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©2014 Waters Corporation 1

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Today’s SpeakerToday’s Speaker


Bill Warren has been with Waters Corporation for more than 20 years,

having worked in both technical and marketing capacities. He is

currently responsible for strategic and tactical implementation of

programs that support new bioseparations products and technologies

which help accelerate customer productivity in the biopharmaceutical

market segment.


Applications of UPLCApplications of UPLCBioseparationsBioseparations

Time30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00

AU

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

8.0e-2

9.0e-2

30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00

AU

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

6.5e-2

7.0e-2

7.5e-2

HPLC

UPLC

AM

Q

NH

3 His S

er

Arg

Gly A

sp

Glu T

hr

Ala

Pro

De

riv

Pe

ak

Cy

sL

ys

Ty

rM

et

Va

l

Ile

Le

uP

he

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Amino Acid Analysis

40T

2 minutes 11

50T

60T

UV 260 nm

Oligonucleotides1 G02 G0F

6

11

2


Peptide MappingOligonucleotides2 G0F

3 Man54 G0FGN5 G16 G1Fa7 G1Fb8 G1FGN9 Man610 G211 G2F12 G1F+SA13 G2F+SA

1

4

5

78

9

10

3

12

13

Glycan Analysis

cytochrome c

MonoclonalAntibody

BSAMyoglobin

Enolase

Phosphorylase b

Protein Reversed Phase

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Protein Size Exclusion

AU

0.000

0.002

0.004

0.006

0.008

AU

0.000

0.002

0.004

0.006

AU

0.000

0.005

0.010

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

pH 6.4

pH 6.6

pH 6.8

Protein Ion Exchange

Topics

� Customer Requested Attributes for Bioseparation Columns

� What Goes into Obtaining “Improved” LC-Based Bioseparations

� Concerns with UPLC to HPLC Method Transferability

� Synthetic Oligonucleotide Analyses

� Amino Acid Analyses

� Peptide Analyses


� Peptide Analyses

� Protein Analyses

� Released Glycan Analyses

Customer Requested Attributes for Customer Requested Attributes for BioseparationBioseparation IEX ColumnsIEX Columns

Type Votes

Weighted

Average Rank

Resolution 91 2.07 1

Column-to-column reproducibility 83 2.52 2

Batch-to-batch reproducibility 79 2.53 3

Volume and mass loading capacity 73 2.64 4

Separation speed 80 2.70 5


Source:Waters Web Survey

(4,760 contacted / 196 responses)

QC tested with biomolecules (e.g. proteins) 56 2.75 6

Column lifetime 77 2.92 7

Price 73 2.96 8

Non-metallic hardware 55 3.00 9

Matching guard column 53 3.28 10

Other 21 3.48 11

The weighted averages were calculate by: ((#5's*5)+(#4's*4)=(#3's*3)+(#2's*2)+#1's)/5

RED: Particles and System Synergy

GREEN: Particles and Manufactury Synergy

How to Build Resolution:Efficiency with Selectivity


Late 1970’s10µ Irregular micro-porous

1000-2500 psi25,000 plates/meter

3.9 x 300mm

Particle Size EvolutionParticle Size Evolution

Early 1970’s40µ pellicular non-porous coated

100-500 psi1000 plates/meter

1m columns

10 min


10 min

1980’s to present day5 – 2.5µ spherical micro-porous

1500-4000 psi50,000 - 80,000 plates/meter

3.9 x 150mm

10 min

10 min

eÉáÖÜí=bèìáî~

äÉåí=íç

=qÜÉ

çêÉíáÅ~ä=mä~íÉ

içïÉëí=ebqm=Z[=léíáãìã=mä~íÉ=`çìåí

ebqm=======mi q̂bp

`=qÉêã

Particle Size and Flow RateParticle Size and Flow Ratevan van DeemterDeemter EquationEquation

A term + B term + C term

H = a(dp) + b + c(dp)2uu


^=qÉêãEm~êíáÅäÉ=ëáòÉ=~åÇ=Üçï=ïÉää=ÄÉÇ=

ï~ë=é~ÅâÉÇF

eÉáÖÜí=bèìáî~

äÉåí=íç

=qÜÉ

çêÉíáÅ~ä=mä~íÉ

iáåÉ~ê=sÉäçÅáíó

ebqm

u ôÅãLëÉÅõ

e

`=qÉêãEj~ëë=íê~åëÑÉêF

_=qÉêãEiçåÖáíìÇáå~ä=aáÑÑìëáçåF

^ÇÇ=íÜÉ=P=íÉêãë=íç=çÄí~áå=Ñáå~ä=/î~å=aÉÉãíÉê=`ìêîÉÒ

Chromatography PrinciplesChromatography PrinciplesMass Transfer / DiffusionMass Transfer / Diffusion

Analyte Molecules

Mobile Phase

A

Adsorption Equilibria


Porous

ParticleB

CDiffusion-related band broadening

Smaller ParticlesSmaller ParticlesThe enabler of productivityThe enabler of productivity


Where Does Band Spreading / Where Does Band Spreading / System Dispersion Occur?System Dispersion Occur?


Band Spreading: 1) From the Injector2) Into, through and out

of the column3) Into the Detector

Extra ColumnWithin Column

Band Spreading, Peak Height Band Spreading, Peak Height and Resolutionand Resolution

LC systems (column and instrument) capable of producing narrower/sharper bands create narrower/sharper peaks

This results in better resolution, taller peaks and better sensitivity

System withMORE

System with LESS


Better separationMore concentrated “Bands”Higher Sensitivity

Both analytes (blue and red) are not separated [a partial co-

elution – shown as a “purple” band]

MOREBand Spreading

LESSBand Spreading

Narrow PeakIncreased SensitivityIncreased Resolving

Power

UPLC®

TechnologyBroad BandBroad Peak

Less SensitivityLess Resolving Power

HPLC

Impact of Band Spreading on Resolution


Requires Columns and Instrumentation to Minimize

Band Spreading

Sources of Band Spreading –Improper Column Connection

Band SpreadingDead / Void Volume

Improper

Resulting Peak Shape


Proper

PackedBedOf

Particles

No Dead Volume

HPLC HPLC vsvs UPLC SEC Analysis of UPLC SEC Analysis of Insulin Insulin DimerDimer vsvs MonomerMonomer


HPLC HPLC vsvs UPLC ReversedUPLC Reversed--Phase GradientPhase GradientAnalysis of Analysis of DerivitizedDerivitized Amino AcidsAmino Acids

4 µm Particles


1.8 µm Particles

UltraPerformance LCUltraPerformance LC®® TechnologyTechnology

� A class of separation science

– Based on chromatography columns with very small particles

– Based on instruments designed and manufactured to take advantage

of the small particles

� Improves resolution, sensitivity, and speed with no


� Improves resolution, sensitivity, and speed with no

compromises to results

� Suitable for chromatographic applications in general

– Appropriate for developing new methods

– Appropriate for improving existing methods

– SCALEABLE CHEMISTRIES FROM UPLC TO HPLC IF NEEDED

Importance of Method TransferabilityImportance of Method TransferabilityFrom Drug Discovery, Development and From Drug Discovery, Development and ManufacturingManufacturing


Ethylene Bridged Hybrid (BEH) Ethylene Bridged Hybrid (BEH) Particles TechnologyParticles Technology

Bridged Ethanes within a silica matrix

©2014 Waters Corporation 21U.S. Patent No. 6,686,035 B2

Importance of Batch to Batch and Importance of Batch to Batch and Column to Column ReproducibilityColumn to Column Reproducibility


Over 30 tests per batch of media totaling over 300 QC response factors

ApplicationApplication--specific Columnsspecific ColumnsACQUITY UPLC BEH Glycan ColumnACQUITY UPLC BEH Glycan Column

Chromatographic Testwith

Biomolecule Standards

Chemical Tests

Individual Column Tests


Manufacturing ConsistencyManufacturing ConsistencyACQUITY UPLC Glycan BEH Amide Columns ACQUITY UPLC Glycan BEH Amide Columns

ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm


Quality control testing ensures consistent analyses

UPLC and HPLCUPLC and HPLC--based,based,22--AB Labeled Glycan AnalysesAB Labeled Glycan Analyses

XBridge BEH Glycan 2.5 µm XP

ACQUITY BEH Glycan 1.7 µm

EU

0.00

2.00

4.00

6.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

1 2

4

3

5

6

7

8910

11

12

14

20.00

Alliance HPLC

UPLC

13

Pc*half-height = 110

Pc*half-height = 78

2.1 x 150 mm0.50 mL/min

2.1 x 150 mm

UPLC-based

HPLC-based

8700 psi (Column, Max)



EU

0.00

10.00

20.00

15.00 20.00 25.00 30.00 35.00 40.00

XBridge BEH Glycan 3.5 µm

EU

0.00

5.00

10.00

Minutes25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Alliance HPLCPc

*half-height = 55

2.1 x 150 mm0.34 mL/min

2.1 x 150 mm0.24 mL/min

HPLC-based



50.0

20

25

% Abundance

1.7 um, 0.50 mL/min)

2.5 um XP, 0.34 mL/min

3.5 um, 0.24 mL/min

UPLC UPLC vsvs HPLC Relative Abundance HPLC Relative Abundance DeterminationsDeterminations

1

2

3

4

5

6,7

8

9

10

11

12

13

14

15,16


n=30

5

10

15

Peak 1 G0-GN

Peak 2 G0

Peak 3 G0F

Peak 4 Man5

Peak 5 G0FN

Peak 6 G1F

Peak 7 G1F

Peak 8 G1FN

Peak 9 Man6

Peak 10 G2

Peak 11 G2F

Peak 12 G2FN

Peak 13 G1FS1

Peak 14 G2FS1

Peak 15 A3

Peak 16 A3

% Abundance

*Peaks 8 and 9 – 3.5 µm resolution insufficient, accuracy of the integration is poor

Influence of ExtraInfluence of Extra--Column Band Column Band Spreading on Gradient SeparationsSpreading on Gradient Separationswith 1.6um Particleswith 1.6um Particles

12000

14000

16000

18000

20000

Efficiency (Plate Count)

Waters ACQUITY UPLC I-ClassN = 18,315

Waters ACQUITY UPLC H-ClassN = 14,072

Dionex Ultimate

Agilent 1290N = 9,392

CORTECS UPLC C18+2.1 x 50 mm, 1.6 µm


0

2000

4000

6000

8000

10000

0 5 10 15 20 25 30 35 40 45 50

Efficiency (Plate Count)

Band spreading (µL, 5 sigma)

Journal of Chromatography A, 1216 (2009) 5979–5988

Dionex Ultimate 3000

N = 10,974

N = 9,392

Shimadzu NexeraN = 6,913

Agilent 1200N = 6,266

Waters Alliance 2695

N = 2,704

Gradient offset aligns chromatograms Gradient offset aligns chromatograms Different instruments and dwell volumeDifferent instruments and dwell volume

* *

No Gradient Offset Programmed Gradient Offset

HPLC HPLC


Retention Time (min)

10 15 20 25 30 35 40 45


10 15 20 25 30 35 40 45

H-Class Bio

Sample: Waters MassPrep PepmixInjection Vol: 95 µlSolvent A: H2O with 0.1% TFASolvent B: MeCN with 0.1% TFA

H-Class Bio

Peptide Mapping of complex separationsPeptide Mapping of complex separationsTrypsinizedTrypsinized RibonucleaseRibonuclease B B

AU

0.2

0.4

0.6

1

4

7

10

11 12

14

15

13

16

17

18 21

2223 24

25

26

2728

29

30

31

32

A

*

Sample: Ribonuclease BPeptidase: Trypsin V5111Injection Vol: 95µlSolvent A: H2O with 0.1% TFASolvent B: MeCN with 0.1% TFA

Relative Retention Time Comparison

HPLC


Minutes

10 15 20 25 30 35 40

0.0

AU

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1

2 356

7

89 19 20 33

1

2 3

4

56

7

89

1011 12

14

15

13

16

17

18

19 20

21

2223 24

25

26

27

2829

30

31

32

33

B

*

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33

Rela

tiv

e R

ete

nti

on

Tim

e

Peak Number

HPLCH-Class Bio

Relative Retention Time Comparison

H-Class Bio

Peptide Mapping of complex separations Peptide Mapping of complex separations TrypsinizedTrypsinized InfliximabInfliximab

8 15

23

26

29

33

3436

37

38

48

49

51

AU

0.00

0.05

0.10

0.15

AU

0.06

0.08

0.10

0.12

12

34

5 6

7

8

9

10

13

14

15

1617

1819 20

21

22

23

24

25

26

27

28

29

30

3132

33

34

35

36

373839

40

41

42

4344

4546

47

48

4950

51

5253

54 55

56

1112

Sample: InfliximabPeptidase: Trypsin

V5111Inj. Vol: 95µlSolvent A: H2O Solvent B: MeCNSolvent C: 1% TFASolvent D: H2O

H-Class Bio

H-Class Bio

HPLC


�A total of 56 peptide peaks were selected for

monitoring

�Approximately 90 peaks were identified within the

chromatographic space.

12

3

4

56

7

8

9

10

1112

13

14

15

16

17

1819 20

21

22

24

25

27

28

30

31

32

34

35

3638

3940

41

42

4344

4546

47

49

5052

53

54 55

56

AU

0.02

0.04

0.06


12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

UPLCUPLC®® Technology Technology


UPLCUPLC Technology Technology

for Synthetic for Synthetic

Oligonucleotide Oligonucleotide

SeparationsSeparations

� Principles of Ion-Pair, Reversed-Phase Chromatography for

Synthetic Oligonucleotides

– Ion-pairing systems

– Performance, optimization, MS compatibility

� Oligonucleotide Separation Technology (OST)



– Importance of sorbent particle size

Retention Mechanism of “Retention Mechanism of “TritylTrityl--Off” Off” ChromatographyChromatography

RP with ion-pairing agent

- charge-charge interaction (oligo backbone)

- hydrophobicity (nucleobases)

Reversed-phase interaction

- hydrophobicity of the nucleobases

1520

2535

3015

2025

30


+

+++ --

-

-

--

--

+C18 sorbent

0 minutes 100 minutes 10

35

TEA+

TEA+ layer on the column surface

Several Different IonSeveral Different Ion--Pairing Reagents Pairing Reagents Available for Synthetic Oligo RP SeparationsAvailable for Synthetic Oligo RP Separations

Ion pairing agent buffering acid Abbreviation

Triethylammonium acetate TEAA

Triethylammonium bicarbonate TEAB

Dimetylbutylammonium acetate DMBAA


Tributylammonium acetate TBAA

Tripropylammonium acetate TPAA

Hexylammonium acetate HAA

Triethylammonium hexafluoroisopropanol TEA-HFIP

Triethylammonium Acetate Reagent

UV 260 nm 24

11 12 13 14 1516+17

18+19

20

21 22

23

25

28

29

30

Comparison of TEAA vs. TEAComparison of TEAA vs. TEA--HFIP forHFIP forthe IPthe IP--RP Separation of the same RP Separation of the same 30mer Oligo Synthesis Mixture30mer Oligo Synthesis Mixture


Triethylammonium HFIP Reagent

0 30Minutes

13 14 15 16

18

20

2122

26

29

25

28

30

27

24

23

19

171211

10 32Minutes

UV 260 nm

T

G

GG

C

G

A

TTT

C

C

TGTTAAGT

AgendaAgenda

� Principles of Ion-Pair, Reversed-Phase Chromatography for

Synthetic Oligonucleotides

– Ion-pairing systems

– Performance, optimization, MS compatibility



– Importance of sorbent particle size

Importance of Sorbent Particle Size:Importance of Sorbent Particle Size:Smaller C18 particles Yield Improved IPSmaller C18 particles Yield Improved IP--RP RP Synthetic Oligo SeparationsSynthetic Oligo Separations

TEA-HFIP, pH 7.9at 0.2 mL/minat 60°C 5 µm

3.5 µm


BEH, C18 Particles: 2.1 x 50mm column, 15-60T ladder

8 minutes 28

3.5 µm

2.5 µm

UV 260 nm

Ultimate Separation Performance Obtained Ultimate Separation Performance Obtained using 1.7um Particles with using 1.7um Particles with Waters UPLC® TechnologyWaters UPLC® Technology

40T50T

60T

UV 260 nm

1.7 µmTEA-HFIP, pH 7.9at 0.2 mL/minat 60°C


BEH, C18 Particles: 2.1 x 50mm column, 15-60T ladder

2 minutes 11

UV 260 nm

Example of an IPExample of an IP--RP, MS Analysis of a RP, MS Analysis of a

21 mer Synthetic RNA Mixture21 mer Synthetic RNA Mixture

5`5`--UUC UGU AAU CUC UUG UCU ATT UUC UGU AAU CUC UUG UCU ATT --3`3`


UPLCUPLC®® TechnologyTechnology


UPLCUPLC TechnologyTechnology

for Amino Acid for Amino Acid

AnalysisAnalysis

Challenges for Amino Acid Analysis

� Difficult analytical problem

– Separation

o Wide range of properties

o Slight differences between pairs

o Wide range of matrices

– Detection


– Detection

o No chromophore

o Wide concentration range

� Analytical method requirements

– Unequivocal identification, accuracy

– Precision and linearity

– Sensitivity and speed

– Rugged and robust method

Chemistry of AQC DerivatizationChemistry of AQC Derivatization


� Reacts readily with both primary and secondary amines

� Forms stable derivatives

� Requires no vacuum drying, sample prep or extraction

� Amendable to automation

HPLC and UPLCHPLC and UPLC®® Amino Acid Amino Acid Analysis Methods Analysis Methods

AM

Q

Asp

Ser

Glu

Gly

His

NH

3

Arg

Thr

Ala

Pro

Cys

Tyr

Val

Met

Lys

ILe

Leu

Phe

HPLC

50 MinuteCycle Time


AM

Q

NH

3

His

Ser Arg

Gly

Asp G

lu

Thr

Ala P

ro

Cys

Lys

Tyr

Met

Val

NV

a

ILe

Leu

Phe

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Minutes

8.00 10.00 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 38.00

UPLC

10 MinuteCycle Time

UPLC® Application Solution for Amino Acid Analysis

� Turn-key UPLC® Amino Acid Analysis application

– Optimized for the ACQUITY UPLC System

– Dedicated QC-tested column and reagents

– Application-specific Performance


– Application-specific Performance Qualification

– Same result day-to-day, instrument-to-instrument, lab-to-lab, around the world

� Target Applications

– Protein and peptide ID and quantitation

– Monitoring cell culture media

Solution: UPLC™ AAA SolutionSolution: UPLC™ AAA Solution

AM

Q

GA

BA H

yL

ys1

HyL

ys2

Orn

Deri

v P

eak

Cys

Lys

Tyr

Met

Val NV

a

Ile

Leu

Ph

eT

rp

AU

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.075

0.080

0.085

0.090

0.095

0.100


NH

3H

yP

roH

is Asn Tau

Ser

Gln

Arg G

ly Asp G

lu Th

r

Ala

GA

BA

Pro

HyL

ys1

HyL

ys2

AA

BA

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

• 7X improvement in throughput

•Eliminated need for outsourcing

•Same day turnaround of samples

“I was totally amazed that we were able to separate this set of amino acids in nine minutes when, not long ago, it took several hours.” VP, Process Development

Amino Acid Analyses of Amino Acid Analyses of Cell Cell Culture SampleCulture Sample

6 day


Ser

Gln

Arg

Gly Asp

Glu

Thr

Ala

Minutes

2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

1 day

3 day



UPLCUPLC TechnologyTechnology

for Peptide for Peptide

MappingMapping

“Ideal” Protein Digestion

IntactProtein Complete Digestion

without generation of modified peptides


The Reality of Many Protein Digestions

IntactProtein

Complete Digestion withoutgeneration of modified peptides


Non-specificCleavages

IncompleteDigestion

Non-Cleavage

MiscleavagesHydrophobic

Protein that may havesolubility issues

0.0015

0.002

0.0025

250 µµµµL/min2.1 mm Column

25 µµµµL/min2.1 mm Column

Plate Height (H

)

3.5 µµµµm

Increase in chromatographic

van Deemter Plot1500da Peptide


0

0.0005

0.001

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Linear Velocity (mm/sec)

Plate Height (H

)

1.7 µµµµm

chromatographicresolution

AU

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

6.5e-2

7.0e-2

7.5e-2 HPLCPeak Capacity

= 372

HPLC 2.1 x 250 mm, 3.5 µm

Improve ResolutionSame Run Time


Time30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00

AU

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

8.0e-2

9.0e-2

30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00

1.0e-2

1.5e-2

2.0e-2

UPLCPeak Capacity

= 723

UPLC 2.1 x 150 mm, 1.7 µm

AU

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

90 min

HPLC 2.1 x 250 mm, 3.5 µm

Reduce Run TimeComparable Resolution


Time20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00

AU

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

8.0e-2

9.0e-2

1.0e-1

Time30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00

1.0e-2

2.0e-2

55 min

UPLC 2.1 x 150 mm, 1.7 µm

Peptide MapPeptide MapTrace ContaminantTrace Contaminant

2.0e-1

22.39

15.54

34.17

29.65

28.60

23.86


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

9.75

18.00

23.86

24.7127.94

32.84

*

9.5e-2

1.0e-1

1.05e-1

1.1e-1

1.15e-1

1.2e-1

1.25e-1

1.3e-1

1.35e-1

1.4e-1

Peptide MapPeptide MapTrace ContaminantTrace Contaminant


Time28.30 28.35 28.40 28.45 28.50 28.55 28.60 28.65 28.70 28.75 28.80 28.85 28.90 28.95 29.00 29.05

AU

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

6.5e-2

7.0e-2

7.5e-2

8.0e-2

8.5e-2

9.0e-2

0.2%

0.5%

1%

2%**

UPLCUPLC®® Technology Technology


UPLCUPLC Technology Technology

for Protein for Protein

SeparationsSeparations

Challenges for Protein SeparationsChallenges for Protein Separations

� Requires the detection of small chemical differences between

quite large molecules

� Employs a variety of analytical techniques that are sensitive to

a different property of the proteins

– Size exclusion for changes in size or aggregation

– Reversed-phase for detecting a wide range of small changes


– Reversed-phase for detecting a wide range of small changes

– Ion-exchange for changes in net charge

Carbohydrate Groups

Hydrophobic Regions

Disulfide Linkages

Aromatic Groups

HydrogenBonding

Net Charge

HPLC HPLC vsvs UPLC SEC Analysis of UPLC SEC Analysis of Insulin Insulin DimerDimer vsvs MonomerMonomer



for Glycans Analysisfor Glycans Analysis


for Glycans Analysisfor Glycans Analysis

UPLC UPLC vsvs HPLC Analysis ofHPLC Analysis of22--AB Labeled NAB Labeled N--Linked GlycansLinked Glycans

6

3

6

3

Glycan Release

Glycoprotein

6

3

6

3

Oligosaccharides


6

3

6

3

Protease Digestion

Glycopeptides

Glycoprotein Oligosaccharides(Glycans)

Monosaccharides

Separation of 2Separation of 2--AB Labeled IgG on AB Labeled IgG on ACQUITY UPLC Glycan BEH AmideACQUITY UPLC Glycan BEH Amide(HILIC) Column(HILIC) Column


SummarySummary

� Holistic approach to separation science

– UPLC® improves resolution, sensitivity, and speed

– Application specific chemistries

– Low dispersion system

– Better separation benefits downstream detection methods

o UV or PDA

o FLR

o Mass Spectrometry


� UPLC is an innovative technology that can be routinely applied to all aspects of biotherapeutic proteins analysis

– Synthetic Oligonucleotides

– Amino Acid Analysis

– Peptide Separations

– Intact Proteins

– Glycan Analysis

� Waters recognizes need for both UPLC and HPLC Bioseparations Solutions and hasofferings and tools to assist in Worldwide Implementation of selected method(s).

Thank You for Attending!Thank You for Attending!

� Post-Event Landing Page…

� www.waters.com/June12

– 30% Promotional Offer On BioSeparations Columns

– Full Webinar Recording of Today’s Session w/PDF Slide

Deck

– Compilation of TODAY’S KEY Literature, Brochures etc…


– Compilation of TODAY’S KEY Literature, Brochures etc…

� For Questions and to Submit your Ideas for our Next Topic

– Please eMail - [email protected]

Documents

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