webinar: the size-exclusion uplc analysis of biomolecules ...€¦ · this webinar will be...

©2012 Waters Corporation 1

Webinar:

The Size-Exclusion UPLC Analysis of Biomolecules

and the Introduction of a New 450A Column

Waters Corporation

November 29, 2012

Thank you for joining us! The Webinar will begin shortly.


Introduction

About today’s presenter:

This webinar will be presented by Stephan M. Koza, Ph.D. Stephan is a

Principal Applications Chemist within the Chemistry Applied Technology

group at Waters in Milford, MA. Stephan specializes in chromatography

and mass spectrometry as applied to the analysis and characterization

of proteins, peptides, and other biomolecules.

Friendly Reminders… Please use text chat functionality to submit questions during the Webinar. Opportunity for audience participation…Please submit answers ‘live’ during our session to see results Upon conclusion, follow up information will be available:

Recorded version Product specific information Promotional material


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

achieved for the characterization of biological macromolecules with

traditional HPLC.


Liquid Chromatography Protein Separation Modes

Protein Structure

Primary, Secondary, Tertiary Structure

Carbohydrate Groups

Hydrophobic Regions

Disulfide Linkages

Hydrophilic Groups

Aromatic Groups

Net Charge

Quaternary Structure and Aggregation


Agenda

Size-Exclusion Chromatography

– Theory and practice

– ACQUITY UPLC Columns for SEC

o ACQUITY UPLC SEC 200Å, 1.7 µm Columns



– Keys to Method Development

– Selected Applications

– Tips & Tricks


Principles of Size Exclusion Chromatography of Proteins

Separates proteins by their size in solution (Stokes radius)

Separations are Isocratic

Generally a “lower resolving” technique compared to other

methods such as ion-exchange or reversed-phase methods

Tends to be used as a “Polishing” isolation step or as an

analytical technique to determine presence of protein

aggregates


Size Exclusion Separation of Proteins

Principles of Size Exclusion Chromatography of Proteins


SEC vs. Other LC Methods: It’s all about Entropy

ΔG˚ = -RT lnK = ΔH˚- TΔS˚

LC SEC

KLC ≈ ℮ KSEC ≈ ℮ ΔS˚/R -ΔH˚/RT

• ΔH˚ is usually negative for sorption, therefore KLC > 1 • Peaks elute in CV ≥ 1 • High Peak capacities

• ΔS˚ is negative since solute mobility decreases inside pores, therefore KSEC < 1 • Peaks elute in CV ≤ 1 • Limited Peak capacities


Monoclonal Antibodies

Antibody Conjugates

Fc Fusion Proteins

Synthetic Oligonucleotides

Protein Subunit Vaccines

Recombinant Proteins and Peptides

Synthetic Peptides

Common SEC applications: Biotherapeutics Types

Aggregation

Aggregation of proteins may either reveal new epitopes or leads to the formation of

multivalent epitopes, which may stimulate the immune system. …… It is important to

monitor the aggregate content of a product throughout its shelf life.

EMEA: GUIDELINE ON IMMUNOGENICITY ASSESSMENT OF BIOTECHNOLOGY-DERIVED THERAPEUTIC PROTEINS


Agenda



– ACQUITY UPLC Systems and Columns for SEC






– Tips & Tricks


Why UPLC?

0

10

20

30

40

50

60

70

0.00 0.05 0.10 0.15 0.20 0.25

Pla

te H

eig

ht

(µ

m)

Linear velocity, ui (cm/s)

H vs u for IgG

Mobile Phase: 100 mM sodium phosphate, pH 6.0. Column configurations: 4.6mm ID (4µ), 4.6mm ID ACQUITY UPLC BEH200 SEC (1.7µ) Flow rate range: 0.1-1.0 mL/min

4µm

1.7µm


HPLC to UPLC SEC Comparison

Murine monoclonal antibody - Scaled load

Conditions: 0.4 mL/min; 25mM Sodium Phosphate, pH 6.8, 0.15 M NaCl

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2.26 % Aggregate 2.24 %

Aggregate

HPLC 100% Silica-Diol

SEC 250Å 5µm 7.8 x 300 mm

ACQUITY UPLC BEH200 SEC,1.7 µm

4.6 x 300mm

8.00 30.00 8.00 30.00


Requires Columns and Instrumentation to Minimize Band Spreading

Broad Band Broad Peak Less Sensitivity Less Resolving Power

HPLC

Advantages of UPLC Technology for SEC Separations

Narrow Peak Increased Sensitivity Increased Resolving Power

Waters UPLC®

Technology


Effect of System Dispersion on ACQUITY UPLC BEH200 SEC 1.7 µm separation

Large system dispersion decreases resolution

Sample: Human polycolonal IgG

Flow Rate: 0.2 mL/min

ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm

UPLC-SEC

ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm

HPLC-SEC

USP Res= 1.37

USP Res= 2.37

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ACQUITY UPLC BEH200 and BEH125 SEC 1.7 µm Columns

Application Areas

– Molecular weight ranges dependent on pore size:

o ACQUITY UPLC SEC 125Å: 1K to 80K Daltons

o ACQUITY UPLC SEC 250Å: 10K to 450K Daltons

o ACQUITY UPLC PSEC 450Å, 100K to 1800K Daltons

– Determination of protein / peptide molecular weight (size)

– Quantitation of protein / peptide aggregates primarily in

therapeutic monoclonal antibodies, EPO, and Insulin

– Determination of size heterogeneity in a sample


Calibration Curves of ACQUITY UPLC BEH450, BEH200, and BEH125 SEC Columns


Protein Adsorption and Size-Exclusion Chromatography

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 solution)

SEC particles frequently coated with a hydrophilic reagent to

minimize non-desired ionic interactions between proteins and

packing material

Mobile phase additives (e.g. NaCl) decrease non-desired ionic

interactions between proteins and packing material


BEH SEC Particle Overview

The packing material is based on our patented Bridged Ethyl

Hybrid base particle and effective diol bonding, which

provide a stable chemistry with minimal secondary

interactions.


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Influence of Ionic Strength on Peak Shape and Retention

Conventional 100% Silica-Diol Coated SEC Column 4.6 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


Influence of Ionic Strength on Peak Shape and Retention

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ACQUITY BEH200 SEC 1.7 µm column, 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

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100 mM

lysozyme


Lysozyme, pKi = 10.7

Suggestive of DIOL Bleed

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HPLC 100% Silica-Diol SEC 250Å 4µm 4.6 x 300 mm Injection 19 Injection 618

Comparative SEC Column Life (pH 6.8, 150 mM NaCl)


Lysozyme, pI = 10.7


BEH200 shows minimal secondary interactions even after 600 injections

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ACQUITY BEH200 SEC, 1.7 µm 4.6 x 150 mm Injection 19 Injection 618

Lysozyme, pKi = 10.7


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HPLC 100% Silica-Diol SEC 250Å 4µm 4.6 x 300 mm Injection 19 Injection 618


Lysozyme, pI = 10.7

Comparative SEC Column Life (pH 6.8, 150 mM NaCl)


ACQUITY UPLC BEH200 SEC, 1.7 um Column Lifetime

Humanized monoclonal antibody

Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm

Column: 4.6 x 300 mm

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Injection 2

Injection 497

Dimer = 0.46% USP Res = 2.35

mAb

mAb

Dimer = 0.49% USP Res = 2.27

Dimer: 300K

Monomer: 150K

Fragment: 100K

Fragment: 50K


BEH200 SEC, 1.7um Batch-to-Batch Reproducibility


Agenda









– Tips & Tricks


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ACQUITY BEH200 SEC (300mm)

ACQUITY BEH200 SEC and BEH450 SEC (150mm + 150mm)


Compounds: 1. Thyroglobulin Dimer (1,340 KDa), 2. Thyroglobulin (667 KDa), 3. IgG (150 KDa), 4. BSA (66 KDa), 5. Myoglobin (17 KDa), 6. Uracil (112 Da)

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Effect of Pore Size and Combining Pore Sizes for Added Method Development Flexibility


Monoclonal Antibody

Adapted from Alain Beck Center of Immunology


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UV

Ab

so

rb

an

ce @

28

0n

m

150 mM 250 mM 350 mM pH

6.0

6.5

7.0

7.5

[NaCl]

HMW

Monomer

LMW1

LMW2

Developing a Robust SEC Method using AutoBlend Plus (25 mM Phosphate)


150 mM

250 mM

350 mM

1

1.1

1.2

1.3

1.4

1.5

1.6

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1.8

6

6.5

7

7.5

mM NaCl

Monomer USP Tailing

pH

Developing a Robust SEC Method


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1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

% P

eak

Are

a

Monomer USP Peak Tailing

HMW % Area

LMW1 % Area

LMW2 % Area

Developing a Robust SEC Method


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Effect of Column Length: Monoclonal Antibody

Murine monoclonal antibody (load: 6.4 µg - 150mm; 12.7 µg -300mm)

Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8;214 nm

300 mm

150 mm 98.88%

98.76% USP Res= 2.81 1.22%

USP Res=2.07 1.12%

mAb aggregates

mAb aggregates


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0.2 mL/min Rs= 2.4 ~1500 psi

0.4 mL/min Rs= 1.8 ~3000 psi

0.8 mL/min Rs= 1.3 ~6000 psi

IgG

dimer

Effect of Flow Rate on Rs (mAb)

Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm

Column: BEH200 SEC 1.7 µm, 4.6 x 150mm


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3.02 35

2.65 50

USP Injection

Volume

3.78 15

3.02 35

2.65 50

USP

Res

Injection

Volume

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3.15 0.625

3.26 2.5

3.26 10

USP

Res

Concentration

(mg/ mL )

3.23 1.25

3.15 0.625

3.26 2.5

3.26 10

USP

Res

Concentration

(mg/ mL )

Effect of Sample Load : Myoglobin

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

Column: BEH125 SEC 1.7µm, 4.6 x 300 mm column

Myoglobin: 5 mg/mL (volume load), and 20 uL injection volume (concentration)

Increased injection volumes can result in a significant loss of resolution in UPLC-SEC analyses.

Effect of Volume Load Effect of Concentration


Agenda









– Tips & Tricks


Considerations in SEC-MS

Protein structure in solution depends on

– pH

– Ionic strength

– Buffer and salt

– Additives

Good ionization conditions are different from conditions

for biological activity

Validation required when buffer is changed

Special uses are valuable

– Fast desalting

– Clips


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LC/MS Compatible Mobile Phase on ACQUITY UPLC BEH200 SEC, 1.7um

Similar retention time/ peak shape observed with MS compatible mobile phases

100mM Ammonium Formate

PBS


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Time 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

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2.5e-3

5.0e-3

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1.75e-2 26.00

17.05

20.02

Humanized Monoclonal Antibody: MS Compatible/Native Mobile Phase

Flow Rates: 100mM Ammonium Formate - 0.15mL/min, PBS- 0.4 mL/min

Lower flow rate for MS compatibility

100mM Ammonium Formate

PBS


SEC-MS Humanized Monoclonal Antibody

MS: Xevo G2 Q Tof

Conditions: 100mM Ammonium Formate, Flow rate: 0.15 mL/min

Post UV detection additive: ACN, 0.8% Formic acid

UV @ 280

TIC

Scan 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

%

4

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

AU

0.0

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4.0e-3

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1.0e-2

1.2e-2

1.4e-2

1.6e-2

1.8e-2

2: Diode Array 280 0.0500Da

Range: 6.757e-1

13.22

25.27

16.58

19.50

1: TOF MS ES+ TIC

7.58e6 19.49

15.35

16.62

23.74

25.06

1 2

3

1

2

3


Herceptin 50%ACN, .4% FA_100mm Amm Form_0.15 mL/min_40CV_AutoQua

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

%

0

100

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

%

0

100

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

%

0

100

7Oct11_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+ 4.34e33448.1404

3025.9878

2907.2991

2797.6379

2601.3782

2471.37262353.8545

2353.5356

3530.1348

3706.4951

3801.6843

3901.6064

3905.8140

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+ 1.97e3

2652.8584

2648.56692460.4124

1251.3574

2801.4185

2968.8967

3154.96903370.0889

3616.4006

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

1702.3831

1765.3279

1985.9363

2056.2769

2166.35232382.8904

2647.5525

Extracted Spectrum

Deconvoluted molecular weight determined using MaxEnt1

Intact IgG MW 148,221 Peak 1

IgG - 1 FAb MW 100,764 Peak 2

Fab and Fc MWs 47270 & 47637 Peak 3


SEC-UV-MS: A generic methodology for screening reduced antibodies

Desalting LC/HC Resolution Detect Clips • No Sample Concentration required

HC

Mass Spectrum

LC

Mass Spectrum

LC

HC

HC - HC

LC

HC - HC

HC

UV 280

TIC

Conditions: System, ACQUITY UPLCTM with TUV optical detector and Synapt G2 QTof MS

Flow Rate: 0.2 ml/min 0.1%TFA and 0.1%FA in 30% ACN


-

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BioSuite 125 4µm UHR

4.6 x 300 mm column

ACQUITY UPLC BEH125 SEC 1.7µm

4.6 x 300 mm column

USP Rs = 1.94

USP Rs = 3.29

Resolution of Small Protein (Myoglobin, 17 kDa)

Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min, sample 2 mg/mL

USP monomer/aggregate resolution was 1.7 times greater on the BEH125 1.7µm SEC column as compared to 4 µm pore diol-coated silica column.


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0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

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

AU

0.00

0.10

0.20

0.30

0.40

0.50

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

Rs = 2.1

USP Plate Count = 3K

Flow Rate = 0.5 mL/min

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Rs = 3.7

USP Plate Count = 15K

Flow Rate = 0.4 mL/min

ACQUITY UPLC BEH125 1.7µm

(4.6 x 300 mm)

HMWP SEC 10µm

(7.8 x 300 mm)

HPLC/UPLC Column Comparison: Insulin

Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Injection volume:

(Waters HMWP) tested to perform in the European Pharmacopoeial method.

Increase in HMW resolution observed in shorter run-times

4 mL mobile phase per analysis vs. 12 mL for HPLC method

10 min

24 min


AU

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

Minutes

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

Comparable absolute retention time change observed for both columns

Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Sample: Human Insulin ( 4mg/mL), Injection volume: 5 µL

AU

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

Minutes

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

Injection 853

Injection 26

BEH125 SEC, 1.7um Column Life Insulin Analysis

ACQUITY UPLC BEH125, SEC 1.7µm 4.6 x 300 mm


1a

1b

2

3

4

5

1a 1b

2 3

4

5

Compounds: 1a. IgM Dipentamer (1.8MDa), 1b. IgM Pentamer (900 Kda), 2. Thyroglobulin (667 KDa), 3. Apoferritin (443KDa), 4. β-Amylase (200 Kda), 5. IgG (150 KDa). Sample injection volumes and flow rate were normalized for column geometry.

ACQUITY UPLC BEH450 SEC (4.6 x 300 mm, 2.5µm) Flow Rate = 0.35 mL/minute Injection Volume = 5µL

BioSuite 450 SEC (7.8x 300 mm, 8µm) Flow Rate = 1.0 mL/minute Injection Volume = 14µL

Comparison of the ACQUITY UPLC BEH450 SEC column to the BioSuite 450 HR column


ACQUITY UPLC BEH450 SEC (4.6 x 300 mm, 2.5µm) Flow Rate = 0.35 mL/minute Injection Volume = 5µL

HPLC-SEC , 450Å (7.8x 300 mm, 8µm) Flow Rate = 1.0 mL/minute Injection Volume = 14µL

Monomer

Dimer

Rs=2.49

Monomer

Dimer

Rs=1.42

UPLC BEH450 SEC 450 Column Compared to 8µ Particle Size 450 Å Column for the Separation of Apoferritin (400 Kda)


0

1

2

3

4

5

6

7

1.5 2 2.5 3 3.5 4 4.5

Lo

g M

W

Elution Volume (mL)

BEH200 BEH200+BEH450 BEH450

Calibration Curve Comparison for BEH200, BEH450, and BEH200 & BEH450 Columns (300 mm total column lengths)


AU

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

Minutes

4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

AU

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

Minutes

3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

AU

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

0.011

Minutes

5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00


ACQUITY BEH200 SEC and BEH450 SEC (150mm + 150mm)


Comparison of the ACQUITY UPLC BEH450 and BEH200 SEC Columns: Cross-Linked IgG Multimers

Monomer

Dimer Trimer Multimers


Agenda









– Tips & Tricks


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

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50

Effect of Column Guard on Lifetime : Monoclonal Antibody

mAb formulation with excipients (Tween 80)

Improved mAb peak tailing with use of guard column

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

Minutes

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Injection 2 Injection 902

Injection 6 Injection 488

No Guards Guards Replaced every 200 injections


ACQUITY UPLC BEH SEC, Care and Use: (Ways to extend column life)

Preparation of SEC Mobile Phase and Needle Wash

– Pre filter through <0 .2 um filter or smaller (i.e, Don’t inject particulates)

– Sterile filtration can decrease contamination problems

– Use high purity water

– Replace mobile phases regularly and do not “top off”

Ramp up and down flow to column over 1min to minimize “bed shock”

Attention to SEC Eluent Inlet Filters

– Use titanium, NOT stainless steel

– Inlet filters 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% of the

maximum recommended flow rate

- Extended: Store in the HPLC grade water with 10% methanol


Method Performance and Transfer: Critical fittings and components


Sources of Band Spreading – Improper Column Connection

Band Spreading Dead / Void Volume

Proper

Improper

Resulting Peak Shape

Packed Bed Of

Particles

No Dead

Volume


The Effect of a Void Gap on the Separation of a mAb by SE-UPLC

1.22% LMW1

1.13% LMW1

0.51% HMW

0.50% HMW

0.33% LMW2

0.33% LMW2

Void Gap ≈ 600 µm

UV

(280 n

m)

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Minutes

4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Reference Material

Care and Use

– Size Exclusion and Ion-Exchange Chromatography of Proteins using

the ACQUITY UPLC™ System,” 715002147, REV. A

– “Size Exclusion and Ion-Exchange Chromatography of Proteins using

the ACQUITY UPLC H-Class System, ” 715002909, Rev A

– “Controlling contamination in LC/MS and HPLC/MS Systems,”

715001307

– “Improving the Lifetime of UPLC Size-Exclusion Chromatography

Columns Using Short Guard Columns,” Waters Technical Brief,

720004034en

– “Guidelines for Routine Use and Maintenance of Ultra-Performance

Size-Exclusion and Ion-Exchange Chromatography Systems”,

Waters Technical Brief, 720004182en


Summary: Waters ACQUITY UPLC SEC System Solution

SEC column chemistries in 125, 200, and 450Å pore size based on

BEH particles

– Reduced secondary interaction

– Improved physical and chemical column lifetime

– Improved column-to-column reproducibility

– Improved resolution

– Improved throughput

– Reduced Mobile Phase Use

UPLC-SEC provides improved resolution, sensitivity, and higher

throughput as compared to tradition HPLC

– Improved resolution of monoclonal antibody aggregates and clipped forms

Complete system solution includes column chemistry and system

– UPLC columns specifically designed for bioseparations

– ACQUITY H-Class Bio System designed for the bioapplications

– Auto●Blend Plus™ Technology provides convenience and efficiency


Thank You!

Questions?

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– Full Webinar Recording of Today’s Session

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