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About Today’s Presenter…

Stephan M. Koza, Ph.D., Principal Applications Chemist,

Waters Corporation

This webinar will be presented by Dr. 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.

©2014 Waters Corporation 4

Expand your HPLC SEC Capabilities with

Bridged Ethyl Hybrid Particles

Stephan M. Koza

Waters Corporation


Seminar Outline

Introduction to SEC

– BEH Particle Technology for SEC

XBridge HPLC Size-Exclusion Columns

Method Transfer (UPLC <-> HPLC)

ACQUITY UPLC Size-Exclusion Columns

Selected Applications

Summary


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


Separates proteins by their size in

solution (Stokes radius)

Separations are Isocratic

Ideally no adsorption to surface of

particles. Autoblend + for method

optimization

Analytes elute in CV ≤ 1 resulting in

low peak capacities as compared to

other methods such as RP methods

where analyte elutes in CV ≥ 1

Principles of Size Exclusion Chromatography of Proteins


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.

These interactions create undesired and unpredictable

retention of proteins (i.e. proteins not separated by size in

solution)


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

AU

0.00

0.02

0.04

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0.12

0.14

0.16

0.18

0.20

0.22

Minutes

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ACQUITY UPLC Protein BEH SEC 200Å, 1.7 µm, 4.6 x 300 mm Injection 19 Injection 618

Lysozyme, pKi = 10.7

Suggestive of DIOL Bleed

AU

0.00

0.10

0.20

0.30

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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

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

Suggestive of DIOL Bleed

Lysozyme, pI = 10.7

BEH200 SEC shows minimal secondary interactions even after 600 injections


Application Areas of XBridge HPLC and ACQUITY UPLC Size-Exclusion Columns

– Molecular weight ranges dependent on pore size:

– Determination of protein / peptide molecular weight (size)

– Quantitation of protein / peptide aggregates primarily in therapeutic

monoclonal antibodies, EPO, and Insulin

1K 10K 100K 1M 10M

200Å: 10KDa to

450KDa

UPLC ONLY HPLC Due Q1 2015


Advantages of XBridge Protein BEH SEC 3.5um HPLC Columns


0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

UV

Ab

so

rban

ce (

280 n

m)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

1b 2

3

4

5

250Å, 5 µm, Silica-Based SEC

200Å, 3.5 µm, XBridge SEC

SEC Molecular Weight Standard

1a

0.00

0.20

0.40

0.60

0.80

4.00 6.00 8.00 10.00 12.00

0.00

0.20

0.40

0.60

0.80

1.00

1.20

4.00 6.00 8.00 10.00 12.00

0.000

0.010

0.020

0.030

0.040

0.050

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

Minutes

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

UV

Ab

so

rban

ce (

214 n

m)

IgG dimer

IgG

monomer

mAb Standard

Comparison of XBridge Protein BEH SEC 200Å, 3.5um and Silica-Based SEC 250Å, 5µm Columns (7.8 X 300 mm)

Benefits Include 1. Improved resolution for all components except for thyroglobulin

dimer and monomer (Peak 1A and 1b) which is better for 5µm due to larger pore size

2. Improved sensitivity (peak height)


Analysis of Infliximab – HPLC comparison

Benefits Include 1. Improved resolution between

HMW and monomer

2. Improved resolution of monomer and LMW forms

3. Higher pressure tolerance allows for running 2 Xbridge 300mm columns in series at 1.0 mL/minute to obtain higher resolution separations with the same analysis time as running a single column at 0.5 mL/minute

UV

Ab

so

rban

ce (

214 n

m)

Minutes

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00

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0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

HMW LMW

Monomer Competitor- Silica SEC, 300Å, 3 µm, (7.8 X 300 mm, 0.5 mL/minute)

XBridge Protein BEH SEC, 200 Å 3.5 µm, (7.8 X 300 mm, 0.5 mL/minute)

Two, XBridge Protein BEH SEC 200Å, 3.5 µm, (7.8 X 600 mm, 1.0 mL/minute)


0.000

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Minutes

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0.018

0.020

UV

Ab

so

rba

nc

e (

280 n

m)

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Minutes

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UV

Ab

so

rban

ce (

214 n

m)

1b

2 3

4

5

IgG dimer

IgG

monomer

IgG dimer

IgG

monomer

IgG

multimer

Buffer

Buffer

1a

450Å, 8 µm, Silica-Based SEC

450Å, 3.5 µm, BEH SEC

SEC Molecular Weight Standard mAb Standard

Note : improved resolution and sensitivity for all components.

HPLC Comparison – 450Å


UV

Ab

so

rba

nc

e (

28

0 n

m)

Time (minutes)

4

0.000

0.010

0.020

0.030

0.040

0.050

1

2 3

5

0.0 18.0

Flow Rate= 1.0 mL/minute

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.0 7.0

Flow Rate= 2.0 mL/minute

250Å, 5 µm, Silica-Based SEC (300 mm)

200Å, 3.5 µm, XBridge SEC (300 mm)

Flow Rate (linear velocity) is 2-fold higher for X-Bridge resulting in an ~50% decrease in analysis time with comparable resolution.

Increased Sample Throughput


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Minutes

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0.05

UV

Ab

so

rban

ce (2

80 n

m)

0.00

0.05

Minutes

6.00 8.00 10.00 12.00 14.00

Batch 1, Column 1

Batch 1, Column 2

Batch 2, Column 1

Batch 2, Column 2

Batch 3, Column 1

Batch 3, Column 2

Batch 1, Column 1

Batch 1, Column 2

Batch 2, Column 1

Batch 2, Column 2

Batch 3, Column 1

Batch 3, Column 2

XBridge 200Å XBridge 450Å

1b 2

3 4

5

1a 1b

2 3

4

5

1a

Data representing two columns packed from 3 individual manufacturing batches of particles to evaluate both column-to-column and batch-to-batch reproducibility

Column-to-Column Reproducibility


0.00

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0.08

UV

Ab

so

rban

ce (

280 n

m)

0.00

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0.08

Minutes

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0.000

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0.015

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0.010

0.015

Minutes

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UV

Ab

so

rban

ce (

214 n

m)

Molecular Weight Standard Injection #2

Molecular Weight Standard Injection #610

mAb Standard Injection #6

mAb Standard Injection #611

1 2 3

4

5

IgG dimer

IgG

monomer Rs=2.55

Rs=2.42

Rs=1.87

Rs=1.83

Mobile phase: 20 mM sodium phosphate, 50 mM NaCl, pH 7.2 Flow rate: 0.84 mL/min Xbridge Protein BEH SEC 200A 3.5 µm (7.8x300mm)

Column Lifetime (@ 600 injections)


Advantages of XBridge Protein BEH SEC Columns

A significant increase in resolution compared to currently

available SE-HPLC columns

An approximately two-fold higher potential sample throughput

for modern HPLC chromatographic systems

Outstanding column-to-column reproducibility and stability

And coming up next…

Capability to directly scale methods between SE-HPLC and SE-

UPLC


Transferring SEC Separations Between UPLC and HPLC

Transferring UPLC SEC method to lab with HPLC instrumentation

Scaling up a UPLC method for fraction collection

(structure/function studies)

Scaling from HPLC to UPLC to increase sample throughput,

reduce solvent use, or analyze volume limited samples

The chemical comparability of the XBridge Protein BEH SEC

HPLC and ACQUITY UPLC BEH SEC particles allows for method

transfer between UPLC and HPLC without re-optimizing mobile

phase conditions


SEC Method Scaling Calculations

Match ~ L/dp

Match Flow Rate (reduced linear velocity)

Correct Injection Volume

Where: L is column length, dp is particle diameter, F is flow rate, D is column ID, and V is injection volumn

L HPLC = L UPLC × d p , HPLC

d p , UPLC

F HPLC = F UPLC × d p , UPLC × D HPLC

2

d p , HPLC × D UPLC 2

V HPLC = V UPLC × L HPLC × D HPLC

2

L UPLC × D UPLC 2


Method Transfer Example: 200Å UPLC (1.7 µm) to HPLC (3.5 µm) Separation of Infliximab

UV

Ab

sorb

an

ce (

21

4 n

m)

0.000

0.004

0.008

0.012

0.016

0.020

0.024

0.028

0.032

0.036

0.040

Minutes

22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 37.00 21.00

0.000

0.005

0.010

0.015

0.020

4.50 5.00 5.50 6.00 6.50 7.00 7.50

HMW

Monomer

LMW

ACQUITY UPLC H-Class Bio

Alliance HPLC XBridge Protein BEH SEC 200Å, 3.5µm (7.8 x 600mm total)

ACQUITY UPLC Protein BEH SEC 200Å, 1.7µm (4.6 x 300mm)

• ~2-fold increase in particle size( 3.5 vs 1.7 µm) therefore HPLC column length will need to be 2X that of the 30 cm UPLC column (i.e. two 30 cm length HPLC columns in series) •The flow rate should be 1.4 times greater for the HPLC analysis •The injection volume should be 5.75 times greater for the HPLC analysis •Run Time is ~ 4X longer for HPLC vs UPLC

Mobile Phase: 20 mM sodium phosphate, 150 mM NaCl, pH 7.2


Method Transfer Example: 450Å UPLC (2.5 µm) to HPLC (3.5 µm) Separation of IgM

• ~1.5-fold (1.4X) increase in particle size( 3.5 vs 2.5 µm) therefore HPLC column length will need to be 1.5X that of the 30 cm UPLC column (i.e. 30 cm + 15 cm length HPLC columns in series) •The flow rate should be 2.05 times greater for the HPLC analysis •The injection volume should be 4.3 times greater for the HPLC analysis •Run Time is only ~ 2X longer for HPLC vs UPLC

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0.30

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Minutes

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UV

Ab

sorb

an

ce (

21

4 n

m)

Aggregates

IgM Dipentamer

IgM Pentamer

XBridge Protein BEH SEC 450Å, 3.5µm (7.8 x 450mm total)

ACQUITY UPLC Protein BEH SEC 450Å, 2.5µm (4.6 x 300mm)

ACQUITY UPLC H-Class Bio

Alliance HPLC


Some manufacturers offer SE-HPLC columns with 4.6 mm ID which is not

ideal for use with HPLC systems

When system dispersion is high (i.e. HPLC vs UPLC system) the resolution

provided by a 7.8 mm ID column will be significantly better than that

provided by a 4.6 mm ID column at an equivalent linear velocity

Why 7.8 mm ID for SEC using HPLC (Alliance)?

UV

Ab

so

rb

an

ce (

28

0 n

m)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

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0.06

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0.12

Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Rs= 3.1

7.8 X 150 mm

0.000

0.007

0.014

0.021

0.028 4.6 X 150 mm Rs= 2.4 1

2 3

4 5

Both columns packed to comparable efficiencies with 3.5µm, 200Å particles


Why 7.8 mm ID for SEC using HPLC (Alliance)?

In SEC, resolution is practically independent of column ID if extra-column dispersion

(pink+red) is low relative to on-column dispersion or variance (green) as depicted for the

7.8 mm ID column on an HPLC and the 4.6 mm ID column on a UPLC scenarios (top and

bottom).

However, if extra-column dispersion becomes significant relative to on-column dispersion a

measurable loss of resolution will be observed as shown for the use the 4.6 mm ID column

on an HPLC scenario (middle). Note that the retention time difference is still equivalent to

that of the 4.6 mm ID column on a UPLC separation.

Additionally, in SEC pre-column and post-column band dispersion are additive because it is a

non-binding technique, however, in binding methods (e.g. RP or IEX) pre-concentration of

the analyte at the head of the column minimizes the impact of pre-column dispersion.

= + +

= + +

= + +

7.8 mm ID on HPLC

4.6 mm ID on HPLC

4.6 mm ID on UPLC


Advantages of ACQUITY Protein BEH SEC 1.7 and 2.5um UPLC Columns


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)

4µm

1.7µm

Classic Small-Molecule Van Deemter Curve

Why are the HPLC Separation Analysis Times so Much Longer?

Theoretical Plate Height (H) at different Linear Velocity Flow Rates (u) for a 150,000 Dalton IgG Separation Smaller H Value = Better Separations

H

~5X Linear Velocity Increase


- -

-

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ACQUITY UPLC BEH125 SEC 1.7um

4.6 x 300mm

BioSuite125 UHR SEC 4.6 x 300mm

A 2

14

A

U

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ACQUITY UPLC BEH125 SEC 1.7um

4.6 x 300mm

BioSuite125 UHR SEC 4um

4.6 x 300mm

A 2

14

Highest Resolution of Peptides Using 125Å UPLC (Aqueous) in the Same Analysis Time

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).


Developing and Using Robust SEC Methods

(HPLC and UPLC)


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2 1

3 4 5

6

ACQUITY UPLC Protein BEH SEC 200Å, 1.7µm

ACQUITY UPLC BEH SEC 200Å, 1.7µm and 450Å, 2.5µm (150mm + 150mm)

ACQUITY UPLC BEH SEC 450Å, 2.5 µm (300mm)

2

1

3 4 5

6

2

1

3 4 5

6

Selecting the Optimal Pore Size and Combining Pore Sizes for Added Method Development Flexibility

BEH200 SEC Standard: 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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UV

Ab

sorb

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 Waters AutoBlend Plus (AB+, 25 mM Phosphate)

ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm (300mm)

SMK


AU

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Effect of Column Length: Monoclonal Antibody

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

Conditions: 0.4 mL/min, 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 3 min.

6 min.

ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm


Effect of Flow Rate on Rs (mAb)

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

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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


SMK


Effect of Sample Load : Myoglobin

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

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.

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3.78 15

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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0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

Minutes

3.50 4.0

0

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

3.23 1.25

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 Volume Load Effect of Concentration



Pump

Autosampler

Detector

Colu

mn

1 1 1 2

1 3

1

2

3

4

UPLC Method Performance and Transfer: Critical Fittings and Components to Minimize Extra-Column Dispersion

Also important for SE-HPLC, however, the larger column ID makes the tolerances for extra-column dispersion less critical.


AU

-0.005

0.000

0.005

0.010

0.015

AU

-0.005

0.000

0.005

0.010

0.015

Minutes

3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

ACQUITY UPLC Protein BEH SEC 200Å, 1.7 µm 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

Injection 2

Injection 497

Dimer = 0.46% USP Res = 2.35

mAb

mAb

Dimer = 0.49% USP Res = 2.27

HMW (300K)

Monomer (150K)

LMW1 (100K)

LMW2 (50K)


Batch-to-Batch Reproducibility

BEH200 SEC Protein Standard


Applications


Monomer

Dimer

Trimer

Multimers

0.00

0.10

0.20

0.30

0.40

Minutes

0.00 5.00 10.00

Tetramer

Fragments

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Minutes

5.00 6.00 7.00 8.00 9.00 10.00

0.00

0.20

0.40

Minutes

0.00 5.00 10.00

Monomer

Dimer

Trimer

Tetramer & Multimers

0 5 10 15

Fragments

0.000

0.002

0.004

0.006

0.008

0.010

4.00 5.00 6.00 7.00 8.00 9.00

Freeze-Thaw Cycle Number

UV

Ab

so

rba

nc

e (

28

0 n

m)


ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm and 450Å, 2.5um (150mm + 150mm)

Analysis of IgG Multimer Aggregate Forms


AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

AU

-0.005

0.000

0.005

0.010

0.015

0.020

Minutes

4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Time 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

AU

0.0

2.5e-3

5.0e-3

7.5e-3

1.0e-2

1.25e-2

1.5e-2

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

2.0e-3

4.0e-3

6.0e-3

8.0e-3

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



SEC-UV-MS: A generic methodology for screening cysteine conjugated ADCs



Summary: Waters HPLC and UPLC Size-Exclusion Columns

XBridge SE-HPLC column chemistries with BEH particles can provide:

– Higher resolution SEC separations on HPLC systems

– Higher potential sample throughput for modern HPLC chromatographic

systems

– Capability to directly scale methods between SE-HPLC and SE-UPLC

As compared to SE-HPLC, SE-UPLC column chemistries with BEH particles

provide:

– Faster analyses for improved sample throughput

– Higher sensitivity

– Reduced mobile phase use

Both HPLC and UPLC BEH SEC columns provide:

– Outstanding column-to-column reproducibility and stability

– Application specific testing using protein/peptide standards, which are

available to the scientist through Waters ASR, of all batches of particles

– Individual testing of the packing quality of every column produced


Thank You for Attending!

Post-Event Home Page http://www.waters.com/Dec4Webinar

30% Promotional Offer On HPLC / UPLC SEC Columns

and Standards

30% Offer on OBD Prep Columns

30% Offer on LC Sample Vials

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

Deck

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

For Questions and to Submit your Ideas for our Next Topic

– Please eMail - [email protected]


mailto:[email protected]


Thank You For Your Time and Attention

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