Agilent AdvanceBio Peptide Plus BioLC Columns

ADVANCE YOUR CONFIDENCEFOR PROTEIN/PEPTIDE ANALYSIS

Sidebar treatment

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LC/MS peptide mapping can simultaneously assess multiple critical attributes with high sensitivity and reproducibility. However, this technique requires a reversed-phase column that delivers robust, reproducible results with MS-friendly additives, such as formic acid.

Confidently identify multiple attributes by LC/MS with NEW AdvanceBio Peptide Plus columns—part of the InfinityLab family

Based on Agilent superficially porous Poroshell technology, AdvanceBio Peptide Plus columns feature a hybrid endcapped C-18 stationary phase on a 120 Å pore size, 2.7 μm particle modified to have a charged surface.

These innovative columns let you quickly resolve and identify amino acid modifications in primary structure. What’s more, they can be used for both early discovery and transfer to QA/QC—eliminating the need for costly, time-consuming method redevelopment. Other advantages include:• Quality assurance: Every column is batch tested with the Agilent peptide

standard mix • High sensitivity—even as the mass load increases—enabling you to identify

critical modifications like oxidation and deamidation • Formic acid compatibility and multiple geometries that let you choose a

single column for early development through QA/QC, and across multiple system platforms

• A charge surface that provides sharp, symmetrical peaks using only formic acid as the mobile phase additive

RELIABLY CHARACTERIZE PRIMARY SEQUENCE AND DETECT POST-TRANSLATIONAL MODIFICATIONS (PTMS)

AGILENT ADVANCEBIO PEPTIDE PLUS BIOLC COLUMNS

One column for discovery and QC AdvanceBio Peptide Plus delivers the speed, sensitivity, and efficiency you need to quickly confirm and identify target proteins and peptides.

AdvanceBio Peptide Plus columns are the latest addition to the Agilent AdvanceBio column portfolio, and are backed by our 60-day warranty. All Agilent BioLC columns are designed for optimum performance with InfinityLab bio-inert LC instruments and supplies.

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Quality assurance: Ensure confidence in your resultsEvery batch of AdvanceBio Peptide Plus media is tested using the Agilent peptide standard. The mixture contains eight hydrophilic, hydrophobic, and basic peptides, ranging in molecular weight from 757 to 2845 Da. Each column is also tested with a small-molecule probe to ensure efficiency.

AdvanceBio Peptide Plus: An integral part of your characterization workflow

SAMPLE PREP SEPARATION IDENTIFICATION DATA ANALYSIS

00

1

1

2

3

1 2 3 4 5 6 7 8 9 100 111 122 13 144 15 16 17 18 19

1

2

3

7

65

4x108

Acquistion Time (min)

Coun

ts

TIC

1. AAFTE[CAM][CAM]QAADK 2. YLYEIAR 3. LVNEVTEFAK 4. KVPQVSTPTLVEVSR 5. RP[CAM]FSALEVDETYVPK 6. AVMDDFAAFVEK 7. HPYFYAPELLFFAK

With the highly efficient Agilent peptide mapping workflow, you can get more done, faster, without sacrificing precision.

The secret? Easy-to-implement automation that takes the tedium (and human error) out of preparing samples… software that’s easy to master… instruments that produce precise, repeatable results… and reversed-phase columns that deliver lot-to-lot reproducibility.

• Sample Prep: The Agilent AssayMAP Bravo protein and peptide sample prep platform combines the benefits of automation with an optimized workflow interface to enable superior efficiency and reproducibility.

• Separation: Enhance the accuracy and speed of your biomolecule characterization with Agilent AdvanceBio Peptide Plus columns.

• Identification: The Agilent 6545XT AdvanceBio LC/Q-TOF system is designed to handle multiple workflows in biomolecule characterization. Agilent MassHunter software efficiently extracts all available information from your samples.

• Data Analysis: Make data review more objective and less complex with Agilent OpenLAB Chromatography Data System and MatchCompare software.

To learn more go to: www.agilent.com/chem/advancebio

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Combined with Agilent analytical LC/MS instruments, AdvanceBio Peptide Plus columns provide the speed, sensitivity, and efficiency you need to confirm and identify target proteins and peptides.

THE SOLUTION FOR BIOMOLECULE CHARACTERIZATION

ADVANCED PEPTIDE PLUS BIOLC COLUMNS

LC/MS conditions for peptide identificationParameter Agilent 1290 Infinity II LC

Column AdvanceBio Peptide Plus 120 Å 2.1 x 150 mm, 2.7 µm

Mobile phase A: 0.1% FA B: 0.1% FA in ACN

Gradient Time (min) %B

0 3 1 3 31 40 33 95 34 95 34.1 3

Post time 10 minInjection volume 1 µLSample thermostat 5 ºCPost time 5 minColumn temperature 55 ºCFlow rate 0.5 mL/min

Parameter Agilent 6545XT AdvanceBio LC/Q-TOF

Ion mode Positive ion mode, dual AJS ESI (profile)Drying gas temperature 325 ºCDrying gas flow 13 L/minSheath gas temperature 275 ºCSheath gas flow 12 L/minNebulizer 35 psiCapillary voltage 4000 VFragmentor voltage 175 VSkimmer voltage 65 VOct RF Vpp 750 VAcquisition parameters Data were acquired at 2 GHz Extended Dynamic Range

MS mass range 100-1700 m/z MS/MS mass range 50-1700 MS scan rate (spectra/second): 8 MS/MS scan rate (spectra/second): 3

Ramped collision energy Charge state Slope Offset 2 3.1 1 3 and >3 3.6 -4.8

Data analysis BioConfirm software B.08

0

1

2

3

4

5x108

Coun

ts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 155 16 17 18 19

20 21 22 23 24 25 Acquistion Time (min)

12

3

8

7

6

54

9

%RSDPeak no. RT FWHM Area Height

1 0.27 1.41 0.40 1.922 0.09 0.43 0.67 0.863 0.06 0.73 0.59 1.104 0.07 0.90 3.58 0.695 0.04 0.48 0.36 0.736 0.05 0.82 0.67 0.417 0.05 0.50 0.94 0.348 0.02 0.70 0.89 1.279 0.03 0.40 1.18 0.39

Five replicate injections of a tryptic mAb digest using an Agilent AdvanceBio Peptide Plus column showing precision of peptide mapping critical attributes. Arrows depict peaks selected for %RSD calculations.

Achieve accurate method reproducibility with LC/MS peptide mappingPrecision of peptide mapping critical attributes

5

Cpd

45: B

(60-

65)

Cpd

81: A

(146

-149

)

Cpd

106:

B(29

7-30

5)Cp

d 87

: A(1

9-24

)

Cpd

123:

B(29

7-30

5)

Cpd

191:

B(66

-76)

Cpd

247:

B(44

4-45

1)

Cpd

277:

A(18

9-20

7)Cp

d 29

8: B(

253-

259) Cp

d 37

1: B(

68-7

6)Cp

d 45

2: B(

331-

338)

Cpd

588:

B(39

-50)

Cpd

259:

B(51

-59)

Cpd

675:

B(25

3-25

9)Cp

d 72

9: A(

25-4

5)Cp

d 79

1: A(

191-

207)

Cpd

874:

B(20

-30)

Cpd

972:

B(44

-50)

Cpd

1068

: A(2

5-42

)Cp

d 12

13: B

(137

-150

)

Cpd

1347

: B(7

7-87

)

Cpd

1425

: B(3

65-3

74)

Cpd

1486

: B(4

21-4

43)

Cpd

1573

: B(1

25-1

36)

Cpd

1696

: B(2

79-2

92)

Cpd

1834

: A(1

-18)

Cpd

1938

: 6(3

65-3

74)

Cpd

2053

: B(2

60-2

78)

Cpd

2217

: B(1

-19)

Cpd

2372

: A(1

70-1

83)

Cpd

2445

: A(1

50-1

83)

Cpd

2549

: A(1

46-1

83)

Cpd

2617

: A(1

08-1

26)

Cpd

2679

: B(2

23-2

52)

Cpd

2848

: B(2

27-2

52)

Cpd

2988

: B(3

06-3

24)

Cpd

3104

: B(3

06-3

24)

Cpd

3158

: B(3

97-4

13)

Cpd

3238

: B(3

06-3

21)

Cpd

3334

: A(1

09-1

26)

Cpd

3429

: B(3

06-3

21)

Cpd

3518

: B(3

06-3

21)

Cpd

3648

: A(4

6-61

)

Cpd

3881

: B(3

06-3

21)

Cpd

3902

: A(6

7-10

7)Cp

d 39

45: A

(67-

103)

Cpd

4026

: A(6

7-10

3)Cp

d 41

52: B

(99-

124)

Cpd

4231

: B(3

75-4

13)

Cpd

4291

: B(3

75-4

13)

Cpd

4353

: B(1

51-2

17)

Cpd

4454

: B(1

51-2

17)

Cpd

4577

: B(9

9-13

6)Cp

d 47

25: B

(151

-213

)Cp

d 48

79: B

(151

-213

)Cp

d 49

40: A

(46-

61)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22Counts vs. Aquisition Time (min)

1.71.61.51.41.31.21.1

10.90.80.70.60.50.40.30.20.1

0

x107

Detect critical PTMs that occur during manufacturing, storage, or handlingInnovator/biosimilar comparison

Innovator/biosimilar comparison Mirror image of LC/MS peptide maps showing the innovator (blue trace) and biosimilar (red trace). The highlighted region (3.73 minutes) displays the difference of SLSLSPGK peptide (enriched in biosimilar), which can be attributed to additional charge (basic) variants.

7x10

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

HC 2

97-3

05 HC 4

44-3

05

LC 1

49-1

68HC

253

-259

LC 1

90-2

06HC

339

-342

HC 6

8-74

HC 1

38-1

51

HC 1

26-1

37 HC 2

79-2

92HC

24-

38HC

279

-305

HC 1

-19

LC 1

69-1

82

HC 2

27-2

52

HC 3

06-3

21HC

44-

63HC

218

-222

HC 9

9-12

5

LC 1

45-1

82HC

152

-214

HC 1

38-2

17

HC 6

6-98

Biosimilar

Innovator

Acquisition Time (min)

Coun

ts

Oxidized peptide

Unmodified peptide x106

0

1

2

3

4

5

6

7

8

3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 Acquisition Time (min)

Coun

ts

DLTMISR

NTAY

LQM

NSLR

1

5.9%

NTAY

LQM

NSLR

5

.5%

NTAY

LQM

NSLR

0.23%

NTAY

LQM

NSLR

0

.97%

NTAY

LQM

NSLR

2.5%

NTAY

LQM

NSLR

0.24%

NTAY

LQM

NSLR

0

.21%

NTAY

LQM

NSLR

Deamidated peptides Nondeamidated peptide6 x10

0

1

2

3

4

Acquisition Time (min) 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2

Coun

ts

NTAYLQMNSLR

Oxidation Deamidation

Separation of nonoxidized and oxidized DLTMISR peptide using an AdvanceBio Peptide Plus column. Relative % oxidation = 1.6 %.

Overlaid extracted compounds for the matched peptides. (Some identified peptides are not labeled)

This overlaid extracted compound chromatogram shows the separation of nondeamidated and deamidated peptide using AdvanceBio Peptide Plus columns. (All modified sites are marked in red.) Total relative % deamidation = 25.52 %.

Successfully separate and identify a wide range of peptidesAdvanceBio peptide identification

AdvanceBio Peptide Plus columns are part of the innovative family of AdvanceBio columns, which deliver consistent performance for protein and mAb characterization.

Agilent AdvanceBio columns are designed to advance accuracy and speed for your characterization, SEC aggregation, IEX charge variants, intact mass, primary structure, and impurities profiles. They are suitable for techniques such as reversed-phase, cleaved glycans by hydrophilic interaction, and mAb titer determination by affinity chromatography.

ACCURATE, EFFICIENT BIOPHARMACEUTICAL ANALYSIS

AGILENT ADVANCEBIO COLUMNS

Agilent BioLC columns

Protein identification and impurity

profiling

Charged variant analysis

Aggregation analysis Glycan analysis Amino acid

Titer determination

and purificationOligonucleotides Desalting

Cartridges

Reversed-Phase

Ion Exchange

Size Exclusion HILIC Reversed-

Phase Affinity Reversed-Phase

Reversed-Phase

PLRP-S Bio MAb AdvanceBio SEC AdvanceBio Glycan Mapping

AdvanceBio Amino Acid Analysis

Bio-Monolith Protein A

AdvanceBio Oligonucleotides

AdvanceBio Desalting-RP

AdvanceBio RP-mAb

Bio IEX (SAX, SCX, WAX, WCX) Bio SEC-3 ZORBAX RRHD

300-HILICBio-Monolith

Protein G

AdvanceBio Peptide Mapping PL-SAX Bio SEC-5

AdvanceBio Peptide Plus PL-SCX ProSEC 300S

ZORBAX RRHD 300Å 1.8 µm

Bio-Monolith (QA, DEAE, SO3)

Poroshell 300

ZORBAX 300SB

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Agilent 6545XT AdvanceBio LC/Q-TOF A new standard for sensitivity and productivityWhen you need detailed information about intact proteins, protein sequences at the peptide level, or post-translational modifications, the 6545XT AdvanceBio LC/Q-TOF delivers results you can trust.

• Characterize intact protein variations with MassHunter BioConfirm software• Adapt your instrument to your needs with SWARM autotune • Be sure of your results with mass accuracies within 10 ppm for glycosylated intact proteins• Dig deeper into your digested samples with powerful iterative MS/MS• Maximize uptime with a system tested to perform through thousands of protein injections

PAIR WITH ADVANCEBIO COLUMNS FOR EFFECTIVE PROTEIN/PEPTIDE ANALYSIS

AGILENT INSTRUMENTS AND SUPPLIES

Agilent 1260 Infinity II Bio-inert LC Protect your biomolecules with a biologically inert flowpath The Agilent InfinityLab Bio-inert LC Solution takes you to the next level of confidence in bio-analysis.

The corrosion resistant titanium solvent delivery system and metal-free sample flow path ensure full integrity of biomolecules in all your applications. Plus, the latest technology gives you the capability to increase your efficiency—every day, in every way.

Agilent supplies Meet your everyday challenges and get tasks done right the first time

InfinityLab Quick Connect fittingsThese re-useable fittings are the only true “finger-tight” fittings stable to 1200 bar. There are no tools needed… just use your fingers to close the lever for a perfect connection every time.

Agilent AdvanceBio columns are part of the Agilent InfinityLab family and are designed to work in perfect harmony with the InfinityLab Bio-inert LC Solution and InfinityLab bio-inert supplies. Together this InfinityLab solution gives you unmatched bioseparation power and the highest efficiency in your biomolecule analysis.

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Accelerate productivity and improve outcomes Avoid the time and costs of developing new methods from scratch, restoring methods after a repair, and transitioning methods to new instruments. Agilent CrossLab application support can help you confidently deploy the latest productivity and usability enhancements and ensure the best outcomes—from sample preparation to final report.

www.agilent.com/crosslab

Learn morewww.agilent.com/chem/advancebio

Find local Agilent customer centerwww.agilent.com/chem/contactus

USA and Canada1-800-227-9770 agilent_inquiries@agilent.com

Europeinfo_agilent@agilent.com

Asia Pacificinquiry_lsca@agilent.com

For Research Use Only. Not for use in diagnostic procedures. This information is subject to change without notice.

© Agilent Technologies, Inc., 2018Printed in the USA, February 5, 2018 5991-7873EN

Ordering informationDescription Part NumberAdvanceBio Peptide Plus, 4.6 x 150 mm, 2.7 µm 671950-902AdvanceBio Peptide Plus, 3.0 x 150 mm, 2.7 µm 671950-302AdvanceBio Peptide Plus, 2.1 x 250 mm, 2.7 µm 677950-902AdvanceBio Peptide Plus, 2.1 x 150 mm, 2.7 µm 675950-902AdvanceBio Peptide Plus, 2.1 x 50 mm, 2.7 µm 673750-902AdvanceBio Peptide Plus, 4.6 mm guard (3/pk) 820750-932AdvanceBio Peptide Plus, 3.0 mm guard (3/pk) 823750-947AdvanceBio Peptide Plus, 2.1 mm guard (3/pk) 821725-946HSA Peptide Standard Mix G2455-85001

All AdvanceBio columns are rigorously tested to ensure reproducibility, and are backed by our 60-day warranty.