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©2015 Waters Corporation 1
Adding Mass Detection to Routine
Peptide Level Biotherapeutic Analysis
©2015 Waters Corporation 2
Presentation Outline
 The unmet need
 The ACQUITY® QDa® Detector – a brief overview
 Demonst...
©2015 Waters Corporation 3
What we’ve been hearing…
“We’re doing a lot of routine MS monitoring in support of pilot
operat...
©2015 Waters Corporation 4
The ACQUITY® QDa® Mass Detector
A pioneering product with mass appeal
 Innovative design focus...
©2015 Waters Corporation 5
Easily Add to Existing LC Systems
Existing stack
Easy to deploy
Fully integrated with
Empower® ...
©2015 Waters Corporation 6
Familiar Graphical User Interface for
Ease-Of-Use and Fast Adoption
Empower GUI identical to
th...
©2015 Waters Corporation 7
Automated Start Up Provides
Robust, Reproducible Performance
 Automated resolution and calibra...
©2015 Waters Corporation 8
Disposable Sample Aperture and
Capillary for Easy Maintenance
Sample Aperture:
As simple as rep...
©2015 Waters Corporation 9
ACQUITY QDa Detector in summary
 A pioneering product that is empowering analytical chemists
a...
©2015 Waters Corporation 10
ACQUITY QDa for Peptides
The following data demonstrates that this easy-to-sue
mass detector c...
©2015 Waters Corporation 11
Extracted spectrum
ACQUITY QDa
ACQUITY QDa Detects Peptides
Over a Wide Mass Range
3 µg inject...
©2015 Waters Corporation 12
ACQUITY QDa Mass Chromatograms
of Trastuzumab Digest
ACQUITY UPLC BEH Column
0.1% TFA, 4-µg in...
©2015 Waters Corporation 13
Trastuzumab – Heavy Chain Peptides
with TFA vs. FA as Acid Modifier
Fragment
Average
Mass [CH+...
©2015 Waters Corporation 14
Trastuzumab – Light Chain Peptides
with TFA vs. FA as Acid Modifier
Fragment
Average
Mass [CH+...
©2015 Waters Corporation 15
HC Peptide T15* Mass Spectrum
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
...
©2015 Waters Corporation 16
Mass Measurement Accuracy
0.00
20.00
40.00
60.00
80.00
100.00
0.00 0.05 0.10 0.15 0.20
Cumulat...
©2015 Waters Corporation 17
Specificity:
Accurate Peak Area Determinations
T10
T26
T26: Area 9.1105 (59%)
T10: Area 6.31...
©2015 Waters Corporation 18
Specificity:
Resolving Coelutions
XIC
SIR
Peak 2
Peak 2
Peak 1
Peak 1&2
Peak 1 [M+4H]+4
536.3 ...
©2015 Waters Corporation 19
ACQUITY QDa Greatly Extends Linear Dynamic
Range (ACQUITY UPLC BEH w/ 0.1% TFA)
Mass Load
Dilu...
©2015 Waters Corporation 20
Extend the Linear Dynamic Range Farther
Using ACQUITY UPLC CSH w/ 0.1% FA
SIR
Mass Load
Diluti...
©2015 Waters Corporation 21
Quantification
T21-Oxidized T21
5.5%
94.5%
SIR
TUV
TIC
Key Takeaway
MS provides the specificit...
©2015 Waters Corporation 22
CDR Peptide Monitoring
TIC
XIC
1
2
3
4
5
6
7
Key Takeaway
MS enables targeted monitoring of
pe...
©2015 Waters Corporation 23
Deamidation Monitoring
(CDR peptide)
TIC
XIC
572.8 m/z
Peptide
Average
Mass
XIC
m/z
T3-4 2,287...
©2015 Waters Corporation 24
 Detect and monitor peptides
over a wide molecular weight
range
 Quantify peptide variants w...
©2015 Waters Corporation 25
Addendum: User Case Study
 Customer X – a large biopharmaceutical developer and
manufacturer ...
©2015 Waters Corporation 26
ACQUITY QDa Detector
LysC digest of mAb* (120 min run)
ACQUITY UPLC TUV DetectorMethod Summary...
©2015 Waters Corporation 27
Zoom-in
comparison:
0-30 min
LysC digest of mAb (0-30 minutes)
TUV
Mass
©2015 Waters Corporation 28
Zoom-in
comparison:
30-60 min
LysC digest of mAb (30-60 minutes)
TUV
Mas
s
©2015 Waters Corporation 29
Zoom-in
comparison:
60-90 min
LysC digest of mAb (60-90 minutes)
TUV
Mass
©2015 Waters Corporation 30
Zoom-in
comparison:
90-120 min
LysC digest of mAb (90-120 minutes)
TUV
Mass
©2015 Waters Corporation 31
LysC digest: SYNAPT vs. ACQUITY QDa
(0-30 minutes)
ACQUITY QDa
SYNAPT (data offset by 2.4 min ...
©2015 Waters Corporation 32
LysC digest: SYNAPT vs. ACQUITY QDa
(30-60 minutes)
ACQUITY QDa
SYNAPT
SYNAPT vs.
ACQUITY QDa
...
©2015 Waters Corporation 33
LysC digest: SYNAPT vs. ACQUITY QDa
(60-90 minutes)
ACQUITY QDa
SYNAPT
SYNAPT vs.
ACQUITY QDa
...
©2015 Waters Corporation 34
LysC digest: SYNAPT vs. ACQUITY QDa
(90-120 minutes)
ACQUITY
QDa
SYNAPT K7 Peptide
(6,714 kDa)...
©2015 Waters Corporation 35
Consistent Repeatable Performance
[M+6H]+6
[M+7H]+7
[M+8H]+8
[M+9H]+9
[M+10H]+10
[M+11H]+11
m/...
©2015 Waters Corporation 36
QDa = 96.8%
(X = 97.5%)
QDa = 3.2%
(X =2.5%)
H21
H21-Ox
SIR
H21: M+2 [418.4]
H21-Ox: M+2 [426....
©2015 Waters Corporation 37
H6
Relative Quantification
Deamidated vs. Non-Deamidated (1)
H6D
H6D
SIR m/z
M+3 [362.4]
M+2 [...
©2015 Waters Corporation 38
Relative Quantification
Deamidated vs. Non-Deamidated (2)
Chromatographic
Resolution
m/z
Resol...
©2015 Waters Corporation 39
IdeS Digest: Humira (adalimumab)
Time
Flow
(mL/min)
% A % B %C % D
Initial 0.200 0 0 95 5
3.00...
©2015 Waters Corporation 40
IdeS Digest: Humira (2)
Focused gradient on FC fragment
1
2
3
4 5
1
2
3
4
5
©2015 Waters Corporation 41
Trypsin digest: Glycopeptides
Peptide
Average
mass
M+3
H25 1189.2 397.4
G0F 2633.7 878.9
G1F 2...
©2015 Waters Corporation 42
 Detect and monitor peptides
over a wide molecular weight
range
 Quantify peptide variants w...
©2015 Waters Corporation 43
More info on the ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm
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Adding Mass Detection to Monitor Peptides in Biopharmaceutical Development & QC

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Learn how the Waters ACQUITY QDa Detector is a powerful tool for mass detection in monitoring peptides in HPLC or UPLC assays, in biopharmaceutical late development and quality control. http://www.waters.com/qdabiopharm

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Adding Mass Detection to Monitor Peptides in Biopharmaceutical Development & QC

  1. 1. ©2015 Waters Corporation 1 Adding Mass Detection to Routine Peptide Level Biotherapeutic Analysis
  2. 2. ©2015 Waters Corporation 2 Presentation Outline  The unmet need  The ACQUITY® QDa® Detector – a brief overview  Demonstrating broad utility for peptide monitoring  A clear path to increased productivity
  3. 3. ©2015 Waters Corporation 3 What we’ve been hearing… “We’re doing a lot of routine MS monitoring in support of pilot operations, manufacturing and QC, and foresee doing more in the future.” “Relying on our core MS resources and high end equipment for this testing is expensive and creates a bottleneck.” “We’d like to empower traditional LC analytical support labs to generate this data, thus reducing the burden on core MS resources and improving our overall workflow and productivity.” The bottom line: Biopharmaceutical laboratories want to better utilize analytical resources and improve productivity.
  4. 4. ©2015 Waters Corporation 4 The ACQUITY® QDa® Mass Detector A pioneering product with mass appeal  Innovative design focused on ease-of-use for chromatographers  Empowering analytical chemists everywhere with orthogonal mass detection – added information with every sample  Compact, robust and affordable: Built for constant use with a wide variety of chromatographic conditions  Seamlessly integrates with HPLC & UPLC® systems that use Empower ® Chromatography Data Software www.waters.com/qda
  5. 5. ©2015 Waters Corporation 5 Easily Add to Existing LC Systems Existing stack Easy to deploy Fully integrated with Empower® CDS Software Minimal training required Qualification documents / procedures ready-to-go 110/220V operation Minimal maintenance ACQUITY QDa Existing stack +1upgrade
  6. 6. ©2015 Waters Corporation 6 Familiar Graphical User Interface for Ease-Of-Use and Fast Adoption Empower GUI identical to that of a PDA for setup, data viewing and reporting  Very little training needed  Quick addendum to current lab SOPs
  7. 7. ©2015 Waters Corporation 7 Automated Start Up Provides Robust, Reproducible Performance  Automated resolution and calibration occurs with each start-up, ensuring mass information is accurate and precise  ESI interface optimized for UPLC performance to ensure chromatographic resolution, sensitivity and throughput is preserved The Graphic ACQUITY QDa monitor display enables easy viewing and adjustment of system parameters
  8. 8. ©2015 Waters Corporation 8 Disposable Sample Aperture and Capillary for Easy Maintenance Sample Aperture: As simple as replacing a detector lamp Capillary: No cutting or assembly required
  9. 9. ©2015 Waters Corporation 9 ACQUITY QDa Detector in summary  A pioneering product that is empowering analytical chemists and chromatographers everywhere to take advantage of the power of mass detection  Brings greater insight into every peak, for enhanced and streamlined monitoring workflows for improved productivity  Easy to deploy, simple to use and maintain, compact, robust and affordable! www.waters.com/qdabiopharm
  10. 10. ©2015 Waters Corporation 10 ACQUITY QDa for Peptides The following data demonstrates that this easy-to-sue mass detector can:  Detect and monitor peptides over a wide molecular weight range  Quantify peptide variants with enhanced specificity  Monitor components below optical detector sensitivity  Selectively detect and monitor coeluting components  Work with both TFA and Formic Acid based separations
  11. 11. ©2015 Waters Corporation 11 Extracted spectrum ACQUITY QDa ACQUITY QDa Detects Peptides Over a Wide Mass Range 3 µg injection Peak Peptide average mass [M+] M+2] [M+3] [M+4] [M+5] 1 Angiotensin frag. 1-7 899.0 900.0 450.5 300.6 225.7 180.8 2 Bradykinin 1060.2 1061.2 531.1 354.4 266.0 213.0 3 Angiotensin II 1046.2 1047.1 524.1 349.7 262.5 210.2 4 Angiotensin I 1296.5 1297.4 649.2 433.1 325.1 260.3 5 Renin substrate 1759.0 1760.0 880.5 587.3 440.7 352.8 6 Enolase T35 1873.2 1874.2 937.6 625.4 469.3 375.6 7 Melittin 2847.5 2848.4 1424.7 950.1 712.8 570.5 Charge State 1 2 3 4 5 6 7 [M+4] [M+3] 712.7 949.9 [M+5] 570.4
  12. 12. ©2015 Waters Corporation 12 ACQUITY QDa Mass Chromatograms of Trastuzumab Digest ACQUITY UPLC BEH Column 0.1% TFA, 4-µg injection ACQUITY UPLC BEH Column 0.1% FA, 4-µg injection Digest 5 BEH 130Å 100mm Gradient: 97-65% A Digest 1 BEH 300Å 150mm Gradient: 97-65% A
  13. 13. ©2015 Waters Corporation 13 Trastuzumab – Heavy Chain Peptides with TFA vs. FA as Acid Modifier Fragment Average Mass [CH+1H]+1 [CH+2H]+2 [CH+3H]+3 [CH+4H]+4 [CH+5H]+5 [CH+6H]+6 [CH+7H]+7 [CH+8H]+8 [CH+9H]+9 [CH+10H]+10 T39 574.3 575.3 288.2 192.4 144.6 115.9 96.7 83.0 72.8 64.8 58.4 T7 681.3 682.3 341.7 228.1 171.3 137.3 114.6 98.3 86.2 76.7 69.1 T5 830.0 831.0 416.0 277.7 208.5 167.0 139.3 119.6 104.7 93.2 84.0 T21 835.0 836.0 418.5 279.3 209.7 168.0 140.2 120.3 105.4 93.8 84.5 T30 838.0 839.0 420.0 280.3 210.5 168.6 140.7 120.7 105.8 94.1 84.8 T9 969.1 970.1 485.5 324.0 243.3 194.8 162.5 139.4 122.1 108.7 97.9 T6 1084.2 1085.2 543.1 362.4 272.1 217.8 181.7 155.9 136.5 121.5 109.4 T3 1089.2 1090.2 545.6 364.1 273.3 218.8 182.5 156.6 137.2 122.0 109.9 T36* 1161.4 1162.4 581.7 388.1 291.3 233.3 194.6 166.9 146.2 130.0 117.1 T2* 1167.4 1168.4 584.7 390.1 292.8 234.5 195.6 167.8 146.9 130.7 117.7 T8-9 1182.3 1183.3 592.2 395.1 296.6 237.5 198.1 169.9 148.8 132.4 119.2 T13 1186.4 1187.4 594.2 396.5 297.6 238.3 198.7 170.5 149.3 132.8 119.6 T10 1310.5 1311.5 656.3 437.8 328.6 263.1 219.4 188.2 164.8 146.6 132.1 T4-5 1311.5 1312.5 656.8 438.2 328.9 263.3 219.6 188.4 164.9 146.7 132.2 T14* 1321.5 1322.5 661.8 441.5 331.4 265.3 221.3 189.8 166.2 147.8 133.2 T11* 1334.4 1335.4 668.2 445.8 334.6 267.9 223.4 191.6 167.8 149.3 134.4 T23 1677.8 1678.8 839.9 560.3 420.5 336.6 280.6 240.7 210.7 187.4 168.8 T33-34 1724.9 1725.9 863.5 576.0 432.2 346.0 288.5 247.4 216.6 192.7 173.5 T26 1808.1 1809.1 905.1 603.7 453.0 362.6 302.4 259.3 227.0 201.9 181.8 T38 1874.1 1875.1 938.0 625.7 469.5 375.8 313.3 268.7 235.3 209.2 188.4 T1 1882.1 1883.1 942.1 628.4 471.5 377.4 314.7 269.9 236.3 210.1 189.2 T22* 2139.4 2140.4 1070.7 714.1 535.8 428.9 357.6 306.6 268.4 238.7 214.9 T26-27 2228.6 2229.6 1115.3 743.9 558.1 446.7 372.4 319.4 279.6 248.6 223.9 T2-3* 2238.6 2239.6 1120.3 747.2 560.6 448.7 374.1 320.8 280.8 249.7 224.9 T37 2544.7 2545.7 1273.3 849.2 637.2 509.9 425.1 364.5 319.1 283.7 255.5 T12 2785.0 2786.0 1393.5 929.3 697.3 558.0 465.2 398.9 349.1 310.4 279.5 T41* 2802.1 2803.1 1402.1 935.0 701.5 561.4 468.0 401.3 351.3 312.3 281.2 T19-20* 3335.9 3336.9 1669.0 1113.0 835.0 668.2 557.0 477.6 418.0 371.7 334.6 T15* 6716.5 6717.5 3359.2 2239.8 1680.1 1344.3 1120.4 960.5 840.6 747.3 672.6 T15-16* 7058.9 7059.9 3530.4 2354.0 1765.7 1412.8 1177.5 1009.4 883.4 785.3 706.9 T15-17* 7187.0 7188.0 3594.5 2396.7 1797.8 1438.4 1198.8 1027.7 899.4 799.6 719.7 Trifluoroacetic acid Formic acid = not observed Legend: 90 % Coverage
  14. 14. ©2015 Waters Corporation 14 Trastuzumab – Light Chain Peptides with TFA vs. FA as Acid Modifier Fragment Average Mass [CH+1H]+1 [CH+2H]+2 [CH+3H]+3 [CH+4H]+4 [CH+5H]+5 [CH+6H]+6 [CH+7H]+7 [CH+8H]+8 [CH+9H]+9 [CH+10H]+10 T2* 748.9 749.9 375.5 250.6 188.2 150.8 125.8 108.0 94.6 84.2 75.9 T19-20* 868.9 869.9 435.5 290.6 218.2 174.8 145.8 125.1 109.6 97.5 87.9 T15 1502.6 1503.6 752.3 501.9 376.7 301.5 251.4 215.7 188.8 168.0 151.3 T5 1773.1 1774.1 887.5 592.0 444.3 355.6 296.5 254.3 222.6 198.0 178.3 T11* 1798.1 1799.1 900.0 600.4 450.5 360.6 300.7 257.9 225.8 200.8 180.8 T18* 1876.1 1877.1 939.1 626.4 470.0 376.2 313.7 269.0 235.5 209.5 188.6 T1 1879.0 1880.0 940.5 627.3 470.8 376.8 314.2 269.4 235.9 209.8 188.9 T10 1946.2 1947.2 974.1 649.7 487.6 390.2 325.4 279.0 244.3 217.2 195.6 T3 1991.2 1992.2 996.6 664.7 498.8 399.2 332.9 285.5 249.9 222.2 200.1 T14 2136.2 2137.2 1069.1 713.1 535.0 428.2 357.0 306.2 268.0 238.4 214.6 T17-18* 2141.4 2142.4 1071.7 714.8 536.4 429.3 357.9 306.9 268.7 238.9 215.1 T3-4 2287.6 2288.6 1144.8 763.5 572.9 458.5 382.3 327.8 286.9 255.2 229.8 T7* 4189.5 4190.5 2095.8 1397.5 1048.4 838.9 699.3 599.5 524.7 466.5 420.0 Charge State Trifluoroacetic acid Formic acid = not observed Legend: Key Takeaways:  Compatible with TFA and FA based separations  Multiple charge states detected /peptide 92% Coverage
  15. 15. ©2015 Waters Corporation 15 HC Peptide T15* Mass Spectrum DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK Average mass = 6716.5 Da [M+6H]+6 [M+7H]+7 [M+8H]+8 [M+9H]+9 [M+10H]+10 [M+11H]+11 m/z Intensity Key Takeaway ~7,000 Da peptide detected with multiple charge states.
  16. 16. ©2015 Waters Corporation 16 Mass Measurement Accuracy 0.00 20.00 40.00 60.00 80.00 100.00 0.00 0.05 0.10 0.15 0.20 CumulativeError(%) Absolute Mass Error Cumulative Error Distribution Plot -0.30 -0.20 -0.10 0.00 0.10 0.20 0.30 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 MassDifference Theoretical Average Molecular Weight (Da) Mass Error Vs. Molecular Weight InstrumentSpec. Key Takeaway Mass measurement accuracy of all reported peptides within instrument specification (+/- 0.2 Da).
  17. 17. ©2015 Waters Corporation 17 Specificity: Accurate Peak Area Determinations T10 T26 T26: Area 9.1105 (59%) T10: Area 6.3105 (41%) T26: Area 1.6108 (62%) T10: Area 9.9107 (38%) TUV XIC T10 T26 Key Takeaway MS detection reduces variability when integrating closely eluting components.
  18. 18. ©2015 Waters Corporation 18 Specificity: Resolving Coelutions XIC SIR Peak 2 Peak 2 Peak 1 Peak 1&2 Peak 1 [M+4H]+4 536.3 m/z Peak 2 [M+2H]+2 426.5 m/z TUV Peak 2 426.5 m/z Key Takeaway MS reveals coelutions and enables you to independently monitor both species.
  19. 19. ©2015 Waters Corporation 19 ACQUITY QDa Greatly Extends Linear Dynamic Range (ACQUITY UPLC BEH w/ 0.1% TFA) Mass Load Dilution ng pmol 1:1 1025.00 6961.19 1:2 512.50 3480.59 1:4 256.25 1740.30 1:8 128.13 870.15 1:16 64.06 435.07 1:32 32.03 217.54 1:64 16.02 108.77 1:128 8.01 54.38 1:256 4.00 27.19 1:512 2.00 13.60 1:1,024 1.00 6.80 y = 74103x - 6636.1 R² = 0.9995 0.00 0.04 0.08 0.12 0.00 5.00 10.00 15.00 Area x10000000 Mass Load (ng) BEH SIR Area Vs. Mass Load SIRTUV Mass Load Dilution ng pmol 1:1 1025.00 6961.19 1:2 512.50 3480.59 1:4 256.25 1740.30 1:8 128.13 870.15 1:16 64.06 435.07 1:32 32.03 217.54 1:64 16.02 108.77 1:128 8.01 54.38 1:256 4.00 27.19 1:512 2.00 13.60 1:1,024 1.00 6.80 Mass Load Dilution ng pmol 1:1 1025.00 6961.19 1:2 512.50 3480.59 1:4 256.25 1740.30 1:8 128.13 870.15 1:16 64.06 435.07 1:32 32.03 217.54 1:64 16.02 108.77 1:128 8.01 54.38 1:256 4.00 27.19 1:512 2.00 13.60 1:1,024 1.00 6.80 1:2,048 0.50 3.40 1:4,096 0.25 1.70 1:8,192 0.13 0.85 1:16,384 0.06 0.42 TIC Linear Linear Linear
  20. 20. ©2015 Waters Corporation 20 Extend the Linear Dynamic Range Farther Using ACQUITY UPLC CSH w/ 0.1% FA SIR Mass Load Dilution ng pmol 1:64 16 109 1:128 8 54.4 1:256 4 27.2 1:512 2 13.6 1:1024 1 6.80 1:2,048 0.50 3.40 1:4,096 0.25 1.70 1:8,192 0.12 0.849 1:16,384 0.063 0.425 1:32,768 0.031 0.212 1:65,536 0.016 0.106 1:131,072 0.0078 0.0531 1:262,144 0.0039 0.0266 1:524,288 0.0020 0.0133 Linear R² = 0.9993 0 4 8 12 16 20 0.00 2.00 4.00 6.00 8.00 10.00 Area Millions Mass Load (ng) CSH 0.1% FA SIR Area Vs. Mass Load 2.0 pg (13fmol) 7.8 pg (53fmol) 16 pg (106fmol) 31 pg (212fmol) SIR 1:32,7 68 1:65,5 36 1:131,0 72 1:524,28 8 S/N 171 S/N 88 S/N 28 S/N 15 dilution
  21. 21. ©2015 Waters Corporation 21 Quantification T21-Oxidized T21 5.5% 94.5% SIR TUV TIC Key Takeaway MS provides the specificity and sensitivity for relative quantification of peptides.
  22. 22. ©2015 Waters Corporation 22 CDR Peptide Monitoring TIC XIC 1 2 3 4 5 6 7 Key Takeaway MS enables targeted monitoring of peptides for product ID testing. Light Chain 1: ASQDVNTAVAWYQQKPGK 2: LLIYSASFLYSGVPSR 3: SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK Heavy Chain 4: DTYIHWVR 5: IYPTNGYTR/(6)YADSVKG 7: WGGDGFYAMDYWGQGTLVTVSSASTK Harris, R.J., Kabakoff, B., Macchi, F.D., Shen, F.J. Kwong, M., Andya, J.D., Shire, S.J., Bjork, N., Totpal, K., Chen, A.B., Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B 752 (2001) 233-245.
  23. 23. ©2015 Waters Corporation 23 Deamidation Monitoring (CDR peptide) TIC XIC 572.8 m/z Peptide Average Mass XIC m/z T3-4 2,287.5 572.8 T18* 1,876.1 626.3 T2* 1,167.3 584.6 T5 8,29.9 415.9 T3-4* 2,288.5 573.1 T5 T3-4 T2* T18* T3-4: ASQDVNTAVAWYQQKPGKAPK Deamidated XIC T3-4 573.1 m/z T3-4*(D) Key Takeaway When chromatographically resolved, deamidated species can be monitored. Harris, R.J., Kabakoff, B., Macchi, F.D., Shen, F.J. Kwong, M., Andya, J.D., Shire, S.J., Bjork, N., Totpal, K., Chen, A.B., Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B 752 (2001) 233-245. T3-4*(iso) 573.1 m/z
  24. 24. ©2015 Waters Corporation 24  Detect and monitor peptides over a wide molecular weight range  Quantify peptide variants with enhanced specificity  Monitor components below optical detector sensitivity  Selectively detect and monitor coeluting components  Compatible with both TFA and Formic Acid based separations ACQUITY QDa – Fit for Purpose When incorporated into Empower-based chromatographic workflows, the ACQUITY QDa Detector provides enhanced specificity, selectivity, and quantification for greater productivity in biotherapeutic development, production and QC. www.waters.com/qdabiopharm
  25. 25. ©2015 Waters Corporation 25 Addendum: User Case Study  Customer X – a large biopharmaceutical developer and manufacturer – wanted to further explore the potential of the QDa to meet their analytical needs.  We were supplied with a range of samples unknown to us at the time and were requested to analyze these samples so they could compare to their own internal results.  The following slides show ACQUITY QDa data we generated from these samples and how the data compared to customer generated results shared with us afterwards.
  26. 26. ©2015 Waters Corporation 26 ACQUITY QDa Detector LysC digest of mAb* (120 min run) ACQUITY UPLC TUV DetectorMethod Summary**  ACQUITY UPLC H-Class with TUV and ACQUITY QDa detectors run with Empower 3, SR2  ACQUITY UPLC CSH C18 130Å 1.7-µm column, 2.1x100 mm  Acid Modifier: FA Notes: * Later revealed to be Humira® ** See speaker notes for gradient info and LC-MS settings Comments: Good agreement between UV trace and TIC trace
  27. 27. ©2015 Waters Corporation 27 Zoom-in comparison: 0-30 min LysC digest of mAb (0-30 minutes) TUV Mass
  28. 28. ©2015 Waters Corporation 28 Zoom-in comparison: 30-60 min LysC digest of mAb (30-60 minutes) TUV Mas s
  29. 29. ©2015 Waters Corporation 29 Zoom-in comparison: 60-90 min LysC digest of mAb (60-90 minutes) TUV Mass
  30. 30. ©2015 Waters Corporation 30 Zoom-in comparison: 90-120 min LysC digest of mAb (90-120 minutes) TUV Mass
  31. 31. ©2015 Waters Corporation 31 LysC digest: SYNAPT vs. ACQUITY QDa (0-30 minutes) ACQUITY QDa SYNAPT (data offset by 2.4 min for comparison) SYNAPT vs. ACQUITY QDa Zoom-in comparison: 0-30 min
  32. 32. ©2015 Waters Corporation 32 LysC digest: SYNAPT vs. ACQUITY QDa (30-60 minutes) ACQUITY QDa SYNAPT SYNAPT vs. ACQUITY QDa Zoom-in comparison: 30-60 min
  33. 33. ©2015 Waters Corporation 33 LysC digest: SYNAPT vs. ACQUITY QDa (60-90 minutes) ACQUITY QDa SYNAPT SYNAPT vs. ACQUITY QDa Zoom-in comparison: 60-90 min
  34. 34. ©2015 Waters Corporation 34 LysC digest: SYNAPT vs. ACQUITY QDa (90-120 minutes) ACQUITY QDa SYNAPT K7 Peptide (6,714 kDa) +6 +7 +8 +9 +10 SYANPT vs. ACQUITY QDa Zoom in comparison: 90-120min Raw spectrum
  35. 35. ©2015 Waters Corporation 35 Consistent Repeatable Performance [M+6H]+6 [M+7H]+7 [M+8H]+8 [M+9H]+9 [M+10H]+10 [M+11H]+11 m/z IntensitySame peptide analyzed from two different samples on different days using different mobile phases… Same Result!  LysC Digest  HC Peptide: K7  FA modifier  Trypsin Digest  HC Peptide: T15*  TFA modifier
  36. 36. ©2015 Waters Corporation 36 QDa = 96.8% (X = 97.5%) QDa = 3.2% (X =2.5%) H21 H21-Ox SIR H21: M+2 [418.4] H21-Ox: M+2 [426.4] Relative Quantification Native vs. Oxidized Peptide Chromatographic Resolution m/z Resolution + + Scenario 1:  Large Δ in m/z  Single charge state  Well resolved Comments:  Ideal scenario  Integration on same data channel
  37. 37. ©2015 Waters Corporation 37 H6 Relative Quantification Deamidated vs. Non-Deamidated (1) H6D H6D SIR m/z M+3 [362.4] M+2 [543.1] Non-deamidated SIR m/z M+3 [362.7] M+2 [543.6] DeamidatedCombined: QDa = 43.2% (X = 43.6%) 31.9% QDa = 56.8% (X = 56.4%) 11.3% Chromatographic Resolution m/z Resolution + - Scenario 2:  Small Δ in m/z  Multiple charge states  Well resolved Comments:  Run w/two SIR channels  Record area from individual SIRs  Calculate % area  Cross channel processing enabled through Empower
  38. 38. ©2015 Waters Corporation 38 Relative Quantification Deamidated vs. Non-Deamidated (2) Chromatographic Resolution m/z Resolution + - Scenario 2(B):  Small Δ in m/z  Single charge state  Well resolved Comments:  Run single SIR channel, but with time dependant windows  Record area from individual SIRs  Calculate % area H37 0-65 min Non-deamidated SIR: [849.2] 65-140 min Deamidated SIR: [849.6] QDa = 89.1% (X = 89.2%) QDa = 10.9% (X = 10.8%)H37D
  39. 39. ©2015 Waters Corporation 39 IdeS Digest: Humira (adalimumab) Time Flow (mL/min) % A % B %C % D Initial 0.200 0 0 95 5 3.00 0.200 0 0 77 23 5.00 0.200 0 0 77 23 35.00 0.200 0 0 67 33 38.00 0.200 0 0 20 80 40.00 0.200 0 0 20 80 43.00 0.200 0 0 95 5 46.00 0.200 0 0 95 5 Mobile phase: A: H2O, 0.1 % TFA B: Acetonitrile, 0.1 % TFA C: H2O, 0.1 % FA D: Acetonitrile, 0.1 % FA TUV Mass FC Fab FC Fab Notes: See speaker notes for LC-MS settings
  40. 40. ©2015 Waters Corporation 40 IdeS Digest: Humira (2) Focused gradient on FC fragment 1 2 3 4 5 1 2 3 4 5
  41. 41. ©2015 Waters Corporation 41 Trypsin digest: Glycopeptides Peptide Average mass M+3 H25 1189.2 397.4 G0F 2633.7 878.9 G1F 2795.8 932.9 G2F 2957.8 986.9 G0 2487.7 830.2 G1 2649.7 884.2 Man5 2405.6 802.9 G2F +NeuAc 3248.9 1084.0 G0F 51.1% (X 50.2*) * Adjusted percent G1F 42.8% (X 43.7*) G2F 6.1% (X 6.2*) G0 G1 G1 Man5 Man5 H25 Zoom-in ACQUITY QDa: SIR full scan G2F +NeuAC
  42. 42. ©2015 Waters Corporation 42  Detect and monitor peptides over a wide molecular weight range  Quantify peptide variants with enhanced specificity Monitor components below optical detector sensitivity  Selectively detect and monitor coeluting components  Compatible with both TFA and Formic Acid based separations ACQUITY QDa – Fit for Purpose When incorporated into Empower-based chromatographic workflows, the ACQUITY QDa Detector provides enhanced specificity, selectivity, and quantification for greater productivity in biotherapeutic development, production and QC. www.waters.com/qdabiopharm
  43. 43. ©2015 Waters Corporation 43 More info on the ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm Details on the ACQUITY QDa Detector: www.waters.com/qda

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