Conquering t he ChromatographiC Challenges of pept ide map ping

Peptide mapping is an important technique for the comprehensive characterization of

biopharmaceutical products. In a peptide map, each peptide fragment must be resolved

into a single peak – representing a significant

chromatographic challenge due to the

inherent complexity of protein digests.

In addition to the large number of peptide

fragments that are generated from the

enzymatic digestion of a protein, the number

of alternative peptide structures (post-

translational modifications, oxidations, etc.)

can be equally daunting. Achieving adequate

resolution in peptide maps is a tedious

process. Good methods often have long run

times and may not be as rugged as desired.

Now you can easily obtain the resolution

your lab needs. The Waters® UPLC® Peptide

Analysis Solution, an Assured Performance

Solution (APS) enabled by UltraPerformance

LC® technology, seamlessly combines

analytical instrumentation with chemistry.

This optimized and comprehensive system

solution delivers the clear information you

need – so you will know as much as possible

about your proteins.

uplC pept ide analYsis solut ion

uplC peptide analYsis solution:

n aCquitY uplC® system

n peptide separation technology

chemistries, 130Å or 300Å

n flexible detection capabilities,

with tunable uV, photodiode

array, or mass spectrometry

n robust software options for

processing and managing data

Time50.00 52.00 54.00 56.00 58.00 60.00 62.00 64.00 66.00 68.00 70.00 72.00 74.00 76.00 78.00

AU

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

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1.0e-1

50.00 52.00 54.00 56.00 58.00 60.00 62.00 64.00 66.00 68.00 70.00 72.00 74.00 76.00 78.00

AU

4.0e-2

6.0e-2

8.0e-2

1.0e-1

1.2e-1

HPLC

UPLC

improved resolving power in peptide maps

More small peaks are resolved when the tryptic digest of Alcohol Dehydrogenase is separated with UPLC.

ex panding t he boundaries – resolut ion, rep roduCibil it Y, and sensit iV it Y

The UPLC Peptide Analysis Solution brings a new level of resolution and sensitivity to peptide analysis

that allows for more complete characterization of proteins.

significantly higher resolution

UPLC delivers remarkable improvements in peptide mapping compared against HPLC. Exceptional

resolution is possible because separations obtained through the use of UPLC’s small particles result

in dramatically narrower peaks. Just as significant, the surface chemistry of our Peptide Separation

Technology packing material provides exceptional peak shape for a wide range of peptide structures.

This remarkable column performance can only be achieved with the optimized instrumentation of the

ACQUITY UPLC® System, which features reduced system volumes, minimal detector band-broadening

and accelerated data acquisition rates required to preserve high-efficiency separations – while

maximizing sensitivity.

intelligently designed for optimum performance

Waters scientists considered every aspect of peptide analysis – from chemistries, instrumentation,

software and methodology to usability, documentation, application support, and remote services –

to create this complete application solution.

The UPLC Peptide Analysis Solution, based on our award-winning ACQUITY UPLC System separation

technology and new Peptide Separation Technology Columns, provides the ultimate in high-resolution

chromatographic performance. And the system offers flexible detection options – such as Tunable UV

(TUV), Photodiode Array (PDA), and Mass Spectrometry (MS) – that are all optimized for UPLC.

assured performanCe solutions

Waters assured performance solutions

(aps) are a family of systems that

bring together industry-leading

technologies – instrumentation,

chemistries, software, qualification

tools, and services – that are

specifically optimized for exceptional,

application-focused performance.

designed by scientists for scientists,

these solutions remove the guesswork

in choosing the best solution for your

application’s needs.

Whether you work in pharmaceuticals,

proteomics, chemical analysis, or

beyond, with Waters aps, you can

be assured that whenever a new

application challenge arises, Waters

will have the technology and expertise

to help you conquer it.

Time10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00

AU

5.0e-2

1.0e-1

1.5e-1

2.0e-1

22.39

15.54

9.7518.00

34.1729.65

28.60

23.86

24.7127.94

32.84

*

*

Time28.30 28.40 28.50 28.60 28.70 28.80 28.90 29.00

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0.0

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2.0e-2

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1.0e-1

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1.4e-1

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1.8e-1

1.9e-1

robust quantitation of trace peptides

The protein digest, spiked with a trace peptide, was separated with a Peptide Separation Technology Column. A small trace peptide can be identified and quantitated as shown here down to a 0.2% level. To achieve this quantitation, sensitivity, and resolution, peak shape must be maintained over a wide mass load.

reproducibility

Advanced fluidic control of flow rate and gradient formulation combines

with stable column chemistry to provide reproducible maps. Long-term

assay stability is ensured with columns that are quality-control tested

using a peptide map.

qualitative and quantitative analysis of variant peptides

All elements of the UPLC Peptide Analysis Solution contribute to the

successful qualitative and quantitative analysis of trace peptides. With

this combination, variant proteins can be detected and measured with the

greatest of confidence.

n UPLC-based fluidic performance ensures retention time reproducibility.

n High-resolution UPLC separations of both large and minor peaks

enable accurate integration.

n Peptide Separation Technology particles deliver chromatographic

resolution and undistorted peak shape at extreme molar ratios.

n The system’s advanced optical and mass detection technologies have

a wide dynamic range ensuring that peptides, at these extreme molar

ratios, are detected even at the lowest limits of quantitation.

empower™ software provides control of

the aCquitY uplC system, including data

acquisition, processing, and reporting, all in the

market-leading chromatography data software

(Cds) platform. empower software is scalable

from a single workstation to an enterprise-wide

Cds solution, and offers advanced features for

method development, reporting, and custom

calculations, as well as enterprise options

for data management and automated method

validation management.

masslynx™ software, for advanced mass

spectrometry applications, enables researchers

to acquire, analyze, manage, and distribute

information. masslynx software offers flexible

control over complex instrument configurations,

and application managers that are tailored for

specific ms analyses.

Whether your peptide analysis configuration

requires Cds software for lC-based analysis,

or is ms-based and can take advantage of

application managers such as biopharmalynx™

for customized uplC/ms biopharmaceutical data

processing, you will be better able to manage

that information with nugenesis® sdms. sdms

is a scientific information management platform

that automatically captures and stores data.

sdms makes information easier to access,

facilitates regulatory compliance, and aids in

the administration of intellectual property.

sdms converts scientific results into accessible

and valuable corporate knowledge.

do more With Your results

Waters software enables you to capitalize on your UPLC results. The Waters Laboratory Informatics Suite includes proven information

management solutions that integrate all your lab resources in a consistent, scalable, and seamless application environment.

Time5.00 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 75.00 80.00

AU

1.0e-2

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

8.0e-2

9.0e-2

UPLC

Time10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00

AU

1.0e-2

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4.0e-2

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UPLC

ap plYing uplC to Your pept ide maps

The UPLC Peptide Analysis Solution can be used the way you want to work.

flexibility in improving peptide mapping

Peptide mapping is an essential technique for characterizing proteins. While most labs want to improve their assays, each has different criteria for what

improved peptide mapping means. Whether you require more resolution, faster throughput, or need to focus on a particular aspect of a protein, the UPLC

Peptide Analysis Solution can be used, without modification, to improve the peptide maps in the manner in which you choose.

Conventional peptide map

Separation of a trypic digest of Phosphoylase b by HPLC.

more resolution – in same time Separation of a trypic digest of Phosphoylase b by UPLC with same gradient and same analysis time – useful when more resolution is required – without an increase in analysis time.

similar resolution – in less time

Separation of a trypic digest of Phosphoylase b by UPLC with a shorter column and scaled gradient provides the ability to reduce run times while maintaining resolution.

Time10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00

AU

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2

3.0e-2

3.5e-2

4.0e-2

4.5e-2

5.0e-2

5.5e-2

6.0e-2

HPLC

enhanced ms detection

UPLC is an ideal inlet to MS. Separation based on

small particles yields MS spectra that are more

easily interpreted because the components are

better resolved. Small particles result in small

peak volumes which provide an increase in signal-

to-noise. It is easier, therefore, to detect and

identify trace components of your peptide digest.

focused assay Modified peptides need to be resolved from native peptides for quantitiation to be acheived. A focused assay utilizing UPLC allows the run times to be reduced without any loss in resolution or quantitative information of the modified peptides.

Focused assay at 12.5 to 16% B in 7.67 min, 50 mm, at 34 °C, total run time 14.33 min.

m/z400 600 800 1000 1200 1400

%

0

100669.63

573.88

1004.45

759.77

m/z400 600 800 1000 1200 1400

%

0

100

832.40

573.88

m/z400 600 800 1000 1200 1400

%

0

100527.71

1054.42

214 nm

T54, Oxidized

T44

T40

Time6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20

%

1

6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20

%

1

6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20

%

1

6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20

AU

5.0e-3

6.0e-3

7.0e-3

8.0e-3

9.0e-3

1.0e-2

Waters Peptide Separation Technology Columns are an integral part of the UPLC Peptide Analysis Solution. Their unique surface chemistry delivers exceptional peak shape for peptides over a wide

range of molecular properties.

Time47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00

AU

9.0e-3

1.0e-2

1.1e-2

1.2e-2

1.3e-2

1.4e-2

1.5e-2

1.6e-2

1.7e-2

1.8e-2

1.9e-2

G0

G1BG1A

G2

922.00G1 (M+H)+3

867.99G0 (M+H)+3

976.02G2 (M+H)+3

Time47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00

%

1

47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00

%

1

47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00

%

2

IgG Glycopeptide UPLC UV

IgG Glycopeptide UPLC/MS

successful separation of glycoforms

A tryptic digest of mouse IgG was separated using a Peptide Separation Technology Column. The glycoforms were observed in both the UV and MS traces. The extracted ion chromatogram provides identification of the glycoforms and show that the two positional isomers of the G1 were separated.

Time20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00

0.0

1.0e-2

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00

0.0

2.0e-2

4.0e-2

6.0e-2

8.0e-2

1.0e-1

1.2e-1

ACQUITY UPLC BEH130Å

ACQUITY UPLC BEH300Å

BA

AC

B

C

AU

AU

Fragment Size MW

A 4 residues 507.3

B 25 residues 2447.2

C 20 residues 1565.8

Comparison of the separation of tryptic digest of Phosphorylase b on ACQUITY UPLC BEH130 and BEH300 1.7 µm Peptide Separation Technology Columns.

two pore sizes for flexible method development

“ We use the Waters aCquitY uplC system for determining the primary sequences of antibodies as well as glycosylation and

other secondary modifications. the uplC separation provides sharp peaks, even when the quantities available are small.”

dingYi Wen, ph.d., principle scientist, biogen idec

Dr. Wen investigates antibody candidates by digesting them with endoproteases, followed by separation, and finally identification of peptides in

the mass spectrometer. HPLC does not provide the resolution or sensitivity needed for the research protocols, and its use of splitting can result in

loss of material, she explained.

improved glycopeptide mapping

The analysis of site-specific glycosylation is challenging because glyco-

peptides often elute as broad, overlapping peaks. The surface chemistry

of Peptide Separation Technology Columns yields sharper and more

symmetrical peaks, simplifying this analysis. When this chromatographic

separation is interfaced with a mass spectrometer, structurally informative

fingerprints can now be routinely obtained.

Choices for method development with peptide separation technology chemistry

The columns are available with either 130 or 300Å pore sizes and

are optimized for either trifluoroacetic acid (TFA) or formic acid (FA)

modified mobile phases. All Peptide Separation Technology Columns

are quality control tested and certified using a peptide digest to

ensure reproducibility.

good sCienC e means good business

To achieve your goals, your laboratories must operate at their best. You are balancing short-term financial performance against long-term

business goals. You need to meet ever-aggressive product availability and delivery timetables, which require shorter turnaround times from

product development through production. The Waters UPLC Peptide Analysis Solution simultaneously addresses laboratory productivity,

efficiency, and analytical quality.

accelerate protein characterization

An inherent degree of structural heterogeneity occurs in proteins. You

need to isolate and identify all the protein’s variants and degradation

products. Utilizing peptide chromatography to describe protein

heterogeneity patterns is a critical tool supporting effective discovery

and lead development efforts. Waters technologies help you characterize

proteins more comprehensively and efficiently, potentially accelerating

your submissions to regulatory agencies.

manage risk

Are you concerned by the demands to document product quality

throughout the development process? The requirements are stringent,

and remain applicable throughout the product’s life cycle. Waters

combines instruments, chemistries and software into performance

assured solutions that you can rely on to give you consistent results for

your validated analytical protocols. And because your scientists know

more about their proteins, you can be more confident of your product

and avoid costly surprises during regulatory review.

Confirm quality

Your objective is to demonstrate process consistency. Yet you must

manage the fact that the product is heterogeneous, so your analytical

methods are often complex. Concurrently, you have a production

schedule to meet, so any delays in your part of the process are

expensive. The UPLC Peptide Analysis Solution provides the flexibility

you need to implement effective methods. You can rely on Waters to

build in the product consistency you need for validated protocols. And

when your analyses are complete, you can utilize Waters informatics

solutions to archive the results for easy future access.

avoid costly retesting

What is the impact of a failed analysis? The cost of a failed manufacturing

process could be substantial. It may result in improperly discarding an

entire process run. Or worse, it could lead to unnecessary checks to

ensure that the manufacturing process has not developed issues. You

need analytical methods that are robust and reliable. By testing our

separation chemistries on ACQUITY UPLC Systems, following the same

protocols Waters engineers used to qualify your system, Waters assures

the reproducibility and ruggedness of your system. You will obtain

the best information about your product, enabling you to support lot

release requirements.

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Waters Corporation 34 Maple StreetMilford, MA 01757 U.S.A.T: 508 478 2000F: 508 872 1990www.waters.com

Waters, ACQUITY, ACQUITY UPLC, UltraPerformance LC, UPLC, and NuGenesis are registered trademarks of Waters Corporation. BiopharmaLynx, Empower, MassLynx, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2006-2007 Waters Corporation. Printed in the U.S.A. January 2008 720001836EN LB-CP