Guanidinated protein internal standard for immunoaffinity-liquid chromatography/tandem mass spectrometry quantitation of protein therapeutics

Article abstract of DOI:10.1002/rcm.6924

RATIONALEA protein internal standard (IS) is essential and superior to a peptide IS to achieve reproducible results in the quantitation of protein therapeutics using immunoaffinity-liquid chromatography/tandem mass spectrometry (LC/MS/MS). Guanidination has been used as a protein post-modification technique for more than half a century. A decade ago, the modification was applied to lysine-ending peptides to enhance their MALDI responses and peptide sequencing coverage. However, rarely has tryptic digestion of guanidinated proteins been investigated, likely due to the early conclusion that trypsin did not hydrolyze peptide bonds involving homoarginine in guanidinated proteins. In this study, the opposite was observed. Guanidinated lysine residues of proteins did not hinder the access of trypsin allowing for proteolytic digestion. Based on this observation, a new concept of internal standard, named Guanidinated Protein Internal Standard (GP-IS), was proposed for LC/MS/MS quantitation of protein therapeutics. METHODSThe GP-IS is prepared by treating a portion of the therapeutic protein (analyte) with guanidine to convert arginine residues in the protein into homoarginine residues. After tryptic digestion, the GP-IS produces a series of homoarginine-ending peptides plus another series of arginine-ending peptides. One of the homoarginine-ending peptides, which corresponds to the analyte surrogate (lysine-ending) peptide, was chosen as a peptide internal standard (GP-PIS) for LC/MS/MS quantitation. RESULTSUsing this GP-IS approach, a sensitive and robust immunoaffinity-LC/MS/MS assay was developed and fully validated with a linearity range from 10 to 1000 ng/mL using 200 μL of human serum for the quantitation of an Astellas protein drug in clinical development. CONCLUSIONSThe proposed strategy allows LC/MS/MS to play an ever-increasing role in bioanalytical support for protein therapeutics development because of its capability of completely tracking all variations from the beginning to the end of sample analysis, easier preparation compared to isotope-labeled protein-IS, and greater flexibility for changing to alternate analyte surrogate peptides. Copyright

Full text of DOI:10.1002/rcm.6924

Research Article
Received: 20 September 2013
Revised: 14 April 2014
Accepted: 15 April 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1489–1500
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6924
Guanidinated protein internal standard for immunoaffinity-
liquid chromatography/tandem mass spectrometry quantitation
of protein therapeutics
*
Wenchu Yang , Robert Kernstock, Neal Simmons and Ala Alak
Bioanalysis-US, Astellas Research Institute of America, Skokie, IL 60077, USA
RATIONALE: A protein internal standard (IS) is essential and superior to a peptide IS to achieve reproducible results in
the quantitation of protein therapeutics using immunoaffinity-liquid chromatography/tandem mass spectrometry
(LC/MS/MS). Guanidination has been used as a protein post-modification technique for more than half a century. A
decade ago, the modification was applied to lysine-ending peptides to enhance their MALDI responses and peptide
sequencing coverage. However, rarely has tryptic digestion of guanidinated proteins been investigated, likely due to
the early conclusion that trypsin did not hydrolyze peptide bonds involving homoarginine in guanidinated proteins.
In this study, the opposite was observed. Guanidinated lysine residues of proteins did not hinder the access of trypsin
allowing for proteolytic digestion. Based on this observation, a new concept of internal standard, named Guanidinated
Protein Internal Standard (GP-IS), was proposed for LC/MS/MS quantitation of protein therapeutics.
METHODS: The GP-IS is prepared by treating a portion of the therapeutic protein (analyte) with guanidine to convert
arginine residues in the protein into homoarginine residues. After tryptic digestion, the GP-IS produces a series of
homoarginine-ending peptides plus another series of arginine-ending peptides. One of the homoarginine-ending
peptides, which corresponds to the analyte surrogate (lysine-ending) peptide, was chosen as a peptide internal standard
(GP-PIS) for LC/MS/MS quantitation.
RESULTS: Using this GP-IS approach, a sensitive and robust immunoaffinity-LC/MS/MS assay was developed and
fully validated with a linearity range from 10 to 1000 ng/mL using 200 μL of human serum for the quantitation of an
Astellas protein drug in clinical development.
CONCLUSIONS: The proposed strategy allows LC/MS/MS to play an ever-increasing role in bioanalytical support for
protein therapeutics development because of its capability of completely tracking all variations from the beginning to the
end of sample analysis, easier preparation compared to isotope-labeled protein-IS, and greater flexibility for changing to
alternate analyte surrogate peptides. Copyright © 2014 John Wiley & Sons, Ltd.
The development of protein therapeutics in the pharmaceutical
industry is increasing rapidly. For example, the development of
new therapeutic proteins involved more than 200 companies
sharing a strong growth market of £90 billion in 2010.[1] In
2012, among 39 FDA drug approvals six were biologics.[2]
According to IMS Health, it is expected that biologics will
account for approximately 17% of total global spending on
medicines by 2016, and seven of the top ten global medicines
by spending will be biologics by mid-2017.[3] This rapid growth
highlights the need for the development of quantitative
bioanalytical methods with sufficient accuracy, precision and
selectivity in biological matrices to support preclinical and
clinical pharmacokinetic and toxicokinetic studies, as well as
drug characterization at the early stage of discovery and
development.
Ligand binding assays (LBAs) are currently the primary
means of quantifying protein therapeutics in biological fluids.
The most common format is enzyme-linked immunosorbent
assay (ELISA); however, alternative technologies are also
available (e.g. Luminex, Gyros, and MSD) with their own
advantages and disadvantages. LBAs are popular due to their
simplicity, sensitivity, and high-throughput capabilities.
However, they sometimes suffer from selectivity/specificity
issues or high background noise resulting from matrix
interference by endogenous. Another disadvantage is lengthy
and costly assay development because of the antibody
production and selection process.[4]
Liquid chromatography/tandem mass spectrometry
(LC/MS/MS) is the gold-standard technology for
quantifying small molecule drugs in the pharmaceutical
industry. Recently, it has become an increasingly popular
approach to LBAs for protein quantitation. LC/MS/MS
offers many inherent features such as robustness,
selectivity/specificity, transferability, the ability to multiplex,
relatively fast method development, and the accumulated
and adaptable knowledge from small molecule analysis and
proteomics. However, common approaches for LC/MS/MS
* Correspondence to: W. Yang, Bioanalysis-US, Astellas
Research Institute of America, Skokie, IL 60077, USA.
E-mail: Wenchu.yang@astellas.com
Rapid Commun. Mass Spectrom. 2014, 28, 1489–1500
Copyright © 2014 John Wiley & Sons, Ltd.

W. Yang et al.
analysis of small molecules are not readily applicable to
macromolecules because of the basic differences in their
chemistry and biology. Quantifying protein therapeutics
using LC/MS/MS involves some unique bioanalytical
challenges.[5,6] One of these challenges is the proper selection
of an internal standard (IS).
Furthermore, a SIL-peptide IS is incapable of compensating
for variations that result from sample cleanup/enrichment
prior to digestion. Common biological matrices, such as
serum or plasma, contain very large amounts of endogenous
proteins which are physicochemically similar to therapeutic
protein drugs circulating at low concentration. Without
sample cleanup or enrichment, direct digestion of protein
therapeutics within the matrix is impractical. It would require
a large amount of digestion enzyme and generate numerous
enzymatic peptide interferences that would overwhelm both
LC and MS systems, leading to ionization suppression of
the analytes and ultimately compromising the entire assay
performance. Therefore, some practical sample cleanup/
enrichment strategies such as protein depletion[19,20] or
immunoaffinity using magnetic beads[10,20,21] are often
employed in order to achieve quantitation limits (ng/mL or
less) necessary to support clinical or preclinical studies.
Extensive sample preparation can cause sample loss resulting
in irreproducible quantitation results if a proper IS is not
used.[22,23]
For the reasons discussed above, a suitable protein internal
standard is an ideal solution when it is introduced from the
beginning of sample preparation. A full-length SIL-protein
IS is the best choice for absolute protein quantitation.[24,25] It
completely tracks the variations introduced by sample
pretreatment, digestion, and LC/MS/MS analysis to produce
accurate and reproducible results. This strategy was
adopted from metabolic labeling in comparative
proteomics.[26,27] However, a SIL-protein IS may not be
widely adopted, especially during the early stages of drug
development, because of cost, departmental resources, and
slow production.
Including an IS in quantitative LC/MS/MS assays is
common practice for small molecule drugs, especially in a
regulated environment. Typically, a stable-isotope-labeled
internal standard (SIL-IS), an isotopically labeled version of
the analyte, is used to better compensate for variations
introduced from sample preparation and the analytical
system to achieve accurate, precise and reproducible results.
An IS is especially critical for protein therapeutics where
sample preparation is more complicated compared to small
molecule drugs.[7] Quantifying proteins by LC/MS/MS
typically involves the selection and analysis of surrogate
peptides generated by enzymatic digestion. In 1996, Barr[8]
demonstrated the use of a chemically synthesized, stable-
isotope-labeled surrogate peptide as an IS (SIL-peptide IS).
It was further generalized by Gygi in 2005 as an absolute
protein quantitation strategy (AQUA),[9] and is still widely
used due to its simplicity.[10,11]
One drawback with using a synthesized SIL-peptide IS is
that the approach involves chemical synthesis using isotope-
labeled amino acids. A new synthesis would be required if
the surrogate peptide is changed, which may happen
frequently over the course of method development. To
overcome this issue, a universal strategy was developed
utilizing differential dimethyl labeling of surrogate peptides.[12]
It provides a fast and low-cost approach to generate a peptide
IS for method development. Similar approaches, such as
isotope-coded affinity tags (ICAT),[13] enzymatic 18O
labeling,[14] isobaric tags for relative and absolute quantitation
(iTRAQ)[15] and tandem mass tags (TMT),[16] have been
developed in comparative proteomics, although they have not
been used for quantitation of protein therapeutics.[17]
To overcome these disadvantages, we propose and
demonstrate a novel internal standard for accurate LC/MS/MS
quantitation of protein therapeutics named Guanidinated
Protein Internal Standard (GP-IS). A GP-IS is prepared from
the target protein through
a simple post-translational
Another disadvantage of using a SIL-peptide IS is that it
does not address variations in protein digestion. This issue
could be overcome by using an enzyme-cleavable SIL-peptide
IS. However, an investigation suggested that this type of IS
did not show significant improvement in accuracy and
precision,[18] likely because it could not completely mimic
the behavior of the intact protein during enzymatic digestion.
modification, guanidination,[28] as illustrated in Fig. 1. The
GP-IS still retains, or at least partially retains, the biological
activity of the original protein drug allowing it to serve as an
ideal internal standard throughout the entire analysis. During
sample processing, tryptic digestion of the GP-IS generates
two series of tryptic peptides: (A) guanidinated lysine (also
called homoarginine)-ending peptides and (B) arginine-ending
Figure 1. Scheme of the generation of guanidinated protein internal standard
(GP-IS) and its surrogate peptide (GP-ISP) for absolute LC/MS/MS quantitation
of proteins. Lysyl residues on proteins react with O-methylisourea to form
homoarginyl residues. After tryptic digestion is applied, a series of peptides with a
homoarginyl residue at the C-terminal are generated, one of which is selected as
an internal standard (GP-ISP) for absolute LC/MS/MS quantitation of the protein
therapeutic.
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Guanidinated protein internal standard
peptides. From Series A, a peptide matching the analyte
surrogate peptide sequence except for the terminal
guanidinated lysine is selected as a GP-IS surrogate peptide
(GP-ISP) for LC/MS/MS monitoring. This approach is
demonstrated through a case study involving development of
an immunoaffinity-LC/MS/MS assay for quantitation of
Astellas protein drug ASP-P1.
samples (QCs) were prepared from the pooled human serum
at 10 ng/mL (LLOQ), 30 ng/mL (LQC), 160 ng/mL (MQC),
and 800 ng/mL (HQC) and stored at –20 °C before use.
Immunocapture
The following procedures were used to prepare magnetic
beads coated with capture antibody, anti-ASP-P1, for the
capture of ASP-P1 from a 200 μL human serum sample. A
30 μL aliquot of M-280 tosylactivated beads in a Protein
Lobind tube was washed twice with 0.5 mL of 0.1 mol/L
borate buffer, pH 9.5 (Buffer A). The beads were separated
from solution using a magnetic particle concentrator for
microcentrifuge tubes (Life Technologies). After removal of
the washing solution, the beads were added to 10 μL of Buffer
A and 5 μL of anti-ASP-P1 at 1 mg/mL and incubated for 4 h
at 37 °C with shaking at 1000 rpm for covalent binding to the
beads. The beads were washed three times with 0.5 mL of
PBS buffer containing 0.1% BSA (Buffer B) to remove
unbound capture antibody. The beads were resuspended in
0.5 mL of Buffer B and incubated at 37 °C with shaking at
1000 rpm for 2 h to block and prevent non-specific binding.
The anti-ASP-P1-coated beads were harvested and stored at
4 °C with 10 μL of Buffer B. The beads were functionally
stable for at least 1 month at this condition.
EXPERIMENTAL
Materials and reagents
Model proteins including α-lactalbumin from bovine milk,
papain from papaya latex, myoglobin from equine heart,
and bovine serum albumin (BSA) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Chemicals such as
dithiothreitol (DTT), iodoacetamide (IAA), ammonium
bicarbonate, O-methylisourea hemisulfate, boric acid and
sodium hydroxide were also purchased from Sigma-Aldrich.
HPLC grade acetonitrile, methanol, acetic acid and formic
acid were from Fisher Scientific (Hampton, NH, USA). 10 ×
phosphate-buffered saline (PBS) was from Teknova (Hollister,
CA, USA). Sequencing grade trypsin was from Promega
(Madison, WI, USA). Tosylactivated magnetic beads
(Dynabeads M-280) were from Life Technologies (Carlsbad,
CA, USA). Zeba spin columns were from Pierce (Rockford,
IL, USA). The proprietary therapeutic protein drug, ASP-P1,
and its monoclonal antibody, anti-ASP-P1, were provided
by Astellas Pharma, Inc. (Tokyo, Japan). The sequence of
ASP-P1 used in these studies may be found in US Patent
8,496,935 under the name D3-69-IgG2.[29]
Immunocapture of ASP-P1 and GP-IS was performed using
the following procedures. 200 μL of serum samples were
incubated at 37 °C with shaking at 1000 rpm for 1.5 h with
10 μL of the functionalized beads and 10 μL of GP-IS
prepared above. After removal of the supernatant, the beads
were washed three times with Buffer B and were ready for
digestion after complete removal of the buffer.
Guanidination procedure
Digestion
O-Methylisourea hemisulfate (677 mg) was dissolved in pure
water (10 mL) to make O-methylisourea stock solution with a
concentration of 0.55 mol/L. Guanidination working solution
was prepared fresh before use by combining 2.2 mL of the
stock solution and 0.3 mL of 1 mol/L sodium hydroxide
solution.
For model proteins, a 450 μL aliquot of this fresh working
solution was added to 50 μL of 10 mg/mL protein solutions
prepared in water. The tube was gently rotated at room
temperature for 12 h. The protein was purified using Zeba
spin column against 0.1 mol/L ammonium bicarbonate
buffer following the manufacturer’s instructions. The purified
solutions were ready for trypsin digestion.
For ASP-P1, an 80 μL aliquot of guanidination working
solution was added to 20 μL of the 10 mg/mL protein
solution prepared in PBS. The reaction was conducted for
4 days at room temperature, and 100 μL of guanidination
working solution was added after every 24 h of incubation.
The protein was purified using Zeba spin column against
0.1 mol/L ammonium bicarbonate buffer following the
manufacturer’s instructions.
After the immunocapture step, 50 μL of 0.5 mol/L
ammonium bicarbonate buffer (Buffer C) was added to the
beads. Digestion was started with thermal denaturation by
heating the sample plate at 95 °C for 30 min. After cooling
and centrifugation, the solutions were spiked with 15 μL of
10 mmol/L DTT prepared in Buffer C and incubated at 55 °C
for 60 min for disulfide bond cleavage. Alkylation was
performed by adding 30 μL of 50 mmol/L IAA prepared in
Buffer C and incubating for 60 min at room temperature with
light protection. The digestion was conducted by incubating
10 μL of 0.1 μg/μL trypsin at 37 °C for 10 h. The reaction was
stopped by adding 10 μL of 5% formic acid.
For model proteins, 50 μL of purified guanidinated solution
or native protein solution was used and the same procedure
as above was followed except for myoglobin, for which the
cleavage and alkylation steps were omitted.
LC/MS/MS instrumentation and conditions
Qualitative and quantitative experiments were performed
using a Shimadzu system comprised of LC20AD XR HPLC
pumps and SIL-20AC XR autosampler coupled with an AB
Sciex 5500 QTrap mass spectrometer. Separation was
achieved on an Aeris Peptide XB-C18 analytical column
(150 × 2.1 mm, 3.6 μm) with a UHPLC C18-Peptide column
guard cartridge (Phenomenex), using the mobile phases
consisting of (A) 0.1% formic acid in water and (B) 0.1%
formic acid in ACN. Elution was programmed by increasing
Sample preparation for ASP-P1 quantitative method
validation
Calibration standards were prepared fresh daily by diluting
10 mg/mL ASP-P1 stock solution in pooled human serum
to 10, 20, 40, 100, 200, 400, 850, 1000 ng/mL. Quality control
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W. Yang et al.
mobile phase B from 2% at 0 min to 40% at 10 min with a flow
rate of 0.35 mL/min. 10 μL of digested tryptic peptide
solution, stored at 10 °C in the autosampler, was injected into
the system.
technology available at that time, such as paper
chromatography and chemical analysis of amino acids.
Therefore, we decided to reinvestigate tryptic digestion of
guanidinated α-lactalbumin using state-of-the-art technology,
LC/MS/MS.
The mass spectrometer was operated in positive
electrospray ionization (ESI) mode. For the characterization
of guanidinated model proteins, mass spectrometer
parameters were optimized through the MRM/Enhanced
Product Scan IDA experiment. For the quantitation of the
drug, the mass spectrometer was extensively optimized
using Instrument Optimization mode and Compound
Optimization mode through infusing tryptic peptide solution
of the standard purified by C-18 cartridges. The optimized
conditions for quantitation were as follows: ionspray voltage
5500 V, source temperature 550 °C, GS1 50, GS2 60, curtain
gas 10, CAD gas medium, EP 10, unit resolution for both
Q1 and Q3, MRM mass transition for analyte surrogate
peptide 772.4 (+2) → 969.4 (CE 33, CXP 24, DP 61, dwell time
150 ms), MRM mass transition for GP-ISP 793.4 (+2) → 1101.4
(CE 34, CXP 24, DP 61, dwell time 150 ms).
The protein was guanidinated as described in the
Experimental section. The guanidinated protein was digested
with the native protein placed side by side as a control to
verify the digestion performance. In silico digestion using
the ExPASy online program was first employed on the native
protein with the setting of zero miss cleavage to predict
theoretical peptides. Lysine-ending peptides with a +2 charge
and m/z value within the mass spectrometer detection
window (<1250) were selected for further evaluation. To
verify the existence of a theoretical peptide, all the possible
MRM mass transitions from the precursor ion (+2) to possible
y-ions (+1) were monitored by LC/MS/MS. The existence of
a particular peptide was confirmed if all its MRM scan peaks
aligned and by analyzing the MS/MS spectrum which was
acquired by MRM-Enhanced Product Ion (EPI)-IDA
experiment. To search for guanidinated tryptic peptides, the
same procedure was applied but MRM mass transitions were
set as ’native precursor ion + 21 (+2) → native product y-ion +
42 (+1)’ per guanidination modification. The results are
presented in Table 1 and Figs. 2 and 3. Clearly, all the
theoretical tryptic guanidinated peptides from guanidinated
α-lactalbumin, corresponding to the native forms, were
present. As an example, Fig. 3 shows the MS/MS spectra of
Peptide 6 from native and guanidinated α-lactalbumin, which
was acquired in MRM-EPI-IDA mode. The identity of the
guanidinated form is confirmed because all its y-ions gained
42 m/z (+1) and its precursor ions gained 21 m/z (+2)
compared to the native form resulting from guanidination
of the C-terminal lysine.
Method validation
Full validation of the LC/MS/MS method for the quantitation
of ASP-P1 in human serum was performed in three batches
according to the FDA guidance for bioanalytical validation.[30]
The calibration standard range and linearity were evaluated
using Analyst 1.5.2 software and least-squares linear regression
analysis with a weighting factor of 1/x2.
RESULTS AND DISCUSSION
Protein guanidination and tryptic digestion
Tryptic digestion of guanidinated proteins was further
substantiated with papain, BSA and myoglobin. The data is
included in Table 1. An extensive search also revealed a few
literature references which describe this tryptic hydrolysis of
guanidinated proteins, although this topic is not their main
focus.[40–43] The data in Table 1 can lead to several conclusions
related to guanidination of proteins and tryptic hydrolysis of
peptide bonds involving homoarginine generated from
guanidination, although further investigation may be needed
for the confirmation. (1) Most lysine residues were completely
guanidinated and completely digested resulting in >95%
yield of homoarginine peptides. (2) Because of steric
hindrance, not all homoarginine residues were equally
accessible to trypsin. This was reflected in Table 1 by the
various yields of homoarginine peptides produced by
digestion. An extreme example in this case was labeled as
’Not Found, Type A’, meaning all lysine residues were
guanidinated to form homoarginine residues, but none were
digested. (3) The various yields might also reflect that the
degree of guanidination varied with the location of lysine
residues in a protein. An extreme example of this case was
labeled as ’Not Found, Type B’, meaning no lysine residues
were guanidinated. (4) Compared to the lysine-ending
peptide, the corresponding homoarginine-ending peptide
signal intensity was significantly reduced (Fig. 2) even for
those with high conversion yield. This probably indicates
sample loss during processing or protein hydrolysis during
guanidination in high pH environment because changing
Guanidination of proteins through lysine residues became a
standardized procedure in the late 1960s to study the
environment and essential nature of lysine side chains in
proteins.[28] A decade ago this mature technology was
adapted and became popular in proteomics research. It was
initially used for enhancing the signal intensities of lysine-
ending tryptic peptide ions in MALDI mass spectrometry to
facilitate protein sequence coverage.[31,32] Later, the
applications were extended into the fields of N-terminal
peptide sequencing[33,34] and quantitative proteomics.[35,36]
In addition, a guanidination kit has been commercialized.[37]
However, guanidination was mostly applied to tryptic
peptides post-digestion, rather than intact proteins prior to
digestion. This situation may exist because of the general
perception that modified lysine residues in proteins would
no longer be recognized by trypsin. Indeed, it was concluded
50 years ago that trypsin did not hydrolyze peptide bonds
involving homoarginine in guanidinated α-lactalbumin.[38]
The conclusion was reaffirmed by experiments involving
papain,[39] and later this inertness to trypsin was further
generalized.[28]
Our intention to conduct more exploration was based on
the fact that arginine is recognized by trypsin and it only
differs from homoarginine by one CH2 unit in its side chain.
Trypsin may not have the specificity to distinguish these
two residues in
a complicated protein structure. The
conclusion made 50 years ago could be based on the
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Guanidinated protein internal standard
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Copyright © 2014 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 1489–1500

Guanidinated protein internal standard
A
B
C
Retention time , min
Figure 2. MRM chromatograms of (A) tryptic lysine-peptides from native
α-lactalbumin, (B) tryptic homoarginine-peptides from guanidinated
α-lactalbumin, and (C) remaining tryptic lysine-peptides from guanidinated
α-lactalbumin. The peaks are labeled per ID numbers from Table 1. MRM mass
transitions for each peptide are given in Table 1. Other conditions were as
described in the Experimental section.
M+2H
y7
A: VGINYWLAHK
y5
y6
y3
y4
y2
y1
M+2H
B: VGINYWLAHK*
y5
y4
y7
y3
y6
y1
y2
Figure 3. MS/MS spectra of Peptide 6 from native α-lactalbumin (A) and
guanidinated α-lactalbumin (B). The y-ions are labeled beside the peaks in the
figure. All y-ions of guanidinated α-lactalbumin gained 42 amu due to
guanidination. Ions beyond y7 were outside the detection window (<1000 Da)
for the MRM-Enhanced Product Ion IDA scan.
the lysine residue to a homoarginine residue should have
native peptide. Typically, the impact on hydrophilic peptides
(early eluted peptides) is greater than on hydrophobic
peptides (late eluted peptides).
resulted in signal enhancement, as seen in both MALDI[29,30]
and electrospray ionization (ESI).[44] (5) It is evidenced in
Figs. 2(A) and 2(B) that the retention times of tryptic peptides
increased after guanidination. This observation was also
reported in the literature.[31,44] This reflects guanidination
could increase the hydrophobicity of tryptic peptides.
Furthermore, the increase in retention time varied from
peptide to peptide, indicating that the degree of increase in
the retention time depends on the hydrophobicity of the
Strategic use of guanidinated protein internal standard
(GP-IS) for development of immunoaffinity-LC/MS/MS
assay for quantitation of ASP-P1 in human serum
ASP-P1 is a therapeutic protein being developed at Astellas.
Initially, a LBA was developed to support the clinical trials,
but the further implementation of this assay was hindered
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W. Yang et al.
by its inadequate sensitivity in a low dosing study. LC/MS/
MS coupled with immunocapture sample preparation
(immunoaffinity-LC/MS/MS) using capture antibody-coated
magnetic beads was therefore investigated because of its
proven capability in sample cleanup and enrichment to
enhance assay sensitivity. Due to extensive bead washing
and sample processing, it is essential to have a proper IS to
mimic the analyte behavior and compensate for the variation
during sample processing. GP-IS was introduced in this study
to fulfill the above requirement.
In order to use GP-IS, the following three characteristics must be
met. (1) The analyte surrogate peptide must end with lysine. (2)
The contribution from GP-ISP to the analyte channel, due to
incomplete guanidination, should be less than 20% of LLOQ,
requiring a high yield of GP-ISP from guanidination and digestion.
(3) Most importantly for immunocapture, the GP-IS must retain
sufficient affinity to the capture antibody. Detailed discussions
on these three aspects will be given in the following sections.
the degrees of guanidination vary between different proteins
and different lysine residues. Although nearly complete
guanidination was generally achieved for these model
proteins in less than 12 h, conversion of the specific lysine
residue located in the surrogate peptide of ASP-P1 into
homoarginine was not fully completed even with thorough
optimization of conditions.
After 4-day guanidination and 10-h tryptic digestion, the
remaining non-guanidinated peptide was about 3% of the
guanidinated peptide in terms of their MRM peak intensities.
No further purification was attempted to remove this non-
guanidinated portion. The spin column purified mixture was
directly used as the IS spiking solution. The residual non-
guanidinated peptide is essentially an impurity in the IS,
contributing a response in the analyte surrogate peptide MRM
channel which could interfere with quantitation. The amount
of GP-IS added to each sample was reduced to a level where
the interference is minimal. As shown in Figs. 4(A) and 4(B),
spiking 10 μL of 1 mg/mL GP-IS into 200 μL of blank serum
produces no detectable peak at the analyte surrogate peptide
retention time, and the IS peak is suitable for quantitation.
Selection of analyte surrogate peptide and GP-ISP
While considerations for the selection of analyte surrogate
peptides have been widely discussed by many authors and
good general recommendations were provided by Li,[7] some
case-by-case limitations should still be taken into account. For
the use of GP-IS, the analyte surrogate peptide selected must
be a lysine-ending peptide. It is preferable to choose the
surrogate peptide that is chromatographically well retained
in order to have close elution with GP-ISP. ASP-P1 is a
recombinant fusion protein with a molecular weight over
75 kDa. In silico digestion yields 30 tryptic peptides; 24 of
which possess m/z (+2) within the detection window (≤1250).
After a Swissprot database search, 21 peptides were excluded
because they overlapped with other endogenous human
proteins. For the three remaining candidates, two peptides
have lysine at the C-terminal which meets the requirement for
GP-IS. Finally, a peptide with the sequence ***SPGK (partially
masked for proprietary reasons) was selected because it
exhibited good chromatographic behavior and ESI response.
This surrogate peptide contains 14 amino acids with m/z 772.4
(+2). Its y7 ion (m/z 969.4 (+1)) was a dominant ion and was
selected as a fragment for MRM monitoring. The GP-ISP
MRM transition is 793.4 (+2) → 1101.4 (+1). Under optimized
HPLC conditions, the analyte surrogate peptide and GP-ISP
eluted approximately 0.1 min apart (retention times 6.4 and
6.5 min, respectively), which enables the IS to sufficiently track
the analytical system variations.
Optimization of immunocapture
Immunocapture using capture antibody-conjugated beads
has been accepted as a sample extraction/enrichment means
in protein drug LC/MS/MS quantitation. The technique is
typically multistep and a long process. To minimize sample
preparation time and maximize enrichment efficiency, the
process was extensively optimized in this study. The
optimized conditions as described in the Experimental
section enable the assay to be completed in one work day,
compared to 2 or 3 days of sample preparation reported in
the literature.[10,18–21] The assay also achieved a very low
LLOQ of 10 ng/mL from a sample volume of 200 μL. It is
especially worth mentioning two observations here. First,
capture antibody-conjugated beads could be prepared ahead
in bulk and stored at 4°C for future use. We have validated
that these beads are stable at 4°C for at least 1 month. Second,
it is commonly believed that the captured drug should be
dissociated from the beads prior to digestion. However, in
this study we found that digestion on the beads was feasible.
The recovery of the immunocapture process for guanidinated
ASP-P1 (GP-IS) was determined to be roughly 20%, compared
to greater than 72% for ASP-P1. This suggests that there may
be reduced affinity of the GP-IS to the capture antibody due
to guanidination modification of lysine residues. Consequently,
the spiking amount of the GP-IS has to be optimized as
discussed above to ensure the assay has sufficient IS signal for
reliable quantitation without significant contribution (less than
20% of LLOQ) to the drug MRM channel. Other than this, the
guanidination modification should have no significant impact
on the process after the capture step.
Optimization of guanidination
In the literature, optimization for guanidination of peptides
has been comprehensively investigated. Using optimized
conditions, which involved heating to 65°C, the complete
conversion of lysines into homoarginines was accomplished
in 5 min.[45] However, these optimized conditions may not
directly apply to protein guanidination because milder
conditions for proteins are preferred in order to retain their
tertiary structure allowing for immunocapture after
guanidination. Moreover, guanidination of proteins could be
more difficult than for peptides because lysine residues could
be buried within the 3D structure of a protein. It has been
shown from the studies of the model proteins (above) that
Optimization of digestion
Optimization of the tryptic digestion time was performed on
samples comprised of both ASP-P1and GP-IS in serum matrix
after the immunocapture step. As shown in Fig. 5, digestion
for 6 h at 37 °C was sufficient to completely digest native
ASP-P1. Further increases in digestion time resulted in more
background noise and reduced signal/noise ratio. However,
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Copyright © 2014 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 1489–1500

Guanidinated protein internal standard
Figure 4. MRM chromatograms: (A) analyte channel and (B) IS channel of Control 0 (Blank + IS); (C) analyte channel and (D) IS
channel of the pooled blank human serum; (E) analyte at LLOQ (10 ng/mL) and (F) analyte at ULOQ (1000 ng/mL). The peak
highlighted in (B) at retention time of 6.55 min is the GP-ISP. No peak was observed at analyte retention time 6.4 min and IS
retention time 6.5 min in blank human serum.
for GP-IS, the homoarginine peptide yield increased with
digestion time up to 8 h. This indicates that the digestion
efficiency of the homoarginine residue was low compared to
the lysine residue. Further increases in digestion time resulted
in slightly decreased yields of both guanidinated and non-
guanidinated peptides. The yield ratio was highest and
almost constant from 8 to 12 h; therefore, 10 h was selected
as the optimal digestion time.
present quantifiable peaks in the analyte or IS MRM channels
at their respective retention times. Assay selectivity was
further validated using six individual lots of human serum.
As shown in Fig. 4(E), the S/N was >5 at the 10 ng/mL LLOQ.
The recovery of the assay was >72% as determined at the
high, middle, and low QC levels. The matrix effect was
investigated by preparing QCs at low and high QC levels in
human serum from six individual donors. Compared to the
nominal concentrations, the measured concentrations had
acceptable accuracy (relative error <20%), indicating
lot-to-lot matrix variability was acceptable. No measurable
carryover, which was evaluated by placing a matrix blank
after the highest calibration standard, was observed. The
validation also demonstrated ASP-P1 in human serum had
Method validation
Three validation batches were conducted on three different
days using QCs and calibrators prepared from pooled human
serum. As shown in Figs. 4(C) and 4(D), this matrix does not
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wileyonlinelibrary.com/journal/rcm

W. Yang et al.
Table 3. Intra- and inter-day accuracy and precision of
quality control sample data for ASP-P1 in human serum
Quality control Nominal conc.
Accuracy
(%RE)
Precision
(%CV)
samples
(ng/mL)
Intra-day
(6 replicates, 3 days)
LLOQ
10
30
160
800
–13.5 to 6.3
7.0–15.0
LQC
–7.5 to –13.3 11.2–14.4
MQC
HQC
–10.5 to –1.9
–5.6 to 7.4
4.6–12.4
5.7–13.3
Inter-day (n = 3)
LLOQ
10
30
160
800
–6.9
1.7
–6.2
0.1
14.0
Figure 5. Effect of digestion time on the yield of tryptic
peptides of guanidinated ASP-P1 and native ASP-P1. (×)
Guanidinated surrogate peptide. (■), Remaining non-
guanidinated surrogate peptide. (○) Ratio of guanidinated to
remaining non-guanidinated surrogate peptide. Line × and
Line ■ use left y-axis and Line 3 uses right y-axis. The data
for Line ■ was amplified 20-fold to make the change more
visible.
LQC
13.3
9.2
MQC
HQC
10.8
calibrators and 18 out of 90 QCs failed the acceptance criteria
individually. The accuracy and precision data for standard
curves and QCs are provided in the Supporting Information.
Compared to these results, the current assay with GP-IS is
more favorable in terms of assay accuracy, precision and ease
of performance.
Table 2. Inter-day calibration curve (n = 3) statistics for
ASP-P1 in human serum
Calibration
standards
Nominal conc.
(ng/mL)
Accuracy
(%RE)
Precision
(%CV)
CONCLUSIONS
As the rapid development of biotech pharmaceuticals
increases, demands for capable bioanalytical assays are
expected. While LBA is still dominating this area,
LC/MS/MS has emerged as a promising approach because
of its advantages as discussed earlier. However, application
of LC/MS/MS is challenging in situations where assay
sensitivity is a concern. It is difficult to have highly
sensitive serum assays without comprehensive sample
preparation such as immunocapture. To improve the
accuracy and precision for this sample preparation
approach, a proper internal standard is desired to off-set
the variations from the entire sample analysis process.
The GP-IS strategy proposed in this work has been proven
for this purpose. Its advantages include: (1) better able to
track assay variation than stable-isotope-labeled peptide-
IS, or other protein analog-IS, since it can be spiked at
the beginning of sample processing and has very similar
functionality as the analyte; (2) easier preparation than
stable-isotope-labeled protein-IS because it only involves
Cal 1
Cal 2
Cal 3
Cal 4
Cal 5
Cal 6
Cal 7
Cal 8
10
20
2.5
–7.5
2.5
–8.7
–0.2
14.4
–3.0
–2.5
3.7
10.1
9.8
40
100
200
400
850
1000
9.1
13.2
0.5
6.8
15.2
Correlation coefficient (r): 0.9911À0.9940
at least 24 h of bench-top stability, six cycles of freeze/thaw
stability, 27 h of reinjection stability at +10°C, and 49 h of
processed sample stability at +10°C.
The statistical analysis results for the calibration curves
and QCs are listed in Tables 2 and 3, respectively. Because
this assay included immunocapture, we adopted the
typical LBA validation acceptance criteria. Accuracy must
be within 20% ( 25% at the LLOQ and ULOQ). Precision
must not exceed 20% (25% at the LLOQ and ULOQ).
Calibration curves must have a correlation coefficient (r)
≥0.99. All data presented in Tables 2 and 3 meet these
acceptance criteria. Over three validation batches, only
one out of 24 calibrators and one out of 72 QCs failed
the acceptance criteria.
a
simple chemical reaction; (3) it provides greater
flexibility for changing to alternate analyte surrogate
peptides. If the selected analyte surrogate peptide has to
be changed during method development, it is possible to
select a different corresponding peptide as an IS (GP-ISP)
from the digested GP-IS. One restriction when using the
GP-IS approach is that the analyte surrogate peptide must
have a lysine residue at the C-terminal, because only this
type of peptide can be guanidinated to generate a GP-ISP.
Overall, the strategy presented in this work allows
LC/MS/MS to play an ever-increasing role in bioanalytical
support for protein therapeutics.
Additionally, a very similar assay had been developed and
validated by using a SIL-peptide IS. In that validation, one
out of five batches failed the acceptance criteria and was
rejected. In the four successful batches, three out of 32
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Copyright © 2014 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 1489–1500

Guanidinated protein internal standard
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