Drug Development and Quality Control

BIOPHARMA WORKFLOW SOLUTIONS

HOW AGILENT HELPS RESOLVE
COMPLEX ANALYTICAL CHALLENGES

+
+
 COMPLEX CHALLENGES
The work you do is extraordinarily complicated and labor-intensive; we can help
We 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.

Cell Culture Scale-up and Production

Samples

High-Throughput Clone Screening and Selection

Samples

+
+ Analytical Groups
Develop analytical methods;
>>

analyze samples from other

functions, method transfers
G
ERIN
GINE

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

PROTEIN EN

Samples

Biomarker ID and Detection

2
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

Downstream Processing and Purification

DEVELOP Formulation and Stability

MEN Samples
T>
>

Samples Preclinical
PR
OC
ES
SS

Clinical
CAL
E-UP
>>

Manufacturing
QA/QC
Methods

Market

3
 INTEGRATED SOLUTIONS
Life is easier when everything works together
Biopharma 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.

Sample Purification Sample Preparation

Cell culture fluid

Papain, FabRICATOR
pepsin

Titer Failed
check Reject
Peptide
digestion
Passed

Prep Protein A C-Terminal lys

capture of mAb removal by CpB

Stability
Fraction collection
studies

Glycan release
Neutralization
and labeling

HOS sample prep

Dilution
(e.g., H/D exchange)

4
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

LC Characterization LC/MS Characterization

Size Intact mass

analysis and glycoforms

Fragment Middle-up,
analysis -down analysis

Peptide Amino acid

mapping sequencing

Charge variant Impurity Clone

analysis analysis selection

PTM (oxidation, PTM

deamidation) analysis

Glycan Glycan ID
analysis and quant

Higher order structure Higher order structure

analysis analysis

5
 TRULY BIO-INERT, FAST METHOD DEVELOPMENT
System and software combine for better analysis, faster method development
When 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.

Increase the quality of characterization

The 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.

Leverage techniques that improve

your characterization without compromise

NEW detectors 2000

(improved specifications)
1800

1600
NEW column compartment (easier 1400
to install bio-inert heat-exchanger,
QuickChange bio-inert fittings) 1200

1000

800

600
NEW bio-inert multisampler (ideal
400
for microtiter plate based workflows,
high capacity, faster, lowest carry 200
over) 0
0 5 10 15 20 25

HIC is routinely used to determine the ADC drug-to-antibody ratio. It is a gentle

NEW bio-inert pump (improved method that retains mAb structure lacking normal disulfide bonds as found in some
usability, full backwards method conjugates. Harsh HIC 2.0 M (NH4)2 SO4 conditions necessitate the use of a fully
compatibility) bio-inert LC system.

6
Reduce analysis time and increase accuracy with Agilent Buffer Advisor
The 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.

Base pH Ionic Strength (mM)

Preset pH
Actual pH 500
7.05
Ionic Strength
Acid 400
7.00
300

Water 6.95
200

6.90 100

NaCl
0 5 10 15 20
Time (min)

pH
7.0 Preset pH
pH 9 Actual pH
6.5
6.0

pH 3 5.5
5.0
4.5
Water
4.0
0 5 10 15 20 25 30 35 6 8 10 12 14 16
Time (min) Time (min)
Salt and pH gradients are easily created from stock solutions. mAb charge heterogeneity analysis by pH
gradient using Agilent Buffer Advisor.

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.

Inert Ceramic Needle PEEK Needle Seat

PEEK seat (inside) Metal-clad PEEK

capillary
SST housing for robustness

PEEK and
ceramic for metal
free sample path

SST holder and screw for robustness

7
 SUPERFAST SEPARATIONS
The Agilent 1290 Infinity II LC offers unique advantages for protein separations
If 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.

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

8
Rapid monoclonal antibody fragment analysis

mAU 0.534
3.399
350

300

250

200
2.759
3.287
150
0.464
100
0.406 2.404
0.333
50 2.956
0.229 4.462 4.855

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5.0
Time (min)

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.

Faster, better separations using the Agilent 1200 Infinity Series LC system
2.476

LU
mAU Isomer
separation
1
3.304

70

60 0.8 +
4.411

50 0.6 2-AB
3.460

labeling
40 0.4 reagent
2.840
0.363

6.167
3.624
0.345

30 Separation
0.2 < 10 min
5.214
2.093

4.636
3.923
2.992

3.770

7.382
5.680

7.819
7.991
1.965

6.480

20 123 4 5 6
0
10 0 1 2 3 4 5 6 7 8 9 min
0 Column Rs 2,1 Rs 3,2 Rs 4,3 Rs 6,5 avg. PW (min) Peak capacity
0 2.5 5 7.5 10 12.5 15 17.5 20 AdvancedBio Glycan 1.63 1.70 3.05 2.09 0.059 135
Mapping Column, 1.8 µm
Time (min)

Fast and efficient high-resolution peptide mapping for 100% sequence coverage using Fast-sub-10-minute separation of N-linked glycans of human IgG using Agilent
Agilent AdvanceBio peptide mapping column. AdvanceBio glycan mapping column, 1.8 μm on an Agilent 1290 Infinity
Quaternary LC system.

9
 MULTIPLY YOUR ANALYTICAL ABILITIES
Imagine how much more you could do if run times were cut in half
At 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.

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

Position 1 Column 2 Position 2 Column 2

1 10 1
Eluent Pump / Autosampler Waste Eluent Pump / Autosampler 10 Waste
2 2
9 9
3 3
8 8
Regeneration Regeneration
Pump 4 Detector Pump 4 Detector
7 7
5 6 5 6
Column 1 Column 1

Analysis 1 Regenerate Analysis 2 Regenerate Analysis 3

With SEQUENTIAL regeneration

With ALTERNATING regeneration

Analysis 1 Regenerate Analysis 3

Analysis 2 Regenerate

Pump/Autosampler

1
Automated method scouting and application switching
2

The process of using alternate bioseparation techniques (e.g., 3

6’ 5´
4´
3´
IEX, SEC, HIC, or RP) can be accelerated significantly using novel IN
1´
2´

valve technology. The same setups can be used for automated 4

application switching for multiple attribute analysis from the 5

6 OUT
Detector

same sample plate, saving time and resources. Column 6

Column 5
Column 4
Column 3
Column 2
Column 1

10
COMPLEXITY SIMPLIFIED
Add a new dimension to your analysis, with unexpected ease
Two-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.

Perform automated online impurity analysis

Agilent’s innovative and exceptionally easy to use Agilent 1290 Setup is simple with one screen
Infinity II 2D-LC solution allows product and impurity analysis from One simplified user screen for the entire system setup
harsh bioseparation methods like IEX, HIC, SEC, and Protein A to makes the Agilent 2D-LC system truly easy to use.
be fully automated.
Loop 1
Fill-direction
Analyze-dir. Define Select Valve
1
¹D-Column
2
8 Pump
3 Waste Second Dimension
²D-Column 7
4 ²D-Pump
5
6
Pump
Select
Fill-direction
Analyze-dir. Define Configuration
Loop 2

First Dimension
Detector
Detector (optional –
Pump Autosampler for 1D chromatogram Detector (optional –
and peak triggering) Column Compartment to monitor waste line)

Detector
Define Peak Graphical
Detector Representation

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

D chromatogram
1
1

2
6´ 5
Waste
´4´ Deck 1
3´

Waste
3

D-Column
2´
IN
1´ 1 #6: 8.67 min
D sampling
2

=
4 #7: 8.98 min
2 OUT 2
5 #8: 10.75 min
3 6 3
1 1 #9: 14.30 min
1
D-Column 4 4

D gradient
8 8
2
D-Pump
Deck 2
2

1
5

6
7 #3: 4.79 min
5

6
7
2
D-Pump
#4: 6.10 min
Comprehensive 2D-LC 2
6´ 5
´4´ D-Column
2
#5: 6.83 min

3´
3
1´ ´
IN 2

5
OUT
2
D-Column
D chromatogram
6
1

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

Heart-cutting 2D-LC

11
 AUTOMATE YOUR SUCCESS
Do it fast and do it right, every time
Biopharma 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.

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

12
Automated sample prep solutions
Glycan
Profiling

At some point, you cannot do it

without automation
Biological System • Maintain data quality with increased
sample capacity
• Consistency scales better with automation

Peptide
Mapping

CrudeCrude
Sample PrepPrep
Sample Method Specific

Oxidation
Characterization

Manufacturing
Process

Sample
ample
ter
Titer

High
Automated Manual
Result Quality

Quality
Threshold
Requires
Automation
Low
Few Samples to Process Many

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

13
 A COMPLETE TOOLSET TO CHARACTERIZE YOUR PROTEIN
Improve productivity for reversed-phase, peptide mapping, size exclusion,
ion-exchange, and affinity techniques
Agilent 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.

Heavy chain

Fab
s s
Light chain s s

Glycosylation site

IgG2 Lambda Intact

mAU
200
150 AdvanceBio RP-mAb Diphenyl
Fc 100
50
0
0 0.5 1 1.5 2 2.5 min

AdvanceBio RP-mAb diphenyl provides an optimal

level of detail for this IgG2 separation.

Intact protein analysis: heavy chains, light chains, Fc region and absolute mass
New 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
M

14
 MAb titers from cell culture broth
BioMonolith Protein A column, an AdvanceBio column
• Captures mAbs fast
• Long column life: minimal clogging

native herceptin
heat/pH-stressed Herceptin

Dimers and higher aggregates mAU

monomer
35
Size exclusion chromatography, using Agilent AdvanceBio SEC
• Reliable performance: hydrophilic layer assures minimal 30
secondary interactions
25

20

15 aggregate
degraded
herceptin
10

Charge variant analysis 0

Ion-exchange chromatography, using Agilent Bio MAb 1 2 3 4 min
and Agilent Bio SCX
• Hydrophilic coating eliminates most nonspecific interactions 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.
- +

Glycosylation
AdvanceBio glycan mapping columns
• Fast, high resolution, reproducible glycan mapping
LU 2
• Available in two UHPLC configurations: 2.7 µm 1.2
superficially porous for high resolution, lower
4
backpressure and 1.8 µm for highest resolutions 1

• Each media lot is tested with a glycan reference mix 0.8

to ensure performance 9

0.6 5

3
0.4 6

Peptide mapping
13
8
1 10
0.2 7 11 12
AdvanceBio peptide mapping columns
• Fast, high resolution, reproducible peptide mapping 10 12 14 16 18 20 22 24 26 min
• Each media lot is tested with a challenging peptide
mix to ensure performance 2.7 µm AdvanceBio glycan mapping column enables fast, high resolution glycan
N
B R mapping with lower backpressure.
B R
N T
L
A P D
P D R I K
N A
H A
R T
T N P

15
 END-TO-END SOLUTIONS
From preparing samples to analyzing results, Agilent has you covered
Confirmation 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 antibodies

Top-down, middle-down and -up, bottom-up.

Intact antibody analysis Antibody fragment analysis Antibody peptide mapping

and glycan profiling
Agilent can deliver
the highest analytical
IdeS
2 LC
DTT (25 kDa) sensitivity available
using 2.1 mm ID
F(ab)’2 + 2 Fc/2 2 Fd columns (replacing
(100 kDa) (25 kDa) (25 kDa)
capillary LC) with no
loss of sensitivity!
2 Fc/2
Papain Enz. (25 kDa)

Fab + Fc
(50 kDa) (50 kDa) Enz.

IgG
(150 kDa) Enz. Peptide/glycopeptide maps
-/+ PNGase F DTT 2 HC
(50 kDa) (LC/MS, LC/MS/MS, CE-MS,
2 LC CE-MS/MS)
(25 kDa)

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)

16
 ACCURATE MASS YOU CAN COUNT ON
Agilent’s mass accuracy is exceptional, as demonstrated by the consistency in these triplicate-run spectra.

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

x102 AB1 x102 AB2
1
1
0.9
0.9
0.8
0.8
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
2000 2400 2800 3200 3600 4000 4400 4800 5200 2000 2400 2800 3200 3600 4000 4400 4800 5200
Counts (%) vs. Mass-to-charge (m/z) Counts (%) vs. Mass-to-charge (m/z)

Deconvolution by Maximum Entropy

x102 x102
1 1
0.9 0.9
0.8 0.8
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
146800 147200 147600 148000 148400 148800 149200 146800 147200 147600 148000 148400 148800 149200
Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu)

×106 N G Y I L

5.5 ×10 3
A(48-63)

227.1755 766.3856
3 628.3125 709.8431
Counts

b2
y 12,b 12-NH 32+ y 132+ y 142+
340.2608 586.6502
5 2 b3 511.2581
y 143+ 822.9272
4.5 1 277.1549
542.7799
y 102+
599.8006
y 112+
y 152+
387.1989 473.5691

4 0
D G Y I L
×10 3
3.5 1.5 227.1741 710.3377 766.8799
Counts

b2 628.8048 y 132+ y 142+

Counts

340.2605 y 122+
3 1 b3
586.97
511.5826 68
2.5 0.5 249.1592 y 14 3+ 542.7818
y 102+ 600.2981
823.4211
y 152+
390.2353 447.2619
y 112+
2 0
D G Y I L
A(48-63)

1.5 ×10 3 227.1761

628.8038
y 122+ 710.3353 766.8816
y 132+
2 b2 y 142+
Counts

1 511.5849
A(48-63)

340.2612 y 143+ 586.9786

b3

0.5 1 542.7781
y 102+ 600.2898 823.4254
249.1630 y 112+ y 152+

0 0 390.2355 475.3254

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 8.6 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880
Acquisition Time (min) Mass-to-charge (m/z)

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.

17
 ENVISION PRECISE PROTEIN CONFIRMATION
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.

Intact Protein Workflow

Qualitative Compound Report

Data File Intact_MyoglobinNewMyo-010012.d Sample Name NewMyo-01

Sample Type Sample Position P1-B-03
Instrument Name MH Instrument User Name wadmin
Acq Method Intact_MyoglobinNovartis.m Acquired Time 3/4/2014 2:08:13 PM
IRM Calibration Status Success DA Method BioConfirmIntactProteinHighMass-Default.m
Comment IntProt
Column2 Column3 Column2 Column3
Sample Group Info.
Protein Sequence horse_myo.txt Mod Profile ||| Myo
Mass 16951 Protein Sequence 1 Myoglobin2.psq
Mod Profile1 ||| Phospho&Ox Mass1 16951
Custom A,B,C Custom1 test
Walkup Method Intact Acq + Myo-DA Stream Name S1
Description
Acquisition SW 6200 series TOF/6500 series
Version Q-TOF B.06.00 (B502)

Compound Table
Mass Sequence Name Target Mass Delta ppm Height MinZ MaxZ Z Count Fit Score Uncertainty Significance Algorithm # Hits Data File Rule
16951.778 Myoglobin (horse) 16951.6073 10.07 201650689 6 18 13 0 0.05 100 Peak Modeling Deconvolution 3 Intact_MyoglobinNewMyo-010012.d Intact protein
Spectrum

Spectrum

Algorithm Mass Sequence Name Target mass Delta ppm MinZ MaxZ Z Count Fit Score Uncertainty Significance Rule
Peak Modeling Deconvolution 16951.778 Myoglobin (horse) 16951.6073 10.07 6 18 13 0 0.05 100 Intact protein

Match protein
Integrate and Generate
Acquire data Deconvolute sequences and
MS Spectrum

extract MS report
predict PTMs MS Spectrum Peak List
m/z Calc m/z Diff(ppm) z Abund
998.1989 998.1704 -28.52 17 23654.5
1060.5234 1060.4931 -28.53 16 64051.1
1131.1578 1131.1255 -28.53 15 121533.45
1211.8829 1211.8483 -28.53 14 159260.59
1305.0272 1304.9899 -28.53 13 169176.91
1413.6955 1413.6552 -28.53 12 153094.25
1542.1217 1542.0777 -28.53 11 129325.84
1696.2332 1696.1848 -28.54 10 76638.5
1884.5917 1884.5379 -28.54 9 32788.9
2120.0398 2119.9793 -28.54 8 13695.6
--- End Of Report ---

Maximum entropy deconvolution provides x105 (a) Maximum Entropy Page 1 of 1 Printed at: 3:13 PM on:3/14/2014

5.5
rapid transformation of multiple charged 5
mass spec data into accurate protein mass. 4.5
The peak modeling (pMod) algorithm 4
3.5
employs additional steps to reduce artifacts 3
and enhance resolution, which helps to 2.5
resolve overlapping peaks and provide 2
1.5
cleaner data, so you can be more confident
1
in your answer. 0.5
0
x105
8 (b) Peak Modeling
7
6
5
4
3
2
1
0
144500 144700 144900 145100 145300 145500
Counts vs. Deconvoluted Mass (amu)

18 Deconvoluted spectra via maximum entropy and pMod algorithms.

ENVISION POWERFUL PEPTIDE MAPPING CAPABILITIES
BioConfirm provides enhanced processing of MS/MS data for streamlined mapping and
data interpretation.

Peptide Mapping Workflow

Match protein Visualize

Extract Generate
Acquire data sequences and sequence
compounds report
identify PTMs coverage

BioConfirm identifies peptides and PTMs x104

based on peptide masses and product ions 1
(b, y, and immonium ions) in the MS/MS 0.9
spectra. This allows for faster and more 0.75
streamlined processing of MS/MS data 0.6
for peptide mapping. 0.45
0.3
0.15
0
200 300 400 500 600 700 800 900 1,000 1,100 1,200
Counts (%) vs. Mass-to-Charge (m/z)

Peptide MS/MS spectrum product ion assignment.

19
 WE PUT THE CAN IN GLYCAN CHARACTERIZATION
Take your pick of fast, fully automated analysis strategies
Glycan 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.

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

Sialic acid

Release APTS
Monosaccharides
glycans
LIF

Oligosaccharides

GlykoPrep

Glycosylation
Glycoprotein Digested site profiling
or mAb glycopeptides (LC/MS/MS) 2-AB label

FLD

Digestion

Intact Glycoform
glycoprotein profiling by
LC/MS

Released glycans with no labeling are also run with MS.

20
GETTING GLYCAN IDENTIFICATION
AND QUANTITATION AT THE SAME TIME
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.

N-glycan identification and quantitation workflow

A B
Instant PC Labeled FLD FLD
Zoom
mAb N-glycans

9 14 19 24 29 34 39 9 14 19 24 29 34 39

C D
Agilent 1290 LC with MS MS
Zoom
Fluorescence Detection

9 14 19 24 29 34 39 9 14 19 24 29 34 39

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
Agilent AdvanceBio mAb 1 glycans. D) Zoom of MS chromatogram of mAb 1 glycans.
Glycan Mapping Column
Intensity (counts)

A
Compound 14
precursor

Agilent 6550 iFunnel

Q-TOF LC/MS 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z
precursor
Intensity (counts)

B
Compound 47

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z
Agilent Mass Profiler
Software
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.

21
 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 x103

and reliable comparison of two samples, 2.4
such as two batches of an engineered
1.8
protein or biosimilars. Samples in the
mirror plot can be switched quickly without 1.2
reprocessing the data. 0.6

0
Monitor biosimilars and -0.6
batch-to-batch variation by
-1.2
quickly comparing samples
-1.8
and reference
-2.4
The comparative analysis module facilitates
the identification and visualization of the -3.0

differences between two batches of data for -3.6

easy inspection. Samples can be compared
to a reference using chromatograms, MS, 147100 1474000 147700 148000 148300 148600 148900
and MS/MS spectra. Counts vs. Deconvoluted Mass (amu)

Mirror plot of two engineered proteins.

1221.9906

x102
0.8
322.0481

0.5
1521.9681
989.5324
850.2981

922.0081

1171.9874
663.4531

1628.9761
1697.1212
363.2525

429.0617

791.8275

0.2
473.2702
473.2723

-0.1 x102
1069.9092

1.05
1221.9907

0.9
544.3041
966.8238

x102 0.75
(M+4H)+4
939.7506

661.3540
661.3466

1217.1748
548.2585

86.0959

0.8 0.6
966.4801
(M+3H)+3

1217.6493
1252.6621

0.45
804.4230
322.0481

360.1914
804.4168

1311.7077

0.5
261.1243
887.4673
1521.9693

1449.7396
242.1548

0.3
360.1884

887.4836
1324.0247
663.4523
363.2529
429.0619

993.2734
762.7855

893.9943

0.2 0.15
-0.1 0
300 450 600 750 900 1050 1,200 1,350 1,500 1,650 100 400 700 1,000 1,300 100 400 700 1,000 1,300
Counts vs. Mass-to-Charge (m/z) Counts vs. Mass-to-Charge (m/z)

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

22
WALKUP ACCESS FOR ALL
Let each of your biologists unleash the power of LC/MS characterization
The 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.

Administrators tailor user Users with different skill levels

access to instrument parameters LC + Q-TOF/TOF/SQ Agilent MassHunter Walkup software perform their own automated
to user access level analyses

Simplified MassHunter Walkup user interface. Three steps to results.

1 2 3
Choose Place Receive
Login
Method Sample Report

Enter username and Enter sample Place sample in the

number samples to information and position as directed
be run. select from a list of by the software.
available methods.

Support virtually any analysis that can be automated.

23
 CE AND CE/MS TO THE RESCUE FOR GLYCANS,
CHARGE VARIANTS, AND PEPTIDES
Agilent solution provides extra power for complex apps
CE/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.

Examples of applications of capillary and microfluidic electrophoresis

Charge heterogeneity analysis by cIEF Glycan analysis by CZE-MS Q-TOF

5.5 x103
80
2.0
70 7.6 7.2 IgG 1.8
60 1.6
50 1.4
1.2
40
mAU

1.0
30 0.8
20 0.6 G2F-1NANA
G2F-2NANA

G2-1NANA
G2-2NANA

10 0.4

G2F
0.2
0
G2
0
-10
10 12 14 16 18 20 22 24 26 28 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Time (min) Counts vs. Acquisition Time (min)
Truly pi-based, high resolution monoclonal antibody charge heterogeneity analysis Mixture of neutral glycan and sialylated glycan separation based on mass-to-charge
by capillary isoelectric focusing ratio using the Agilent 7100 CE system.

Antibody drug conjugate analysis by CE/MS Peptide mapping analysis

×10 4 x105
7.2 = scFv
898.00 = Cy5.5
6.4 29,304.80 30,203.82 7
899.00
5.6
4.8 5
4.0
Counts

3.2
3
2.4
898.00
1.6 31,102.52
1
0.8
28,893.72 29,792.04 32,001.26
0
28,000 28,800 29,600 30,400 31,200 32,000 32,800 13 14 14 16 17 18 19 20 21 22 23 24 25 26 27 28
Deconvoluted mass (amu) Counts vs. Acquisition Time (min)

Deconvoluted mass spectrum of scFv-A conjugate. The assigned structures are based Peptide mapping by CE/MS routinely provides 100 percent sequence coverage and
on deconvoluted mass. is regularly employed as an orthogonal method of LC/MS peptide mapping.
24
 The technology behind Agilent CE/MS

Sample CE capillary Agilent iFunnel MS TOF-MS analysis

Complex mixtures of charged Separation based on mobility Combines Agilent Jet Stream Flight tube with vacuum-insulated
molecules, ranging from in an electrical field takes place (precision sprayer to desolvate shell eliminates thermal mass drift
inorganic ions to native proteins. in 50-100 cm (usually fused and concentrate ions), hexabore due to temperature changes, for
CE offers the smallest injection silica) capillaries filled with capillary (array of capillaries for excellent mass accuracy.
volumes (nL range) with little aqueous buffers. sampling more ions) and dual- Reflectron and long flight tube
sample preparation. stage ion funnel (to efficiently improve mass resolution.
remove gas while focusing ions).

Agilent 7100 CE System Agilent 6550 Accurate-Mass Q-TOF

CE capillary

CE/MS cassette CE-ESI/MS-sprayer HPLC

sheath
First part of the CE/MS interface; Triple-tube interface combines liquid
houses separation capillary and CE capillary with sheath liquid
controls temperature, providing a for stable flow rates and
UV-detection window and an exit electrical contact, and isolates Data analysis
to external detectors. separation chemistry from Nitrogen
nebulizing Integrated software packages
MS ionization. Nebulization
gas control the CE/MS system and
Airstream gas facilitates ESI.
interpret and filter the massive
amount of data created during
UV detection analysis.

MS detection
capillary
on ground
Inlet vial
high voltage

25
 AUTOMATED ELECTROPHORESIS
Quality control of samples has never been so easy
The 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.

Applications
• Antibody QA/QC
• Protein purification
analysis
• Protein expression
analysis

Introducing the 4200 TapeStation System

Agilent 2100 Bioanalyzer system Agilent 4200 Tapestation system

[FU] Reducing conditions Intact

2000 Nonreducing conditions
ngHC
1500 Heavy chain
240
Lower marker
150 1000 Light chain
95 0.0x%
63
46 500
28
15
5 0
5 15 28 46 63 95 150 240 [kDa]

The High Sensitivity Protein 250 kit on the 2100 Bioanalyzer system provides antibody Results can be viewed in familiar gel view or Electropherogram view.
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.

For more information about these systems, visit

www.agilent.com/genomics/bioanalyzer
www.agilent.com/genomics/tapestation

26
A FULL RANGE OF SOLUTIONS
We have what you need for raw material, media, amino acid, and
leachable and extractable analysis via UHPLC, GC, and ICP-MS
It’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.

mAU
mAU Control
Day 1 Control 800
Glutamine

800 Day 1
Day 2 Day 2 600
Day 3
600 Day 3 400
Complete Cell Media
200
Arginine

400
Threonine

0
Alanine
Glycine

-200
Histidine

200 Day 3
Day 2 -400
Day 1
Control 800µM Amino Acid Standard
0 -600
6 6.5 7 7.5 8 8.5 min 0 2 4 6 8 10 12 14 min

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.

Tune Step 1: 0099MPL.D

Mg & AI Ca Fe & Mn Rh (ISTD) Tb (ISTD) Ir (ISTD)
x105 Sc (ISTD) Ga & In (ISTD)
Ge (ISTD)
4 Ti &
Cr Rb &Sr
CPS

2 Ba & REE U
B Zr & Mo Sn Pb & Bi
I
0
50 100 150 200
Mass

The Agilent 7700x ICP-MS is well-suited for analyzing This full mass can of a commercial antacid sample illustrates the screening capability of the Agilent
digested samples and analysis of elemental impurities 7700x ICP-MS in helium mode.
according to USP <232>/<233>.

27
Learn more
www.agilent.com/chem/biologics

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www.agilent.com/chem/store

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www.agilent.com/chem/contactus

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1-800-227-9770
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For Research Use Only. Not for use in diagnostic

procedures. This information is subject to change
without notice.
© Agilent Technologies. Inc. 2017
Printed in the USA, February 13, 2017
5991-5235EN