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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation MKT13072 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 Miura 1 ; Taiji Kawase 2 ; Kenji Hirose 2 1 Japan blood products organization, Tokyo, Japan; 2 Nihon 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 VIII N. 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 FVIII A 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 + FVIII Load onto Q-Sepharose column （Φ1cm×1cm） (pre-equillibration with 25mM Citric acid, 5mM CaCl 2 , 85mM NaCl, pH6.0) Concentrate with ultra filtration 10K at 1900g for 10min (Amicon Ultra, MERCK Millipore) Wash with 25mM Citric acid, 5mM CaCl 2 , 85mM NaCl, pH6.0 Wash with 2.0mL of 25mM Citric acid, 5mM CaCl 2 , 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 : 40 o C ・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 : 55 o C ・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 Time 0.00 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 2.17e5 70.81 638.4 2.39 365.1 65.05 474.0 37.23 616.8 20.01 495.3 3.34 407.1 19.30 432.2 14.17 407.2 6.91 604.3 35.12 485.8 26.80 524.8 26.58 470.9 20.63 504.2 27.16 665.3 33.74 530.6 30.97 476.3 63.02 679.0 58.93 561.0 51.10 532.3 41.72 626.8 49.80 521.3 55.45 482.3 67.32 849.1 72.58 517.3 79.77 768.4 75.89 545.0 80.24;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 MS E 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 VIII The MS E 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 Response b/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 A2 The 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, R 2 >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. min 14.00 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+005 DFPILPGEIFK 15.24 min % 0 100 F6:MRM of 2 channels,ES+ 638.4 > 690.5 20120710_37 Smooth(Mn,2x2) 3.469e+004 DFPILPGEIFK 15.24 min % 0 100 F6:MRM of 2 channels,ES+ 638.4 > 690.5 20120710_36 Smooth(Mn,2x2) 2.513e+004 DFPILPGEIFK 15.23 min % 0 100 F6:MRM of 2 channels,ES+ 638.4 > 690.5 20120710_35 Smooth(Mn,2x2) 2.791e+003 DFPILPGEIFK 15.22 13.51 14.09 15.00 14.40 16.03 16.56 min % 0 100 F6:MRM of 2 channels,ES+ 638.4 > 690.5 20120710_34 Smooth(Mn,2x2) 1.316e+003 DFPILPGEIFK 15.22 13.51 14.09 15.01 16.03 15.37 16.60 1 U/mL 2 U/mL 13 U/mL 24 U/mL 104 U/mL B) Compound name: DFPILPGEIFK Correlation coefficient: r = 0.999739, r^2 = 0.999479 Calibration curve: 109.878 * x + -2.68378 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None U/mL 0 10 20 30 40 50 60 70 80 90 100 Response 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 A) The Verify E 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@60 o C for 15min Add 100mM DTT, incubate@60 o C for 30min Add 200mM DTT, [email protected]. for 30min Add Trypsin, incubate@37 o C for 18.5hr Add TFA, incubate@37 o C 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 plasma A) 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 min 8.00 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 DFPILPGEIFK 9.60 min % 0 100 F6:MRM of 2 channels,ES+ 638.4 > 690.5 20120824_37 Smooth(Mn,2x1) 6.007e+006 DFPILPGEIFK 9.61 min % 0 100 F6:MRM of 2 channels,ES+ 638.4 > 690.5 20120824_33 Smooth(Mn,2x1) 6.007e+006 DFPILPGEIFK 9.60 min % 0 100 F6:MRM of 2 channels,ES+ 638.4 > 690.5 20120824_25 Smooth(Mn,2x1) 6.007e+006 DFPILPGEIFK 9.60 5min 30min 120min 300min B) A) Monitoring point (min) 300 250 200 150 100 50 0 MRM Bioassay ● ▲ 600 500 400 300 200 100 0 MRM (IU/mL) 160 140 120 100 80 60 0 Bioassay (IU/mL) 20 40 MRM monitoring assay <UPLC/MS system> Acquity UPLC I-Class FTN Xevo TQ-S MS <Informatics> TargetLynx Peptide mapping and selection <UPLC/MS system> Acquity UPLC BSM Xevo G2 Qtof MS <Informatics> BiopahamaLynx VerifyE Peptide identification with accurate mass High-sensitive quantification with selectivity, linearity and wide range
