A Sensitive and Reproducible Signature Peptide MRM
Based Quantitation Method for Pegylated Interferon α-2b in
Serum
SCIEX QTRAP® 6500 LC-MS/MS System
Faraz Rashid1,Dipankar Malakar1, Anoop Kumar1,Manoj Pillai1,Praveen Kumar V2, Manoj Bob2, Ravisekhar K2
1
SCIEX, 121, Udyog Vihar, Phase IV, Gurgaon, Haryana, India
2
Lupin bioresearch Center, Pune Maharasthra, India
Key Challenges of Pegylated Interferon α-2b
Quantitation
•
•
•
Sensitivity: The overlapping protein charge state
distribution and the poly-dispersity of the
pegylation complicate the ESI-MS spectrum and
dilute signal intensity making intact quantitation at
sub pg levels difficult to achieve
Sample Clean-up: Selective isolation of the low
concentration protein drug from the abundant
plasma endogenous proteins without loss is
difficult
Interferences: Immunological approaches to this
assay are available however, imprecision and
matrix interferences are common issues
Unique Features of this Procedure for
Quantification of Pegylated Interferon α-2b from
Serum
•
•
•
High Sensitivity: An LLOQ of 246 pg/mL in
serum is achieved using signature peptide
detection and selective sample cleanup.
Maximum sensitivity is achieved by the patented
TM
®
IonDrive technology of the SCIEX QTRAP
6500 System.
Linear Dynamic Range (LDR): A dynamic range
from 246 to 50,000 pg/mL from serum samples is
supported by the IonDrive™ High Energy Detector
technology.
Easy Sample Processing: Removal of high
abundance endogenous proteins is achieved by
selective protein precipitation followed by
enzymatic digestion.
Figure 1. The SCIEX QTRAP® 6500 System.
lysine residues. Pegylation is one of the most effective
and acceptable procedures for stabilization of therapeutic
proteins. This stabilization procedure is important to
sustain absorption, generate a longer half-life and shorten
1,2,3
renal clearance . In this technical note, we report an
LC-MS/MS signature peptide based method for the
quantification of PEG-IFN- α-2b in serum that can be used
in bioanalytical labs.
As reported earlier by Yang Z, et al, LOQ of 3.6ng/ml was
achieved for pegylated-interferon-α-2a. To achieve
maximum sensitivity several parameters related to sample
processing, chromatography and mass spectrometric
parameters were optimized. Extraction of PEG-IFN- α-2b
was obtained through precipitation of endogenous
proteins in serum followed by enzymatic digestion of
supernatant. A surrogate tryptic peptide generated from
the pegylated protein was used for MRM based
quantitation at picogram per mL levels in serum using the
SCIEX QTRAP® 6500 system.
INTRODUCTION
Pegylated drug conjugate Interferon α-2b (PEG-IFN- α-2b)
is a 165 amino acid therapeutic protein used for the
treatment of chronic hepatitis C infection. Pegylated
interferon α-2b is produced by covalent attachment of 40
kDa PEG (polyethylene-glycol) to interferon-α2b via the
p1
TM
Materials and Methods
PEG-IFN- α-2b was obtained commercially (lyophilized
powder) and diluted in aqueous buffer containing 150mM
ammonium formate pH 4.5.
Table 1. MS Parameters for MRM Quantitation
Name
Q1
Q3
Dwell DP
CE
CXP
Peptide 1 741.4 1047.5 25
85
35
12
Peptide 2 741.4 615.5
25
85
30
14
Peptide 3 741.4 745.4
25
85
32
12
Protein Pilot 5.0. Additionally, Skyline software was
used to identify the right precursor and its fragments
followed by method creation. Peptide sequence
2+
SFSLSTNLQESLR (m/z 741.38 >1047.5) was identified
as most stable and ionized sequence ion from the PEG2+
IFN- α-2b. Three transitions (741.38 >1047.5, 615.5 and
745.4) were selected for data acquisition but only
2+
(741.38 >1047.5) transition was used for quantitation. To
achieve maximum sensitivity both the source and
compound related parameters were optimized
Table 3. Sample Preparation
Step 1
Isopropyl alcohol (600 uL) contaiuning 0.1%
formic acid was added to 200 uL of plasma and
gently mixed for 10 minutes at room temperature.
Step 2
Samples were centrifuged at 4000 rpm for 10 min
at 5 C to collect the precipitate and the
supernatant was then removed to a fresh set of
tubes and dried under N2.
Step 3
The dried supernatant was reconstituted in 300
uL of digest buffer of 10 mM ammonium
bicarbonate, 50% methanol and 5 ul BSA
Step 4
Trypsin 10 ul, 1 mg/ml was addded and the
digestion was left overnight at 37 C
Step 5
The digestion reaction was quenched with 10 uL
of 10% formic acid.
Step 6
After centrifugation to collect any precipitate the
samples were injected.
Table 2. Gradient profile for MRM Quantitation
Column
Atlantis dC18, 100x 2.1 mm, 3.5μm
Mobile Phase A
Water, 0.1% formic acid
Mobile Phase B
Methanol, 0.1% formic acid
Flow rate
400 µL/min
Column temperature
45 °C
Injection volume
30 µL
Gradient profile
Time (min)
%B
0.0
20
0.5
20
15.0
60
16.0
80
18.0
80
18.5
20
20.0
20
Gradient Conditions for Signature Peptide Selection
A Shimadzu Nexera LC system was used with an
Atlantis-dC18, 100x 2.1 mm, 3.5μm column to separate
the tryptic digest at a flow rate of 300 µl/min. The tryptic
peptides were chromatographically separated with a
gradient starting at 5% acetonitrile (0.1% formic acid) to
40% of acetonitrile (0.1% formic acid) in 35 min run at
40°C.
Mass Spectrometry Conditions
The SCIEX QTRAP® 6500 system with Turbo V™ Ion
Drive source and electrospray ionization (ESI) probe was
used for peptide selection and quantitation experiments.
The QTRAP based Information Dependent Acquisition
(IDA) workflow was used to identify a unique peptide from
PEG-IFN- α-2b and sequence confirmation was done by
Results and Discussion
Improving sensitivity is one of the major challenges for the
application of MS-based assays for protein bioanalysis.
The existence of highly abundant endogenous proteins in
biological matrices may cause high background noise and
ion suppression during MS detection, which will
significantly reduce the sensitivity of the assay. Methods
for sample preparation that can remove the high
abundance background proteins and selectively extract
the target protein are critical to overcome these
challenges. To improve the assay sensitivity and
selectivity a variety of sample preparation strategies have
been applied to the purification of the target protein prior
to digestion. Protein precipitation can be used for the
extraction of organic soluble proteins (e.g., pegylated
proteins), since they are retained in the supernatant and
can be separated from other precipitated proteins (Wu et.
al., 2011). The advantage of this approach is that protein
precipitation can provide some degree of sample cleanup
by removing soluble proteins, salts, and a significant
portion of phospholipids. We have optimized the method
with IPA with formic acid assisted protein precipitation that
can efficiently remove albumin, the most abundant
endogenous protein in serum samples, while retain the
p2
target protein, thereby obtaining cleaner samples and
achieving improved sensitivity.
ELISAs are the most popular analytical method for protein
quantification, but can be challenging to use for
PEGylated because epitope-masking may limit sensitivity
and accuracy. LC-MS/MS based quantitation is becoming
more popular because of its advantage of selecting
specific signature peptides in complex drug conjugate
protein molecules. A sensitive and reproducible LCMS/MS method for PEG-IFN- α-2b has been developed in
this study. The sample extraction and cleanup workflow is
described in Table 3. The total ion chromatogram (TIC) of
tryptic digested PEG-IFN- α-2b and extracted ion
chromatogram (XIC) of the selected signature peptide
SFSLSTNLQESLR using a non-targeted IDA workflow
with QTRAP technology is depicted in Figure 2.
The calibration curve in serum extracted samples showed
excellent linearity with an r value of >0.99 for the linear
range between 246-50,000 pg/mL. Chromatograms of
various calibration samples are given in Figure 3. The
chromatograms of extracted serum blank, HQC and low
limit of quantitation (LLOQ) are shown in Figure 4. The
LLOQ has an excellent signal to noise ratio of 27.0. Three
precision and accuracy batches were processed and
tested. The QC sample statistics are summarized in Table
4. All the three batches were within the recommended
acceptance criteria for protein LC-MS/MS of %CV ± 25%
6
of LLOQ level and ±20% at other levels. The recovery of
the PEG-IFN- α-2b signature peptide was 76% across all
QC concentrations.
Table 4. The accuracy of PEG-IFN- α-2b at different
concentration levels in serum.
% Accuracy
PEG-IFN- α-2b
Statistics
N
Mean
S.D
% C.V
% Accuracy
LLQC
(pg/ml)
6
268.7
57.13
21.26
103.8
LQC
(pg/ml)
6
809.1
137.97
17.05
92.3
MQC
(pg/ml)
6
40079
3022
7.54
97.6
HQC
(pg/ml)
6
56225
3127
5.56
105.0
serum matrix. This sample preparation approach is
less time consuming and more affordable in CRO
business.
•
Linearity within a concentration range of 246-50,000
pg/ml of PEG-IFN- α-2b was achieved in serum matrix
2
with regression coefficients r 0.992.
References
1. Rinat R. et al., Structural characterization of protein–
polymer conjugates. I. Assessing heterogeneity of a
small PEGylated protein and mapping conjugation
sites using ion exchange chromatography and topdown tandem mass spectrometry. International
Journal of Mass Spectrometry 312 (2012) 135– 143.
2. Wu S, et al, A strategy for liquid chromatography
/tandem mass spectrometry based quantitation of
pegylated protein drugs in serum using serum protein
precipitation with water-miscible organic solvents and
subsequent trypsin digestion to generate surrogate
peptides for detection. Rapid Commun Mass
Spectrom. 2011; 25(2):281-90.
3. Yang Z, et al., A sensitive and high-throughput LCMS/MS method for the quantification of pegylatedinterferon-alpha2a in human serum using monolithic
C18 solid phase extraction for enrichment. J
Chromatogr B. 2009; 877(18-19):1737-42.
4. Long Yuan
and Mingshe Zhu, Quantitative
Bioanalysis of Proteins by Mass Spectrometry.
Materials
and
Methods
2015;5:133.
http://www.labome.com/method/QuantitativeBioanalysis-of-Proteins-by-Mass-Spectrometry.html
5. Catherine François, et al., Quantification of Different
Human Alpha Interferon Subtypes and Pegylated
Interferon Activities by Measuring MxA Promoter
Activation. Antimicrob Agents Chemother. 2005 Sep;
49(9): 3770–3775.
6. Rand Jenkins, et al., Recommendations for Validation
of LC-MS/MS Bioanalytical Methods for Protein
Biotherapeutics. AAPS J. 2015 Jan; 17(1): 1–16.
Conclusions
•
A highly specific and robust LC-MS/MS based
quantitation method has been developed for the
pegylated drug conjugate Interferon-α2b in serum.
•
A doubly charged signature peptide was selected for
better and robust ionization and used for MRM
2+
quantitation m/z 741.4 =>1047.5
•
A liquid-liquid extraction (LLE) based method was
optimized for the quantitation of PEG-IFN- α-2b from
p3
Figure 2: Total ion chromatogram (TIC) of tryptic digested Pegylated Drug Conjugate Interferon-α2b (a) and extracted ion
chromatogram (XIC) of selected peptide SFSLSTNLQESLR (b) using non-targeted IDA workflow on QTRAP ® technology.
Figure 3: Chromatograms of extracted peptide “SFSLSTNLQESLR” in serum.
p4
BLANK
HQC (60,000 pg/ml)
LLOQ (246.0 pg/ml)
Figure 4: Chromatograms of blank serum, LLOQ and HQC samples.
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Document number: RUO-MKT-02-4277-B
p5