Webinar: The Size-Exclusion UPLC Analysis of Biomolecules ...€¦ · This webinar will be...
Transcript of 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.
©2012 Waters Corporation 2
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
©2012 Waters Corporation 3
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.
©2012 Waters Corporation 4
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
©2012 Waters Corporation 5
Agenda
Size-Exclusion Chromatography
– Theory and practice
– ACQUITY UPLC Columns for SEC
o ACQUITY UPLC SEC 200Å, 1.7 µm Columns
o ACQUITY UPLC SEC 125Å, 1.7 µm Columns
o ACQUITY UPLC SEC 450Å, 2.5 µm Columns
– Keys to Method Development
– Selected Applications
– Tips & Tricks
©2012 Waters Corporation 6
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
©2012 Waters Corporation 7
Size Exclusion Separation of Proteins
Principles of Size Exclusion Chromatography of Proteins
©2012 Waters Corporation 8
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
©2012 Waters Corporation 9
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
©2012 Waters Corporation 10
Agenda
Size-Exclusion Chromatography
– Theory and practice
– ACQUITY UPLC Systems and Columns for SEC
o ACQUITY UPLC SEC 125Å, 1.7 µm Columns
o ACQUITY UPLC SEC 200Å, 1.7 µm Columns
o ACQUITY UPLC SEC 450Å, 2.5 µm Columns
– Keys to Method Development
– Selected Applications
– Tips & Tricks
©2012 Waters Corporation 11
Why UPLC?
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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
©2012 Waters Corporation 12
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
©2012 Waters Corporation 13
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
©2012 Waters Corporation 14
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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©2012 Waters Corporation 15
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
©2012 Waters Corporation 16
Calibration Curves of ACQUITY UPLC BEH450, BEH200, and BEH125 SEC Columns
©2012 Waters Corporation 17
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
©2012 Waters Corporation 18
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.
©2012 Waters Corporation 19
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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
©2012 Waters Corporation 20
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
©2012 Waters Corporation 21
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)
Suggestive of DIOL Bleed
Lysozyme, pI = 10.7
©2012 Waters Corporation 22
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
Suggestive of DIOL Bleed
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HPLC 100% Silica-Diol SEC 250Å 4µm 4.6 x 300 mm Injection 19 Injection 618
Suggestive of DIOL Bleed
Lysozyme, pI = 10.7
Comparative SEC Column Life (pH 6.8, 150 mM NaCl)
©2012 Waters Corporation 23
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
©2012 Waters Corporation 24
BEH200 SEC, 1.7um Batch-to-Batch Reproducibility
©2012 Waters Corporation 25
Agenda
Size-Exclusion Chromatography
– Theory and practice
– ACQUITY UPLC Systems and Columns for SEC
o ACQUITY UPLC SEC 125Å, 1.7 µm Columns
o ACQUITY UPLC SEC 200Å, 1.7 µm Columns
o ACQUITY UPLC SEC 450Å, 2.5 µm Columns
– Keys to Method Development
– Selected Applications
– Tips & Tricks
©2012 Waters Corporation 26
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ACQUITY BEH200 SEC (300mm)
ACQUITY BEH200 SEC and BEH450 SEC (150mm + 150mm)
ACQUITY BEH450 SEC (300mm)
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
©2012 Waters Corporation 27
Monoclonal Antibody
Adapted from Alain Beck Center of Immunology
©2012 Waters Corporation 28
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UV
Ab
so
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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)
©2012 Waters Corporation 29
150 mM
250 mM
350 mM
1
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1.2
1.3
1.4
1.5
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1.7
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6.5
7
7.5
mM NaCl
Monomer USP Tailing
pH
Developing a Robust SEC Method
©2012 Waters Corporation 30
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% P
eak
Are
a
Monomer USP Peak Tailing
HMW % Area
LMW1 % Area
LMW2 % Area
Developing a Robust SEC Method
©2012 Waters Corporation 31
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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
©2012 Waters Corporation 32
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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
©2012 Waters Corporation 33
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USP Injection
Volume
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3.02 35
2.65 50
USP
Res
Injection
Volume
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Res
Concentration
(mg/ mL )
3.23 1.25
3.15 0.625
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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
©2012 Waters Corporation 34
Agenda
Size-Exclusion Chromatography
– Theory and practice
– ACQUITY UPLC Systems and Columns for SEC
o ACQUITY UPLC SEC 125Å, 1.7 µm Columns
o ACQUITY UPLC SEC 200Å, 1.7 µm Columns
o ACQUITY UPLC SEC 450Å, 2.5 µm Columns
– Keys to Method Development
– Selected Applications
– Tips & Tricks
©2012 Waters Corporation 35
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
©2012 Waters Corporation 36
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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
©2012 Waters Corporation 37
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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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5.0e-3
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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
©2012 Waters Corporation 38
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
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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
©2012 Waters Corporation 39
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
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0
100
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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
©2012 Waters Corporation 40
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
©2012 Waters Corporation 41
-
-
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00
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.
©2012 Waters Corporation 42
.
AU
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.010
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
AU
0.000
0.002
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
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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
©2012 Waters Corporation 43
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
©2012 Waters Corporation 44
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
©2012 Waters Corporation 45
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)
©2012 Waters Corporation 46
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)
©2012 Waters Corporation 47
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 (300mm)
ACQUITY BEH200 SEC and BEH450 SEC (150mm + 150mm)
ACQUITY BEH450 SEC (300mm)
Comparison of the ACQUITY UPLC BEH450 and BEH200 SEC Columns: Cross-Linked IgG Multimers
Monomer
Dimer Trimer Multimers
©2012 Waters Corporation 48
Agenda
Size-Exclusion Chromatography
– Theory and practice
– ACQUITY UPLC Systems and Columns for SEC
o ACQUITY UPLC SEC 125Å, 1.7 µm Columns
o ACQUITY UPLC SEC 200Å, 1.7 µm Columns
o ACQUITY UPLC SEC 450Å, 2.5 µm Columns
– Keys to Method Development
– Selected Applications
– Tips & Tricks
©2012 Waters Corporation 49
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
©2012 Waters Corporation 50
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
©2012 Waters Corporation 51
Method Performance and Transfer: Critical fittings and components
©2012 Waters Corporation 52
Sources of Band Spreading – Improper Column Connection
Band Spreading Dead / Void Volume
Proper
Improper
Resulting Peak Shape
Packed Bed Of
Particles
No Dead
Volume
©2012 Waters Corporation 53
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
©2012 Waters Corporation 54
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
©2012 Waters Corporation 55
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
©2012 Waters Corporation 56
Thank You!
Questions?
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