analysis of biomolecules by sec and iex uplc. · pdf fileanalysis of biomolecules by sec and...

Analysis of biomolecules bySEC and Ion-Exchange UPLC

Anders Feldthus Waters Nordic

©2011 Waters Corporation 1

Anders Feldthus, Waters Nordic

Waters Commitment

To develop, commercialize and market columns that when used on Waters ACQUITY UPLC® systems, give the speed, sensitivity, resolution and method reproducibility that has not been previously resolution, and method reproducibility that has not been previously achieved for the characterization of biological macromolecules with traditional HPLC.


Agendag

Ion-Exchange Chromatography— Theory and practice Theory and practice — Protein-Pak Hi Res IEX Columns — Method Development Strategies

o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio SystemSystem

Size-Exclusion Chromatography— Theory and practice— ACQUITY UPLC for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution— Ways to Maximize SEC Column LifeWays to Maximize SEC Column Life


Ion-Exchange Chromatographyg g p y

Na +

Cl -Na +Cl -

Anion-Exchange

Na +

Na +Cl -

Cl

Na +

Cl -Na +Cl

Cl -

Anion-Exchangeg

Particle

Cl -Na +

Cl -

Na +

Na +Cl -Cl -

Cl -

gParticle

Elute with Step or Continuous

Na +Na +

Cl -

Binds at Low Ionic StrengthElute with Step or Continuous

Gradients of Increasing Ionic StrengthSeparations are based on net surface charge on protein with oppositely charged groups on ion-exchanger


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

Ion-Exchange Gradient: Starting pointg p

Sample High Equilibration Injection Gradient Salt Re-Equilibration

~ 5 cv

Volume Wash

unbound proteinsproteins

10-20 cv

5-10 cv 5-10 cv


Typical linear gradient for ion-exchange chromatography

Ion-Exchangeg

Advantages Limitations

S ti b h d Moderate resolution

Concentrating technique

Separation by charge and charge distribution; insensitive to other properties

— Can load large volumes of a dilute sample For salt gradients, time-

consuming pH optimization required for best resolution

Non-denaturing protein elution techniques

required for best resolution.

Fractions may need to be desalted prior to the next

Preferred first step when a cost-effective affinity purification is not available

desalted prior to the next purification or characterization step (e.g., Mass Spectrometry)


Protein Isoelectric Points and IEX

ParticleSupport

ParticleSupport


Strong versus Weak Ion Exchangersg g

Strong and weak DO NOT indicate how tightly the protein is bound

A t h i l i i d hil k h ’ A strong exchanger is always ionized while a weak exchanger’s ionization varies with pH

— Weak Cation Exchange column- pka 3.5-4.5

— Weak Anion Exchange column – pka ~9

Strong exchangers are recommended where there is a need to run at H ( id i )extreme pH (wider operating range)

The ion exchange capacity of a weak ion exchanger varies with pH

— Sample loading (binding) capacity can vary with pH due to loss of charge from the exchanger


Selectivity differences exist between weak and strong exchangers— Binding constants are a function of the IEX functional group

Agendag

Ion-Exchange Chromatography— Theory and practice y p

— Protein-Pak Hi Res IEX Columns

— Method Development Strategies

o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio gy QSystem

Size-Exclusion Chromatography— Theory and practice

— ACQUITY UPLC for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution


Protein-Pak Hi Res IEX


Attributes of Protein-Pak™ Hi Res IEX 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


Batch to Batch Reproducibility:Protein-Pak Hi Res Q Column

0.035

Q

Std Dev %RSD1 - Myoglobin 0.06 1.282 T f i 0 03 0 53

Retention Time

0.025

0.0301

2 3

2 - apo-Transferrrin 0.03 0.533 - Trypsin Inhibitor 0.03 0.27

AU

0.015

0.020 Batch A

0 005

0.010

Batch C

Batch B

0.000

0.005

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Batch C


Minutes

Reproducible retention time observed for multiple batches

Carryover

0.16

y

1st Repeat Gradient

2nd Repeat Gradient

0.12

0.14

Gradient Gradient

AU

0 06

0.08

0.10

0.02

0.04

0.06

Expected Elution Points

0.00

Minutes0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00 150.00 160.00


No detectable carryover of lysozyme (100 µg injection)

Ion-Exchange Analysis of Antibodies on Weak Cation Exchange Column

AU0.020

0.030

0.040

gA

0.000

0.010

0.020

Humanized IgG1

AU0.010

0 0 0

Humanized IgG2

0.000

U

0.010

0.015

AU

0.000

0.005

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Chimeric IgG1


Minutes

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

Confirmation of c-terminal Lysine Variants of mAb

0 00 Untreated mAb

AU 0.002

0.003

0.004 Untreated mAb

KKK

0.000

0.001

0.008

AU

0.002

0.004

0.006 mAB treated with carboxypeptidase B, 30 °C, 20 minutes

0.000

Minutes24.00 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 58.00


IEX can be used to confirm the presence of mAb lysine variantsColumn: Protein-Pak Hi Res CM, 4.6 x 100mm

Agendag







o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution— Ways to Maximize SEC Column Life


Strategies to Developing an Ion-Exchange Protein Separationg p

Retention is optimized by adjustment of ionic strength

Selectivity is most conveniently optimized with pH

Changing buffer and counter ion may improve selectivity

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


Affect of Salt Gradient Slope % 24.00

36.00

48.00

p

0-0.15M NaCl

Elutes in high salt wash step1

3

25 Ovalbumin 1

Myoglobin 2

28 00

0.00

12.00

Minutes3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80

1

3

2

4 Ribonuclease A 3

Cytochrome C 4

Lysozyme 5

%

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 proteinsHigh salt wash may be needed in shallower gradients to elute tightly bound proteinsColumn: Protein-Pak Hi Res CM 4.6 mm x 100mm

Affect of Salt Gradient Slope: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


Retention is optimized by adjustment of ionic strengthRetention is optimized by adjustment of ionic strength



Methods may require adjustment if the temperature is changed


Effect of pH on Selectivity

0.040

0.050

p y

pH 6.6 α-Chymotrypsinogen 1

Ribonuclease A 2

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

Effect of Buffer on Selectivity

0.025

y

20mM Sodium Phosphate α-Chymotrypsinogen 1

Ribonuclease A 21 2

3

AU

0.010

0.015

0.020 cytochrome c 32

0.000

0.005

20 M MES1

3

AU

0 010

0.015

0.020

0.025 20mM MESMorpholino 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 Effects

NaClRibonuclease A 1

cytochrome c 21

2

AU 0.010

0.020Aprotinin 3

13

0.000

0.020KCl

AU

0.000

0.010

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 Effectsp

45 °C

AU 0.005

40 °C

0.000

AU 0.005

35 °C

0.000

AU 0.005

30 °C

A

0.000

U 0 005 KKK

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


Temperature may effect selectivity and retentionChanges may be similar to those observed with pH changeColumn: ProteinP-ak Hi Res CM 4.6 x 100 mm column

Waters Auto•Blend Plus™ Technologygy

12.5% 100mM NaH 2PO 4

12.5% 100mM Na 2HPO 4

25mM Sodium Phosphate, 150mM NaCl pH 6.8

15% 1000 mM NaCl

60% H20NaCl


Auto•Blend Plus™ Technology

Enter Buffer

Enter Buffer System

Concentration

Enter pH andSalt Gradient


Salt Gradient

Effect of pH on Selectivity pH 6

0.020

1 2

p y

pH 6 0

Ribonuclease A 1

cytochrome c 2

3

AU

0.000

0.010

1 3

20.020

pH 6.0Aprotinin 3

2

1 32

AU

0.000

0.010 pH 6.2

2

1 3

AU

0.000

0.010

0.020

pH 6.4

1 3

2

AU

0.000

0.010

0.020

pH 7.2


Minutes10.00 15.00 20.00 25.00 30.00 35.00

pH can be used to alter selectivity of proteinsColumn: Protein-Pak Hi Res CM 4.6 x 100 mm column

AutoBlend Plus Buffer Management forProtein Ion Exchange Method Developmentg p

Fluid Path of ACQUITY UPLC H-Class Bio System configured for multi-buffer use

— The optional solvent select valve provides 6 selectable choices that are associated with particular method names.

— The proportions of the four selected reservoirs are calculated with Auto•Blend Plus™ Technology to meet the requirements of the method using the defined buffer system specified with the method


meet the requirements of the method, using the defined buffer system specified with the method.

— The four physical reservoirs, labeled A, B, C, and D1-D6 can be assigned as needed as the Acid, Base, Salt , and Aqueous components.

Enhanced Method Development viaAutoBlend Plus and Automated Column ManagementManagement

d l f h


Automated Column Management for Protein Ion Exchange.

The method specifies the column to be used and the bypass (B) permits matching the buffer to the column.

Use of AutoBlend Plus and Automated Column Switching for mAb Separation Method Scouting

0.055

0.060

i k i

AU

0.020

0.025

0.030

0.035

0.040

0.045

0.050 Protein-Pak™ HiRes Q

0.000

0.005

0.010

0.015

0.050

0.055

0.060

AU

0.015

0.020

0.025

0.030

0.035

0.040

0.045

Protein-Pak™ HiRes SP

0.000

0.005

0.010

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 25.00 26.00 27.00 28.00


IEX Summaryy

Total system solution with combination of ACQUITY UPLC H Class Bio System and column

— Auto•Blend Plus™ Technology allows for change of pH and ionic strengths without preparation of different buffers

— Simplifies methods developmentp p

Protein-Pak Hi Res IEX column benefits

— Consistent batch-to-batch performance (tested with protein — Consistent batch-to-batch performance (tested with protein standards)

— Minimal column related carryover

Stable over a wide pH range— Stable over a wide pH range


Agendag









Principles of Size ExclusionChromatography of Proteins

Separates proteins by their size in solution (Stokes radius)

Separations are Isocratic

Tends to be used as a “Polishing” isolation step or as an analytical technique to determine presence of protein aggregates

Generally a “lower resolving” technique compared to other methods such as ion-exchange or reversed-phase methods


Size Exclusion Separation of Proteinsp


Common 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 pressure—Increased pressure

—Changes in resolution

Tailing of specific proteins

Resolution

Throughput


Agendag







o ACQUITY BEH200 SEC, 1.7 µm Columns— Factors Influencing Component Resolution— Ways to Maximize SEC Column Life


Advantages of UPLC Technologyfor SEC Separations

Requires Columns and Instrumentation to Minimize Band Spreading

Waters UPLC®HPLC

p

Narrow PeakIncreased Sensitivity

Waters UPLC®

TechnologyBroad BandBroad PeakLess Sensitivity

HPLC

Increased Resolving Power

Less SensitivityLess Resolving Power


Effect of LC System Dispersion onBEH200 SEC mAb Separationp

Larger system dispersion decreases component resolution

0.20

0.30 BEH200 SEC 1.7um Column (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

UPLC-SEC vs HPLC-SECof mAb monomer and aggregatesgg 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

ACQUITY BEH200 SEC 1.7 µm ColumnsQ µ

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 Overview

The packing material is based on our patented Bridged Ethyl H b id b ti l d ff ti di l b di hi h Hybrid base particle and effective diol bonding, which provide a stable chemistry with minimal secondary interactions.


BEH200 SEC, 1.7um Column Batch Test

0 20

0.22

Analyte pI MW

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

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

0.16

0.18

0.20

2

4

2. IgG, 2 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

0.12

0.14 35

0.06

0.08

0.10

1

0.02

0.04


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 Calibration BEH200 SEC, 1.7um Calibration CurveCurve

10000000

Analyte MWBlue Dextran 2,000,000Thyroglobulin 669,000β Amylase 200 000

100000

1000000IgG (~150,000 Da)

β-Amylase 200,000IgG, 150,000Amyloglucosidase 97000Conalbumin 75000BSA 66,400Ovalbumin 44 000

10000

100000

MW

Ovalbumin 44,000Carbonic Anhydrase 29,000Myoglobin 17,000Lysozyme 14,400Ribonuclease A 13,700Aprotinin 6 500

100

1000

Aprotinin 6,500Uracil 112

10

100

1 1.5 2 2.5 3 3.5 4 4.5 5


Elution Volume

Protein Adsorption and SizeProtein Adsorption and Size--Exclusion Exclusion ChromatographyChromatographyg p yg p y

Proteins can interact or adsorb onto the SEC packing material

These interactions create undesired and unpredictable retention of proteins (i.e. proteins not separated by size in p ( p p ysolution)

SEC particles frequently coated with a hydrophilic reagent toSEC particles frequently coated with a hydrophilic reagent tominimize non-desired ionic interactions between proteins and packing material

Mobile phase additives (e.g., 150mM NaCl) may decrease non-desired ionic interactions between proteins and packing

l


material

DIOL coating used to minimizenon-desired ionic interactionsbetween proteins and packing material

Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins

p p g

SEC

-

SEC Particle


DIOL Loss increases non-desired ionic interactions between proteins and packing material packing material

SEC Particle

-

Particle


Comparative SEC Column Life

0.60

0.70

HPLC 100% Silica-DiolLysozyme, pKi = 10.7

AU

0 20

0.30

0.40

0.50 SEC 250Å 4µm4.6 x 300 mm

Injection 19

Suggestive of DIOL Bleed

0.00

0.10

0.20

Minutes5.50 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

0.22

jInjection 618

AU

0.10

0.12

0.14

0.16

0.18

0.20

ACQUITY BEH200 SEC, 1.7 µm4.6 x 150 mm

I j ti 19

0.00

0.02

0.04

0.06

0.08

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

Injection 19Injection 618


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 shows minimal secondary interactions even after 600 injections

NaCl will decrease ionic interactions between proteins and packing material

Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins

p p g

Na +

Cl -

Na + Cl -

Na +

Na +

Cl - Cl -

SEC Na +

Na +

Na +

-

ParticleNa + Cl -

Na +

Na +

Na +

Cl

Cl -Cl -

Na +


Cl -

Na + Cl -Cl -

Influence of Ionic Strength on Peak Shape and Retention on Silica-based SEC

0 06

0.08

Conventional 100% Silica-Diol CoatedSEC Column 4.6 x 300 mm 10mM

N Cl

and Retention on Silica based SECA

U

0.00

0.02

0.04

0.06

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

NaClLysozyme, pKi = 10.7

Minutes

AU

0.04

0.06

0.08 25mMNaCl

lysozyme

0.00

0.02

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

0 08

lysozyme

AU

0 00

0.02

0.04

0.06

0.08

100mMNaCllysozyme


0.00

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

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

Influence of Ionic Strength on Peak Shape and Retention on BEH200 SEC

AU

0 12

0.18

0.24

0 12

0.18

0.24 ACQUITY BEH200 SEC 1.7 µm Column, 4.6 x 150mm

10mMN Cl

and Retention on BEH200 SEC A

0.00

0.06

0.12

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

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

NaClLysozyme, pKi = 10.7

AU

0.12

0.18

0.24

0.12

0.18

0.24

25mMNaCl

0.00

0.06

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

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

0.240.24

AU

0.06

0.12

0.18

0.06

0.12

0.18 100mMNaCl

©2011 Waters Corporation 52Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8

0.00

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

0.005.00 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

Agendag









Factors Influencing Resolutiong

Resolution decreases with increasing volume and mass l dloads

Resolution decreases with increasing flow rateId l fl i l h i ll i h —Ideal flow rate is lower than typically running, however run time will be longer

l h l l hResolution increases with column length

Baseline resolution typically achieved at 50%-100% molecular weight difference—Proteins with less than 100% molecular weight difference

may not be acceptably resolved


Mass Loading Capacity onBEH200 SEC, 1.7um 4.6 x 150mm,

3.125 µg Total LoadRs = 1.7

400 µg Total LoadRs = 1.5

0 010

0.012

0 60

0.70

BSA

BSA

0.012 0.70

AU 0.006

0.008

0.010

AU

0.40

0.50

0.60

BSA dimer BSA dimer

0.002

0.004

0.10

0.20

0.30BSA dimer

0.000

Minutes1.00 2.00 3.00 4.00 5.00 6.00

0.00

Minutes1.00 2.00 3.00 4.00 5.00 6.00


Constant injection volume, 20 µL Resolution decreases for mass loads of 3- 400 µg

Volume Load Capacity onBEH200 SEC, 1.7um 4.6 x 150mm

1.60

,

Injection Volume

USP Resolution (Monomer-Dimer)

1.00

1.20

1.40 5 1.70

10 1.54

15 1.43

20 µL

AU

0.60

0.8020 1.27

5 µL

0 00

0.20

0.405 µL

0.00

Minutes1.80 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

Constant BSA concentration, 20 mg/mL


Resolution decreases for higher injection volumePoor peak shape observed at higher volumes

Effect of Column Length on mAb Rsg

0.015 BEH200, SEC, 1.7um4 6 x 150 mm

20.1% Aggregate

AU

0.005

0.0104.6 x 150 mm

USP Res= 1.44

0.000

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

0.020

BEH200 SEC 1 719 9%

AU

0 005

0.010

0.015BEH200, SEC, 1.7um4.6 x 300 mm

19.9% Aggregate

USP Res= 1.72

0.000

0.005

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00


Comparable aggregate quantitation Human IgG (Sigma), (load: 10 µg - 150mm; 20 µg -300mm): TUV: 280 nm

Effect of Flow Rate on mAb Rs

AU 0.010

0.020

0.030

0.4 mL/min BEH200, SEC, 1.7um4.6 x 150 mm

0.000

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

0.030

AU 0.010

0.020

0.030

AU 0.010

0.020

0.030

AU

0.000

0.010

0.0200.35 mL/min

0.000

Minutes2.00 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

0.000

Minutes2.00 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

0 020

0.030

Minutes2.00 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

AU

0.000

0.010

0.020

Mi t3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

0.2 mL/min


Triplicate injectionsNo observable trend in aggregation with flow rate

Minutes

Effect of Temperature onmAb SEC Peak Shapep

45 °CBEH200, SEC, 1.7um4 6 x 150 mm

0.63

0.70

0.7740 °C

4.6 x 150 mm

AU

0.35

0.42

0.49

0.56

30 °C

0.14

0.21

0.28

0.00

0.07

2.10 2.45 2.80 3.15 3.50


Chimeric IgG 0.4 mL/min, 25 mM Sodium Phosphate, 0.15m NaCl, pH 6.8

Effect of Salt Cation on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shapep

Different cations of chloride salt additive


Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil

Agendag









BEH200 SEC, 1.7um Care and Use:(Ways to extend column life)( y )

Preparation of SEC Mobile Phase and Needle Wash— Pre filter through 0 2 or 0 45 um filter (i e Don’t inject particulates)— Pre filter through 0.2 or 0.45 um filter (i.e, Don t inject particulates)— Use high purity water— Replace mobile phases weekly and do not “top off”

Use of BEH200 SEC, 1.7um Guard Column

Attention to SEC Eluent SinkersAttention to SEC Eluent Sinkers— Use titanium sinkers NOT stainless steel— Sinkers can be major source of bacterial contamination

o Consider occasional sinker replacement or 70% alcohol “pull through” to prevent problems

Column Storage Considerations- Overnight: Continuously flush with the mobile phase at 10 - 20% of the


g y pmaximum recommended flow rate- Extended: Store in the HPLC grade water with 20% methanol

BEH200 SEC, 1.7um Guard E(4.6 x 30mm) Extends UPLC SEC Column Life


QC Protein Standards Mix onBacterial Contaminated, BEH200 SEC Column

BEH200, SEC, 1.7um4 6 x 150 mm4.6 x 150 mm


Summary:Waters ACQUITY UPLC SEC System SolutionQ y

New SEC column chemistry based on BEH particles

T UPLC tiTrue UPLC separation

Application benefits

— Reduced secondary interaction

— Improved physical and chemical column lifetime

— Improved column-to-column reproducibility

— Improved resolutionp

— Improved throughput

Synergistic combination of UPLC system and column

Higher throughput compared to traditional HPLC Higher throughput compared to traditional HPLC


analysis of biomolecules by sec and iex uplc. · pdf fileanalysis of biomolecules by sec and...

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