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1 The world leader in serving science
Easy One-Click Access to Powerful and Complete Workflows for LC and LC-MS
2
Your Challenges
• Work faster with better results • Produce more data with fewer injections • Use fewer methods and less equipment • Be always ready • Someone is always happy to audit
3
Agenda
1. Thermo Scientific™ UHPLC Capabilities
2. Unsurpassed UHPLC Solutions
3. Beyond Conventional UHPLC Experiences
4. The Apps Library of the Future
4
Our UHPLC Portfolio at the Heart of Your Workflows
620 bar
Basic Automated • Highly economic & reliable • 620 bar UHPLC compatible • Flow rates up to 10 mL/min
Standard • Highest flexibility with Dual
solutions • 620 bar UHPLC compatible • Flow rates up to 10 mL/min
Up to 1000 bar
Thermo Scientific EASY-nLC/RSLCnano Systems • UHPLC systems for
Nano/Cap/Micro range • 20 nL/min – 50 µL/min up to
800 bar with RSLCnano • 20 – 2000 nL/min up to 1000 bar
with Easy-nLC • Direct flow (splitless) delivery
Thermo Scientific™ Dionex™ UltiMate™ 3000 RS/BioRS Systems • Advanced workflows with Dual
systems • 1000 bar up to 5 mL/min • 200 Hz data rate • Binary and Quaternary UHPLC
systems
1250 bar
UltiMate 3000 XRS System • Highly LC/MS optimized
quaternary UHPLC system • Pressures up to 1250 bar • Flow rates of up to 2 mL/min • Open Autosampler available
Thermo Scientific™ Vanquish™ UHPLC System • Highest pressure capability up to 1500 bar • Lowest system dispersion by optimized
system setup and flow path • Extended sample capacity:
4 sample containers in the sampler • Space for 9 racks or deep well plates /
20 shallow well plates in the charger • Two thermostatting modes to face diverse
application challenges • Unmatched detection sensitivity and linearity
up to 3 AU
1500 bar
Answers Samples
Preparation
Results
5
Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System Software
System Setup, Control, Data Review and Results
Chromeleon CDS – The Gold Standard in Chromatography Software
6
Chromeleon CDS eWorkflows
• eWorkflow ™ • Facilitate method setup • All relevant materials in one place • Methods, data processing, additional info (SOP) • Convenience, accuracy and traceability
1. Select eWorkflow
2. Select Instrument
3. Click Launch
7
Overlay of all XIC‘s
Component list
Quantitation and confirmation ions
Complex Data or MS Data Processing
8
Thermo Scientific IonCount Solution • Pharmaceutical counterion
analysis • Simultaneous detection of
cations and anions
Thermo Scientific XPert Troubleshooting Solution • Automated HPLC
troubleshooting • First aid kit to quickly identify
cause of deviations from expected results
Unsurpassed UHPLC Solutions
Thermo Scientific BeerNHop Solutions
• Isohumulones in beer analysis
Bio applications • Dedicated system with
dedicated columns • Advanced workflows
powered by Thermo Scientific™ Orbitrap™ mass accuracy technology
9
IonCount Solution uses trimode column technology to allow simultaneous analysis of cat- and anions No longer need 2 different assays
Need to analyze pharmaceutical counterions? Require different assays for cations and anions?
The IonCount Solution
10
IonCount —The Complete Solution
UltiMate 3000 SD System
Thermo Scientific™ Dionex™ Corona™ Veo™ Charged Aerosol Detector
System Package
Starter Kit
Thermo Scientific™ Acclaim™ Trinity™ P1 and P2 Columns
Eluents and Buffers
Test Standards
eWorkflows Guide & CD
eWorkflows for different API and Counterion Analyses
Installation and Operation Guides
+
+ +
+
11
Reference eWorkflows and Smart Reporting
Column: Acclaim Trinity P1, 3 x 50 mm System: UltiMate 3000 Quaternary SD Mobile phase: A - H2O B - CH3CN C -100 mM NH4HCO3, pH=3.65 Gradient: 0-7 min 40-70% B, 7-10 min 70% B,
10-10.5 min 70 - 40% B, 10.5-15 40% B, 0-15 min 30% C
Flow rate: 600 µL/min Power Func.: 1.5 Temperature: 30 ºC Injection: 3 µL Detection: Corona Veo Charged
Aerosol Detection Analytes: IonCount Test mix 1. Sodium 2. Chloride 3. Tosylate
Routine system readiness checks Smart Chromeleon CDS reporting if test passes or fails
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
1
2
3
min.
pA 1. Sodium 2. Chloride 3. Tosylate
12
Pre-defined eWorkflows – Counterion Screening
0 2.0 4.0 6.0 8.0 10.0 12.0
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1
2
3
4
5
6
7 8
9
min
pA Column: Acclaim Trinity P2, 3 x 50 mm System: UltiMate 3000 Quaternary SD Mobile phase: A - H2O B - CH3CN C -100 mM NH4HCO3, pH=3.65 Gradient: 0-1 min 10% C, 1-11 min 10-100% C,
11-20 min 100% C, 20-21 100-10% C, 21-29 min 10% C
Flow rate: 600 µL/min Power Func.: 1.15 Temperature: 30 ºC Injection: 1 µL Detection: Corona Veo Charged Aerosol Detector Analytes: all 100 µg/mL 1. Phosphate 2. Sodium 3. Potassium 4. Chloride 5. Bromide 6. Nitrate 7. Tosylate 8. Sulfate 9. Magnesium eWorkflow available on Solution CD for reduced startup time
13
Pre-defined eWorkflows – API Analysis
0 2.0 4.0 6.0 8.0 10.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
2
1
3 4
5
min
pA Column: Acclaim Trinity P1, 3 x 50 mm System: UltiMate 3000 Quaternary SD Mobile phase: A - H2O B - CH3CN C -100 mM NH4HCO3, pH=3.65 Gradient: 0-15 min 5-80% B, 15-19.5 min 80% B,
19.5-20.5 80-5% B, 20.5-25 min 5% B 0-25 min 15% C
Flow rate: 700 µL/min Power Func.: 1.3 Temperature: 30 ºC Injection: 1 µL Detection: Corona Veo Charged Aerosol Detector Analytes: 1. Sodium 2. Potassium 3. Chloride 4. Nitrate 5. API (Cefazolin)
• eWorkflow for simultaneous analysis of API and counterion • Alternative instrument methods for fast method development available
14
Acclaim Trinity P1 & P2 Mixed Mode Columns
Acclaim Trinity P1 Column
• RP / WCX / WAX • Monovalent ions
Nanopolymer Silica Hybrid (NSH) technology
Acclaim Trinity P2 Column
• HILIC / WCX / SAX • Multivalent ions
Advantages of the IonCount Solution: • Simultaneous analysis of anions, cations and API’s • Complete chromatography starter kit with quality standards, required eluent additives and columns • Pre-defined eWorkflows for typical use cases and quality standards • A tailored UHPLC system including UV detection and the universal charged aerosol detection
15
BeerNHop Solutions allows brewers to create new innovative brands and flavors
Determine the isohumulones in beer for occasional beer analyses, direct injection of untreated beer samples or for high throughput analysis
The BeerNHop Solutions
Foam stability
Bitterness
Antimicrobial
16
Questionable assay results? Out-of control situations? Want to avoid downtime while waiting for service?
XPert Solution is a first-aid kit to easily find out: Is the column or the instrument the source of deviation?
… and it assists with: preventive maintenance - column replacement - application fine tuning complete confidence in your analysis!
Ask the XPert Troubleshooting Solution
17
Biopharma Solutions - UltiMate 3000 BioRS System
Complete bio-system solution • Extendable to 2D-LC • Unique columns for glycan analysis • Innovative pH buffer kits and high resolution
columns; seamless pH gradients for improved separation
• pH, conductivity, UV/Vis and MS data in one chromatography data system (CDS) package
18
Proteins Ion Exchange Mechanisms
Lysine Variants
Y Y K K
Y Y K
Y Y
Acidic Variants Basic Variants
+
0
–
3 4 5 6 7 8 9 10
Buffer/System pH
Protein net charge vs. pH
Buffer pH typically > pI
Anion-Exchange Chromatography
Buffer pH typically < pI
Cation-Exchange Chromatography
NH2R
COO -
NH3R +
COOH
Isoelectric Point (pI)
NH3R +
COO -
2
Cationic protein binds to
negatively charged cation exchanger
+ ++
++
+ ++
Anionic proteinbinds to
positively chargedanion exchanger
- -- -
-
- - -
• Release from IEX columns is typically done by salt displacement.
• At pI the protein still bears charges, but the overall charge is neutral
• Therefore the protein will release from an IEX column at the pI
19
Salt vs. pH Gradient
pH gradient in many cases provide better resolution
Easy method development for mAb charge variants
0-100 %B
25-50 %B
0.0 10.0 20.0 30.0
0.0
15.0
min %B: 10.0
30.0
Salt gradient
0.0 10.0 20.0 30.0
0.0
15.0
min %B: 25.0
50.0
25.0
pH gradient
0 10 20 30 40 5.00
6.00
7.00
8.00
9.00
10.50
min
pH trace
0 10 20 30 40 6.60
7.00
7.25
7.50
7.75
8.00
min
pH trace
20
pH Gradient Ion Exchange of Proteins
Examples: Separation of 4 proteins and related charge variants
pH trace
0 5 10 15 20 25 30 35 40 -10
20
40
60
90
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Abso
rban
ce (m
AU)
Retention Time (min)
1
Lectin-1 – pI 6.1
Lectin-2 - pI 6.3
Lectin-3 - pI 6.4
Trypsinogen - pI 7.7
Ribonuclease A - pI 8.7
Cytochrome C - pI 10.1
2
pH
Perfect pH gradient linearity from pH 5.6 to pH 10.2 Excellent resolution Very good correlation retention time – isoelectric point
21
Glycan Analysis Solutions
Thermo Scientific™ Accucore™ 150-Amide-HILIC Column
5.0 10.0 15.0 20.0 25.0 30.0
GU2
GU3
GU4
GU5 GU6
GU7 GU8
min
counts
Dextran ladder
Unknown sample
2-AB-Glycans
Thermo Scientific™ GlycanPac™ AXH-1 Column
2-AB-Glycans
2-AA-glycans
Native glycans
GlycanPac AXR-1 Column
2-AB-Glycans
2-AA-glycans
Native glycans
The bio-pharma work-horse for fluorescent glycans
Mixed mode HILIC WAX for separation based on charge and size
Mixed mode RP WAX for separation based on charge, isomeric structure and size Unsurpassed selectivity and resolution for glycan analysis!
Glycans control functionality of many proteins
Structures are not linear and often hard to detect
UHPLC-Fluorescence (labelled) and UHPLC-MS (labeled and native) are most commonly used in LC
22
GlycanPac AXR-1Column
More than 70 peaks - 2x the peaks resolved with existing amide columns!
• GlycanPac AXR-1 1.9 μm column, 2.1x150 mm
• Sample: Fetuin 2-AB-glycans
• GlycanPac AXR-1 1.9 μm column, 2.1x150 mm
• Sample: Human IgG 2-AA-glycans
Separation of IgG Glycans
Reversed-phase and anion exchange combined for unrivaled separations
• Size
• Isomers
• Sialic acid content
The Vanquish UHPLC System
Go Beyond Your Conventional UHPLC Experience
24
Rapid Separation of 18 Herbicides Using Accucore Vanquish 1.5 μm Columns
-2.5
5.0
10.0
15.0
20.0
25.0 mAU
1
WVL:230 nm
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
-2.5
5.0
10.0
15.0
20.0
25.0 2
mAU
min
2
WVL:230 nm
1
2 3
4
5 6
7
8
9 10
11
12
13
14 15 16
17 18
1
2 3
4
5 6
7
8
9 10
11
12
13
14 15 16
17 18
Accucore Vanquish 1.5 µm
2.6 µm solid core
Time (min) %A %B 0 80 20
6.93 60 40 12.13 20 80 13.00 80 20 20.80 80 20 Gradient conditions for the 2.6 µm
solid core column
Gradient Time: 20.8 min Flow Rate: 0.38 mL/min
Gradient Time: 12 min Flow Rate: 0.65 mL/min
Advantages from Accucore Vanquish columns: The Vanquish system combined with Accucore Vanquish columns provides higher pressure capabilities for faster analyses
25
Rapid Separation of 18 Herbicides Using Accucore Vanquish 1.5 μm Columns
-5.0
10.0
25.0 mAU
1
-5.0
10.0
25.0 2 mAU
2
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 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 5.20 5.40 5.60 5.80 6.00 -5.0
10.0
25.0 3 mAU
min
3
1
2 3
4 5 6
7 8
9 10 11 12 13
14 15 16
17 18
1
2 3
4 5 6
7 8
9 10 & 11
12 13
14 15 16
17 18
1
2 3 4 5 6
7 8
9 10 & 11
12 13 14 15 16
17 18
Accucore Vanquish 1.5 µm
Competitor I 1.6 µm
Competitor II 1.7 µm
Mobile phase A: Water Mobile phase B: Acetonitrile Column temperature: 43 °C Injection details: 0.5 µL Detection: UV at 230 nm (0.1 s rise time, 50 Hz, 8 nm slit width) Viper connections: Autosampler to column: 0.1 x 250 mm (6040.2225) Column to detector: 0.075 x 350 mm (6041.5735) UHPLC Column 1: Accucore Vanquish C18, 1.5 µm, 2.1 x 100 mm Flow rate: 0.65 mL/min
Time (min) %A %B 0 80 20
4.00 60 40 7.00 20 80 7.50 80 20
12.00 80 20
Gradient conditions for the Accucore Vanquish 1.5 µm and
competitor columns I and II
Advantages from using Accucore Vanquish columns: Only with the Accucore Vanquish columns can all peaks be resolved
Pressure: ~1340bar
26
There Is More Than Pressure to a Vanquish System
Hippuric acid: metabolite that may indicate presence of aromatic molecules (toluene, benzene) e.g. in glue sniffing intoxication
C18 columns cannot separate hippuric acid isomers. Hypercarb can separate the isomers at isocratic condition (25% ACN in water)
Hippuric acid Methyl-hippuric acid positional isomers
27
Separation of Hippuric Acid Positional Isomers With Hypercarb
0 3.5 7 min
mA
U
60 °C, 680 bar
120 °C, 380 bar 1
2
3 4
1 – 2-Methyl Hippuric Acid 2 – Hippuric Acid 3 – 3-Methyl Hippuric Acid 4 – 4-Methyl Hippuric Acid
Pressure limit of Hypercarb is 400 bar
Decrease flow rate? This will increase retention time and reduce sensitivity (lower peak height)
Increase temperature? • Sufficient resolution • Short analysis time • High sensitivity • Column stability preserved
28
Accucore Vanquish C18 column, 1.5 µm, 2.1 x 100 mm Overlaid selected reaction monitoring chromatograms showing detection of 36 antibiotics within a 5 minute detection window, binary Vanquish system and a Thermo Scientific™ TSQ Vantage™ Mass Spectrometer
Time (min) %B
0 10
4.375 90
5.000 90
5.125 10
8.750 10
Mobile phase A: 0.1% formic acid / Water Mobile phase B: 0.1% formic acid / MeOH Flow rate: 400 µL/min Column temperature: 40 °C, active pre-heating Injection volume: 2 µL Table 1. LC gradient conditions
Key Application –Targeted Screening of 36 Antibiotics
29
Accucore Vanquish C18 column 1.5 µm, 2.1 x 100 mm
Overlaid selected reaction monitoring chromatograms showing detection of 47 drugs within a 4 minute detection window, binary Vanquish system and TSQ Vantage Mass Spectrometer
Time (min) %B 0 10
0.16 10 2.88 90 3.20 90 3.28 10 5.60 10
Mobile phase A: 10 mM ammonium acetate in water Mobile phase B: 0.1% formic acid in methanol Flow rate: 500 µL/min Column temperature: 50 °C, with active pre-heating Injection volume: 2 µL Table 1. LC gradient conditions
Key Application – 47 Drugs in 4 Minutes
30
Biopharma Analysis Using the Vanquish System
High-end system for demanding analyses for characterizing biotherapeutics
31
What If You Need To Analyze 1000 Clones?
• Salt gradient ion exchange loss of resolution for fast separation • pH gradient with short columns will provide sufficient resolution • Need a system with small extra column volume low gradient delay and
faster column re-equilibration • Need a system suitable for high throughput
Example: charge variants of mAb produced in large number of cell culture need to be screened in short time Typical run time 30 min 5 min < 1 min Analyzing 1000 samples 1000 samples 1000 samples Time 21days 4 days 17 hours
32
pH Gradient Ion Exchange of Monoclonal Antibodies
High-throughput separation of mAb charge variants example: infliximab (Remicade)
0 0.50 1.00 1.50 2.00 2.50 3.00
1
2
3
4
5
min
1
2
3
4
5 0.5 mL/min 5 min gradient
1.2 mL/min 0.8 min gradient
Charge variants separation accomplished in 1 minute!
33
Peptide Mapping of Therapeutics Monoclonal Antibody
Digestion
RT: 0.00 - 60.00
0 5 10 15 20 25 30 35 40 45 50 5Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
25.9920.752.12
23.1733.83
19.32
26.7418.64
16.57 27.00 38.57
39.1139.20
14.24
2.37 31.7413.53 36.553.7912.77
4.208.48
8.4029.92 39.638.72 51.17
48.516.25 9.22 40.72 48.03 52.50 5
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
• Peptide Map • PTMs • Impurities
Intact Ab
Peptides
Bottom up
Sequence/PTMs unknown or need to be confirmed
Sequence and PTMs known. No further information required Stability studies, QA/QC, batch release)
Peptide identification by unknown and reference sample chromatogram comparison (retention time comparison)
High degree of confidence on retention time determination is
required!
34
Peptide Mapping of Therapeutics Monoclonal Antibody
Peptide mapping with LC-UV peak assignment based on retention time comparison with reference chromatogram (biopharma: stability study, QA/QC, process control)
HIGH RETENTION TIME PRECISION REQUIRED!
2.5 6.0 10.0 14.0 18.0 22.0 26.0 30.0 min
mA
U
Overlay of 13 injections
35
24.50 25.00 26.00 27.00 28.00 28.10 0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
min
mAU
18.10 19.00 20.00 20.60 -5 0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
min
mAU
7.30 8.00 9.00 10.00 11.00 11.40 2.00 2.50
3.75
5.00
6.25
7.50
8.75
10.00
11.25
12.50
13.75
15.00
16.25
17.50
18.75
20.00
21.25
22.50
23.75
25.00
26.00
min
mAU
Peptide Mapping: What You Can Do When You Are Confident on the RT
5.0 10.0 15.0 20.0 25.0 30.0 min
mAU
Peptide separation: comparison of mAb digested at different conditions
36
Thermo Scientific AppsLab – The Apps Library of the Future
• … an on-line application research engine with rich information and ready-to-run analytical methods (‘one click workflows’).
• … a unique, easy-to-use starting point for method development challenges.
• … a central repository for Thermo Scientific chromatography and MS applications.
37
Your Partner…
…For All Analytes & All Applications
38
Useful Links:
Method Development Application Search Revolutionary Next Generation SPE Accucore Column Technology Accurately Linear pH Gradients Chromatography Resource Center - Separated by Experience All About Mass Spectrometry - Planet Orbitrap Smart Phone Method Speed-Up Calculator iPhone HPLC Trouble Shooter Thermo Scientific Dionex UltiMate 3000 BioRS Thermo Scientific Vanquish UHPLC System
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