++

BIOPHARMA WORKFLOW SOLUTIONSHOW AGILENT HELPS RESOLVE COMPLEX ANALYTICAL CHALLENGES

Drug Development and Quality Control

++

SamplesSamples

SamplesSamples

SamplesSamplesSamplesSamples

SamplesSamples

MethodsMethods

Biomarker ID and Detection

MAb Generation Platform (e.g., Transgenic Mice)

Manufacturing

Clinical

Preclinical

Market

QA/QC

Formulation and Stability

Downstream Processing and Purification

High-Throughput Clone Screening and Selection

Cell Culture Scale-up and Production

Analytical GroupsDevelop analytical methods; analyze samples from other functions, method transfers

PROT

EIN

ENGI

NEER

ING

>>

DEVELOPMENT > >

PROCESS SCALE-UP>

>

COMPLEX CHALLENGES

The work you do is extraordinarily complicated and labor-intensive; we can helpWe know you have a lot of samples to analyze, and we know what an understatement that is. The samples come to you from a wide range of sources involved in a wide range of projects—and everyone wants results right away. We understand, and we can help you get the results you need.

2

++

SamplesSamples

SamplesSamples

SamplesSamplesSamplesSamples

SamplesSamples

MethodsMethods

Biomarker ID and Detection

MAb Generation Platform (e.g., Transgenic Mice)

Manufacturing

Clinical

Preclinical

Market

QA/QC

Formulation and Stability

Downstream Processing and Purification

High-Throughput Clone Screening and Selection

Cell Culture Scale-up and Production

Analytical GroupsDevelop analytical methods; analyze samples from other functions, method transfers

PROT

EIN

ENGI

NEER

ING

>>

DEVELOPMENT > >

PROCESS SCALE-UP>

>

The throughput and productivity challenge• Increasing number of mAb candidates in the pipeline• Increasing number of samples to analyze with limited resources• Driving adoption of high-throughput and high-productivity methods

3

Cloneselection

Charge variantanalysis

Peptidemapping

Fragmentanalysis

PTM (oxidation,deamidation)

Glycananalysis

Higher order structure analysis

RejectFailed

Passed

Prep Protein Acapture of mAb

Cell culture fluid

Fraction collection

Neutralization

Dilution

Sizeanalysis

Impurityanalysis

Amino acidsequencing

Middle-up,-down analysis

PTManalysis

Glycan IDand quant

Higher order structureanalysis

LC/MS CharacterizationLC CharacterizationSample PreparationSample Purification

Intact massand glycoforms

C-Terminal lysremoval by CpB

Peptidedigestion

Papain, FabRICATORpepsin

Stabilitystudies

Glycan releaseand labeling

HOS sample prep(e.g., H/D exchange)

Titer check

Life is easier when everything works togetherBiopharma workflows have become increasingly complex, labor intensive, and time consuming. So how can you speed things up and be more productive, without sacrificing accuracy? We’ll work with you to address your most complex workflows with fully integrated solutions that span from sample prep to final analysis.

INTEGRATED SOLUTIONS

4

Cloneselection

Charge variantanalysis

Peptidemapping

Fragmentanalysis

PTM (oxidation,deamidation)

Glycananalysis

Higher order structure analysis

RejectFailed

Passed

Prep Protein Acapture of mAb

Cell culture fluid

Fraction collection

Neutralization

Dilution

Sizeanalysis

Impurityanalysis

Amino acidsequencing

Middle-up,-down analysis

PTManalysis

Glycan IDand quant

Higher order structureanalysis

LC/MS CharacterizationLC CharacterizationSample PreparationSample Purification

Intact massand glycoforms

C-Terminal lysremoval by CpB

Peptidedigestion

Papain, FabRICATORpepsin

Stabilitystudies

Glycan releaseand labeling

HOS sample prep(e.g., H/D exchange)

Titer check

Agilent biopharma analytical solutions• Best-in-class HPLC and UHPLC solutions for higher throughput and productivity• Walkup sample prep automation• Best-in-class biocolumns• High-resolution accurate mass TOF and Q-TOF for routine and walkup LC/MS applications• High-resolution 2D-LC solutions for glycan analysis • CE and simple plug-and-spray CE/ME solution• Sample QC by automated microfluidic chip and next-generation sequencing electrophoresis

5

2000

1800

1600

1400

1200

1000

800

600

400

200

00 5 10 15 20 25

6

System and software combine for better analysis, faster method developmentWhen you’re analyzing biomolecules, you don’t want extraneous materials showing up in your results, so our Bio-inert LC system ensures that you’re analyzing just the sample and no trace elements from the system itself.

TRULY BIO-INERT, FAST METHOD DEVELOPMENT

Increase the quality of characterizationThe Agilent 1260 Infinity II Bio-inert LC system is designed to work under even the harshest bioseparation conditions such as ADC analysis by HIC (hydrophobic interaction chromatography) using 2M ammonium sulfate. The system is truly metal-free, starting from the point of sample entry. Only bio-inert titanium is used in the pump head—no other questionable alloys.

NEW detectors (improved specifications)

NEW column compartment (easier to install bio-inert heat-exchanger,QuickChange bio-inert fittings)

NEW bio-inert multisampler (ideal for microtiter plate based workflows,high capacity, faster, lowest carryover)

NEW bio-inert pump (improvedusability, full backwards methodcompatibility)

Leverage techniques that improve your characterization without compromise

HIC is routinely used to determine the ADC drug-to-antibody ratio. It is a gentle method that retains mAb structure lacking normal disulfide bonds as found in some conjugates. Harsh HIC 2.0 M (NH4)2 SO4 conditions necessitate the use of a fully bio-inert LC system.

6 8 10 12 14 16Time (min)

pH 9

pH 3

Water

0 5 10 15 20 25 30 354.0

4.5

5.0

5.5

6.0

6.5

7.0pH

Time (min)

Preset pH Actual pH

Base

Acid

Water

NaCl 0 5 10 15 20Time (min)

6.90

6.95

7.00

7.05

pH Ionic Strength (mM)

100

200

300

400

500 Preset pH Actual pH Ionic Strength

7

SST housing for robustness

What makes the 1260 Infinity II Bio-Inert LC truly bio-inert?New capillary and fitting technology for robust and secure operation—day in, day out.

Salt and pH gradients are easily created from stock solutions.

Reduce analysis time and increase accuracy with Agilent Buffer AdvisorThe example below shows monoclonal antibody charge variant analysis by means of pH gradient ion-exchange chromatography using Agilent Bio-MAb 5 μm, 4.6 x 50 mm column.

mAb charge heterogeneity analysis by pH gradient using Agilent Buffer Advisor.

Inert Ceramic Needle PEEK Needle Seat

PEEK and ceramic for metal free sample path

PEEK seat (inside) Metal-clad PEEK capillary

SST holder and screw for robustness

8

The Agilent 1290 Infinity II LC offers unique advantages for protein separationsIf long separation times are keeping you from getting through an extensive list of samples, we have an ultra-high-performance LC for you. With our 1290 Infinity II system, you can achieve exceptional throughput and resolution. Our shorter separation methods will give you an advantage you can’t get anywhere else.

SUPERFAST SEPARATIONS

1290 Infinity II LC and 1290 Infinity II Flexible Pump This system offers performance combined with flexibility. The 1290 Infinity II Flexible Pump is the only low-pressure mixing quaternary UHPLC pump with binary-like accuracy and precision. Other advantages include:

• UHPLC power range with up to 1300 bar and 5 mL/min for super-fast run times

• BlendAssist, the easiest tool for accurate buffer and additive blending—a big time saver!

• UHPLC productivity with HPLC ownership costs

Use for method development or walkup systems with accurate buffer blending

For a closer look at these advanced systems, visit efficientuhplc.agilent.com

0 2.5 5 7.5 10 12.5 15 17.5 20

0

10

20

30

40

50

60

70mAU

Time (min)

1 2 3 4 5 6

0.3

45 0

.363

1.9

65 2.0

93

2.4

76

2.8

40 2

.992

3.3

04 3

.460

3.6

24 3

.770

3.9

23

4.4

11

4.6

36

5.2

14

5.6

80

6.1

67

6.4

80

7.3

82

7.8

19 7

.991

2-AB labeling reagent

+

Separation

Isomerseparation

< 10 min

9 min0

Column Rs 2,1 Rs 3,2 Rs 4,3 Rs 6,5 avg. PW (min) Peak capacityAdvancedBio Glycan 1.63 1.70 3.05 2.09 0.059 135Mapping Column, 1.8 µm

1 2 3 4 5 6 7 8

LU

0

0.2

0.4

0.6

0.8

1

00

50

100

150

200

250

300

350

mAU

0.229

0.534

2.404

2.759

2.956

3.287

3.399

4.462 4.855

0.3330.406

0.464

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5.0Time (min)

9

Faster, better separations using the Agilent 1200 Infinity Series LC system

Fast-sub-10-minute separation of N-linked glycans of human IgG using Agilent AdvanceBio glycan mapping column, 1.8 μm on an Agilent 1290 Infinity Quaternary LC system.

Fast and efficient high-resolution peptide mapping for 100% sequence coverage using Agilent AdvanceBio peptide mapping column.

Rapid monoclonal antibody fragment analysis

Reversed-phase separation of a reduced monoclonal antibody using Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300SB-C8, 2.1 x 50 mm column on Agilent 1290 Infinity UHPLC. The separation was achieved in under 5 minutes.

IN

OUTDetector

Pump/Autosampler

Column 6Column 5

Column 4Column 3

Column 2Column 1

4

5 6

2

3

1

6’ 5́ 3́ 4́

2́ 1́

Detector

Waste

Column 2

RegenerationPump

Eluent Pump / Autosampler

Column 1

12

3

4

5 67

8

9

10 12

3

4

5 67

8

9

10

Detector

Waste

Column 2

RegenerationPump

Eluent Pump / Autosampler

Column 1

With SEQUENTIAL regenerationAnalysis 1 Analysis 2 Analysis 3

Analysis 2

Analysis 3

Regenerate

Regenerate

Regenerate

Regenerate

Analysis 1With ALTERNATING regeneration

10

Imagine how much more you could do if run times were cut in halfAt Agilent, we’re fully focused on making your lab more productive. We do it with ultra-high-pressure instruments and with applications that address your most pressing needs: From offline column regeneration (which can shorten run times by as much as 50 percent) to method scouting and application switching.

MULTIPLY YOUR ANALYTICAL ABILITIES

Automated method scouting and application switchingThe process of using alternate bioseparation techniques (e.g., IEX, SEC, HIC, or RP) can be accelerated significantly using novel valve technology. The same setups can be used for automated application switching for multiple attribute analysis from the same sample plate, saving time and resources.

Position 2

Shorten run times by 20-50% with automated offline column regeneration

Position 1

First Dimension

Second Dimension

Pump AutosamplerDetector (optional – for 1D chromatogram and peak triggering) Column Compartment

Detector (optional – to monitor waste line)

Pump

Detector

8¹D-ColumnWaste

²D-Pump²D-Column

Fill-directionAnalyze-dir.

3

4 5 6

2

7

Loop 2

1

Fill-directionAnalyze-dir.

Loop 1

Select ValveDefine Pump

Select ConfigurationDefine

Detector

Define Peak Detector

Graphical Representation

Heart-cutting 2D-LC

Comprehensive 2D-LC

1D chromatogram

2D sampling

2D gradient

1D chromatogram

1

4

5

67

2 3

8

IN

OUT4

5 6

2

3

1

IN

OUT4

5 6

2

3

1

6́ 5́ 4́ 3́

2́ 1́

6́ 5́ 4́ 3́

2́ 1́

WasteWaste

=1D-Column

1D-Column

2D-Column2D-Column

2D-Pump 2D-Pump

1

4

5

67

2 3

8

Deck 1

#6: 8.67 min #7: 8.98 min #8: 10.75 min #9: 14.30 min

Deck 2

#3: 4.79 min #4: 6.10 min #5: 6.83 min

11

Add a new dimension to your analysis, with unexpected easeTwo-dimensional liquid chromatography is proving to be an effective tool in biopharma, but it is often considered too complex, which has kept many labs from implementing the technology. Agilent’s ready-to-go 2D-LC is the first commercial product of its kind on the market—and it provides an intuitive interface that makes it easy to set up.

COMPLEXITY SIMPLIFIED

Perform automated online impurity analysisAgilent’s innovative and exceptionally easy to use Agilent 1290 Infinity II 2D-LC solution allows product and impurity analysis from harsh bioseparation methods like IEX, HIC, SEC, and Protein A to be fully automated.

Setup is simple with one screenOne simplified user screen for the entire system setup makes the Agilent 2D-LC system truly easy to use.

Multiple heart-cutting configuration with novel parking deck cluster (PDC) allows parking of up to twelve peaks for subsequent analysis in the second dimension.

Flexibility with both comprehensive and heart-cutting 2D-LC

12

Do it fast and do it right, every timeBiopharma sample prep is highly complex, requiring numerous timed steps—and the reproducibility of the method often depends on the analyst. Unless you automate. Agilent offers state-of-the-art sample prep automation, with kits and simplified interfaces for a far better user experience.

AUTOMATE YOUR SUCCESS

Only Agilent AssayMAP Automated Protein Sample Prep technology successfully integrates multiple labor-intensive operations such as affinity purification, digestion, and desalting into high-precision workflows designed to enhance analysis by mass spectrometry.

Automate: • Antibody and peptide

enrichment

• Digestion

• Glycan mapping

• Fractionation

Samples to Process

Resu

lt Qu

ality

Few Many

High

Low

Automated Manual

QualityThreshold

RequiresAutomation

Crude Sample Prep

Manufacturing Process

Peptide Mapping

At some point, you cannot do itwithout automation• Maintain data quality with increased sample capacity• Consistency scales better with automation

Biological System

Glycan Profiling

Method SpecificCrude Sample Prep

Sample Titer

Oxidation Characterization

ample ter

13

Automated sample prep solutions

As sample capacity increases, only automation scales to maintain consistency and data quality.

Automate: • Antibody and peptide

enrichment

• Digestion

• Glycan mapping

• Fractionation

MAb titers from cell culture brothBioMonolith Protein A column, an AdvanceBio column • Captures mAbs fast• Long column life: minimal clogging

GlycosylationAdvanceBio glycan mapping columns• Fast, high resolution, reproducible glycan mapping• Available in two UHPLC configurations: 2.7 µm superficially porous for high resolution, lower backpressure and 1.8 µm for highest resolutions• Each media lot is tested with a glycan reference mix to ensure performance

Intact protein analysis: heavy chains, light chains, Fc region and absolute massNew AdvanceBio RP-mAb features a 450Å pore size and Poroshell technology to deliver high resolution, high efficiency mAb characterization. Agilent has the largest selection of reversed-phase biocolumns available. Learn more at www.agilent.com/chem/advancebio

Fc

Fab

Light chain

Glycosylation site

Heavy chain

s s

ss

Dimers and higher aggregatesSize exclusion chromatography, using Agilent AdvanceBio SEC• Reliable performance: hydrophilic layer assures minimal secondary interactions

Charge variant analysisIon-exchange chromatography, using Agilent Bio MAb and Agilent Bio SCX• Hydrophilic coating eliminates most nonspecific interactions

-+

+

+

Peptide mappingAdvanceBio peptide mapping columns• Fast, high resolution, reproducible peptide mapping• Each media lot is tested with a challenging peptide mix to ensure performance

AN

N P

T

I

L

KA

N

DP

MH

T

B R

R A

N

DP

T

B R

R

AdvanceBio RP-mAb diphenyl provides an optimal level of detail for this IgG2 separation.

min0 0.5 1 1.5 2 2.5

mAU

AdvanceBio RP-mAb Diphenyl

IgG2 Lambda Intact

050

100150200

min

monomer

native herceptinheat/pH-stressed Herceptin

aggregatedegradedherceptin

1 2 3 4

mAU

0

5

10

15

20

25

30

35

2.7 µm AdvanceBio glycan mapping column enables fast, high resolution glycan mapping with lower backpressure.

min10 12 14 16 18 20 22 24 26

LU

0.2

0.4

0.6

0.8

1

1.2

1

2

3

4

5

6

7

8

9

10 11 12

13

Chromatogram of native (control; red trace) innovator mAb, herceptin and ADC overlaid with heat/pH stressed (blue trace) using an Agilent AdvanceBio SEC 300Å, 4.6 × 150mm, 2.7 µm column.

Improve productivity for reversed-phase, peptide mapping, size exclusion, ion-exchange, and affinity techniquesAgilent offers the industry’s widest range of biocolumns, providing leading-edge technology for every major technique. Agilent AdvanceBio columns are designed to advance accuracy and productivity for biomolecule separations.

A COMPLETE TOOLSET TO CHARACTERIZE YOUR PROTEIN

14

MAb titers from cell culture brothBioMonolith Protein A column, an AdvanceBio column • Captures mAbs fast• Long column life: minimal clogging

GlycosylationAdvanceBio glycan mapping columns• Fast, high resolution, reproducible glycan mapping• Available in two UHPLC configurations: 2.7 µm superficially porous for high resolution, lower backpressure and 1.8 µm for highest resolutions• Each media lot is tested with a glycan reference mix to ensure performance

Intact protein analysis: heavy chains, light chains, Fc region and absolute massNew AdvanceBio RP-mAb features a 450Å pore size and Poroshell technology to deliver high resolution, high efficiency mAb characterization. Agilent has the largest selection of reversed-phase biocolumns available. Learn more at www.agilent.com/chem/advancebio

Fc

Fab

Light chain

Glycosylation site

Heavy chain

s s

ss

Dimers and higher aggregatesSize exclusion chromatography, using Agilent AdvanceBio SEC• Reliable performance: hydrophilic layer assures minimal secondary interactions

Charge variant analysisIon-exchange chromatography, using Agilent Bio MAb and Agilent Bio SCX• Hydrophilic coating eliminates most nonspecific interactions

-+

+

+

Peptide mappingAdvanceBio peptide mapping columns• Fast, high resolution, reproducible peptide mapping• Each media lot is tested with a challenging peptide mix to ensure performance

AN

N P

T

I

L

KA

N

DP

MH

T

B R

R A

N

DP

T

B R

R

AdvanceBio RP-mAb diphenyl provides an optimal level of detail for this IgG2 separation.

min0 0.5 1 1.5 2 2.5

mAU

AdvanceBio RP-mAb Diphenyl

IgG2 Lambda Intact

050

100150200

min

monomer

native herceptinheat/pH-stressed Herceptin

aggregatedegradedherceptin

1 2 3 4

mAU

0

5

10

15

20

25

30

35

2.7 µm AdvanceBio glycan mapping column enables fast, high resolution glycan mapping with lower backpressure.

min10 12 14 16 18 20 22 24 26

LU

0.2

0.4

0.6

0.8

1

1.2

1

2

3

4

5

6

7

8

9

10 11 12

13

Chromatogram of native (control; red trace) innovator mAb, herceptin and ADC overlaid with heat/pH stressed (blue trace) using an Agilent AdvanceBio SEC 300Å, 4.6 × 150mm, 2.7 µm column.

15

Intact antibody analysis Antibody fragment analysisand glycan profiling

Antibody peptide mapping

IgGPeptide/glycopeptide maps

IdeS DTT

Enz.

Enz.

Enz.

2 LC2 HC

FcFab

F(ab)’2 2 Fc/2 2 Fd

2 Fc/2

2 LC

Papain

DTT(150 kDa)

-/+ PNGase F

+

+(25 kDa)

(25 kDa)

(25 kDa)

(25 kDa)

(25 kDa)

(100 kDa)

(50 kDa)(50 kDa)

(50 kDa) (LC/MS, LC/MS/MS, CE-MS, CE-MS/MS)

16

From preparing samples to analyzing results, Agilent has you coveredConfirmation of intact protein mass, major glycoforms, and other post-translational modifications (PTMs) are all critical measurements for characterizing proteins and understanding their efficacy and stability. Mass spectrometry is the primary tool that enables all of these measurements on a single platform with high mass accuracy, analytical specificity and sensitivity.

Agilent offers highly accurate solutions developed for routine measurements of intact protein mass and common PTMs using accurate-mass time-of-flight LC/MS and accurate-mass quadrupole time-of-flight LC/MS platforms.

MS strategies for the characterization of antibodiesTop-down, middle-down and -up, bottom-up.

END-TO-END SOLUTIONS

IgG are characterized as whole antibodies, fragments (including light chains, heavy chains, glycans, Fc regions) and by peptide/glycopeptide mapping (LC/MS and LC/MS/MS)

Agilent can deliver the highest analytical sensitivity available using 2.1 mm ID columns (replacing capillary LC) with no loss of sensitivity!

AB1 AB2

Deconvolution by Maximum Entropy

Overlay of Triplicate Injections of Antibody1 (AB1) vs. Antibody2 (AB2)

x102

10.90.80.70.60.50.40.30.20.1

0

Counts (%) vs. Mass-to-charge (m/z) 2000 2400 2800 3200 3600 4000 4400 4800 5200

x102

10.90.80.70.60.50.40.30.20.1

0

Counts (%) vs. Mass-to-charge (m/z) 2000 2400 2800 3200 3600 4000 4400 4800 5200

x102

10.90.80.70.60.50.40.30.20.1

0

Counts vs. Deconvoluted Mass (amu) 146800 147200 147600 148000 148400 148800 149200

Counts vs. Deconvoluted Mass (amu) 146800 147200 147600 148000 148400 148800 149200

x102

10.90.80.70.60.50.40.30.20.1

0

×106

0

0.51

1.52

2.53

3.54

4.55

5.5

A(48

-63)

A(48

-63)

A(48

-63)

Acquisition Time (min)

Coun

ts

5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6

×103

×103

×103

0123 227.1755

b2

b2

b2

b3

b3

b3

709.8431 766.3856628.3125

586.6502340.2608 511.2581y14

3+ 822.9272

277.1549 387.1989 473.5691

00.5

11.5

249.1592

447.2619

390.2353

0

1

2586.9786

249.1630 475.3254

390.2355

200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880

599.8006

600.2981

600.2898

511.5849

511.5826586.9768

542.7818

542.7799

542.7781

340.2605

340.2612

227.1761

227.1741

628.8038

628.8048710.3377

710.3353

766.8799

766.8816

823.4211

823.4254

N G Y I L

D G Y I L

D G Y I L

Mass-to-charge (m/z)

Coun

tsCo

unts

Coun

ts

y102+

y102+

y102+

y132+

y132+

y132+

y142+

y142+

y142+

y152+

y152+

y152+

y112+

y112+

y112+

y122+

y122+

y12,b12-NH32+

y143+

y143+

17

Agilent’s mass accuracy is exceptional, as demonstrated by the consistency in these triplicate-run spectra.

ACCURATE MASS YOU CAN COUNT ON

Deamidation is an important post-translational modification to monitor in peptide mapping. Deamidation can occur during storage, purification, and sample manipulation. Deamidation can be identified by an LC/MS/MS peptide mapping experiment.

Intact Protein Workflow

DeconvoluteMatch proteinsequences and predict PTMs

Generate report

Integrate and extract MSAcquire data

Qualitative Compound Report

MS Spectrum

IRM Calibration Status Success DA Method BioConfirmIntactProteinHighMass-Default.m

Data File Intact_MyoglobinNewMyo-010012.d Sample Name NewMyo-01Sample Type Sample Position P1-B-03

Comment IntProt

Instrument Name MH Instrument User Name wadminAcq Method Intact_MyoglobinNovartis.m Acquired Time 3/4/2014 2:08:13 PM

Intact Acq + Myo-DA

6200 series TOF/6500 series Q-TOF B.06.00 (B502)

Column2Sample Group

Protein Sequence

Mass

Mod Pro�le1

Custom

Column3

horse_myo.txt16951||| Phospho&OxA,B,C

S1

Column2Info.

Mod Pro�le

Protein Sequence 1

Mass1

Custom1

Stream NameWalkup Method Description

Acquisition SW Version

Column3

||| MyoMyoglobin2.psq16951test

Compound TableMass

16951.778Sequence Name

Myoglobin (horse)Target Mass

16951.6073MaxZ

18Z Count

13Fit Score

0

RuleIntact protein

Rule

Intact proteinUncertainty

0.05Signi�cance

100Algorithm

Peak Modeling Deconvolution# Hits

3Data File

Intact_MyoglobinNewMyo-010012.dDelta ppm

10.07Height

201650689MinZ

6

998.1704

Spectrum

Spectrum

AlgorithmPeak Modeling Deconvolution

Mass16951.778

Sequence NameMyoglobin (horse)

Target mass16951.6073

Delta ppm10.07

MinZ6

MaxZ18

Z Count13

Fit Score0

Uncertainty0.05

Signi�cance100

1131.15781211.88291305.02721413.69551542.1217

MS Spectrum Peak Listm/z

998.19891060.5234

Calc m/z

1060.49311131.12551211.84831304.98991413.65521542.0777

-28.54

z

1716151413

Di�(ppm)

-28.52-28.53

Abund

23654.564051.1

121533.45159260.59

-28.53

-28.53-28.53-28.53-28.53

98

1696.18481884.53792119.9793

1884.59172120.0398

1696.2332 -28.54-28.54

--- End Of Report ---

169176.91153094.25129325.84

76638.532788.913695.6

121110

Page 1 of 1 Printed at: 3:13 PM on:3/14/2014

Counts vs. Deconvoluted Mass (amu)

876543210

x105

5.55

4.54

3.53

2.52

1.51

0.50

x105

144500 144700 144900 145100 145300 145500

(b) Peak Modeling

(a) Maximum Entropy

18

BioConfirm provides both classical maximum entropy deconvolution and enhanced peak modeling (pMod) deconvolution to determine the molecular weight of intact proteins. ADC DAR calculator automatically calculates drug-to-antibody ratio and streamlines the data analysis process.

ENVISION PRECISE PROTEIN CONFIRMATION

Deconvoluted spectra via maximum entropy and pMod algorithms.

Maximum entropy deconvolution provides rapid transformation of multiple charged mass spec data into accurate protein mass. The peak modeling (pMod) algorithm employs additional steps to reduce artifacts and enhance resolution, which helps to resolve overlapping peaks and provide cleaner data, so you can be more confident in your answer.

Counts (%) vs. Mass-to-Charge (m/z)

1

0.9

0.75

0.6

0.45

0.3

0.15

0

x104

200 300 400 500 600 700 800 900 1,000 1,100 1,200

Peptide Mapping Workflow

Match protein sequences and identify PTMs

Visualize sequence coverage

Generate report

Extract compoundsAcquire data

19

BioConfirm identifies peptides and PTMs based on peptide masses and product ions (b, y, and immonium ions) in the MS/MS spectra. This allows for faster and more streamlined processing of MS/MS data for peptide mapping.

Peptide MS/MS spectrum product ion assignment.

BioConfirm provides enhanced processing of MS/MS data for streamlined mapping and data interpretation.

ENVISION POWERFUL PEPTIDE MAPPING CAPABILITIES

Digestion

GlykoPrep

Releaseglycans

Digested glycopeptides

Glycosylationsite profiling(LC/MS/MS)

LIF

FLD

APTS

2-AB label

Intactglycoprotein

Glycoformprofiling by

LC/MS

Glycoproteinor mAb

Sialic acid

Monosaccharides

Oligosaccharides

20

Take your pick of fast, fully automated analysis strategiesGlycan analysis is so complex it can be difficult to determine which analytical strategy will work best. Rest assured that Agilent has proven solutions to address every facet of your glycan analysis.

WE PUT THE CAN IN GLYCAN CHARACTERIZATION

Strategies for intact glycoform profiling, glycopeptide and glycosylation site identification, and release glycan analysis

Released glycans with no labeling are also run with MS.

Instant PC LabeledmAb N-glycans

Agilent 1290 LC with Fluorescence Detection

Agilent AdvanceBioGlycan Mapping Column

Agilent 6550 iFunnelQ-TOF LC/MS

Agilent Mass ProfilerSoftware

149

AFLD

BFLDZoom

19 24 29 34 39 149 19 24 29 34 39

149

CMS

DMSZoom

19 24 29 34 39 149 19 24 29 34 39

ACompound 14 precursor

m/zprecursorB

Compound 47

Inte

nsity

(cou

nts)

Inte

nsity

(cou

nts)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

21

Agilent LC/FLD system offers accurate quantitation for glycan profiles, while LC/MS system provides the researcher with the capability to perform both identification and MS-based quantification of glycans. High quality Q-TOF MS data greatly facilitate peak assignment by offering accurate mass and tandem mass information for each of the glycans detected using FLD.

GETTING GLYCAN IDENTIFICATION AND QUANTITATION AT THE SAME TIME

Tandem MS data were acquired for all glycans. MS/MS aided in compound identifi cation when accurate mass was insuffi cient. The two examples above are consistent with gal-gal and outer arm fucose (A) and NeuGC (B) modifi cations.

Comparison of FLD and MS chromatograms for mAb 1. A) FLD chromatogram of mAb 1 glycans. B) Zoom of FLD chromatogram of mAb 1 glycans. C) MS chromatogram of mAb 1 glycans. D) Zoom of MS chromatogram of mAb 1 glycans.

N-glycan identification and quantitation workflow

242.1

548

360.1

884

548.2

585

661.3

466

1217

.1748

1311

.7077

661.3

540

1449

.7396

261.1

243

360.1

914

887.4

836

1069

.9092

1217

.6493

544.3

041

804.4

230

966.4

801

473.2

702

86.09

59

473.2

723

804.4

168

887.4

673

966.8

238

Counts vs. Mass-to-Charge (m/z)

1.050.9

0.750.6

0.450.3

0.150

x102

100 400 700 1,000 1,300 100 400 700 1,000 1,300

322.0

481

363.2

529

429.0

619

663.4

523

762.7

855

893.9

943

993.2

734

1221

.9907

1324

.0247

1521

.9693

939.7

506

(M+4

H)+4

1252

.6621

(M+3

H)+3

322.0

481

363.2

525

429.0

617

663.4

531

791.8

275

850.2

981

922.0

081

989.5

324

1171

.9874

1221

.9906

1521

.9681

1628

.9761

1697

.1212

Counts vs. Mass-to-Charge (m/z)

0.8

0.5

0.2

-0.1

x102

0.8

0.5

0.2

-0.1

x102

300 450 600 750 900 1050 1,200 1,350 1,500 1,650

Counts vs. Deconvoluted Mass (amu)

2.4

1.8

1.2

0.6

0

-0.6

-1.2

-1.8

-2.4

-3.0

-3.6

x103

147100 1474000 147700 148000 148300 148600 148900

22

ENVISION EASY BATCH-TO-BATCH COMPARISON

MassHunter BioConfirm provides easy visual comparisons among samples, allowing for fast batch-to-batch analysis on both protein and peptide levels.

Mirror plot functionality enables rapid and reliable comparison of two samples, such as two batches of an engineered protein or biosimilars. Samples in the mirror plot can be switched quickly without reprocessing the data.

Monitor biosimilars and batch-to-batch variation by quickly comparing samples and referenceThe comparative analysis module facilitates the identification and visualization of the differences between two batches of data for easy inspection. Samples can be compared to a reference using chromatograms, MS, and MS/MS spectra.

Mirror plot of two engineered proteins.

MS/MS spectrum comparison results.MS spectrum comparison results.

Administrators tailor user access to instrument parameters to user access level

Agilent MassHunter Walkup softwareLC + Q-TOF/TOF/SQUsers with different skill levelsperform their own automated analyses

1

Enter username and number samples to be run.

Enter sample information and select from a list of available methods.

Place sample in the position as directed by the software.

Choose Method

Place Sample

Receive ReportLogin

2 3

23

Let each of your biologists unleash the power of LC/MS characterizationThe value of any resource is directly tied to the ease with which people can access and benefit from it. That’s why we created Agilent MassHunter Walkup software: So more people can access your lab’s LC/MS instrument, with ease. Now even untrained users can benefit, without assistance from your busy staff. This user-friendly software allows people of different skill levels to perform their own automated LC and LC/MS analyses. All they have to do is input some basic information, choose a method, and insert samples as directed. Results show up in the submitter’s in-box automatically.

WALKUP ACCESS FOR ALL

Simplified MassHunter Walkup user interface. Three steps to results.

Support virtually any analysis that can be automated.

1

3

5

7

13 14 14 16 18 1917 20 21 22 23 24 25 26 27 28Counts vs. Acquisition Time (min)

x105

x103

00.20.40.60.81.01.21.41.61.82.0

Counts vs. Acquisition Time (min)7 8 9 10 11 12 13 14 15 16 17 18 19 20

G2

G2F

G2-

1NA

NA

G2-

2NA

NA

G2F

-2N

AN

A

G2F

-1N

AN

A

30,203.82

028,000 28,800

28,893.72 29,792.04

29,304.80

31,102.52

32,001.2629,600 30,400 31,200 32,000 32,800

0.81.62.43.24.04.85.66.47.2 = scFv

= Cy5.5

×104

898.00

899.00

898.00

Deconvoluted mass (amu)

Coun

ts

-10

0

10

20

30

40

50

60

70

80

10 12 14 16 18 20 22 24 26 28

mAU

Time (min)

7.27.6

5.5

IgG

24

Mixture of neutral glycan and sialylated glycan separation based on mass-to-charge ratio using the Agilent 7100 CE system.

Truly pi-based, high resolution monoclonal antibody charge heterogeneity analysis by capillary isoelectric focusing

Peptide mapping by CE/MS routinely provides 100 percent sequence coverage and is regularly employed as an orthogonal method of LC/MS peptide mapping.

Deconvoluted mass spectrum of scFv-A conjugate. The assigned structures are based on deconvoluted mass.

Examples of applications of capillary and microfluidic electrophoresis

Peptide mapping analysisAntibody drug conjugate analysis by CE/MS

Agilent solution provides extra power for complex appsCE/MS combines the short analysis time and high separation efficiency of capillary electrophoresis with the molecular weight and structural information of mass spectrometry. The technique has been successfully used to analyze various compounds in complex sample matrices.

CE AND CE/MS TO THE RESCUE FOR GLYCANS, CHARGE VARIANTS, AND PEPTIDES

Glycan analysis by CZE-MS Q-TOFCharge heterogeneity analysis by cIEF

The technology behind Agilent CE/MS

Agilent 6550 Accurate-Mass Q-TOFAgilent 7100 CE System

Data analysis Integrated software packages control the CE/MS system and interpret and filter the massive amount of data created during analysis.

CE capillarySeparation based on mobility in an electrical field takes place in 50-100 cm (usually fused silica) capillaries filled with aqueous buffers.

SampleComplex mixtures of chargedmolecules, ranging from inorganic ions to native proteins. CE offers the smallest injection volumes (nL range) with little sample preparation.

CE/MS cassette First part of the CE/MS interface; houses separation capillary and controls temperature, providing a UV-detection window and an exit to external detectors.

CE-ESI/MS-sprayerTriple-tube interface combines CE capillary with sheath liquid for stable flow rates and electrical contact, and isolates separation chemistry from MS ionization. Nebulization gas facilitates ESI.

Agilent iFunnel MSCombines Agilent Jet Stream (precision sprayer to desolvate and concentrate ions), hexabore capillary (array of capillaries for sampling more ions) and dual-stage ion funnel (to efficiently remove gas while focusing ions).

TOF-MS analysisFlight tube with vacuum-insulated shell eliminates thermal mass drift due to temperature changes, for excellent mass accuracy. Reflectron and long flight tube improve mass resolution.

Nitrogennebulizinggas

CE capillary

HPLC sheath liquid

Inlet vialhigh voltage

MS detectioncapillary on ground

UV detection

Airstream

25

240

15095634628155

Reducing conditionsNonreducing conditions

Intact

ngHC

0.0x%

Heavy chain

Light chainLower marker

2000[FU]

1500

1000

500

05 46 63 95 150 240 [kDa]15 28

26

AUTOMATED ELECTROPHORESIS

Quality control of samples has never been so easyThe Agilent 2100 Bioanalyzer system with Protein 80, Protein 230, and High Sensitivity Protein 250 kits offers an easy to use, benchtop platform to reliably assess protein concentration, identity, and purity. The DNA ScreenTape assays with the Agilent 4200 TapeStation and Agilent 2200 TapeStation system were developed for the separation and analysis of DNA fragments and libraries up to 5000 base pairs. The Agilent RNA ScreenTape assay provides efficient and reliable analysis of total RNA samples from eukaryotic or prokaryotic origin, providing quality, quantity, and sizing information. DNA or RNA samples are automatically loaded, separated, imaged, and analyzed at the press of a button.

Agilent 2100 Bioanalyzer system Introducing the 4200 TapeStation System

The High Sensitivity Protein 250 kit on the 2100 Bioanalyzer system provides antibody analysis at the highest sensitivity, equivalent or better than SDS-PAGE silver stains. The dynamic range facilitates impurity detection down to the pg/uL range. Reduced (blue) and nonreduced (red) conditions can be analyzed in parallel on the same chip.

Applications• Antibody QA/QC

• Protein purification analysis

• Protein expression analysis

For more information about these systems, visit www.agilent.com/genomics/bioanalyzer www.agilent.com/genomics/tapestation

Agilent 4200 Tapestation system

Results can be viewed in familiar gel view or Electropherogram view.

4

2

0

x105

Mass

CPS

50

Mg & AI

B

Ti & Cr

Zr & Mo Sn I

Ba & REEPb & Bi

U

Ca Fe & Mn Rh (ISTD)Sc (ISTD) Ga &

Ge (ISTD)Rb &Sr

Tb (ISTD)In (ISTD)

Ir (ISTD)Tune Step 1: 0099MPL.D

100 150 200

mAU

0

200

400

600

800

Control

Glut

amine

Hist

idine Th

reon

ine Argin

ine

Alan

ine

Glyc

ine

Day 1Day 2

Day 3

ControlDay 1Day 2Day 3

ControlDay 1Day 2Day 3

800µM Amino Acid Standard

Complete Cell Media

6 6.5 7 7.5 8 8.5 min

mAU

0

200

400

600

-200

-400

-600

800

0 2 4 6 8 10 12 14 min

27

We have what you need for raw material, media, amino acid, and leachable and extractable analysis via UHPLC, GC, and ICP-MSIt’s absolutely vital that you monitor various parameters in your fermentation broth—amino acids, sugars, organic acids—and use robust, well-established methods for analysis. You must be sure your final product is free of any leachable from any source. Metal contaminants from stainless steel fermenters, for example. Agilent has a comprehensive set of solutions that include GC, ICP-MS, and UHPLC instruments and software.

A FULL RANGE OF SOLUTIONS

Agilent 1200 Infinity II UHPLC Series provides the power range and performance you require for amino acid analysis. Agilent ZORBAX Amino Acid Analysis (AAA) columns provide high resolution separations for amino acids.

This full mass can of a commercial antacid sample illustrates the screening capability of the Agilent 7700x ICP-MS in helium mode.

The Agilent 7700x ICP-MS is well-suited for analyzing digested samples and analysis of elemental impurities according to USP <232>/<233>.

Learn more www.agilent.com/chem/biologics

Buy online www.agilent.com/chem/store

Find a local Agilent customer center www.agilent.com/chem/contactus

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For Research Use Only. Not for use in diagnosticprocedures. This information is subject to changewithout notice.

© Agilent Technologies. Inc. 2017 Printed in the USA, February 13, 2017 5991-5235EN