample load
volume, and flow rate. The effect ofsample dilution on recovery from RP-W
cartridges was tested using a serial
dilution of denatured, reduced, alkylated
hIgG1 diluted to 5.0, 2.5, or 1.25 M

12guanidine. The RP-W cartridges were
loaded with sample, washed, and thebound antibody was eluted. Sample
dilution had little effect on recovery up to
approximately 75 µg. Dilution is necessary
for maximum binding capacity, but thisrequires a larger sample volume and acorrespondingly longer time to load thesamples (Figure 4).
The effect of alkylation on the binding ofdenatured and reduced antibodies was
examined by denaturing, reducing, andeither alkylating with iodoacetamide, or
not alkylating a serial dilution of hIgG1.
The samples were loaded onto RP-W
cartridges. The cartridges were washedand eluted with various concentrations ofacetonitrile. The results in Figure 5 showthat alkylation is required for the best
recovery under the standard conditions
used in this study. The magnitude ofthis effect is antibody-dependent (datanot shown). Alkylation has the effect of
reducing the hydrophobicity of modified
cysteines3. Nonalkylated proteins may
require optimized conditions different
from those described here for alkylated
antibodies.
The effect of sample volume and eluent
evaporation on recovery was tested
with 25, 50, or 75 µg hIgG1 that had
been denatured, reduced, alkylated, anddiluted to 200, 300, or 1,000 µL. These
volumes reflect the capacity limits ofthe three 96-well labware options: PCR
plate, round-bottom plate, and 1 mL
deepwell plate, respectively. The samples
were desalted using a two‑step Protein
Cleanup protocol where the eluent wasadded immediately before elution, or
they were desalted using the Protein
Cleanup protocol where the eluent was
put on the deck at the beginning of run,then the protocol was conducted in one
continuous run. If a two-step approach isused, the recovery is high regardless ofsample volume (Figure 6A). If the elution
is on the deck for the entire length of
the run, there may be some decrease
in recovery with large sample volumes(Figures 6B and 6C).

The time the sample is exposed to theresin in the cartridge (residence time)
is a critical factor for efficient binding.
Residence time is inversely related toflow rate. The relationship between
flow rates and recovery was examinedby loading serial dilutions of hIgG1 that
had been denatured, reduced, alkylated,diluted then loaded onto RP-W cartridges
at flow rates of 2, 5, 10, or 20 µL/min.
The cartridges were then washed, andbound antibody was eluted. Antibody
recovered in both the flowthrough andthe eluate was quantified. For RP-W
cartridges, the efficient capture ofdenatured hIgG1 was similar for load flow
rates up to 20 µL/min (Figure 7). Higher
flow rates were not examined since
20 µL/min is sufficient for loading the
maximum sample volume (1 mL) in lessthan 1 hour. The default load flow rate
was set to 5 µL/min to be consistent withthe default flow rate of other AssayMAP
cartridges, which show diminishingrecovery as flow rate increases from 2 to20 µL/min.
The effect of sample acidification
on recovery was also tested using
denatured, reduced, and alkylated hIgG1.
Sample pH did not significantly affectsample recovery for sample loads up to75 µg. It was, however, observed thatsample acidification tended to improve
reproducibility of recovery, and it wasnever observed to negatively impact
recovery (data not shown).

Internal cartridge wash
optimization

The Protein Cleanup application wash
step uses 50 µL (10 column volumes) at
10 µL/min by default. No benefit wasobserved by increasing the wash volume
or decreasing the wash flow rate (datanot shown). Both of these parameters
can be adjusted if desired.

Elute optimization

AssayMAP Bravo applications provide
elution options that give the user control
over the volume and buffer compositionof the eluted sample. The effect of pH,
acetonitrile concentration, and eluent
volume were examined.

To determine the optimal eluent
composition, hIgG1 was denatured,
reduced, alkylated, diluted, thenloaded onto RP-W cartridges. The
cartridges were washed, then eight
eluent conditions (four acetonitrile
concentrations at pH 8 or pH 3) were
tested. Elution with greater than 70 %acetonitrile was not tested due to the riskof protein precipitation. Figure 8 shows
acidification of the eluent with 0.1 % TFA
resulted in maximal recovery at 60–70 %acetonitrile, but at pH 8 recovery waspoor at all acetonitrile concentrations.
This illustrates the importance of
optimization, since even at the lower pH
recovery drops off significantly below
60 % acetonitrile.
Having established the optimal eluent
formulation, the minimum volume
required for recovery of the entire sample
was examined. A 50 µg load of denatured,
reduced, and alkylated hIgG1 was applied
to eight replicate RP-W cartridges, andthe cartridges were washed. The antibody
was eluted from the cartridges using a
specialized protocol that sequentiallydispenses 2 µL of eluate into each wellof a collection plate. Eluted antibody wasquantified to generate the elution profile
shown in Figure 9. The first fraction
represents the column void volume,
which contained no detectable antibody.
The next four to five fractions (8 to 10 µL
total) contained the antibody. The total
recovery was greater than 90 % of theload.

RP-W Reproducibility

Human IgG1 was denatured, reduced,
alkylated, and diluted to give mass loads
of 10, 25, 50, and 75 µg. The samples
were loaded onto RP-W cartridges (n = 12
per mass load), washed, and eulted using
the Protein Cleanup application. The CV
and recovery for each mass load were
determined. The CV was found to be lessthan 5 %, and the recovery greater than
90 %, as shown in Figure 10. CVs below
5 % are consistent with reproducibilities
that have been determined for other
AssayMAP cartridges, highlighting the
precision that can be achived using thisplatform.

13

Performance formultiple antibodies

To determine how well the standard
conditions used in this study translate
to other antibodies, two additional hIgG
antibodies were examined. The first wasa polyclonal hIgG preparation from normal
serum. The second was the monoclonal
standard antibody SILuLite from Sigma.
All three antibodies were serially diluted,
denatured, reduced, alkylated, and 25,
50, or 75 µg was loaded onto RP-W
cartridges. As shown in Figure 11, therecovery for the hIgG1 was greaterthan 90 %, and CVs were below 5 %, as
expected. The recovery and variability
for the other two antibodies were notquite as good as hIgG1 but still worked
well without further optimization. Thesefindings suggest that the standard
conditions presented here can be usedwith a variety of antibodies, and that
optimal conditions may vary for different
antibodies.

Rapid trypsin digestion

Trypsin digestion of denatured proteinsis inefficient and incomplete in thepresence of denaturant. Trypsin digestion
following desalting on RP-W cartridges
or SEC was compared with simply
diluting the denaturant. Human IgG1
was denatured, reduced, alkylated,and either diluted to 0.9 M guanidine,
desalted using RP-W cartridges, ordesalted using a 96-well SEC spin plate.
Quadruplicate samples were digested
with trypsin for 0.5, 1, 2, 4, or 18 hours
at 37 °C in parallel. Figure 12 shows the
TICs of all reactions (20 overlaid traces),for each guanidine reduction method.
The colors red, purple, green, blue, andblack indicate the respective digest
time points of 0.5, 1, 2, 4, and 18 hours.
In the case of dilution‑only reactions,which were digested in the presenceof 0.9 M guanidine, the five colors form
visually distinct groups of quadruplicates,
indicating a gradual progression toward
complete digestion that required up
to 18 hours for most of the peaks. By
contrast, both desalting methods showedmuch less variation over the digestion
time course. This is in agreement withpreviously published results showing thatremoval of guanidine decreases digestion

time and increases the reproducibility oftrypsin digestion1. It was also observedthat SEC desalting resulted in smaller
TIC areas than RP-W desalting or simple
dilution, indicating lower recovery. SEC
is a well-accepted method for desalting
proteins, and it is likely that this is anantibody-specific observation, and thatfurther optimization of this method would
result in better recovery.
Twelve fully tryptic peptides wereidentified that are conserved between
the hIgG1 antibodies used in this study.
Extracted ion chromatograms of all
12 peptides were generated from MS1
data, and peak areas were integrated
and quantified for each of the time points(Figure 13). When guanidine was simply
diluted to 0.9 M (panel A), complete
liberation of nearly all the peptides
required more than 4 hours, as indicatedby the continuous increase in bar heightacross the time course. Only the peptide

beginning with WQQG reached a plateauby 4 hours (compare blue and blackbars). Samples that were desalted prior
to digestion using either RP-W cartridges(panel B) or an SEC spin plate (panel C)
show the majority of peptides reaching
maximum levels by 1 hour.

Workflow reproducibility

A common sample preparation workflow
for biotherapeutic antibody analysis
is the purification of a target antibody
from a complex matrix, denaturationand digestion of the purified antibody,and quantification of selected peptides.
The reproducibility of this workflow
was examined using the AssayMAP
Bravo for the sample preparation steps.
Human IgG1 or SILuLite antibody was
spiked into cell-free medium from anonantibody‑expressing CHO cell culture(n = 16 per antibody). The antibodies
were purified from this matrix using

14

the AssayMAP Affinity Purification
application and protein G (PG-W)
cartridges2. The antibodies were eluted
directly into a solution containing
guanidine and TCEP, and incubated for
60 minutes at 60 °C in a sealed plate. The
AssayMAP Bravo In-Solution Digestion
application was used to alkylate anddilute the samples. The Protein Cleanup
application and RP-W cartridges wereused to desalt the samples, and the
In‑Solution Digestion application wasused to digest the antibodies prior to
LC/MS analysis. Twelve conserved
peptides were quantified for each sample.
Figure 13 shows that CVs for the entire
workflow were less than 10 % for atleast nine out of 12 peptides examined.
The peptides with CVs higher than 10 %were those with the smallest peak areas.
The average CVs for all 12 peptides were
8.4 % for hIgG1 peptides, and 6.0 % for
the SILuLite peptides.

Conclusions

References

The Agilent AssayMAP platform is ascalable and easy-to-use automation
platform that can simultaneously improve
the reproducibility of LC/MS workflows
and reduce labor. The AssayMAP Affinity
Purification, In-Solution Digestion, andProtein Cleanup applications can beused to purify antibodies from complex
matrices, denature, reduce, alkylate, clean
up, and digest the antibodies, all in lessthan 8 hours, with CVs less than 10 %.
This represents a significant improvementin time-to-results compared to overnightdigestion, so you can move projects
forward faster.

1. Ren; et al. An improved trypsin
digestion method minimizes
digestion‑induced modifications onproteins. Analytical Biochemistry 2009,
392, pp. 12-21.
2. Bovee, M.; Russell, J.; Murphy, S.
Automation of Sample Preparation for
Accurate and Scalable Quantification
and Characterization of BiotherapeuticProteins Using the Agilent AssayMAP
Bravo Platform, Agilent Technologies
Application Note, publication number
5991‑4872EN, 2014.
3. Jiang, X.; et al. The effect ofvarious S-alkylating agents on
the chromatographic behavior ofcysteine‑containing peptides inreversed-phase chromatography.
J. Chromatogr. B 2013, 915-916,
pp. 57‑63.

15www.agilent.com/chem
For Research Use Only. Not for use indiagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2016
Published in the USA, March 16, 2016
5991-6478EN