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

Agilent MassHunter BioConfirm Software

ENHANCED PROTEIN AND PEPTIDE CHARACTERIZATION

2

ENVISION HIGHER CONFIDENCE IN YOUR PROTEIN/PEPTIDE CHARACTERIZATION

Agilent’s sophisticated MassHunter BioConfirm Software brings you greater confidence in your protein/peptide analysis. Together with Agilent’s high performance liquid chromatography/mass spectrometry (LC/MS) systems, it provides an intelligent solution for rapid confirmation of synthetic peptides and characterization of proteins and peptides. Now you can elevate your discovery by confirming your data with confidence and ease.

AGILENT MASSHUNTER BIOCONFIRM SOFTWARE

BioConfirm Software Delivers:• Precise and rapid protein confirmation• Easier batch-to-batch analysis• Comprehensive peptide mapping

3

Counts vs. Deconvoluted Mass (amu)

2.82.42.01.61.20.80.4

0

x106

3.22.82.42.01.61.20.80.4

0

x104

144600 144800 145000 145200 145400 145600

144783.28

144946.75

145106.77

145265.67

144582.58

144786.27

144947.23 145107.68

145268.27

145583.41

(a) Maximum

(b) Peak Modeling

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

Deconvoluted spectra. (a) Maximum entropy deconvoluted spectrum. (b) Peak modeling deconvoluted spectrum.

ENVISION PRECISE PROTEIN CONFIRMATION

BioConfirm provides both classical maximum entropy deconvolution and enhanced peak modeling (pMod) to determine the molecular weight of intact proteins.

Maximum entropy deconvolution provides rapid mass resolution, especially for simple protein mixtures. For complex protein mixtures, the enhanced pMod algorithm removes artifacts from the spectrum and helps to resolve overlapping peaks—even severely overlapping peaks—providing cleaner spectra and more precise mass measurement. This enables fast confirmation of intact protein sequences and modifications.

BioConfirm Software Delivers:• Precise and rapid protein confirmation• Easier batch-to-batch analysis• Comprehensive peptide mapping

4

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

7.26.66.05.44.84.23.63.02.41.81.20.6

0

x104

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

Peptide Mapping Workflow

Match Protein sequences and identify PTMs

Visualize sequence coverage

Generate report

Extract compoundsAcquire data

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

ENVISION POWERFUL PEPTIDE MAPPING CAPABILITIES

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.

5

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

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

ENVISION EASY BATCH-TO-BATCH COMPARISON

BioConfirm provides easy visual comparison 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—an essential part of biopharmaceutical analysis. 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.

6

mAb Glycan Workflow

Extract compounds

Search database and identify glycans

Calculate percentage and generate report

Acquire data

N

ENVISION RAPID AND ACCURATE mAb GLYCAN ANALYSIS

BioConfirm enables users to accurately identify and visualize glycan structures in their mAb samples using Agilent’s Glycan Personal Compound Database (PCD) and extensive predefined glycan modifications. Using Agilent’s mAb-Glyco Chip, customers have a comprehensive workflow for glycan analysis including hardware, software, and consumables—a complete workflow that includes on-chip deglycosylation of the mAb as well as enrichment, chromatographic separation, and detection of the cleaved glycans. All activities, including data processing using BioConfirm, can be completed in less than 15 minutes.

BioConfirm provides an easy and effective workflow for the analysis of mAb glycans.

7

Sample Submission in Three Easy Steps

Enter sample information Place vial in ALS

Reports delivered to email inbox

Login

1 2 3

ENVISION WALK UP ANALYSIS FOR NON-EXPERT MS USERS

BioConfirm—part of Agilent’s MassHunter Walkup solution.

MassHunter Walkup provides a simple-to-use interface that provides novice users the ability to analyze protein and peptide samples in three easy steps with minimal training. Users do not need to concern themselves with complex mass spec conditions; instead, they can run their samples with standard acquisition and data processing methods. Results are directly delivered to their email Inbox.

Learn more www.agilent.com/chem/masshunter

Buy online www.agilent.com/chem/store

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

USA and Canada 1-800-227-9770 agilent_inquiries@agilent.com

Europe info_agilent@agilent.com

Asia Pacific inquiry_lsca@agilent.com

Research use only. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material.

© Agilent Technologies, Inc., 2014 Published in the USA, April 7, 2014 5991-3643EN