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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