©2004 Waters Corporation

Development and Evaluation of a LC/UV/MS Peptide Mapping System

Paul Rainville, Da Ren, Himanshu Gadgil, and Jeff Mazzeo Waters Corporation, 34 Maple Street, Milford, MA 01757

Introduction

Methods and Materials BioSuite™ Peptide Mapping System BioSuite™ Peptide Mapping MS System BioSuite™ Peptide Mapping MS/MS System MassPREP™ peptide standard Column: BioSuite™ PA-A C18 3µm 2.1 x 150 mm unless specified Column temperature: 40.0ºC, unless specified Flow rate: 0.2 mL/min Mobile phase: A: 0.1% formic acid unless specified B: 0.1%formic acid/MeCN unless specified Gradient elution see figure legends MS conditions: Mode: ESI + Capillary: 3300 V Cone: 30 V Desolvation gas flow: 500 L/hr Cone gas flow: 50 L/hr Source temperature: 150 ºC Desolvation temperature: 350 ºC MS/MS conditions: Mode: ESI + Capillary: 3300 V Cone: 35 V Desolvation gas flow: 500 L/hr Cone gas flow: 50 L/hr Source temperature: 150 ºC Desolvation temperature: 350 ºC Collision cell: 27eV Sample preparation: IgG1 was obtained from mouse acities and purified using a protein G column (Shodex). The purified IgG1 was concentrated by TCA precipitation and resus-pended in ammonium bicarbonate buffer, pH 7.8, and digested overnight with trypsin (Promega) at 37.0°C MassPREP peptide was dissolved in 150µL of Mobile phase A and mixed well

Characterization and quality control of biotherapeutics relies heavily on the use of peptide mapping. Peptide mapping is used to determine small chemical variations in proteins that can be the difference between active and inactive protein therapeutics. Historically, this technique has been based on UV detection and TFA mobile phase modifiers. Recently, peptide mapping has implemented mass spectrometry to complement traditional UV detection. In order to improve performance of mass spectrometry, it is highly desirable to replace TFA with formic acid as the mobile phase modifier. Waters has therefore developed coumns specific to improve resolution with formic acid modified mobile phases. In addition, we developed highly robust, reproducible peptide mapping systems that are designed both for UV and mass spectrometry detection. Data presented here will show the reproducibility of these peptide mapping systems as well as their capabilities in detecting modifications of IgG1 such as oxidation, deamidation and lysine variants.

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050m/z0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

70.06

530.77175.11112.08

98.06

157.11

115.09

522.27402.21

237.13217.12245.12 305.16 371.20321.65 404.21 521.78452.73

807.39642.32531.27

625.28614.32

555.30

710.37643.31

653.36 711.37748.34

904.43

858.44808.41

840.42859.43

905.44

906.491001.46

70.06

530.77175.12157.12112.09

98.06 140.09

522.27

402.21237.14217.14

245.13 305.15 321.67371.19

452.73 521.76

468.22

807.42642.33531.28

625.31614.31531.78

596.31

710.34643.36

702.38711.34 748.36

904.47

858.46808.44

809.37859.45

905.45

906.521001.58

70.06

530.77175.12112.08

98.07

157.11

115.08

522.27402.21

237.13217.13 245.14 371.20305.16 321.67 452.76 521.76

807.39642.32531.31

625.31614.34531.80

556.34

710.36643.33

702.37711.36748.34

904.48

858.44808.38887.43

905.44

906.471001.46

70.06

530.79175.12112.08

98.06

157.11

115.08

522.25402.21

237.13217.13245.14 305.17 371.21361.17

404.20 452.75 521.77

904.45807.40642.32531.28

625.31614.35531.78

556.28

710.36643.34

653.31 711.36748.39

858.46808.42

808.53 859.43

905.44

906.491001.49

70.06

112.09

98.06

530.77175.12157.11

140.08

522.27

402.20237.14217.13 245.13 305.16 371.22361.20

404.21 452.72 521.80

807.42642.33531.29

624.34614.34531.79

556.30

710.37643.33

653.39 711.34748.36

904.45858.46

858.40809.46

859.48904.53

906.451001.46

70.06

807.39530.79112.08

98.06

175.12157.11

140.08

522.27402.24

237.13217.14245.13 305.16 371.21361.19

404.19 521.77452.73

642.35531.27

614.34555.30

710.33643.33

653.31 711.36748.34

904.47858.44808.38

840.40859.42

905.44

906.471001.48

Figure 3: BioSuite peptide mapping MS system consisting of the 2796 Biosepara-tions module, MassLynx™ software, Waters 2487 dual wavelength detector, the Waters Micromass® ZQ mass spectrometer , BioSuite PA columns and MassPrep standards

Figure 6. BioSuite peptide mapping system consisting of the 2796 Bioseparations module, MassLynx™ software, Waters 2487 dual wavelength detector, BioSuite™ PA columns and MassPrep™ standards

Figure 6: Analysis and identification of spiked deamidation products in a peptide map of IgG1 on BioSuite Peptide Mapping MS System. Tg=0-40%B/180 min

20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00Time-37

100

%

Time (Minutes) 18010

DLDVKDLisoDVKDLNVK

20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00Time-37

100

%

Time (Minutes) 18010

DLDVKDLisoDVKDLNVK

SHSLSPGSHSLSPG

SHSLSPGKSHSLSPGK

Lys 128DaLys 128Da

Figure 5: Separation reproducibility of MAb tryptic digest on BioSuite Peptide Mapping MS Sytem. Tg= 0-40%B/180 min on BioSuite PA-B C18 3.5µm 2.1 x 250mm mm

Figure 7: Fragmentation reproducibility of Bradykinin (doubly charged parent ion 530.79) on BioSuite Peptide Mapping MS/MS System.

Figure 1: BioSuite peptide mapping system consisting of the 2796 Bioseparations module, MassLynx™ software, Waters 2487 dual wavelength detector, BioSuite PA columns and MassPrep standards 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00

Time0

100

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100

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0

100

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1: TOF MS ES+ 776985

22.74

17.5624.63

1: TOF MS ES+ 768

6.66e327.15

1: TOF MS ES+ TIC

5.20e436.35

27.1520.4017.56

10.918.310.06 2.64 13.29

23.60

28.70 35.42

33.6133.01

46.0636.77 43.7950.71 64.8760.43

[M+2H]

[M+2H]

Oxidized

Non-oxidized

Mass shift

Retention time shift

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00Time0

100

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5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00Time0

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1: TOF MS ES+ 776985

0

100

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1: TOF MS ES+ 776985

22.74

17.5624.63

1: TOF MS ES+ 768

6.66e3

22.74

17.5624.63

1: TOF MS ES+ 768

6.66e327.15

1: TOF MS ES+ TIC

5.20e4

27.15

1: TOF MS ES+ TIC

5.20e436.35

27.1520.4017.56

10.918.310.06 2.64 13.29

23.60

28.70 35.42

33.6133.01

46.0636.77 43.7950.71 64.8760.43

[M+2H]

[M+2H]

Oxidized

Non-oxidized

Mass shift

Retention time shift

670 672 674 676 678 680 682 684 686 688 690 692 694 696 698 700 702m/z0

100

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674.3

695.4

20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 200.00 220.00Time0

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167.32

76.595.45 76.52

61.82

56.2048.85

19.27 46.7736.36

62.56

160.32111.16106.41

82.29 89.99

155.15131.70

122.06 136.97

167.64

188.60

174.74

222.64

192.74

199.43218.45

T62T43

674.3695.4

675.3 696.3

670 672 674 676 678 680 682 684 686 688 690 692 694 696 698 700 702m/z0

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674.3

695.4

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167.32

76.595.45 76.52

61.82

56.2048.85

19.27 46.7736.36

62.56

160.32111.16106.41

82.29 89.99

155.15131.70

122.06 136.97

167.64

188.60

174.74

222.64

192.74

199.43218.45

T62T43

674.3695.4

675.3 696.3

Figure 4: Peptide map of human serum albumin demonstrating the BioSuite Peptide Mapping MS System's ability to resolve co-eluting peptide. Tg= 0-40%B/225 min

Figure 8: Identification of an early eluting peptide sequence corresponding to a lysine variant. Tg = 0-40%B/120 min

Figure 10: Identification and de novo sequencing of T19 and oxidized T19 of IgG1 demonstrating peak identification and modification location by MS/MS sequencing.

Figure 9: Separation and identification of oxidized T19 of IgG1

Conclusions •BioSuite™ Peptide mapping systems and BioSuite™ columns showed excellent reproducibility in separating peptides of a IgG1 tryptic digest. • Detection and identification of post translational modifications was accomplished by LC/MS. • Site specific modification of amino acids was determined by Biolynx™ software following fragmentation experiments.

Figure 2: Separation reproducibility of MassPREP peptide standard on BioSuite Peptide Mapping System. Mobile phase A: 0.02%TFA, mobile phase B 0.018%TFAin MeCN Tg= 0-50%B/30 min Water blank spiked with DLDVK

DLisoDVK DLNVK

DLDVK and DLisoDVK

DLNVK and DLisoDVK

5

20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00Time0

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123.86113.86

83.3466.5753.534.41 45.6233.4723.4998.09

145.34

134.89 160.19

123.77113.68

83.2366.5153.4633.424.00 23.4511.1045.57

97.98145.21

134.72 159.99

123.76113.68

83.1866.4853.454.39 45.5433.3523.4097.90

145.20

134.64 160.28

123.62113.50

83.1164.2953.4245.4833.314.00 23.3411.0397.81

145.08

134.57 159.80

123.58113.45

83.1066.3653.3645.4833.304.00 23.3311.1497.72

144.98

134.48 159.67

123.50113.34

83.0764.2153.3133.194.00 23.24 45.4297.65

144.86

134.36 159.61

Peak# 1 2 3 4 5 6 Avg 17.91 53.42 83.17 97.86 123.68 145.11

%RSD 0.42 0.13 0.11 0.15 0.10 0.11

1 2 3 4 6

2_peptideMmix_32901W01

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00Time0

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100

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100

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19.6618.7717.66

15.1510.17

21.67 24.6126.90

28.19

19.6318.7717.62

15.1110.14

21.60 24.6126.86

28.12

19.5918.7317.59

15.1110.14

21.56 24.5326.83

28.08

19.7018.8017.69

15.1910.17

21.63 24.6126.90

28.12

19.6618.7717.66

15.1910.17

21.60 24.6426.90

28.12

19.6618.7717.66

15.1910.17

21.63 24.6126.86 28.12

Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Avg 10.16 15.16 17.65 18.77 19.65 21.62 24.60 26.88 28.13

%RSD 0.15 0.26 0.20 0.12 0.19 0.17 0.15 0.11 0.13

1 2 3 4 5

6 7 8 9