Application Note
Biotherapeutics andBiosimilars

Host Cell Protein Analysis Using
Agilent AssayMAP Bravo and 6545XT
AdvanceBio LC/Q-TOFAuthors
Linfeng Wu, Shuai Wu andTe-Wei Chu
Agilent Technologies, Inc.,
Santa Clara, CA, USA

Introduction
Use of biopharmaceuticals such as monoclonal antibodies (mAbs) has been
growing rapidly. Since biopharmaceuticals are generated from biological sources,
some of the low-level host cell proteins (HCPs) could remain in the final products
even after multiple purification steps. Due to their potential to affect productsafety and efficacy, HCP level must be monitored and controlled in drug products
according to regulatory requirements1. Traditionally, enzyme-linked immunosorbent
assay (ELISA) is the standard method of choice for quantifying HCPs in protein
therapeutics. However, ELISA lacks the specificity and coverage to identify andquantify individual HCPs. Therefore, LC/MS technologies have become a choicefor HCP analysis. The main challenge during LC/MS analysis of HCPs exists in thecoelution of low-abundance HCP peptides with the highly abundant peptides fromthe drug product. This requires better separation of the peptides and broad dynamic
range of the LC/MS system.

This study demonstrates an HCP
analysis workflow including theAgilent AssayMAP Bravo platform forautomated sample preparation, theAgilent 6545XT AdvanceBio LC/Q‑TOF
for LC-MS/MS analysis, and thevendor‑neutral software from Protein
Metrics for data analysis (Figure 1). TheAssayMAP Bravo platform was used
for automated sample preparation,
which included protein digestion,
desalting, and on-cartridge high-pH
reversed-phase (HPRP) fractionation.
Both digested samples with or without
HPRP fractionation were subjected toLC-MS/MS analysis to examine theplatform performance. A new acquisition
method, Iterative MS/MS, has beenshown to improve protein identification.
This standard-flow LC/Q-TOF systemdemonstrates broad dynamic
range, excellent reproducibility, andsemiquantitative capability. Our results
also showed that all HCPs above 2 ppm
were identified with high confidenceby coupling on-cartridge sample
fractionation with Iterative MS/MS.

Sample preparation

Online separation

AssayMAP Bravo

1290 Infinity II LCSystem

Enzymatic digestionand HPRP fractionation

AdvanceBio Peptide
Plus column

Data processing
and report

6545XT AdvanceBio
Q-TOF
Iterative MS/MS

Protein Metrics

Figure 1. Overview of Agilent HCP Workflow.

Experimental
Materials
Human IgG1 mAb (an R&D product froma partner) was produced from Chinese
Hamster Ovary (CHO) cells and purified
with protein A. Proteomics Dynamic
Range Standard Set (UPS2) waspurchased from Sigma-Aldrich.

Instrumentation•Agilent AssayMAP Bravo system•Agilent 1290 Infinity II LC system
including:• Agilent 1290 Infinity II High Speed
Pump G7120A• Agilent 1290 Infinity II
Multisampler G7167B• Agilent 1290 Infinity II
Thermostatted Column
Compartment G7116B

2Detection•Agilent 6545XT AdvanceBio
LC/Q‑TOF•

Agilent Dual Jet Stream ESI source

Sample preparation
UPS2 was spiked into the mAb ata 1:1,000 ratio. A second sample,
comprising the mAb without UPS2
spiking, was prepared and analyzed inparallel as a negative control to ensurethat the identification of UPS2 proteins
was not due to the presence of sequencehomologs in the control sample. Bothsamples were reduced, alkylated,trypsin-digested, and desalted using theAssayMAP Bravo system. The samples
were then aliquoted; one aliquot wassubjected directly to LC-MS/MS analysis
using the Agilent 6545XT AdvanceBio
LC/Q-TOF system, and the other aliquot
was fractionated using HPRP method on
the AssayMAP Bravo system, followed by
LC-MS/MS analysis.

HPRP fractionation
The digested sample was fractionatedinto six fractions on an Agilent Reversed
Phase (RP-S) cartridge using thepreconfigured Protein Sample Prep
WorkBench Fractionation applicationon the AssayMAP Bravo (Figure 2).
RP-S cartridges were first primed
with 70 % acetonitrile (ACN), 0.1 %trifluoroacetic acid (TFA) and equilibrated
with 0.1 % TFA. Then, 150 μg of digested
sample was loaded onto each cartridge,
and eluted into six fractions using 10 mM
ammonium formate buffer (pH 10) witha stepwise increase of ACN (10, 15, 20,25, 30, and 90 %).

LC/MS analysis
LC separation was performed on anAgilent AdvanceBio Peptide Plus column(2.1 × 150 mm, 2.7 µm, p/n 675950‑902)
with 0.4 mL/min flow rate using a60 minute LC method (Table 1). Eachsample was analyzed on the 6545XT
AdvanceBio LC/Q-TOF system using
either a conventional Auto MS/MS
method or an Iterative MS/MS method
as indicated (Table 2).

Table 1. Liquid chromatography parameters.
LC Parameters
Analytical column

AdvanceBio Peptide Plus, 2.1 × 150 mm, 2.7 μm (p/n 675950-902)

Mobile phase A

H2O, 0.1 % formic acid

Mobile phase B

90 % ACN, 0.1 % formic acid

Flow rate

0.4 mL/min

Gradient

1.0 minutes & 3 %B
50.0 minutes & 21 %B
53.0 minutes & 90 %B
55.0 minutes & 90 %B
55.1 minutes & 3 %B

Stop time

60 minutes

Column temperature

60 °C

Table 2. MS parameters.
MS Parameters
Drying gas temperature

290 °C

Drying gas flow

13 L/min

Nebulizer

35 psiSheath gas temperature

275 °C

Sheath gas flow

12 L/minIsolation width

Narrow (~1.3 m/z)

Dynamic exclusion within run

1 spectrum release after 0.2 minutes

MS Mass range

250–1,700 m/z

MS Acquisition rate

10 spectra/second

MS/MS Mass range

50–1,700 m/z

MS/MS Acquisition rate

3 spectra/second

Acquisition mode

Iterative MS/MS or Auto MS/MS as indicated

Iterative MS/MS

Mass error tolerance: 15 ppm
RT exclusion tolerance: –0.2 minutes to +0.4 minutes

Figure 2. Fractionation application user interface with default settings.

3Data processing

Results and discussion
Comparison of Iterative MS/MS andAuto MS/MSThe Agilent 6545XT AdvanceBio Q-TOF
system provides a new data acquisition
method, Iterative MS/MS, which
improves identification of low-abundance
precursors (Figure 3). Using this method,
the protein digest sample was subjected
to multiple LC-MS/MS analyses. The
first analysis was performed as aconventional Auto MS/MS run using
data dependent acquisition. In thefollowing Iterative LC-MS/MS analyses,
precursors that were previously
selected for MS/MS fragmentation wereautomatically excluded on a rolling basiswith customizable mass error tolerance
and retention time exclusion tolerance.
As a result, more precursors wereautomatically interrogated by LC-MS/MS.

4Counts

The raw data files were processedusing software from Protein Metrics Inc.
One raw data file was searched using
Preview to generate Byonic parameters.
All raw data files were searched against
a Uniprot CHO K1 protein database thatwas concatenated with mAb and UPS2
protein sequences using Byonic. The
search parameters included semispecifictrypsin digestion, up to two missedcleavages, 20 ppm precursor masstolerance, 30 ppm fragment masstolerance, fixed cysteine (C) alkylation,
variable methionine (M) oxidation,asparagine (N) and glutamine (Q)deamidation, and other variable
modifications as suggested by thePreview analysis. The Byonic result
files were imported into Byologic forfurther detailed analysis on a smaller
set of protein sequences, and for result
reporting. A minimum score of 150 forMS2 search was used to filter peptides.

×106
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
022

23

2425
26
27
Acquisition time (min)

2829

30

LC/MS
Injection 1m/z

LC/MS
Injection 2m/z

Additional injections …
Precursors selected for MS/MS
Rolling excluded precursors

Figure 3. A diagram of an automated Iterative MS/MS acquisition method.

We first compared Iterative MS/MS toAuto MS/MS using the LC/Q-TOF system
without any offline fractionation—that is,where the purified mAb with spiked-inUPS2 was analyzed in both methods.
UPS2 is the proteomics dynamic range
standard produced by Sigma-Aldrich,
which is a complex protein mixture
containing 48 human proteins at six
concentrations ranging from 500 amoles
to 50 pmoles. Spiking UPS2 into themAb sample at a 1:1,000 ratio (w/w)
before digestion resulted in protein levels
ranging from 0.0004 to 313 ppm, among
which 19 proteins were present above
one ppm. This spiking sample mimics
the wide dynamic range of the HCPs
present in the therapeutic proteins. Italso allows us to evaluate the sensitivity
and dynamic range of different methods
being investigated in this study.

Table 3 shows the total numbersof unique peptide sequences frommAb and UPS2 proteins that wereidentified by each acquisition method.
Peptides with the same amino acidsequence but different modifications
were counted as one unique peptidesequence. The sample loading amountfor each injection was 32 μg, and three
injections were carried out for eachacquisition method. Overall, Iterative
MS/MS identified more unique peptide
sequences per protein across a wide
dynamic range. All the spiked-in proteins
above 8 ppm level were identified withhigh confidence. All additional resultspresented here were performed byIterative MS/MS.

Chromatographic reproducibility anddynamic range
We also assessed the chromatographic
reproducibility and dynamic range
using the above dataset acquired bythree injections of Iterative MS/MS
analyses. Figure 4 shows the overlaid
chromatograms of the base peakchromatogram (BPC) and Extracted
Ion Chromatogram of four coeluting
precursor ions from three Iterative
LC-MS/MS runs. Due to the high sample

Table 3. Number of unique peptide sequences identified by Iterative MS/MS or
Auto MS/MS acquisition methods.
Three injections per method

Molecularweight (kDa)

Protein spiking
level (ppm)

Iterative MS/MS

Auto MS/MSmAb_HC

49.7

NA419382mAb_LC

24.0

NA201186

ALBU_HUMAN_spike

66.4

313.0

4643

CAH2_HUMAN_spike

29.1

137.3

1915

CAH1_HUMAN_spike

28.7

135.6

99

LEP__HUMAN_spike

16.2

76.2

41

HBB_HUMAN_spike

15.9

74.8

1210

HBA_HUMAN_spike

15.1

71.3

76

UBIQ_HUMAN_spike

10.6

50.0

66

CO5_HUMAN_spike

8.6

40.4

44

CATA_HUMAN_spike

59.6

28.1

22

SUMO1_HUMAN_spike

38.8

18.3

31

NQO1_HUMAN_spike

30.7

14.5

20

PRDX1_HUMAN_spike

22.0

10.4

30

PPIA_HUMAN_spike

20.2

9.5

44

MYG_HUMAN_spike

17.1

8.0

2

1

Protein accession

loading amount on the column, many of
the signals from mAb peptides saturated
the MS detector, which showed aplateau in the BPC. Details about thosefour coeluting peptide precursors
are summarized in Table 4. Excellent
chromatographic reproducibility wasobserved for the coeluting peptides, andquantitative reproducibility was excellent

across an extremely broad dynamicrange (peak intensities from 6.76 × 103
to 1.38 × 108). For the lowest-abundance
myoglobin peptide, which was spiked inat 8 ppm, we observed an RSD value of
10.3 %. These data demonstrated theexcellent in-spectrum dynamic range andreproducibility of the 6545XT AdvanceBio
LC/Q-TOF system.

×106
2.4

EPQVYTLPPSR

BPC

Zoom

2.2

SAVTALWGK

2.0

TIAQDYGVLK

1.8Counts

1.6
1.4
1.2
1.0

ALELFR

0.8
0.6
0.4
0.202468101214161820 222426 28 30 32 34 36
Acquisition time (min)384042444648 50

52545658

Figure 4. Overlaid chromatograms of three LC-MS/MS runs for BPC and extracted ion chromatograms of four selected peptide precursors.

5

Table 4. Data of the four selected peptide precursors.
Precursor ion(m/z)

Mass error(ppm)

Intensity

ALELFR

374.7208-1.1

6.76E+03

10.3 %

8

Myoglobin

TIAQDYGVLK

554.3049-1.8

1.51E+05

6.2 %

10.4

Peroxiredoxin 1

SAVTALWGK

466.7659

4.8

1.36E+05

6.0 %

74.8

EPQVYTLPPSR

643.844

1.01.38E+08

1.2 %NAPeptide

Intensity Protein spiking%RSDlevel (ppm)Protein name

Hemoglobin
subunit betamAb

Normalize XIC in mAb control
Normalized XIC in UPS2 spiking sample
Actual level in UPS2 spiking sample (ppm)
0.12
0.10

400
350
300

0.08
0.06
0.04

250200
150
100

0.02
050

Actual UPS2 protein spiked-in level (ppm)

We also explored semiquantitative
analytical capabilities of this workflow
solution using the above data files
acquired by three injections of Iterative
MS/MS analyses. The total extracted ion
chromatogram (XIC) for each identified
UPS2 protein using all the measured
peptides was normalized to the highest
protein in the sample, which is mAb
heavy chain in this study, and the values
were exported directly from Byologic
software. Figure 5 shows the normalized
XIC per protein (Y-axis on the left) inthe mAb control sample (blue) and theUPS2 spiking sample (orange) plottedwith the actual UPS2 protein level inthe spiking sample (green, Y-axis onthe right). As expected, there are no XIC
signals from UPS2 proteins in the mAbcontrol sample, demonstrating the highspecificity of this workflow solution. Inthe UPS2 spiking sample, the normalized
XIC was correlated with the actual
protein spiked-in level in ppm. These
results indicate that the normalized XIC
values reported from Byologic software
offer a semiquantitative estimate of HCPabundance.

Normalized XIC per protein

Semiquantification

0Figure 5. Comparison between normalized XIC per protein and actual UPS2 spike-in level. The XIC perprotein was normalized to mAb heavy chain, and their values in mAb control sample (blue) and UPS2
spiking sample (orange) were plotted with the actual protein levels in the UPS2 spiking sample (green).

6

Improving identification by HPRP
fractionation using AssayMAP Bravo

cartridges and task‑centric automation
protocols2–4. With the same
automation platform, we demonstrated
improvements on identification
sensitivity by HPRP fractionation using
RP-S cartridges. One hundred fifty
micrograms of digested sample wereloaded on each RP-S cartridge and eluted
into six fractions using an easy-to-use

Several core protein sample preparation
tasks have been automated using
the Agilent AssayMAP Bravo sample
preparation platform (for example,protein digestion, peptide cleanup,
immuno-affinity purification, and soforth) with micro-chromatography

high-pH fractionation protocol. Eachfraction was analyzed by two Iterative
MS/MS runs. Figure 6 shows the TIC
signal from the LC-MS/MS analysis ofunfractionated sample compared toHPRP eluates, which demonstrated thechromatographic separation on the RP-Scartridge.

Counts×107
2

UnfractionatedCounts

0×106
5HPRP FlowthroughCounts1Counts2

Counts0×107

1

HPRP Fraction 10×107

HPRP Fraction 20×107

HPRP Fraction 30×10
Counts2Counts72

HPRP Fraction 40×107

HPRP Fraction 5Counts0×107
20

HPRP Fraction 61357

911131517192123 25 27 29 31Acquisition time (min)

33

3537

39

41

43

45

47

4951Figure 6. TIC signal from unfractionated and HPRP fractionated sample.

7The MS/MS spectra of peptides from
low-abundance spiked-in proteins
were manually inspected to ensurehigh spectral quality and no homolog
contamination from endogenous
CHO HCPs. All the spiked-in proteins
above 2 ppm were identified withhigh confidence, demonstrating animprovement in the identification
sensitivity using HPRP fractionation(Table 5, compared to Table 3). Figure 7
shows an example MS/MS spectrum for
peptide IEEIFK from the human KCRM
protein, which was spiked in at 2 ppm.

Table 5. Identification of unique peptide sequences from the UPS2 spiking proteins using
AssayMAP HPRP fractionation coupled with two Iterative MS/MS runs per fraction.
Protein accession

Molecular weight(kDa)

Protein spiking level(ppm)

No. unique peptide
sequences

ALBU_HUMAN_spike

66.4

313.0

79

CAH2_HUMAN_spike

29.1

137.3

32

CAH1_HUMAN_spike

28.7

135.6

15

LEP_HUMAN_spike

16.2

76.2

6

HBB_HUMAN_spike

15.9

74.8

22

HBA_HUMAN_spike

15.1

71.3

14

UBIQ_HUMAN_spike

10.6

50.0

9

CO5_HUMAN_spike

8.6

40.4

6

CATA_HUMAN_spike

59.6

28.1

14

SUMO1_HUMAN_spike

38.8

18.3

11

NQO1_HUMAN_spike

30.7

14.5

8

PRDX1_HUMAN_spike

22.0

10.4

9

PPIA_HUMAN_spike

20.2

9.5

11

MYG_HUMAN_spike

17.1

8.0

2

CYB5_HUMAN_spike

16.0

7.62

EGR_HUMAN_spike

6.4

3.01

SYHC_HUMAN_spike

58.2

2.75

KCRM_HUMAN_spike

43.1

2.032,500

Intensity

2,000
1,500
1,000
500
0100200300
400
Mass-to-charge (m/z)

500600Figure 7. MS/MS spectrum of peptide IEEIFK originated from the KCRM protein spiked at 2 ppm.

8700

160120

A host cell protein analysis workflow
including the AssayMAP Bravo platform
for automated sample preparation,
the 6545XT AdvanceBio LC/Q-TOF
for LC-MS/MS analysis, and thevendor‑neutral software from Protein
Metrics for data analysis, has been
demonstrated.
••The AssayMAP Bravo platformusing microchromatography
cartridges and task-centricautomation protocols has brought
unprecedented reproducibility,scalability, flexibility, and ease-of-use
to sample preparation automation.

100
80
60
40

38

20

All the above results demonstrate
the advantage of on-cartridge HPRP
fractionation in improving identificationof HCPs.

Conclusions

138

140

Number of CHO proteins

The impact of HPRP fractionation onidentifying endogenous CHO HCPs
was also examined. Figure 8 shows
the number of identified CHO proteins
between unfractionated and HPRP
fractionated samples at a 1 % protein
false discovery rate. The unfractionatedsample was analyzed by three Iterative
MS/MS runs, and the HPRP fractionatedsamples were analyzed by two Iterative
MS/MS runs per fraction. The results
show a more than three-fold increaseusing HPRP fractionation coupled withIterative MS/MS (138 versus 38).

0

Nonfractionation withthree Iterative runs

HPRP Fractionation with
two Iterative runs

Figure 8. Comparison of number of identified endogenous CHO HCPs
between unfractionated and HPRP fractionated samples coupled withIterative MS/MS acquisition.

•Broad dynamic range and excellent
reproducibility were demonstrated
on this standard-flow LC/Q-TOFsystem.

•By adding HPRP fractionation using
AssayMAP Bravo, all the spiked-in
proteins above 2 ppm were identified
with high confidence.

•Data files acquired bydata‑dependent acquisition allow
simple and fast protein identificationand semiquantification.

Iterative MS/MS improves protein
identification coverage. Using
LC-MS/MS coupled with Iterative
MS/MS acquisition, all the spiked-instandard proteins above 8 ppm wereidentified with high confidence.

9References
1.

ICH Q6B Specifications: Test
Procedures and Acceptance Criteria
for Biotechnological/Biological
Products.

2.

Automation for LC/MS Sample
Preparation: High Throughput
In-Solution Digestion andPeptide Cleanup Enabled by the
Agilent AssayMAP Bravo Platform.
Agilent Technologies, publication
number 5991-2957EN.

3.

Automation of Sample Preparation
for Accurate and Scalable
Quantification and Characterizationof Biotherapeutic Proteins Using theAgilent AssayMAP Bravo Platform.
Agilent Technologies, publication
number 5991-4872EN.

4.

Agilent AssayMAP Bravo Technology
Enables Reproducible Automated
Phosphopeptide Enrichment from
Complex Mixtures Using HighCapacity Fe(III)-NTA Cartridges.
Agilent Technologies, publication
number 5991-6073EN.

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2018
Printed in the USA, July 2, 20185991-9300EN