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 [email protected]
Asia [email protected]
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.