A New Way to Look at Biomolecules - Waters Corporation · ©2011 Waters Corporation 1 A New Way to...

©2011 Waters Corporation 1

A New Way to Look at Biomolecules

Focused on the unique challenges

and specific needs of biopharmaceutical

development

COMMITTED TO PROVIDING

THE BEST ANALYTICAL METHODS

TO ACHIEVE THE RESULTS YOU REQUIRE


Your Laboratory Requirements

Increase efficiency and reduce costs

Work within regulatory guidelines

Protect IP

Analytical methods capable of reliably

and accurately identifying and measuring

product variants

— Biopharmaceuticals are complex

and heterogeneous

— Potential for multiple product

variants is high

By 2014, five of the top five best-selling

and 50% of the top 100 branded drugs

will be proteins. (Evaluate Pharma)


Your Technology Needs

Accuracy

Sensitivity

Linearity over a broad range

Application-specific column chemistries

Capable of measuring all possible

structural variants of a protein

and higher-order conformation

And enables high efficiency

— Throughput: time to decision

— Maximize use of talent

— Minimize solvent

consumption


Sample Preparation

Separation

Detection

Data Analysis

The Tasks in Your Workflow


Sample PreparationKits & Consumables

UPLC System& Column Technology

Optical Detectors& Mass Spectrometers

Bioinformatics

Your Workflow & Our Technology


2006

Column Manager

Column Heater/Cooler

BEH 300 C18

HSS T3

AccQ•Tag Ultra

SQ Detector

TQ Detector

MS Third Party Control

MassLynx 4.1

PLGS 2.2.5

Amino Acid Analysis

2007

FLR Detector

Improved Column Oven

HSS C18

HSS C18 SB

SYNAPT

SYNAPT HDMS

BioPharmaLynx

Peptide Mapping

2008

Extended Wavelength PDA

Open Access

BEH 300 C4

Xevo TQ MS

BioPharmaLynx1.1

IdentityE

Oligonucleotide Analysis

Intact Protein MS Analysis

2009

Local Console

BEH Amide

Xevo QTof MS

SYNAPT G2

SYNAPT G2 HDMS

BioPharmaLynx1.2

Intact Protein RP Analysis

Glycan Analysis

2010

ACQUITY UPLC H-Class Bio

Column Managers

Auto•Blend Plus

BEH SEC

IEX

Xevo G2 QTof

BioPharmaLynx1.3

Intact Protein SEC and IEX

Analysis

Continuous Technology Evolution


Application-Specific Solutions

UPLC

— AAA

— Peptide Mapping

— Released Glycans

— Intact RP

— Intact SEC

— Intact IEX

— Oligonucleotides

UPLC/MS

— Peptide Mapping


— Intact RP

— HDX

— HCPs


Acquity H-Class Bio

Acquity H-Class

High pressure Quaternary pump

« Flow Through » Injector

Stainless Steel

Corrosion

Unwanted adsoption

Alliage Titane and/or NiCo

Primary & accumulator pump heads

Check-valves

UPLC Valves (Vent, Injector, CM, etc..) &

SSV

Mixer housing, mixer manifold, sinkers

SM Needle Assembly

APH Assembly

UPLC tubing assemblies

PDA & TUV Flow Cells

UPLC performancesBiological solvent and buffer compatibilityBiological separation compatibility


Applications

UPLC


— AAA

— Peptide Mapping


— Intact RP

— Intact SEC

— Intact IEX


DNA

ACQUITY UPLC® OST C18 Up to 60-mer

Acquity PST BEH300 C18, 2.1x50mm, 1.7 µm

from 60-mer


Applications

UPLC


— AAA

— Peptide Mapping


— Intact RP

— Intact SEC

— Intact IEX


Chemistry 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


Ser

Gln

Arg

Gly A

sp

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

Cell Culture Sample

1 day

3 day

6 day


AAA

Cell media dilutionAccQ Tag derivatisationUPLC with AccQ Tag SolventColumn AccQ Tag (2.1X100)mm , 1.7µm

AM

Q

NH

3 His

Se

r

Arg

Gly As

p

Glu T

hr

Ala

Pro

De

riv

Pe

ak

Cy

sL

ys

Ty

r

Me

tV

al

Ile

Le

uP

he

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

0.24

0.26

0.28

0.30

0.32

0.34

0.36

Minutes1.50 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

Monitoring cell culture media


Applications

UPLC


— AAA

— Peptide Mapping


— Intact RP

— Intact SEC

— Intact IEX


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

AU

1.0e-2

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

90 min

55 min

HPLC 2.1 x 250 mm, 3.5 mm

UPLC 2.1 x 150 mm, 1.7 mm

Reduce Run TimeComparable Resolution


Time8.00 10.00 12.00 14.00 16.00 18.00

AU

2.5e-2

5.0e-2

7.5e-2

1.0e-1

1.25e-1

1.5e-1

1.75e-1

2.0e-1

2.25e-1

2.5e-1

2.75e-1

0.25

1.00.5

105.02.0

5020

100

pmol

Peptide QuantitationLinearity


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

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

0.2%

0.5%

1%

2%*

Peptide MapTrace Contaminant


Solution: UPLC Peptide Mapping3-6X Improvement in Productivity

―We were able to get much faster modification analysis with no significant loss in resolution, allowing us to move the samples through the process much faster‖


Separation of PENNY Peptide and its Deamidated Peptides


Peptides mapping

Time8.00 10.00 12.00 14.00 16.00 18.00

AU

2.5e-2

5.0e-2

7.5e-2

1.0e-1

1.25e-1

1.5e-1

1.75e-1

2.0e-1

2.25e-1

2.5e-1

2.75e-1

0.25

1.0

0.5

10

5.0

2.0

50

20

100

pmol

Peptides and PTM’s qualification during development

Peptide (Modified and unmodified) Quantitation during production


Applications

UPLC


— AAA

— Peptide Mapping


— Intact RP

— Intact SEC

— Intact IEX


Murine IgG OligosaccharidesHPLC vs. UPLC BEH Glycan Column

Time30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

EU

x 1

0e4

0.000

5000.000

10000.000

15000.000 G0F

Man6

G2F

Man5

G1F

Time

2 x 150 mm - 3 µm

100.0

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00

EU

x 1

0e4

0.000

2500.000

5000.000

7500.000

10000.000

12500.000

15000.000

35.0

Man5

Man6

G1F

G2F

G0F

2.1 x 150 mm - 1.7 µm

EU

EU


Batch-to-Batch Reproducibility of ACQUITY UPLC®

BEH Glycan Material Using 2-AB Labeled Human IgG N-Linked Glycans

Batch 1

Batch 2

Batch 3

Batch 4

©2009 Waters Corporation | COMPANY CONFIDENTIAL©2011 Waters Corporation

UPLC® SeparationHuman IgG N-linked Glycans

1 G02 G0F3 Man54 G0FGN5 G16 G1Fa7 G1Fb8 G1FGN9 Man610 G211 G2F12 G1F+SA13 G2F+SA

1

4

5

6

78

9

10

11

2

3

12

13

Peak identification was done in LC-MS under same gradient conditions


2-AB Labeled Dextran LadderUPLC Separation

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


New Glycan Found in EPO Exoglycosidase Digestion with UPLC/FLR

Minutes 4 6 8 10 12 14 16 18

GU 5 6 74 8 9 10 11 12 13 14 15

Same digestion pattern and ΔGUs

F(6)A4G4Lac4S4GU ~ 14.8

Dr. Jonathan Bones.

Dublin – Oxford Glycobiology Laboratory,

National Institute for Bioprocessing Research and Training,

University College Dublin.


Glycans analysis

Antibody glycan analysis Structural information with the GU

Release GlycanDerivatisation with 2ABCoilumn ACQUITY UPLC® BEH Amide, 2.1 x 150 mm


Applications

UPLC


— AAA

— Peptide Mapping


— Intact RP

— Intact SEC

— Intact IEX


MeCN

AU

0.00

0.18

0.36

0.54

AU

0.00

0.18

0.36

0.54

AU

0.00

0.18

0.36

0.54

AU

0.00

0.18

0.36

0.54

Minutes

0.00 7.00 14.00 21.00 28.00 35.00

ACN

7:3 IPA:ACN

IPA

MeOH

1. Ribonuclease2. Cytochrome c3. BSA4. Myoglobin5. Enolase6. Phosphorylase b

1

23

45

6

12

34

5

6

1 2

3 45

6

2

34

1

4800 psi

8200 psi

13600 psi

6700 psi

Protein Mix Effect of Solvent on Separation @ 40 ºC

IPA

MeOH


SEC ChromatogramStandards

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

1

2

3

4

5

Analyte pI MW

1. Thyroglobulin, 3 mg/mL 4.6 669,000

2. IgG, 3 mg/mL (Vicam) 6.7 150,000

3. BSA, 5 mg/mL 4.6 66,400

4. Myoglobin, 2 mg/mL 6.8, 7.2 17,000

5. Uracil, 0.1 mg/mL N/A 112


AU

0.00

0.02

0.04

0.06

0.08

Minutes10.00 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 40.00 42.00 44.00 46.00 48.00 50.00

AU

0.00

0.02

0.04

0.06

0.08

Minutes10.00 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 40.00 42.00 44.00 46.00 48.00 50.00

AU

0.00

0.02

0.04

0.06

0.08

Minutes10.00 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 40.00 42.00 44.00 46.00 48.00 50.00

Conventional SEC Column Influence of Ionic Strength on Peak Shape and Retention

Conventional SEC Column4.6 x 300 mm

Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8

10 mM

25 mM

100 mM

lysozyme

lysozyme

lysozyme


ACQUITY BEH200 Influence of Ionic Strength on Peak Shape and Retention

AU

0.00

0.06

0.12

0.18

0.24

Minutes5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

0.00

0.06

0.12

0.18

0.24

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

AU

0.00

0.06

0.12

0.18

0.24

Minutes

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.000.00

0.06

0.12

0.18

0.24

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

ACQUITY BEH200, 1.7 µm, 4.6 x 150mm

Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8

10 mM

25 mM

AU

0.00

0.06

0.12

0.18

0.24

Minutes5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

0.00

0.06

0.12

0.18

0.24

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

100 mM

lysozyme


AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

ACQUITY UPLC BEH200

SEC,1.7 µm

4.6 x 150mm column

Aggregate

10.3%

Detection of Aggregation with SECMonoclonal IgG

Flow rate: 0.4 mL/min; Mobile phase: 25mM Sodium phosphate, pH 6.8, 0.15M NaCl


Column Lifetime: Lysozyme

Flow rate: 0.4 mL/min; Mobile phase: 25mM Sodium phosphate, pH 6.8, 0.15M NaCl

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Minutes

5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

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

HPLC SEC 250Å 4µm4.6 x 300 mm

Injection 19Injection 618

ACQUITY UPLC BEH200 SEC, 1.7 µm4.6 x 150 mm

Injection 19Injection 618


AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

Chimeric Monoclonal Antibody Separation of C- terminal Lysine Variants

KK

K


Protein-Pak™ Hi Res Q Anion ExchangeAnalysis of Chicken Ovalbumin

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

0.130

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00

Grade C

Grade B

Grade A


Protein-Pak™ Hi Res Cation ExchangeComparing Carboxymethyl and Sulfopropyl

AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.02

0.04

0.06

0.08

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

Lysozyme

Ribonuclease A

Cytochrome C

Lysozyme

Ribonuclease A

Cytochrome C

Protein-Pak™ Hi Res SP

Protein-Pak™ Hi Res CM

Conditions- Flow Rate: 1 mL/min; Mobile Phase A: 20mM Sodium Phosphate Buffer, pH 6.6; Mobile Phase B: A + 0.5 M NaCl; Gradient: 0 – 60%B in 34min


AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

IEC : charges differences

SEC

RP : Mapping and quant.

Mapping and quant.

Agregates

Intact protein - agregates – charges state

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

ACQUITY UPLC

BEH200 SEC,1.7

µm

4.6 x 150mm

columnAggregate

10.3%


Auto●Blend

Auto•Blend Technology allows the automatic blending of up to four

solvents in accurate proportions and to run any sequence of

isocratic, binary, ternary, quaternary gradients for routine method

operation, automatic method development, convenient method

transfer, or system flushing

Routine assays become more rugged (less human error)

Water

1%TFAIsopropanol

Acetonitrile Water

1%TFAIsopropanol

Acetonitrile

95%

0%

0%

5%

45%

0%

50%

5%

100% Water

0% Acetonitrile

0.05% TFA

50% Water

50% Acetonitrile

0.05% TFA


Applications

Peptide Mapping with Reversed-phase

Protein Separations

— Reversed-phase

— Ion Exchange

— Size Exclusion

Released glycan analysis


Peptide Map DevelopmentVarying TFA Concentration - %D

AU

0.00

0.05

0.10

0.15

AU

0.10

0.15

0.20

0.25

AU

0.05

0.10

0.15

0.20

Minutes

27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00

0.05% TFA – 5% D

0.1% TFA – 10% D

0.025% TFA – 2.5% D


Conventional Method EditorProgram Percentage Flow


Auto●Blend™ Plus Method Editor Program Required Gradient


Chimeric Monoclonal Antibody,pH Study, Initial 0M NaCl

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

0.0 – 1.0 M NaCl, 20mM Sodium Phosphate in 40 min

0.5 mL/min

pH 6.4

pH 6.6

pH 6.8


Auto●Blend Plus TechnologyA

U

0.00

0.05

0.10

AU

0.00

0.06

0.12

AU

0.00

0.05

0.10

AU

0.00

0.05

0.10

AU

0.00

0.05

0.10

Minutes

0.00 5.00 10.00 15.00

pH 6.1

pH 7.6

pH 7.1

pH 7.0

pH 6.9

A mixture of proteins was separated using cation exchange chromatography- alpha-Chymotrypsinogen A (peak A), Ribonuclease A (peak B), and Cytochrome C (peak C) –

A

A

A+B

A

A

B

B

B

B

C

C

C

C

C


SEC of Humanized Monoclonal AbEffect of AutoBlend Plus pH Adjustment

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

AU

0.00

0.10

0.20

0.30

0.40

0.50

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

A: 17.26%B: 2.74%C: 20.00%D: 60.00%

A: 2.74%B: 17.26%C: 20.00%D: 60.00%

pH6.0

pH7.6

125mM NaH2PO4

125mM Na2H2PO4

1000mM NaCL

H2O


CONCLUSION

ACQUITY H-Class Bio System designed for the application

Complete system solution including column chemistry with

system

Auto●Blend™ and Auto●Blend Plus™ Technology for

convenience and efficiency

A New Way to Look at Biomolecules - Waters Corporation · ©2011 Waters Corporation 1 A New Way to...

Documents

Transcript of A New Way to Look at Biomolecules - Waters Corporation · ©2011 Waters Corporation 1 A New Way to...