A New Way to Look at Biomolecules - Waters Corporation · ©2011 Waters Corporation 1 A New Way to...
Transcript of A New Way to Look at Biomolecules - Waters Corporation · ©2011 Waters Corporation 1 A New Way to...
©2011 Waters Corporation 1
A New Way to Look at Biomolecules
Focused on the unique challenges
and specific needs of biopharmaceutical
development
COMMITTED TO PROVIDING
THE BEST ANALYTICAL METHODS
TO ACHIEVE THE RESULTS YOU REQUIRE
©2011 Waters Corporation 2
Your Laboratory Requirements
Increase efficiency and reduce costs
Work within regulatory guidelines
Protect IP
Analytical methods capable of reliably
and accurately identifying and measuring
product variants
— Biopharmaceuticals are complex
and heterogeneous
— Potential for multiple product
variants is high
By 2014, five of the top five best-selling
and 50% of the top 100 branded drugs
will be proteins. (Evaluate Pharma)
©2011 Waters Corporation 3
Your Technology Needs
Accuracy
Sensitivity
Linearity over a broad range
Application-specific column chemistries
Capable of measuring all possible
structural variants of a protein
and higher-order conformation
And enables high efficiency
— Throughput: time to decision
— Maximize use of talent
— Minimize solvent
consumption
©2011 Waters Corporation 4
Sample Preparation
Separation
Detection
Data Analysis
The Tasks in Your Workflow
©2011 Waters Corporation 5
Sample PreparationKits & Consumables
UPLC System& Column Technology
Optical Detectors& Mass Spectrometers
Bioinformatics
Your Workflow & Our Technology
©2011 Waters Corporation 6
2006
Column Manager
Column Heater/Cooler
BEH 300 C18
HSS T3
AccQ•Tag Ultra
SQ Detector
TQ Detector
MS Third Party Control
MassLynx 4.1
PLGS 2.2.5
Amino Acid Analysis
2007
FLR Detector
Improved Column Oven
HSS C18
HSS C18 SB
SYNAPT
SYNAPT HDMS
BioPharmaLynx
Peptide Mapping
2008
Extended Wavelength PDA
Open Access
BEH 300 C4
Xevo TQ MS
BioPharmaLynx1.1
IdentityE
Oligonucleotide Analysis
Intact Protein MS Analysis
2009
Local Console
BEH Amide
Xevo QTof MS
SYNAPT G2
SYNAPT G2 HDMS
BioPharmaLynx1.2
Intact Protein RP Analysis
Glycan Analysis
2010
ACQUITY UPLC H-Class Bio
Column Managers
Auto•Blend Plus
BEH SEC
IEX
Xevo G2 QTof
BioPharmaLynx1.3
Intact Protein SEC and IEX
Analysis
Continuous Technology Evolution
©2011 Waters Corporation 7
Application-Specific Solutions
UPLC
— AAA
— Peptide Mapping
— Released Glycans
— Intact RP
— Intact SEC
— Intact IEX
— Oligonucleotides
UPLC/MS
— Peptide Mapping
— Released Glycans
— Intact RP
— HDX
— HCPs
©2011 Waters Corporation 8
Acquity H-Class Bio
Acquity H-Class
High pressure Quaternary pump
« Flow Through » Injector
Stainless Steel
Corrosion
Unwanted adsoption
Alliage Titane and/or NiCo
Primary & accumulator pump heads
Check-valves
UPLC Valves (Vent, Injector, CM, etc..) &
SSV
Mixer housing, mixer manifold, sinkers
SM Needle Assembly
APH Assembly
UPLC tubing assemblies
PDA & TUV Flow Cells
UPLC performancesBiological solvent and buffer compatibilityBiological separation compatibility
©2011 Waters Corporation 9
Applications
UPLC
— Oligonucleotides
— AAA
— Peptide Mapping
— Released Glycans
— Intact RP
— Intact SEC
— Intact IEX
©2011 Waters Corporation 10
DNA
ACQUITY UPLC® OST C18 Up to 60-mer
Acquity PST BEH300 C18, 2.1x50mm, 1.7 µm
from 60-mer
©2011 Waters Corporation 11
Applications
UPLC
— Oligonucleotides
— AAA
— Peptide Mapping
— Released Glycans
— Intact RP
— Intact SEC
— Intact IEX
©2011 Waters Corporation 12
Chemistry of AQC Derivatization
Reacts readily with both primary and secondary amines
Forms stable derivatives
Requires no vacuum drying, sample prep or extraction
Amendable to automation
©2011 Waters Corporation 13
Ser
Gln
Arg
Gly A
sp
Glu
Thr
Ala
Minutes
2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
Cell Culture Sample
1 day
3 day
6 day
©2011 Waters Corporation 14
AAA
Cell media dilutionAccQ Tag derivatisationUPLC with AccQ Tag SolventColumn AccQ Tag (2.1X100)mm , 1.7µm
AM
Q
NH
3 His
Se
r
Arg
Gly As
p
Glu T
hr
Ala
Pro
De
riv
Pe
ak
Cy
sL
ys
Ty
r
Me
tV
al
Ile
Le
uP
he
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
0.30
0.32
0.34
0.36
Minutes1.50 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
Monitoring cell culture media
©2011 Waters Corporation 15
Applications
UPLC
— Oligonucleotides
— AAA
— Peptide Mapping
— Released Glycans
— Intact RP
— Intact SEC
— Intact IEX
©2011 Waters Corporation 16
Time20.00 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 52.00 54.00 56.00
AU
2.0e-2
3.0e-2
4.0e-2
5.0e-2
6.0e-2
7.0e-2
8.0e-2
9.0e-2
1.0e-1
Time30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00
AU
1.0e-2
2.0e-2
3.0e-2
4.0e-2
5.0e-2
6.0e-2
7.0e-2
90 min
55 min
HPLC 2.1 x 250 mm, 3.5 mm
UPLC 2.1 x 150 mm, 1.7 mm
Reduce Run TimeComparable Resolution
©2011 Waters Corporation 17
Time8.00 10.00 12.00 14.00 16.00 18.00
AU
2.5e-2
5.0e-2
7.5e-2
1.0e-1
1.25e-1
1.5e-1
1.75e-1
2.0e-1
2.25e-1
2.5e-1
2.75e-1
0.25
1.00.5
105.02.0
5020
100
pmol
Peptide QuantitationLinearity
©2011 Waters Corporation 18
Time28.30 28.35 28.40 28.45 28.50 28.55 28.60 28.65 28.70 28.75 28.80 28.85 28.90 28.95 29.00 29.05
AU
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
4.5e-2
5.0e-2
5.5e-2
6.0e-2
6.5e-2
7.0e-2
7.5e-2
8.0e-2
8.5e-2
9.0e-2
9.5e-2
1.0e-1
1.05e-1
1.1e-1
1.15e-1
1.2e-1
1.25e-1
1.3e-1
1.35e-1
1.4e-1
0.2%
0.5%
1%
2%*
Peptide MapTrace Contaminant
©2011 Waters Corporation 19
Solution: UPLC Peptide Mapping3-6X Improvement in Productivity
―We were able to get much faster modification analysis with no significant loss in resolution, allowing us to move the samples through the process much faster‖
©2011 Waters Corporation 20
Separation of PENNY Peptide and its Deamidated Peptides
©2011 Waters Corporation 21
Peptides mapping
Time8.00 10.00 12.00 14.00 16.00 18.00
AU
2.5e-2
5.0e-2
7.5e-2
1.0e-1
1.25e-1
1.5e-1
1.75e-1
2.0e-1
2.25e-1
2.5e-1
2.75e-1
0.25
1.0
0.5
10
5.0
2.0
50
20
100
pmol
Peptides and PTM’s qualification during development
Peptide (Modified and unmodified) Quantitation during production
©2011 Waters Corporation 22
Applications
UPLC
— Oligonucleotides
— AAA
— Peptide Mapping
— Released Glycans
— Intact RP
— Intact SEC
— Intact IEX
©2011 Waters Corporation 23
Murine IgG OligosaccharidesHPLC vs. UPLC BEH Glycan Column
Time30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
EU
x 1
0e4
0.000
5000.000
10000.000
15000.000 G0F
Man6
G2F
Man5
G1F
Time
2 x 150 mm - 3 µm
100.0
Time5.00 10.00 15.00 20.00 25.00 30.00 35.00
EU
x 1
0e4
0.000
2500.000
5000.000
7500.000
10000.000
12500.000
15000.000
35.0
Man5
Man6
G1F
G2F
G0F
2.1 x 150 mm - 1.7 µm
EU
EU
©2011 Waters Corporation 24
Batch-to-Batch Reproducibility of ACQUITY UPLC®
BEH Glycan Material Using 2-AB Labeled Human IgG N-Linked Glycans
Batch 1
Batch 2
Batch 3
Batch 4
©2009 Waters Corporation | COMPANY CONFIDENTIAL©2011 Waters Corporation
UPLC® SeparationHuman IgG N-linked Glycans
1 G02 G0F3 Man54 G0FGN5 G16 G1Fa7 G1Fb8 G1FGN9 Man610 G211 G2F12 G1F+SA13 G2F+SA
1
4
5
6
78
9
10
11
2
3
12
13
Peak identification was done in LC-MS under same gradient conditions
©2011 Waters Corporation 26
2-AB Labeled Dextran LadderUPLC Separation
ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
©2011 Waters Corporation 27
New Glycan Found in EPO Exoglycosidase Digestion with UPLC/FLR
Minutes 4 6 8 10 12 14 16 18
GU 5 6 74 8 9 10 11 12 13 14 15
Same digestion pattern and ΔGUs
F(6)A4G4Lac4S4GU ~ 14.8
Dr. Jonathan Bones.
Dublin – Oxford Glycobiology Laboratory,
National Institute for Bioprocessing Research and Training,
University College Dublin.
©2011 Waters Corporation 28
Glycans analysis
Antibody glycan analysis Structural information with the GU
Release GlycanDerivatisation with 2ABCoilumn ACQUITY UPLC® BEH Amide, 2.1 x 150 mm
©2011 Waters Corporation 29
Applications
UPLC
— Oligonucleotides
— AAA
— Peptide Mapping
— Released Glycans
— Intact RP
— Intact SEC
— Intact IEX
©2011 Waters Corporation 30
MeCN
AU
0.00
0.18
0.36
0.54
AU
0.00
0.18
0.36
0.54
AU
0.00
0.18
0.36
0.54
AU
0.00
0.18
0.36
0.54
Minutes
0.00 7.00 14.00 21.00 28.00 35.00
ACN
7:3 IPA:ACN
IPA
MeOH
1. Ribonuclease2. Cytochrome c3. BSA4. Myoglobin5. Enolase6. Phosphorylase b
1
23
45
6
12
34
5
6
1 2
3 45
6
2
34
1
4800 psi
8200 psi
13600 psi
6700 psi
Protein Mix Effect of Solvent on Separation @ 40 ºC
IPA
MeOH
©2011 Waters Corporation 31
SEC ChromatogramStandards
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
1
2
3
4
5
Analyte pI MW
1. Thyroglobulin, 3 mg/mL 4.6 669,000
2. IgG, 3 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
©2011 Waters Corporation 32
AU
0.00
0.02
0.04
0.06
0.08
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
AU
0.00
0.02
0.04
0.06
0.08
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
AU
0.00
0.02
0.04
0.06
0.08
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
Conventional SEC Column Influence of Ionic Strength on Peak Shape and Retention
Conventional SEC Column4.6 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
©2011 Waters Corporation 33
ACQUITY BEH200 Influence of Ionic Strength on Peak Shape and Retention
AU
0.00
0.06
0.12
0.18
0.24
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
0.18
0.24
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
AU
0.00
0.06
0.12
0.18
0.24
Minutes
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 15.000.00
0.06
0.12
0.18
0.24
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
ACQUITY BEH200, 1.7 µm, 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
AU
0.00
0.06
0.12
0.18
0.24
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
0.18
0.24
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
100 mM
lysozyme
©2011 Waters Corporation 34
AU
-0.02
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
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
ACQUITY UPLC BEH200
SEC,1.7 µm
4.6 x 150mm column
Aggregate
10.3%
Detection of Aggregation with SECMonoclonal IgG
Flow rate: 0.4 mL/min; Mobile phase: 25mM Sodium phosphate, pH 6.8, 0.15M NaCl
©2011 Waters Corporation 35
Column Lifetime: Lysozyme
Flow rate: 0.4 mL/min; Mobile phase: 25mM Sodium phosphate, pH 6.8, 0.15M NaCl
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
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
HPLC SEC 250Å 4µm4.6 x 300 mm
Injection 19Injection 618
ACQUITY UPLC BEH200 SEC, 1.7 µm4.6 x 150 mm
Injection 19Injection 618
©2011 Waters Corporation 36
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Chimeric Monoclonal Antibody Separation of C- terminal Lysine Variants
KK
K
©2011 Waters Corporation 37
Protein-Pak™ Hi Res Q Anion ExchangeAnalysis of Chicken Ovalbumin
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
0.110
0.120
0.130
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00
Grade C
Grade B
Grade A
©2011 Waters Corporation 38
Protein-Pak™ Hi Res Cation ExchangeComparing Carboxymethyl and Sulfopropyl
AU
0.00
0.02
0.04
0.06
0.08
AU
0.00
0.02
0.04
0.06
0.08
Minutes
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
Lysozyme
Ribonuclease A
Cytochrome C
Lysozyme
Ribonuclease A
Cytochrome C
Protein-Pak™ Hi Res SP
Protein-Pak™ Hi Res CM
Conditions- Flow Rate: 1 mL/min; Mobile Phase A: 20mM Sodium Phosphate Buffer, pH 6.6; Mobile Phase B: A + 0.5 M NaCl; Gradient: 0 – 60%B in 34min
©2011 Waters Corporation 39
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
IEC : charges differences
SEC
RP : Mapping and quant.
Mapping and quant.
Agregates
Intact protein - agregates – charges state
AU
-0.02
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
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
ACQUITY UPLC
BEH200 SEC,1.7
µm
4.6 x 150mm
columnAggregate
10.3%
©2009 Waters Corporation | COMPANY CONFIDENTIAL©2011 Waters Corporation
Auto●Blend™
and
Auto●Blend Plus™ Technology
©2011 Waters Corporation 41
Auto●Blend
Auto•Blend Technology allows the automatic blending of up to four
solvents in accurate proportions and to run any sequence of
isocratic, binary, ternary, quaternary gradients for routine method
operation, automatic method development, convenient method
transfer, or system flushing
Routine assays become more rugged (less human error)
Water
1%TFAIsopropanol
Acetonitrile Water
1%TFAIsopropanol
Acetonitrile
95%
0%
0%
5%
45%
0%
50%
5%
100% Water
0% Acetonitrile
0.05% TFA
50% Water
50% Acetonitrile
0.05% TFA
©2011 Waters Corporation 42
Applications
Peptide Mapping with Reversed-phase
Protein Separations
— Reversed-phase
— Ion Exchange
— Size Exclusion
Released glycan analysis
©2011 Waters Corporation 43
Peptide Map DevelopmentVarying TFA Concentration - %D
AU
0.00
0.05
0.10
0.15
AU
0.10
0.15
0.20
0.25
AU
0.05
0.10
0.15
0.20
Minutes
27.00 27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00
0.05% TFA – 5% D
0.1% TFA – 10% D
0.025% TFA – 2.5% D
©2011 Waters Corporation 44
Conventional Method EditorProgram Percentage Flow
©2011 Waters Corporation 45
Auto●Blend™ Plus Method Editor Program Required Gradient
©2011 Waters Corporation 46
Chimeric Monoclonal Antibody,pH Study, Initial 0M NaCl
AU
0.000
0.002
0.004
0.006
0.008
AU
0.000
0.002
0.004
0.006
AU
0.000
0.005
0.010
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 26.00
0.0 – 1.0 M NaCl, 20mM Sodium Phosphate in 40 min
0.5 mL/min
pH 6.4
pH 6.6
pH 6.8
©2011 Waters Corporation 47
Auto●Blend Plus TechnologyA
U
0.00
0.05
0.10
AU
0.00
0.06
0.12
AU
0.00
0.05
0.10
AU
0.00
0.05
0.10
AU
0.00
0.05
0.10
Minutes
0.00 5.00 10.00 15.00
pH 6.1
pH 7.6
pH 7.1
pH 7.0
pH 6.9
A mixture of proteins was separated using cation exchange chromatography- alpha-Chymotrypsinogen A (peak A), Ribonuclease A (peak B), and Cytochrome C (peak C) –
A
A
A+B
A
A
B
B
B
B
C
C
C
C
C
©2011 Waters Corporation 48
SEC of Humanized Monoclonal AbEffect of AutoBlend Plus pH Adjustment
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
AU
0.00
0.10
0.20
0.30
0.40
0.50
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
A: 17.26%B: 2.74%C: 20.00%D: 60.00%
A: 2.74%B: 17.26%C: 20.00%D: 60.00%
pH6.0
pH7.6
125mM NaH2PO4
125mM Na2H2PO4
1000mM NaCL
H2O
©2011 Waters Corporation 49
CONCLUSION
ACQUITY H-Class Bio System designed for the application
Complete system solution including column chemistry with
system
Auto●Blend™ and Auto●Blend Plus™ Technology for
convenience and efficiency