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INTRODUCTION
The levels of active protein blood coagulation factors in plasma preparations are typically measured using a biological assay. As a result it is difficult to get an accurate quantitative measurement when analogue proteins or inhibitory compounds are present. Liquid chromatography-mass spectrometry (LC/MS) has excellent selectivity, sensitivity reproducibility and linear dynamic range and is the most frequently used quantitative bioanalysis method for small molecule drugs. This technique is also expected to become a key method for the monitoring of protein therapeutics in plasma. In this study, we selected a specific proteolytic peptide derived from the LC/MS generated peptide map of Factor VIII and used this to develop a high sensitivity quantitative LC/MS/MS method for monitoring Factor VIII in plasma.
QUANTITATIVE ANALYSIS OF BLOOD COAGULATION FACTOR VIII THERAPEUTICS IN PLASMA USING UPLC/MS Hiroya Miura1; Taiji Kawase2; Kenji Hirose2 1Japan blood products organization, Tokyo, Japan; 2Nihon Waters K.K., Tokyo, Japan
METHODS
RESULTS
CONCLUSION Sensitivity ;LOQ < 1 unit/mL, Dynamic range ; >1000, Linearity ; R2>0.99
This MRM monitoring assay is highly useful for the high sensitivity and selective quantitative measurement of similar large proteins in plasma (>300kDa).
This can also be easily adapted to study other protein therapeutics.
Structure of factor VIIIN. BOVENSCHEN, D. C. RIJKEN, L . M. HAVEKES, B . J . M. VAN VLIJMEN and K. MERTENS, Journal of Thrombosis and Haemostasis, 3: 1257–1265
Blood coagulation factor FVIIIA large ~ 300kDa molecule (2322AA).
The whole protein is composed of several domains whose structural changes define bio-activity.
The levels in plasma are typically measured using a biological assay.
LC/MS will improve sensitivity, reproducibility and linearity.
Domain-specific measurements will show the role of each domain.
Significance of LC/MS measurement ?
In this study, we developed a LC/MS quantitative assay for this macromolecule.
Plasma 0.5mL + FVIIILoad onto Q-Sepharose column （Φ1cm×1cm）(pre-equillibration with 25mM Citric acid, 5mM CaCl2, 85mM NaCl, pH6.0)
Concentrate with ultra filtration 10K at 1900g for 10min (Amicon Ultra, MERCK Millipore)
Wash with 25mM Citric acid, 5mM CaCl2, 85mM NaCl, pH6.0
Wash with 2.0mL of 25mM Citric acid, 5mM CaCl2, 500mM NaCl, pH6.0
Plasma pretreatment protocol
Then trypsin digestion according to the protocol as shown in “Proteolysis protocol”
LC/MS conditions
<MS>・Ionization : ESI positive・Capillary voltage : 1.0kV・Cone voltage : 40V
Peptide mapping<UPLC>・MP A : 0.1% Formic acid aq.・MP B : 0.1% Formic acid in ACN・Column : BEH300 C18
1.7 µm 2.1x150 mm・Column temperature : 40oC・Gradient : B(1%->35%,0->90min)
@0.2mL/min
Monitoring assay<UPLC>・MP A : 0.1% Formic acid aq.・MP B : 0.1% Formic acid in ACN・Column : BEH300 C18
1.7 µm 2.1x100 mm・Column temperature : 55oC・Gradient : B(5%->18%->31%,
0->0.5->10min)@0.25mL/min
<MS>・Ionization : ESI positive・Capillary voltage : 1.0kV・Cone voltage : 30V・CE (Low) : 6eV・CE (High) : 15-45eV
Time0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
%
0
100
20120425_03 1: TOF MS ES+ BPI
2.17e570.81638.4
2.39365.1
65.05474.0
37.23616.8
20.01495.3
3.34407.1
19.30432.214.17
407.26.91
604.3
35.12485.8
26.80524.8
26.58470.9
20.63504.2
27.16665.3
33.74530.6
30.97476.3
63.02679.0
58.93561.0
51.10532.341.72
626.8
49.80521.3
55.45482.3
67.32849.1
72.58517.3
79.77768.475.89
545.080.24;599.3 96.78
976.2
UPLC was connected to a Xevo G2 quadrupole time-of-flight mass spectrometer and peptide maps were generated using the MSE
mode. More than 400 peptides from Factor VIII were detected from the 100 minute peptide mapping experiment.
Figure 1. BPI Chromatogram of tryptic peptides from recombinant factor VIII therapeutic (Advate) Figure 2. Coverage map display of BiopharmaLynx
for recombinant factor VIIIThe MSE data was processed using the BioPharmaLynx software, allowing the comprehensive mass accurate assignment of peptides and their fragment ions.
Peptide Label Start End Domain RT (Min) m/z Charge State Mass (Da) Intensity
(Counts)ADC
Responseb/y
Found b/y %Mass Error (ppm)
DFPILPGEIFK 1:T053 500 510 A2 70.9 638.35 2 1274.6943 771301 26009750 16 80 2.6
VVFQEFTDGSFTQPLYR 1:T194 1733 1749 A3 63.1 678.67 3 2032.9899 695482 25046680 30 93.8 0.2
GELNEHLGLLGPYIR 1:T195 1750 1764 A3 59.1 560.97 3 1679.9006 551703 23609800 23 82.1 0.7
ETLIQENVVLPQIHTVTGTK 1:T125 1202 1221 B 57.7 740.75 3 2219.2195 435581 17250870 23 60.5 1.5
VDLLAPMIIHGIK 1:T225 2073 2085 C1 65.2 473.95 3 1418.832 416434 19371900 16 66.7 0.1
FDDDNSPSFIQIR 1:T033 360 372 A1 49.8 777.37 2 1552.7183 390832 13578940 18 75 1.6
Figure 3. Calibration curve of specific MRM for domain A2The doubly charged precursor ion at m/z 638 generates a stable singly charged fragment at m/z 690.A) Calibration curve : This MRM transition was used to develop a high sensitivity assay with an LOQ < 1
unit/mL, R2>0.99 for the concentration range of >100. B) Chromatograms of the monitored peptide in plasma : These showed the excellent reproducibility of
this MRM assay.
min14.00 14.50 15.00 15.50 16.00 16.50
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120710_38 Smooth(Mn,2x2)
1.394e+005DFPILPGEIFK
15.24
min
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120710_37 Smooth(Mn,2x2)
3.469e+004DFPILPGEIFK
15.24
min
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120710_36 Smooth(Mn,2x2)
2.513e+004DFPILPGEIFK
15.23
min
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120710_35 Smooth(Mn,2x2)
2.791e+003DFPILPGEIFK
15.2213.51
14.09 15.0014.40 16.03 16.56
min
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120710_34 Smooth(Mn,2x2)
1.316e+003DFPILPGEIFK
15.2213.51
14.09 15.01 16.0315.37 16.601 U/mL
2 U/mL
13 U/mL
24 U/mL
104 U/mL
B)Compound name: DFPILPGEIFKCorrelation coefficient: r = 0.999739, r^2 = 0.999479Calibration curve: 109.878 * x + -2.68378Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
U/mL0 10 20 30 40 50 60 70 80 90 100
Resp
onse
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
A)
The VerifyE software is then used to interrogate the acquired peptide maps and select the peptide T053 (DFPILPGEIFK) according to software determined criteria. The peptide: (i) does not include easily modified amino acids such as Met and Cys; (ii) shows high intensity; (iii) shows a sharp Gaussian distribution in the chromatogram; (iv) shows the most abundance doubly charged ion.
Table 1 Selected peptides for monitoring
FVIII*Add RapiGest, incubate@60oC for 15min
Add 100mM DTT, incubate@60oC for 30min
Add 200mM DTT, [email protected]. for 30min
Add Trypsin, incubate@37oC for 18.5hr
Add TFA, incubate@37oC for 40min
Centrifuge@13,000rpm for 10min and take supernatant
Proteolysis protocol
*for selecting monitoring peptides, recombinant FVIII was used
Figure 4. Results of quantitative assay in mouse plasmaA) Monitoring curve : Compared to traditional bioassays using a synthetic substrate, the time course
monitoring of the Factor VIII therapeutics-treated mouse plasma using this MRM methodology showed very similar concentrations at each monitoring point (5, 30, 120 and 300 minutes).
B) Chromatograms of each monitored point
min8.00 8.50 9.00 9.50 10.00
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120824_41 Smooth(Mn,2x1)
6.007e+006
DFPILPGEIFK9.60
min
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120824_37 Smooth(Mn,2x1)
6.007e+006
DFPILPGEIFK9.61
min
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120824_33 Smooth(Mn,2x1)
6.007e+006
DFPILPGEIFK9.60
min
%
0
100
F6:MRM of 2 channels,ES+638.4 > 690.5
20120824_25 Smooth(Mn,2x1)
6.007e+006DFPILPGEIFK
9.60
5min
30min
120min
300min
B)
0
20
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60
80
100
120
140
160
0 50 100 150 200 250 300
採血時間（min）
合成
基質
（IU
/mL
）
0
100
200
300
400
500
600
Mass
法（IU
/mL
）
処理後
マス
A)
Monitoring point (min)
300250200150100500
MRMBioassay
●
▲
600
500
400
300
200
100
0
MR
M (
IU/
mL)
160
140
120
100
80
60
0
Bio
assa
y (I
U/
mL)
20
40
MRM monitoring assay
<UPLC/MS system>Acquity UPLC I-Class FTNXevo TQ-S MS
<Informatics>TargetLynx
Peptide mapping and selection
<UPLC/MS system>Acquity UPLC BSMXevo G2 Qtof MS
<Informatics>BiopahamaLynxVerifyE
Peptide identification with accurate mass
High-sensitive quantification with selectivity, linearity
and wide range
