View
1
Download
0
Category
Preview:
Citation preview
©2014 Waters Corporation
1
Thank you for joining us! Our session will begin shortly…
While you are waiting, please feel free to browse our library of program content: www.waters.com/meettheexperts
Also, click below to learn more about CORTECS, our newest Solid-Core LC Column platform: www.waters.com/CORTECS
Expand your HPLC SEC Capabilities with Bridged
Ethyl Hybrid Particles
©2014 Waters Corporation
2
Please use text chat functionality to submit your questions today.
Providing ‘Live’ Technical Support during today’s event
Poll Questions – Audience participation
Upon conclusion, follow up information will be available:
http://www.waters.com/Dec4Webinar
Recorded version of today’s presentation
PDF Copy of today’s slides
Application specific discount offers
SEC Columns and Standards, OBD Prep Columns, LC Vials
Product specific information and reference materials
Friendly Reminders…
©2014 Waters Corporation
3
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
©2014 Waters Corporation 5
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
©2014 Waters Corporation 6
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
©2014 Waters Corporation 7
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
©2014 Waters Corporation 8
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)
©2014 Waters Corporation 9
Comparative SEC Column Life (pH 6.8, 150 mM NaCl)
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
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
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
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
©2014 Waters Corporation 10
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
©2014 Waters Corporation 11
Advantages of XBridge Protein BEH SEC 3.5um HPLC Columns
©2014 Waters Corporation 12
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)
©2014 Waters Corporation 13
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
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00
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)
©2014 Waters Corporation 14
0.000
0.005
0.010
0.015
0.020
Minutes
9.00 10.00 11.00 12.00 13.00 14.00
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
UV
Ab
so
rba
nc
e (
280 n
m)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Minutes
6.00 8.00 10.00 12.00 14.00 16.00
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
©2014 Waters Corporation 15
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
©2014 Waters Corporation 16
0.00
0.05
0.10
0.00
0.05
0.10
0.00
0.05
0.00
0.05
0.10
0.00
0.05
0.10
0.00
0.05
0.10
Minutes
7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
0.00
0.05
0.00
0.05
0.00
0.05
0.00
0.05
0.00
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
©2014 Waters Corporation 17
0.00
0.02
0.04
0.06
0.08
UV
Ab
so
rban
ce (
280 n
m)
0.00
0.02
0.04
0.06
0.08
Minutes
6.00 8.00 10.00 12.00 14.00
0.000
0.005
0.010
0.015
0.000
0.005
0.010
0.015
Minutes
6.00 7.00 8.00 9.00 10.00 11.00
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)
©2014 Waters Corporation 18
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
©2014 Waters Corporation 19
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
©2014 Waters Corporation 20
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
©2014 Waters Corporation 21
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
©2014 Waters Corporation 22
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
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
11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes
5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
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
©2014 Waters Corporation 23
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
0.00
0.02
0.04
0.06
0.08
0.10
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
©2014 Waters Corporation 24
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
©2014 Waters Corporation 25
Advantages of ACQUITY Protein BEH SEC 1.7 and 2.5um UPLC Columns
©2014 Waters Corporation 26
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
©2014 Waters Corporation 27
- -
-
AU
0.00
0.50
1.00
1.50
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
ACQUITY UPLC BEH125 SEC 1.7um
4.6 x 300mm
BioSuite125 UHR SEC 4.6 x 300mm
A 2
14
A
U
0.00
0.50
1.00
1.50
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.50
1.00
1.50
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
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).
©2014 Waters Corporation 28
Developing and Using Robust SEC Methods
(HPLC and UPLC)
©2014 Waters Corporation 29
AU
0.00
0.05
0.10
AU
0.00
0.05
0.10
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
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)
©2014 Waters Corporation 30
0.000
0.005
0.010
0.015
0.000
0.005
0.010
0.015
0.000
0.005
0.010
0.015
0.000
0.005
0.010
0.015
Minutes 4.00 5.00 6.00 7.00 8.00
Minutes 4.00 5.00 6.00 7.00 8.00
Minutes 4.00 5.00 6.00 7.00 8.00
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
©2014 Waters Corporation 31
AU
-0.020
-0.010
0.000
0.010
0.020
0.030
0.040
Minutes
3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00
AU
-0.020
-0.010
0.000
0.010
0.020
0.030
0.040
Minutes
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
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
©2014 Waters Corporation 32
Effect of Flow Rate on Rs (mAb)
Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm
AU
0.00
0.10
0.20
0.30 A
U
0.00
0.05
0.10
0.15
0.20
0.25
AU
0.00
0.05
0.10
0.15
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
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
ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm (150mm)
SMK
©2014 Waters Corporation 33
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.
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
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 9.00 9.50 10.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
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 9.00 9.50 10.00
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
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
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
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
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
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
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
ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm (300mm)
©2014 Waters Corporation 34
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.
©2014 Waters Corporation 35
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)
©2014 Waters Corporation 36
Batch-to-Batch Reproducibility
BEH200 SEC Protein Standard
©2014 Waters Corporation 37
Applications
©2014 Waters Corporation 38
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 (300mm)
ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm and 450Å, 2.5um (150mm + 150mm)
Analysis of IgG Multimer Aggregate Forms
©2014 Waters Corporation 39
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
ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm (300mm)
©2014 Waters Corporation 40
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
ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm (300mm)
©2014 Waters Corporation 41
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
©2014 Waters Corporation 42
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
ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm (300mm)
©2014 Waters Corporation 43
SEC-UV-MS: A generic methodology for screening cysteine conjugated ADCs
ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm (150mm)
©2014 Waters Corporation 44
SEC-UV-MS: A generic methodology for screening cysteine conjugated ADCs
©2014 Waters Corporation 45
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
©2014 Waters Corporation 46
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 - mychemrep@waters.com
©2014 Waters Corporation 47
Thank You For Your Time and Attention
Recommended