Principles and Practices for SEC, IEX for Intact Protein ... · 3 cv gradient 1 2 57 ©2012 Waters Corporation 14 Minutes Longer gradient: increased resolution, lower sensitivity

UPLC User meeeting April 2012UPLC User meeeting April 2012

Principles and Practices for SEC, IEX for Principles and Practices for SEC, IEX for Intact Protein Analysis by UPLC Intact Protein Analysis by UPLC

[email protected][email protected]

©2012 Waters Corporation 1

AgendaAgendagg

Ion-Exchange Chromatography– Theory and practice

P t i P k Hi R IEX C l – Protein-Pak Hi Res IEX Columns – Method Development Strategies

Size-Exclusion Chromatographyg p y– ACQUITY UPLC for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7 µm ColumnsInsulin Analysis– Insulin Analysis

– Combination of SEC and MS


IonIon--Exchange ChromatographyExchange Chromatographyg g p yg g p y

Binds at Low Ionic Strength Elute with Step or Continuous Gradients of Increasing Ionic Gradients of Increasing Ionic Strength

Separations are based on net surface charge on protein with oppositely charged groups on ion-exchanger


charged groups on ion exchanger

Proteins elute from column using either a gradient of increasing salt concentration (most common) or changing pH (less common)

Select buffer pHSelect buffer pHIsoelectric Point of a Protein (pI)Isoelectric Point of a Protein (pI)(p )(p )

Isoelectric Isoelectric point (pI)Zero net

charge at this gpH


Select buffer pHSelect buffer pHIsoelectric Point of a Protein (pI)Isoelectric Point of a Protein (pI)(p )(p )

pH below pIp pProtein has net +ve charge pH region cation exchange

pH above pIP t i h t h


Protein has net -ve chargepH range for anion exchange

Ion Exchange Chromatography Ion Exchange Chromatography BuffersBuffers

Select buffer with pKa near to desired pH

Buffer ions should have same charge as functional groups on packing material (PO4

- for cation, Tris+ for anion)


packing material (PO4 for cation, Tris for anion)

Common Customer ConcernsCommon Customer Concerns

Reproducibility between columns

Not getting required resolution from the start

Recovery and carryover


ProteinProtein--Pak Hi Res IEXPak Hi Res IEX


Attributes of ProteinAttributes of Protein--Pak™ Hi Res Pak™ Hi Res IEX ColumnsIEX Columns

Multi-layered network of ion-exchange groups (SP, CM or Q)

Effective diffusion and binding o Effective diffusion and binding

o High sample loading and resolution

o Minimal non-desired interactions

No MW limitations: non-porous material

QC tested with protein samples for batch-to-batch reproducibility

High chemical stability: hydrophilic, polymer-based IEX particles

– Wide pH range (3- 10)

– high salt concentrations (1M)

St d d ( t 1450 i f CEX d 2175 i f AEX) – Standard pressures (up to 1450 psi for CEX and 2175 psi for AEX)

– Can be cleaned with aggressive washing

eCord enabled for data tracking


Strategies to Developing an Strategies to Developing an IonIon--Exchange Protein SeparationExchange Protein Separationg pg p

Selectivity is most conveniently optimized with pH

Retention is optimized by adjustment of ionic strength

Changing buffer and counter ion may improve selectivity

Methods may require adjustment if the temperature is changed Methods may require adjustment if the temperature is changed


Effect of pH on SelectivityEffect of pH on Selectivity

0.040

0.050

p yp y

α-Chymotrypsinogen 1

Ribonuclease A 2pH 6.6

1 3

AU

0.020

0.030cytochrome c 3

2

0.000

0.010

0.050

1 3

AU

0.020

0.030

0.040

pH 5.0

2

0.000

0.010

Minutes4.00 6.00 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


Column: Protein-Pak Hi Res CM 4.6 x 100 mm column

Fine tune pH Fine tune pH pH Effect on mAb SeparationpH Effect on mAb Separationp pp p

0.006

0.008 pH 6.4

AU

0.000

0.002

0.004

0.006

AU

0 000

0.002

0.004

0.006 pH 6.6

0.000

AU 0.005

0.010 pH 6.8

A

0.000

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00


Column: Protein-Pak Hi Res CM 4.6 x 100 mm column Gradient: 0.0 –0.10 M NaCl, 20mM Sodium Phosphate in 40 min Flow: 0.5 mL/min

Effect of Salt Gradient Slope Effect of Salt Gradient Slope % 24.00

36.00

48.00

pp

Ovalbumin 1

Myoglobin 20-0.15M NaCl

Elutes in high salt wash step1

3

25

28 00

0.00

12.00

Minutes3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80

Ribonuclease A 3

Cytochrome C 4

Lysozyme 51

3

2

4

%

7.00

14.00

21.00

28.00

0-0.3M NaCl

3

4 5

0.00

Minutes3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80

36.00

48.00

Unbound proteins

1 24 5

%

0.00

12.00

24.00

Minutes0.00 3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80

0- 0.5M NaCl3


Minutes

Higher salt gradients result in earlier elution of bound proteins High salt wash may be needed in shallower gradients to elute tightly bound proteins Column: Protein-Pak Hi Res CM 4.6 mm x 100mm

Effect of Salt Gradient Slope:Effect of Salt Gradient Slope:Ovalbumin Variants Ovalbumin Variants

8 10 cv gradient

AU 0.02 1 2

3 4

56

79

10

0.00

AU 0.02

5 cv gradient

3 45

6

7

0.00

1 25 7

3 4

6

AU

0.00

0.02

Min tes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

3 cv gradient1 2

3 45 7


Minutes

Longer gradient: increased resolution, lower sensitivity

Column: Protein-Pak HI Res Q, 4.6 x 100 mm

Effect of Buffer on SelectivityEffect of Buffer on Selectivity

0.025

yy

α-Chymotrypsinogen 1

Ribonuclease A 2

20mM Sodium Phosphate

1 2

3

AU

0.010

0.015

0.020 cytochrome c 32

0.000

0.005

20mM MES (Morpholino ethane13

AU

0 010

0.015

0.020

0.02520mM MES (Morpholino ethaneSulfonic acid)

12

0.000

0.005

0.010

Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00


Buffer can alter selectivity and retention of proteins at same pH (6)

Column: Protein-Pak Hi Res CM 4.6 x 100 mm column

Counter Ion EffectsCounter Ion Effects

Ribonuclease A 1

cytochrome c 2

AU 0.010

0.020 12

3

Aprotinin 3

0.000

0020

NaCl

AU

0 000

0.010

0.020

KCl0.000

Minutes14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00

C t i h l ti it d t ti f t i


Counter ion may change selectivity and retention of proteins

Effects tend to be minimal

Temperature EffectsTemperature Effectspp

45 °C

AU 0.005

40 °C

0.000

AU 0.005

35 °C

A

0.000

U 0 005

30 °C

AU

0.000

0.005

KKK

Temperature may effect selectivity and retention

AU

0.000

0.005

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

KKK


p y y Changes may be similar to those observed with pH change Column: ProteinP-ak Hi Res CM 4.6 x 100 mm column

IEX SummaryIEX Summaryyy

Protein-Pak Hi Res IEX column benefits

– Consistent batch-to-batch performance (tested with protein p ( pstandards)

– Minimal column related carryover

– Stable over a wide pH rangeStable over a wide pH range

Method Parameters to optimize areS l ti it i t i tl ti i d ith H– Selectivity is most conveniently optimized with pH

– Retention is optimized by adjustment of ionic strength

– Changing buffer and counter ion may improve selectivity

– Methods may require adjustment if the temperature is changed


AgendaAgendagg

Ion-Exchange Chromatography– Theory and practice

P t i P k Hi R IEX C l – Protein-Pak Hi Res IEX Columns – Method Development Strategies

Size-Exclusion Chromatographyg p y– ACQUITY UPLC for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7 µm ColumnsInsulin Analysis– Insulin Analysis

– Combination of SEC and MS


Common Customer ConcernsCommon Customer Concerns

Column-to-column reproducibility

– Changes in retention timeChanges in retention time

– Changes in spacing between peaks

– Changes in resolution

Column lifetime

– Peak shape deteriorates over time

– Increased pressureIncreased pressure

– Changes in resolution

Tailing of specific proteins

Resolution

Throughput


UPLCUPLC--SEC vs HPLCSEC vs HPLC--SECSECof mAb monomer and aggregatesof mAb monomer and aggregatesgg ggg g

0.065

0.070

0.065

0.070

HPLC 100% ACQUITY BEH200

0.045

0.050

0.055

0.060

0.045

0.050

0.055

0.060Silica-Diol

SEC 250Å 5µm7.8 x 300 mm

ACQUITY BEH200 SEC, 1.7 µm4.6 x 300mm

AU

0.030

0.035

0.040

0.045

AU

0.030

0.035

0.040

0.015

0.020

0.025

0.015

0.020

0.025

2.26 % Aggregate 2.24 %

Aggregate

0.000

0.005

0.010

0.000

0.005

0.010


Minutes2.00 4.00 6.00 8.00 10.00

Minutes5.00 10.00 15.00 20.00 25.00 30.00

8.00 30.008.00 30.00

Effect of LC System Dispersion onEffect of LC System Dispersion onBEH200 SEC mAb SeparationBEH200 SEC mAb Separationpp

Larger system dispersion decreases component resolution

0.20

0.30 BEH200 SEC 1.7umColumn (4.6 x 300mm)

HPLC System

USP Res= 1.37

AU

0 00

0.10

0.00

0.20

0.25 Waters ACQUITY UPLC SystemUSP Res= 2.37 BEH200 SEC 1.7um

AU

0.05

0.10

0.15 Column (4.6 x 300mm)


0.00

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

ACQUITY BEH200 SEC 1.7 µm ACQUITY BEH200 SEC 1.7 µm ColumnsColumns

Application Areas

– Determination of protein molecular weightDetermination of protein molecular weight

– Molecular weight range of 10,000 to 450,000 Daltons

– Determination of size heterogeneity in a protein sample

– Quantitation of protein aggregates primarily in therapeutic monoclonal antibodies.


BEH OverviewBEH Overview

The packing material is based on our patented Bridged Ethyl Hybrid base particle and effective diol bonding, which provide a y p g, pstable chemistry with minimal secondary interactions.


BEH200BEH200 SEC, SEC, 1.7um1.7umColumn Batch Test Column Batch Test

0.22

Analyte pI MW

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

0.16

0.18

0.20

2

4

y g , g/ ,

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

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

U

0.12

0.14 35

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

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

A

0.06

0.08

0.10

1

0.02

0.04

0.06


0.00

Minutes0.00 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

BEH200 SEC, 1.7um BEH200 SEC, 1.7um BatchBatch--toto--Batch ReproducibilityBatch Reproducibilityp yp y

AU

0 00

0.05Batch 1, Column 1

0.00

AU

0 00

0.05Batch 1, Column 2

0.00

AU

0.00

0.05 Batch 1, Column 3

AU

0.00


AU

0.00



Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

What’s new ? What’s new ?

BEH 125 SEC UPLC Column– 15 cm/30 cm/Guard15 cm/30 cm/Guard

– Launched on January 10th

– Linear range from 1.000 to 80.000 Dalton

Reference Description

186006504 ACQUITY UPLC BEH125 SEC 1.7µm 4.6x30 Gd

186006505 ACQUITY UPLC BEH125 SEC 1.7µm 4.6x150mm

186006506 ACQUITY UPLC BEH125 SEC 1.7µm 4.6x300mm


Calibration Curves for Calibration Curves for ACQUITYACQUITY UPLCUPLCBEH200BEH200 and and BEH125BEH125, SEC, 1.7 , SEC, 1.7 μmμm ColumnsColumns

BEH200, SEC, 1.7um

Thyroglobulin (~ 669,000 Da)

IgG (~ 150,000 Da)

Rnase A (~ 13,700 Da)

Conalbumin (~ 75,000 Da)

Ovalbumin dimer(~ 88,000 Da)

O lb i ( 44 000 D )

Aprotinin (~ 6,500 Da)

Ovalbumin (~ 44,000 Da)

BEH125, SEC, 1.7um Uracil (~ 112 Da)

Angiotensin II (~ 1,045 Da)


Resolution of Proteins and Peptides Resolution of Proteins and Peptides pp

1.50ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm1.501.50ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm

AU

0 00

0.50

1.00

AU

0 00

0.50

1.00

AU

0 00

0.50

1.00

0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

0.80

1.00BioSuite125 UHR SEC 4.6 x 300mm

A2

14

0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

0.80

1.00

0.80

1.00BioSuite125 UHR SEC 4.6 x 300mm

A2

14

AU

0.00

0.20

0.40

0.60

AU

0.00

0.20

0.40

0.60

AU

0.00

0.20

0.40

0.60

- -

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min


- BEH125 column provides increased resolution throughout the lower end of the

peptide mass range (132 29,000).

Insulin Analyses by TraditionalInsulin Analyses by TraditionalHPLCHPLC--SEC vs UPLCSEC vs UPLC--SECSEC

Waters Alliance HPLCSystemInsulin HMWP SEC 10 μm(7.8 x 300mm)

Waters ACQUITY UPLCSystemBEH125, SEC, 1.7 μm (4.6 x 300mm)


Column Stability for Insulin Analysis Column Stability for Insulin Analysis

Retention Time USP Resolution

y yy y

3

4

5

6

ime

n)

3.0

4.0

5.0

utio

n

0

1

2

3

Ret

entio

n T

(mi n

0.0

1.0

2.0

USP

Res

olu

0 100 200 300 400 500 600 700 800 900

Injection Number

Over 800 injections the retention time of the insulin monomer peak


and the resolution between insulin monomer and dimer peaks are maintained.

What about SEC – MS ?


SECSEC--MSMSHumanized Monoclonal AntibodyHumanized Monoclonal Antibodyyy

UV @ 2801 2 2

1.4e-2

1.6e-2

1.8e-2

2: Diode Array 280 0.0500Da

Range: 6.757e-125.27

1UV @ 280

AU

4.0e-3

6.0e-3

8.0e-3

1.0e-2

1.2e-2

16.58

2

3

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 17000.0

2.0e-313.22

19.50

1: TOF MS ES+ TIC

7.58e619.493

TIC

%

15.35

16.62

1 2

Scan500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

4

23.7425.06


MS: Xevo G2 Q Tof Conditions: 100mM Ammonium Formate, Flow rate: 0.15 mL/min on BEH200 15 cm Post UV detection additive: ACN, 0.8% Formic acid

Extracted SpectrumExtracted SpectrumHerceptin 50%ACN, .4% FA_100mm Amm Form_0.15 mL/min_40CV_AutoQua

1007Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 907 (15.351) Sm (SG, 10x5.00); Sb (15,2.00 ); Cm (891:932) 1: TOF MS ES+

4.34e33448.14043025.98782907.2991

2797.63792601.3782

2471.37262353.8545

3530.13483706.4951

3801.68433901.6064

3905.8140

pp

Intact IgG MW 148 221

m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800

%

0

2353.5356 4118.3599

7Oct11 PH SEC BEH200 Ext T ACN pt8FA 2 5 982 (16.619) Sm (SG, 10x5.00); Cm (969:996) 1: TOF MS ES+

MW 148,221Peak 1

%

100_ _ _ _ _ _ _p _ _ ( ) ( , ); ( )

1.97e3

2652.85842648.5669

2460.41241251.3574

2801.4185

2968.89673154.9690

3370.08893616.4006

ClipMW 100,764Peak 2

100

m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800

0

7Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 1152 (19.493) Sm (SG, 10x5.00); Cm (1116:1184) 1: TOF MS ES+ 1.22e41537.7053

1489.6832

1478 2386

1643.7091

1702.38311765 3279

Peak 2

%

1478.2386 1765.3279

1985.9363

2056.27692166.3523 2382.8904

2647.5525

Low MW SpeciesPeak 3


m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800

0

Deconvoluted molecular weight determined using MaxEnt1

Summary: Waters Summary: Waters ACQUITYACQUITYUPLCUPLC SEC System SolutionSEC System Solutionyy

New SEC column chemistries based on BEH particlesp

True UPLC separation

Application benefits

– Reduced secondary interaction– Reduced secondary interaction

– Improved physical and chemical column lifetime

– Improved column-to-column reproducibility

I d l i– Improved resolution

– Improved throughput

Synergistic combination of UPLC system and column

Higher throughput compared to traditional HPLC


MultiMulti--Mode ChromatographyMode ChromatographyAutomated with ACQUITY UPLC HAutomated with ACQUITY UPLC H--Class Bio Class Bio Mouse Ascites FluidMouse Ascites Fluid

0.24

0.26

0.28

0.30

SEC

ACQUITY UPLC BEH200 AU

0.10

0.12

0.14

0.16

0.18

0.20

0.22 ACQUITY UPLC BEH200 4.6x150mm0.5mL/min20mM Na phosphate, pH 6.8 150mM NaCl

0.00

0.02

0.04

0.06

0.08

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 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

0.070

0.075

0.080

0.085

0.090

0.095

0.100 Cation Exchange

0.032

0.034

0.036

0.038

0.040

0.042

0.044

0.046

0.048

Anion Exchange

Protein-Pak Hi Res SP 4.6x100mm

Protein-Pak Hi Q4.6x100mm0.5mL/min

AU

0 020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

AU

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

4.6x100mm0.5mL/min20mM phosphate pH 60-250mM NaCl20mins

20mM TrispH 7.50-250mM NaCl20mins


-0.005

0.000

0.005

0.010

0.015

0.020

Minutes0.00 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 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00

-0.002

0.000

0.002

0.004

0.006

0.008

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00

Interesting Application Notes/postersInteresting Application Notes/posterson IEX and SECon IEX and SEC

IEX Method Development of a Monoclonal Antibody and Its Charge Variants 720003836en on www.waters.comg

Method Development for Size-Exclusion Chromatography of Monoclonal Antibodies and Higher Order Aggregates 720004076en on www waters com720004076en on www.waters.com

Multi-Mode Analytical Separations of Proteins 720003854en on www.waters.com

Improving the Lifetime of UPLC Size-Exclusion Chromatography Columns Using Short Guard Columns 720004034en on www.waters.com

Technology Brief with SEC and IEX guidelines 720004182en on www.waters.com


Technology Brief on SEC with MS 720004018en on www.waters.com

Looking for more info ?Looking for more info ?

www.waters.com/biosep

gg

www.waters.com/biopharm

p


Documents

Principles and Practices for SEC, IEX for Intact Protein ... · 3 cv gradient 1 2 57 ©2012 Waters Corporation 14 Minutes Longer gradient: increased resolution, lower sensitivity