Fast and Efficient Peptide Mappingof a Monoclonal Antibody (mAb):UHPLC Performance withSuperficially Porous Particles

Authors

James Martosella, Alex Zhu

Agilent Technologies, Inc.

Wilmington, DE 19808

Ning Tang

Agilent Technologies, Inc.

Santa Clara, CA 95052

Application Note

Biotherapeutics and Biosimilars

Introduction

Peptide mapping by reversed-phase (RP) chromatography is the mainstay techniquein biotherapeutic analysis, delivering comprehensive characterization ofbiopharmaceutical products. When interfaced with a mass spectrometer (MS), itcan deliver the identification of proteins and their variants, determinepost-translational modifications (PTMs) and locations, and confirm proteinsequences. However, peptide mapping represents a significant chromatographicchallenge due to the inherent complexity of protein digests. As a result, manyorganizations struggle with developing robust and reliable peptide maps. In general,peptide maps have suffered from low sensitivity, poor peak shapes, and very longseparation times to achieve the desired resolution.

More recently, Ultra High Performance Liquid Chromatography (UHPLC) has beenemployed to overcome these challenges, demonstrating superior resolution, highersensitivity, and much shorter analysis times compared to traditional HPLC. UHPLCtechnology can provide the basis for a more detailed characterization of proteinbiotherapeutics and takes advantage of faster flow rates, smaller particles, andshorter column lengths to achieve high separation performance during much fasterrun times. However, the pressure requirement becomes increasingly high for theseanalyses and prevents operation on traditional 400 and 600 bar HPLC instruments,thus severely limiting its broader use.

2

To address this limitation, Agilent has introduced a 2.7 µmAdvanceBio Peptide Mapping column to fill a critical gap inbiotherapeutics characterizations, for generating both rapidand highly efficient peptide maps at traditional LC-systempressures. Using superficially porous chromatographic media,AdvanceBio Peptide Mapping columns achieve substantialimprovements in peptide mapping during very fast run timesand low system pressures, while still maintaining high peak-performance efficiency. In this work, an AdvanceBio PeptideMapping column was used for the LC/MS peptide mappinganalysis of a monoclonal antibody (mAb) tryptic digest withreduced analysis times. Additionally, the AdvanceBio columnand methodology was compared to a sub-2 µm non-AgilentUHPLC peptide mapping column for performance comparisons.

Materials and Methods

Sample preparation

Fifty microliters mAb IgG1 (30 mg/mL) were first mixed with75 µL 100 mM ammonium bicarbonate (pH 8). Seventy-fivemicroliters of trifluoroethanol and 3 µL 200 mM dithiothreitol(DTT) were added to the protein sample and heated at 60 °Cfor 1 h to denature and reduce the protein. After the proteinhad cooled to room temperature, 12 µL 200 mMiodoacetamide (IAM) was added to the sample. The samplewas kept at room temperature in the dark for 1 h.Subsequently, 3 µL DTT was added in the sample to reactwith the excess IAM for 1 h. The sample was diluted with900 µL water and 300 µL 100 mM ammonium bicarbonate.Trypsin solution (75 µL) was added to the sample andincubated at 37 °C for 20 h. After overnight incubation, 3 µL ofneat formic acid was added to quench the digest.

ConditionsColumns: AdvanceBio Peptide Mapping 2.1 × 100 mm

(p/n 655750-902), AdvanceBio Peptide Mapping, 2.1 × 150 mm (p/n 653750-902), non-Agilent UHPLC column, 2.1 × 100 mm

Eluent: A: H2O + 0.1% FA (v/v)B: 90% ACN + 0.1% FA (v/v)

Injection volume: 15 µL

Flow rates: various

Temperature: 40 °C

Detection: UV, 215/220 nm

Instrumentation: Agilent 1290 Infinity LC System and an Agilent 6530Accurate-Mass Quadrupole Time-of-Flight (Q-TOF)

Q-TOF MS parametersIon mode: Positive

Source: Agilent Dual Jet Stream

Drying gas temperature: 250 °C

Drying gas flow: 10 L/min

Sheath gas temperature: 250 °C

Sheath gas flow: 12 L/min

Nebulizer: 35 psi

Capillary voltage: 3,500 V

Fragmentor: 200 V

Skimmer: 65 V

Oct 1 RF: 750 V

Nozzle: 0 V

MS range (m/z): 100 to 1600

MS/MS range (m/z): 100 to 1600

MS scan rate (spectra/second): 8

MS/MS scan rate (spectra/second): 3

LC/MS results were analyzed using Agilent MassHunterQualitative Analysis Software B.06 and Agilent MassHunterBioConfirm Software B.06.

3

Figure 1. AdvanceBio Peptide Mapping column optimization for achieveing a faster peptide mapping analysis. Gradient 10-40% B,DAD: 215 nm, 40 °C. Top panel, 75 minute separation on a 2.1 × 150 mm column generated 59 peptide peaks (flow rate 0.2 mL/min,211 bar). Bottom panel, optimized 14 minute separation on a 2.1 × 100 mm column generated 57 peptide peaks (flow rate 0.6 mL/min, 433 bar).

min0 10 20 30 40 50 60

mAU2.1 × 150 mm AdvanceBio Peptide Mapping column

2.1 × 100 mm AdvanceBio Peptide Mapping column

0

20

40

60

80

100

120

140

160%B-50%

%B-50%

min0 2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

140

160

Results and Discussion

Fast peptide mapping optimization Optimization of peptide maps to resolve and analyzepotentially hundreds of peaks is a significant task. Performingthese optimizations with high resolution during shortenedanalysis times can be even more challenging and timeconsuming. Employing long gradients of mAb tryptic peptidemaps to achieve the desired resolution can take 120 minutesor longer, especially for large proteins like mAbs. While fastermaps are highly desirable, resolution must not becompromised, while end test results need to provide the samelevel of information found during an extended run.

To demonstrate the AdvanceBio Peptide Mapping column’sflexibility for increasing analysis speed without sacrificingseparation performance, two UV separations are compared inFigure 1. In this comparison, a mAb tryptic peptide map wasfully optimized at 75 minutes on a 2.1 × 150 mm AdvanceBioPeptide Mapping column (top chromatogram) with an

efficient baseline-resolved peptide map across the entiregradient profile. The 75 minute separation is an excellentexample of the column’s ability to enable faster separationperformance for a 150 mm length column (traditional columnlength), compared to conventional peptide mapping times atthis dimension. However, it also provides a better example forcomparing the column’s flexibility for further increasinganalysis speed without sacrificing separation performancewhen moving to a shorter gradient.

In the bottom 14 minute chromatogram in Figure 1, columnflow rate was increased from 0.2 to 0.6 mL/min while columnlength was decreased from 150 to 100 mm. Volumetric flowduring this change was carefully maintained, keeping thegradient change per column volume equal to ensure similarselectivity. The 14 minute peptide map details excellentseparation performance, maintaining the baseline resolutionand sensitivity while keeping resolving power unchanged. The14 minute separation demonstrates UHPLC- type speed andperformance, yet keeps this rapid analysis attainable on600 bar HPLC instrumentation.

4

Figure 2. Comparison of Agilent AdvanceBio Peptide Mapping column total ion chromatograms (TICs), highlighting peptidemapping performance between 40-min and 14-min runs for an mAb tryptic digest. Left panel, TIC collected during 40-minanalysis (flow rate 0.2 mL/min, 140 bar). Right panel, TIC collected during 14-min analysis. (flow rate 0.6 mL/min, 433 bar).

0

0.6

1.6

2.6

3.6

4.6

1 2 3 4 5 6 7 8 9 10 11 12 13

×108×108

0

0.75

1.75

2.75

3.75

Acquisition time (min)

Counts

Acquisition time (min)

Counts

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

40-min run140 bar0.2 mL/min

14-min run433 bar0.6 mL/min

Gradient, 40 minute Gradient, 14 minute

Time (min) %B Time (min) %B0 3 0 335 35 10 3537 90 12 9040 90 14 90

LC/MS peptide mapping with an AdvanceBio Peptide

Mapping column: steep versus long gradient

In a typical biopharmaceutical peptide mapping workflow,reversed-phase LC is combined with mass spectrometrydetection. Compared with the more traditional approach of UVonly detection, RP LC/MS significantly enhances theinformation content available from the peptide-mappingexperiment. Measurement by RP LC/MS can differentiatecoeluting peptides, locate and identify peptide modifications,and determine the sequence coverage. High sequencecoverage is an indicator that optimal separation of the trypticdigest has been achieved.

During RP LC/MS, long gradients are typically used to enablehigher resolution and ensure modified peptides (referred to aspost translational modifications or PTMs) are detectable fromtheir unmodified (native) forms as well as other close eluting

peptides within the overall digest profile. To reduce run timessteeper gradients are employed, however, this is typically atthe cost of lost resolution and overall mapping quality. It is,therefore, important to not sacrifice separation quality orcompromise mass spectral information when run times arereduced.

Figure 2 compares peptide mapping total ion chromatograms(TIC) from an AdvancedBio Peptide Mapping column during40-min and 14-min runs. In this comparison, columndimensions remained constant, and the gradient slope wasadjusted to keep gradient change/column volume betweenthe columns equal, ensuring that the chromatographicselectivity remained similar. The TIC comparisons showedthat resolution, selectivity, and separation performance hadnot been compromised during the reduced run time of 14 min,while the increase in pressure at the higher flow rate did notrequire > 600 bar UHPLC instrumentation.

5

Additionally, sequence coverage between the 40-min and14-min run was also evaluated using the Molecular FeatureExtractor (MFE) in the MassHunter Qualitative AnalysisSoftware. MFE is an algorithm that finds and extracts a list ofcompounds from complex separation data, such as peptidemaps. The list of compounds is then matched back to themAb protein sequence and provides the sequence coverage.Figure 3 displays the extracted compound chromatograms(ECCs) for the matched peptides and the sequence coverage.

All matched peptides were acquired with at least one MS/MSspectrum for confirmation. The 40-min and the 14-min runsremain relatively unchanged in coverage. The 14-min LC/MSrun resulted in 99.63% sequence coverage of mAb while the40-min analysis resulted in 99.84%. This comparison providesfurther confidence that peptide characterization information isnot sacrificed when peptide mapping analyses times areshortened on the AdvanceBio Peptide Mapping column.

Figure 3. Agilent AdvanceBio Peptide Mapping column extracted compound chromatograms (ECC) to compare mAb tryptic digestresults. The top panel shows an ECC for peptides identified from the 40-min run, resulting in 99.84% sequence coverage of mAb.The bottom panel shows an ECC for peptides identified from the 14-min run, resulting in 99.63% sequence coverage of mAb.

00.40.81.21.6

22.42.83.23.6

44.44.8

Cpd

702

: B(2

98-3

13)

Cpd

384

: B(3

57-3

66)

Cpd

445

: B(2

52-2

70)

Cpd

345

: B(1

30-1

43)

Cpd

424

: B(1

18-1

29)

Cpd

302

: A(4

8-63

)

Cpd

524

: B(4

5-65

)

Cpd

731

: B(2

98-3

13)

Cpd

660

: B(2

19-2

44)

Cpd

775

: A(1

36-1

55)

Cpd

156

: A(1

96-2

10)

Cpd

624

: B(3

89-4

05)

Cpd

106

: A(1

63-1

72)

Cpd

205

: A(1

09-1

16)

Cpd

275

: B(4

36-4

42)

Cpd

240

: B(2

45-2

51)

Cpd

236

: B(3

23-3

30)

Cpd

395

: B(4

13-4

35)

Cpd

483

: A(1

17-1

35)

Cpd

499

: A(1

8-47

)

Cpd

133

: A(2

11-2

18)

Cpd

561

: A(1

78-1

92)

Cpd

120

: B(6

8-74

)

Cpd

291

: A(1

-17)

Cpd

60:

A(1

56-1

62)

Cpd

330

: A(4

8-63

)

Cpd

580

: B(3

67-3

88)

Cpd

454

: B(3

57-3

66)

Cpd

639

: B(2

98-3

18)

Cpd

31:

A(6

4-68

)

Cpd

16:

B(2

10-2

14)

Cpd

749

: A(6

9-10

8)

Cpd

549

: B(4

5-65

)

Cpd

132

: B(2

89-2

97)

Cpd

175

: A(1

96-2

10)

Cpd

221

: A(1

56-1

77)

Cpd

599

: B(2

52-2

88)

Cpd

686

: B(2

98-3

13)

Cpd

768

: B(4

13-4

35)

Cpd

741

: A(1

73-1

95)

Cpd

43:

B(3

37-3

40)

Cpd

780

: A(1

8-47

)

0.6 1 1.4 1.8 2.2 2.6 3 3.4 3.8 4.2 4.6 5 5.4 5.8 6.2 6.6 7 7.4 7.8 8.2 8.6 9 9.4 9.8 10.2 10.6 11

×107

40-min run

14-min run×107

00.40.81.21.6

22.42.83.23.6

44.4 C

pd 9

34: B

(252

-270

)

Cpd

147

9: B

(298

-313

)

Cpd

827

: B(3

57-3

66)

Cpd

626

: A(4

8-63

)

Cpd

741

: B(1

30-1

43)

Cpd

170

9: A

(136

-155

)

Cpd

110

6: B

(45-

65)

Cpd

153

6: B

(298

-313

)

Cpd

871

: B(1

18-1

29)

Cpd

135

0: B

(219

-244

)

Cpd

312

: A(1

96-2

10)

Cpd

128

3: B

(389

-405

)

Cpd

167

: A(1

63-1

72)

Cpd

105

6: A

(18-

47)

Cpd

428

: A(1

09-1

16)

Cpd

494

: B(2

45-2

51)

Cpd

118

3: A

(178

-192

)

Cpd

562

: B(4

36-4

42)

Cpd

475

: B(3

23-3

30)

Cpd

102

7: A

(117

-135

)

Cpd

704

: A(4

8-63

)

Cpd

121

5: B

(367

-388

)

Cpd

187

: B(6

8-74

)

Cpd

230

: A(2

11-2

18)

Cpd

94:

A(1

56-1

62)

Cpd

131

0: B

(298

-318

)

Cpd

675

: A(4

8-63

)

Cpd

44:

A(6

4-68

)

Cpd

157

3: A

(69-

108)

Cpd

963

: B(3

57-3

66)

Cpd

167

3: B

(144

-206

)

Cpd

124

0: B

(367

-388

)

Cpd

104

7: A

(48-

63)

Cpd

25:

B(2

10-2

14)

Cpd

608

: B(3

41-3

51)

Cpd

162

0: B

(1-4

4)

Cpd

137

8: B

(77-

98)

Cpd

100

5: B

(252

-270

)

Cpd

156

2: B

(289

-313

)

Cpd

112

8: A

(18-

47)

Cpd

127

7: B

(99-

117)

Cpd

120

4: B

(252

-288

)

Cpd

141

4: B

(99-

129)

Cpd

147

0: B

(289

-313

)

Cpd

260

: B(2

89-2

97)

Cpd

60:

B(3

37-3

40)

Cpd

365

: B(6

6-67

)

Cpd

792

: B(2

52-2

70)

Cpd

466

: A(1

09-1

16)

Cpd

349

: B(2

45-2

51)

Cpd

108

5: A

(163

-177

)

Cpd

816

: A(4

8-63

)

Acquisition time (min)

Acquisition time (min)

Cou

nts

Cou

nts

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

6

Rapid PTM profiling

Deamidation can cause structural and functional changes andis an important post translational modification to monitorduring mAb discovery, development, and manufacturing.Figure 4 displays an example of how peptide deamidation wasmonitored and conserved during a fast run time of14 minutes, in comparison to a longer run time of 40 minutes.Heavy chain peptide 357-366, which contains Asn 357, wasidentified in both peptide mapping separations. In the top andmiddle ECC figures, the native peptide peak was fullyseparated from its two deamidated forms, displaying as atotal of three separate peaks in the overlaid ECCs. Incomparison to the 40-min run (top), the native anddeamidated species in the 14-min run (bottom) were still wellresolved and readily identified by the QTOF analysis.

In the bottom figure, MS/MS of the native peptide (precursorat m/z = 581.32) and two deamidated forms (precursor atm/z = 581.81) are shown. In this spectra, the y and b-seriesfragments are the predominant ions, and all three peptidesshow the same y4-y8 ions, however the b2 and b3 ions(circled) are 0.98 Da higher for peak 2 and 3 thus validatingthe modified forms from the native species.

The automatic compound extraction (in MFE) also adds all theion intensities (isotopes, charge states, adducts, and so forth)belonging to the peptides. These are listed under the Volumetab in peptide Table 1. The comparative percentage ofmodified peptides can then be easily derived from the twodata sets in Table 1. In summary, the rapid 14 minute peptidemap generated from the AdvanceBio Peptide Mappingcolumn did not compromise PTM (deamidation) informationor the sequence coverage in comparison to the longer run.Using a steeper gradient at increased flow, the columnmaintained excellent separation performance and delivered ahigh degree of confidence in a complete peptide mappingcharacterization.

Figure 4. Overlaid extracted compound chromatograms (ECC)and MS/MS spectra (bottom) of native peptide and itsdeamidated forms. Upper panel displays results from the40-min analysis. Middle panel displays results from the 14-minanalysis. Bottom panel provides MS/MS spectra of the nativeand two deamidated forms shown above.

Acquisition time (min)

Cou

nts

×106

00.5

11.5

22.5

33.5

44.5

55.5

66.5

77.5 Cpd 154: B(357-366)

18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5

Native peptide

Deamidatedform 2

Deamidatedform 1

40 min ECC

00.05

0.10.150.2

0.250.3

0.350.4

0.450.5

0.550.6

0.650.7

0.750.8

0.850.9

0.951

Cpd 132: B(357-366)

Acquisition time (min)

Cou

nts

6.1 6.3 6.5 6.7 6.9 7.1 7.3 7.5 7.7 7.9 8.1 8.3 8.5

Native peptide

×107

Deamidatedform 2

Deamidatedform 1

14 min ECC

×103

×103

×103

012345

820.4576y7

919.5266y8

243.1073

132.0761 342.1757620.3438

y5 733.4290y6

519.2975y4429.2099

0

1

2

3 244.0939 820.4583y7 919.5293

y8133.0605733.4298

y6620.3407

y5343.1553

88.0400

0

1

2

3244.0906b2

b2

b2

820.4558y7 919.5275

y8133.0594 620.3403

y5 733.4199y6343.1612

b3

b3

b3

469.1724

Mass-to-charge (m/z)

Coun

tsCo

unts

Coun

ts

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 10001050

Native

Deamidated

Deamidated

7

Peptide mapping with steep gradients:

HPLC (AdvanceBio Peptide column) versus

UHPLC (non-Agilent UHPLC peptide column)

With the advancement of UHPLC peptide mapping, separationperformance has been significantly improved and analysistime shortened. With sub-2 µm particle technology, peptideresolving power can now be vastly improved during shortenedruntimes; a consequence of reduced diffusional distance insmall particles. This higher resolution and increasedsensitivity becomes particularly important in peptide mapping,especially for the detection of modified peptides. In directcomparison to sub-2 µm column performance, 2.7 µmsuperficially porous particles with a thin porous outer shelland solid inner core can offer exceptional resolving power,which also results in fewer backpressure constraints ascolumn flow rates are increased. This pressure and speedadvantage can obviate the requirement for > 600 bar UHPLCinstrumentation to perform these fast and highly efficientpeptide mapping analyses.

The UV chromatographic comparison for an IgG tryptic digestin Figure 5 highlights the AdvanceBio Peptide Mappingcolumn’s speed and resolving power in direct comparison to atop industry performing UHPLC peptide mapping column. Inthe top chromatogram, the AdvanceBio Peptide Mappingcolumn was optimized for a rapid and highly efficient analysisusing 2.1 × 100 mm dimensions. In this separation, thepeptide peaks were well resolved across the entire gradientprofile, delivering 56 peaks at a pressure of 433 bar. In thebottom chromatogram, a 2.1 × 100 mm UHPLCpeptide-mapping column was compared under the samechromatographic conditions and column dimensions.However, the critical aspect of the peptide-mapping results ishighlighted by the low pressure requirement from theAdvanceBio Peptide Mapping column to achieve the sameseparation results. The UHPLC peptide column resulted in700 bar backpressure and required the use of UHPLCinstrumentation, thus limiting its broader application.

Table 1. Extracted compound list showing the native peptide HC (357-366) and two deamidated forms with their sequence,modification, retention time, mass and volume for the 40-min analysis (top) compared to the 14-min analysis (bottom).

14-min run

40-min run

8

The ability to quickly, routinely and comprehensively runLC/MS analyses for mAb mapping helps to accelerate allfacets of the therapeutic development process. However,these separations are typically performed on high pressureLCs to take full advantage of maximum efficiency and timesavings. Superficially porous separations can offer theflexibility to operate on either UHPLC/MS or HPLC/MSinstrumentation coupled to MS, to deliver rapid and highefficiency separations. To contrast the LC/MS peptidemapping performance, and pressure requirements betweensub-2 µm UHPLC and superficially porous HPLC peptide-mapping columns, a 2.1 × 100 mm AdvanceBio PeptideMapping column was directly compared to a non-AgientUHPLC peptide mapping column of the same dimension.Figure 6 displays the RP LC/MS TIC and ECC performanceresults between these two columns. Again all the identified

peptides were acquired with at least one MS/MS spectrumas confirmation.

In the TIC and ECC comparisons, the AdvanceBio PeptideMapping LC/MS analysis delivered excellent separation andsequence coverage in comparison to the non-Agilent UHPLCpeptide-mapping column. Sequence coverage for theAdvanceBio peptide Mapping column was 99.63%, while theUHPLC coverage was comparable at 99.0%. Both columnsdelivered identity for 76 total tryptic peptides. However, theUHPLC analysis resulted in 700 bar backpressure, while theAdvanceBio separation operated 500 bar. The ability to obtainUHPLC like results on the AdvanceBio Peptide Mappingcolumn can make this column more attractive for obtainingrapid and highly efficient peptide maps with the flexibility ofusing traditional HPLC instrumentation.

min0 2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

140

160AdvanceBio Peptide Mapping column433 bar

non-Agilent UHPLC peptide column700 bar

min0 2 4 6 8 10 12

mAU

0

20

40

60

80

100

120

140

160

Figure 5. 2.1 × 100 mm Agilent and non-Agilent UHPLC peptide maps of an mAb tryptic digest. Gradient: 10-40% B at 0.6 mL/min,DAD: 215 nm, temperature: 40 °C, flow: 0.6 mL/min. Top, HPLC separation with an Agilent AdvanceBio Peptide Mapping columngenerated 56 peptide peaks at 433 bar. Bottom, UHPLC separation with a non-Agilent UHPLC peptide column generated 52 peptidefragment peaks at 700 bar.

9

0

0.6

1.6

2.6

3.6

4.6 non-Agilent UHPLC peptide column AdvanceBio Peptide Mapping column

×108 ×108

0.5

1.5

2.5

3.5

4.5

1 2 3 4 5 6 7 8 9 10 11 12 13

Acquisition time (min)

Cou

nts

Acquisition time (min)

Cou

nts

1 2 3 4 5 6 7 8 9 10 11 12 13

Cpd

702

: B(2

98-3

13)

Cpd

384

: B(3

57-3

66)

Cpd

445

: B(2

52-2

70)

Cpd

345

: B(1

30-1

43)

Cpd

424

: B(1

18-1

29)

Cpd

302

: A(4

8-63

)

Cpd

524

: B(4

5-65

)

Cpd

731

: B(2

98-3

13)

Cpd

660

: B(2

19-2

44)

Cpd

775

: A(1

36-1

55)

Cpd

156

: A(1

96-2

10)

Cpd

624

: B(3

89-4

05)

Cpd

106

: A(1

63-1

72)

Cpd

205

: A(1

09-1

16)

Cpd

275

: B(4

36-4

42)

Cpd

240

: B(2

45-2

51)

Cpd

236

: B(3

23-3

30)

Cpd

395

: B(4

13-4

35)

Cpd

483

: A(1

17-1

35)

Cpd

499

: A(1

8-47

)

Cpd

133

: A(2

11-2

18)

Cpd

561

: A(1

78-1

92)

Cpd

120

: B(6

8-74

)

Cpd

291

: A(1

-17)

Cpd

60:

A(1

56-1

62)

Cpd

330:

A(4

8-63

)

Cpd

580

: B(3

67-3

88)

Cpd

454

: B(3

57-3

66)

Cpd

639

: B(2

98-3

18)

Cpd

31:

A(6

4-68

)

Cpd

16:

B(2

10-2

14)

Cpd

749

: A(6

9-10

8)

Cpd

549

: B(4

5-65

)

Cpd

132

: B(2

89-2

97)

Cpd

175

: A(1

96-2

10)

Cpd

221

: A(1

56-1

77)

Cpd

599

: B(2

52-2

88)

Cpd

686

: B(2

98-3

13)

Cpd

768

: B(4

13-4

35)

Cpd

741

: A(1

73-1

95)

Cpd

43:

B(3

37-3

40)

Cpd

780

: A(1

8-47

)

Cpd

269

2: B

(298

-313

)

Cpd

156

5: B

(252

-270

)

Cpd

114

9: B

(357

-366

)

Cpd

706

: A(4

8-63

)

Cpd

913

: B(1

30-1

43)

Cpd

279

5: B

(298

-313

)

Cpd

128

7: B

(118

-129

)

Cpd

141

6: B

(271

-284

)

Cpd

232

5: B

(219

-244

)

Cpd

374

6: A

(136

-155

)

Cpd

254

2: B

(389

-405

)

Cpd

190

1: B

(45-

65)

Cpd

551

: B(2

45-2

51)

Cpd

797

: B(4

36-4

42)

Cpd

212

7: A

(178

-192

)

Cpd

455

: B(3

23-3

30)

Cpd

324

: A(1

09-1

16)

Cpd

179

0: A

(117

-135

)

Cpd

994

: A(4

8-63

)

Cpd

220

0: B

(99-

117)

Cpd

293

2: B

(298

-313

)

Cpd

94:

A(1

56-1

62)

Cpd

338

7: B

(413

-435

)

Cpd

224

: A(1

96-2

10)

Cpd

164

: A(1

63-1

72)

Cpd

358

2: B

(144

-206

)

Cpd

26:

A(6

4-68

)

Cpd

133

: B(4

0-44

)

Cpd

239

9: B

(75-

98)

×107

00.40.81.21.6

22.42.83.23.6

44.44.8

AdvanceBio Peptide Mapping column

non-Agilent UHPLC peptide column

Acquisition time (min)

Acquisition time (min)

Cou

nts

Cou

nts

1 2 3 4 5 6 7 8 9 10 11

×107

00.5

11.5

22.5

33.5

44.5

5

0.5 1 1.50 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11

Figure 6A. Total ion chromatograms (TIC) for a mAb tryptic digest performed on a 2.1 × 100 mm Agilent AdvanceBio PeptideMapping column, 433 bar (left panel) and 2.1 × 100 mm non-Agilent UHPLC peptide-mapping column, 700 bar (right panel).Chromatographic conditions same as Figure 5 conditions.

Figure 6B. Extracted compound chromatograms (ECC) for a mAb tryptic digest. Top panel, 2.1 × 100 mm Agilent AdvanceBioPeptide Mapping column, 433 bar, yielded 99.63% sequence coverage and 76 tryptic peptides identified. Bottom panel, non-Agilent UHPLC peptide-mapping column, 700 bar, yielded 99.0% sequence coverage and 76 tryptic peptides identified.

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Agilent shall not be liable for errors contained herein or for incidental or consequentialdamages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to changewithout notice.

© Agilent Technologies, Inc., 2013Printed in the USADecember 6, 20135991-3585EN

Conclusions

The AdvanceBio Peptide Mapping column greatly reducedthe analysis time for RP LC/MS peptide mapping, deliveringhigh resolution separations during fast runtimes. Incombination with optimized gradient conditions, thisapplication provides examples of highly efficient peptidemapping analysis for a monoclonal antibody tryptic digest in14 minutes with low LC operating pressures (< 450 bar). Incontrast to a non-Agilent UHPLC peptide-mapping columnand fast analysis conditions, the AdvanceBio PeptideMapping column generated rapid and well resolved peptidepeaks across the entire gradient profile and resulted in highsequence coverage of mAb. Most importantly, theAdvanceBio Peptide Mapping separations were generated atlow system pressures, highlighting the flexibility to run theseseparations on HPLC or UHPLC instrumentation.

For More Information

These data represent typical results. For more information onour products and services, visit our Web site atwww.agilent.com/chem.