Method Transfer Best Practice - Thermo Fisher Scientific · Method Transfer Best Practice . 2 ... cylinder) 0.9 0.8 . 0.6 . 0.7 0.5 0.4 ... Fine-tuning of GDV by the autosampler metering

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Dr. Frank Steiner Scientific Advisor Co-authored by Dr. Susanne Fabel, Dr. Alexandra Manka, Michael Menz, Sabrina Patzelt, Dr. Markus Martin

Method Transfer Best Practice

2

• Important considerations for method transfer

• Direct UltiMate 3000 Vanquish method transfer by instrument similarity

• Method transfer by exchange of gradient mixer

• Unique hardware features of Vanquish systems for method transfer

• Adaptable GDV by autosampler metering device

• Eluent pre-heating and column thermostatting modes

• What is the best practice?

Agenda

3

Important considerations for method transfer

4

Impact on Method Transfer – Instrument Properties and Settings

Pump • Gradient generation principle • Gradient delay volume (GDV) • Solvent mixing characteristics

5



Autosampler • Contribution to GDV • Contribution gradient shape (flush out)

6



Column thermostat • Working principle (still air vs. forced air) • Eluent pre-heater design and working principle


7



System connection tubing • Dimensions • Plumbing (dead volumes)



8



System connection tubing • Dimensions • Plumbing (dead volumes)


Detector • Flow cell design and dimensions • Wavelength settings (bandwidth, reference) • Data collection rate and filter constant


9

Gradient Generation and Mixing Volume

+

H2O CH3OH

= ?

10


+

H2O CH3OH

= !

11


Volume percentage of mixture (organic content)

% o

f res

ultin

g vo

lum

e af

ter m

ixin

g H2O / CH3CN

H2O / CH3OH +

H2O CH3OH

= !

12



% o

f res

ultin

g vo

lum

e af

ter m

ixin

g H2O / CH3CN

H2O / CH3OH +

H2O CH3OH

= !

• Volume contraction has influence on gradient generation

13



% o

f res

ultin

g vo

lum

e af

ter m

ixin

g H2O / CH3CN

H2O / CH3OH +

H2O CH3OH

= !

• Volume contraction has influence on gradient generation

• Effect will differ between HPG and LPG pumps, also in isocratic dial-a-mix mode

14

Mixing Volume Effects with HPG and LPG Instruments

0 2 4 6 8 10

1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0

100

90

80

60

70

50

40

30

20

10

0

% M

ethanol

Flow

rate

/ m

L/m

in

t [min]

Programmed flow rate

0 2 4 6 8 10

100

90

80

60

70

50

40

30

20

10

0 %

Methanol

Flow

rate

/ m

L/m

in

t [min]

Delivered flow rate HPG (at 25 °C)

1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0

15


0 2 4 6 8 10

1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0

100

90

80

60

70

50

40

30

20

10

0

% M

ethanol

Flow

rate

/ m

L/m

in

t [min]


0 2 4 6 8 10

100

90

80

60

70

50

40

30

20

10

0 %

Methanol

Flow

rate

/ m

L/m

in

t [min]


1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0 0 2 4 6 8 10

100

90

80

60

70

50

40

30

20

10

0

% M

ethanol

Flow

rate

/ m

L/m

in

t [min]

Delivered flow rate LPG (complete mixing before pump cylinder)

1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0

16


0 2 4 6 8 10

1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0

100

90

80

60

70

50

40

30

20

10

0

% M

ethanol

Flow

rate

/ m

L/m

in

t [min]


• Method transfer from HPG to HPG straightforward (if GDV matches) • Method transfer from LPG to LPG more challenging (mixing properties must also match) • Better refrain from transfer between LPG and HPG (in applications with critical resolution)

0 2 4 6 8 10

100

90

80

60

70

50

40

30

20

10

0 %

Methanol

Flow

rate

/ m

L/m

in

t [min]


1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0 0 2 4 6 8 10

100

90

80

60

70

50

40

30

20

10

0

% M

ethanol

Flow

rate

/ m

L/m

in

t [min]

Delivered flow rate LPG (complete mixing before pump cylinder)

1.0

0.9

0.8

0.6

0.7

0.5

0.4

0.3

0.2

0.1

0.0

17

UltiMate 3000 Vanquish Flex Method transfer

by instrument similarity

18

Method Transfer: UltiMate 3000 BioRS Vanquish Flex (HIV Medication Example)

Column: Accucore aQ, 2.6 µm, 2.1 × 100 mm System: Vanquish Flex System with DAD (10 mm

light path) BioRS System, VWD with 2.5 µL flow cell (7 mm light path) Mobile Phase: A - 25 mM ammonium acetate buffer, pH 3.8 with acetic acid B - Methanol C - Acetonitrile D - Water Flow rate: 0.600 mL/min

Retention time [min] 0.00 6.00 -20

0

160 -20

0

160

Abs

orba

nce

[mAU

] A

bsor

banc

e [m

AU]

1

1

2

3

2

3

Vanquish Flex

UltiMate 3000 BioRS

1. Uracil 10 µg/mL 2. Tenofovir 100 µg/mL 3. Emtricitabine 100 µg/mL

19

Method Transfer: UltiMate 3000 BioRS Vanquish Flex (HIV Medication Example)

Column: Accucore aQ, 2.6 µm, 2.1 × 100 mm System: Vanquish Flex System with DAD (10 mm

light path) BioRS System, VWD with 2.5 µL flow cell (7 mm light path) Mobile Phase: A - 25 mM ammonium acetate buffer, pH 3.8 with acetic acid B - Methanol C - Acetonitrile D - Water Flow rate: 0.600 mL/min

Retention time [min] 0.00 6.00 -20

0

160 -20

0

160

Abs

orba

nce

[mAU

] A

bsor

banc

e [m

AU]

1

1

2

3

2

3

Vanquish Flex

UltiMate 3000 BioRS

Vanquish Flex enables easy method transfer from

UltiMate 3000 systems.

1. Uracil 10 µg/mL 2. Tenofovir 100 µg/mL 3. Emtricitabine 100 µg/mL

20

Method Transfer – Deamidation of Ribonuclease A

• Seamless method transfer

from UltiMate 3000 BioRS to Vanquish Flex already with identical tubing and mixer (both for DAD and VWD)

• Improved Sensitivity with Vanquish DAD

• Increased peak resolution on Vanquish Flex with VWD

0 5 10 15 20 25

-80

0

80

Vanquish Flex VWD

BioRS DAD

Vanquish Flex DAD

Ion exchange separation of Ribonuclease variants

21

Method Transfer – Antibody Charge Variant Analysis

• Method transfer between

UltiMate 3000 BioRS and Vanquish Flex

• Improved sensitivity with Vanquish DAD

• Identical methods were applied and successful method transfer was achieved by recreating the same hardware conditions on both instrument platforms 0,00 2,50 5,00 7,50 10,00 12,50 15,00 17,50 20,00

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

Retention time [min]

Abso

rban

ce [m

AU] @

280

nm

MabPac SCX-10; 4x250mm, 10 µm, A: 20mM MES, 60mM NaCl, pH 5.7 B: 20mM MES, 300mM NaCl, pH 5.7 Gradient: 15-45% B in 15 min, 30 °C; 0.5mL min-1; UV detection@280nm; Injection volume 4 µL

Vanquish Flex UltiMate 3000 BioRS

1

2

3

Peak Relative RT deviation between systems [%]

1 1.2

2 1.1

3 1.0

22

Method transfer from other vendor’s instrument

by modification of gradient mixer

23

Transfer of a Heart Treatment Drug Analysis from Other Vendor to UltiMate 3000 Column: Thermo Scientific™ Accucore™ XL, C18,

4.6 × 150 mm, 4 µm, P/N 74104-154630 Mobile Phases: A: Water + 0.1% FA B: Methanol + 0.07% FA Gradient: 0-10.0 min from 10 to 80% B, 10.0 -11.5 min 80% B, 11.5-12.0 min 80 to 10% ,12.0-17.0 min 10% B Flow rate: 1.200 mL/min Temperature: 50 ºC still air mode Injection: 25 µL Detection: 214 nm data collection rate: 10 Hz Sample: 1. Hydrochlorthiazide 10 µg/mL 2. Chlorthalidone 20 µg/mL 3. Enalapril 60 µg/mL 4. Impurity 5. Ramipril 60 µg/mL 6. Telmisartan 20 µg/mL 7. Azilsartan 20 µg/mL 8. Valsartan 20 µg/mL

Abso

rban

ce [m

AU]

Vendor A

-100 0

1,100

1

2

3

6

5

7 8

4

1.30 2.50 5.00 7.50 10.00 11.00 -100

0


UltiMate 3000 SD

1

2

3 5

6 7 8

4

1,100

24

Transfer of a Heart Treatment Drug Analysis from Other Vendor to UltiMate 3000 Column: Thermo Scientific™ Accucore™ XL, C18,

4.6 × 150 mm, 4 µm, P/N 74104-154630 Mobile Phases: A: Water + 0.1% FA B: Methanol + 0.07% FA Gradient: 0-10.0 min from 10 to 80% B, 10.0 -11.5 min 80% B, 11.5-12.0 min 80 to 10% ,12.0-17.0 min 10% B Flow rate: 1.200 mL/min Temperature: 50 ºC still air mode Injection: 25 µL Detection: 214 nm data collection rate: 10 Hz Sample: 1. Hydrochlorthiazide 10 µg/mL 2. Chlorthalidone 20 µg/mL 3. Enalapril 60 µg/mL 4. Impurity 5. Ramipril 60 µg/mL 6. Telmisartan 20 µg/mL 7. Azilsartan 20 µg/mL 8. Valsartan 20 µg/mL

Abso

rban

ce [m

AU]

Vendor A

-100 0

1,100

1

2

3

6

5

7 8

4

750 µL mixer instead of the default 350 µL mixer to mimic GDV of the other vendor’s HPLC

1.30 2.50 5.00 7.50 10.00 11.00 -100

0


UltiMate 3000 SD

1

2

3 5

6 7 8

4

1,100

25

Unique hardware features of Vanquish systems

for method transfer

26

Transferring the Analysis of Pain Killer from UltiMate 3000 to Vanquish

3.000 3.500-93

1.500

2.0005

6

m

mAU

∆=0.06 s deviation or ∆=0.03 % relative deviation


3.000 3.500-93

1.500

2.0005

6

m

mAU



Idle Volume: 25 µL (default)

Idle Volume: 15 µL

Vanquish Idle Vol. 15 µL

UltiMate 3000

0 2.5 5.0 -50

3,000 Caffeine

Acetylsalicylic acid

Salicylic acid

Ibuprofen

Valerophenone


Abso

rban

ce [m

AU]

27

Transferring the Analysis of Pain Killer from UltiMate 3000 to Vanquish

3.000 3.500-93

1.500

2.0005

6

m

mAU



3.000 3.500-93

1.500

2.0005

6

m

mAU



Idle Volume: 25 µL (default)

Idle Volume: 15 µL

Vanquish Idle Vol. 15 µL

UltiMate 3000

0 2.5 5.0 -50

3,000 Caffeine

Acetylsalicylic acid

Salicylic acid

Ibuprofen

Valerophenone


Abso

rban

ce [m

AU]

Fine-tuning of GDV by the autosampler metering device makes the perfect match!

28

Column Thermostatting Effects – Analysis of Preservatives

Column: Acclaim RSLC PA2, Polar Advantage II, 2.1 x 150 mm, 2.2 µm Mobile Phase: isocratic

20 mM phosphate buffer pH 7/methanol (35/65, v/v, dial-a-mix)

Flow rate: 0.55 mL/min, resulting in 760 bar back pressure Temperature: 40 ºC Injection: 1 µL Detection: UV, 2.5 µL flow cell, 254 nm, 50 Hz (VWD) Sample: 1. Uracil, 2. Dimethyl phthalate (DMP), 3.

Methyl parabene (MP), 4. Methyl benzoate (MB)

0

200

450 1

2 3

4

min

mAU

0

200

450 1 2 3

4

min

mAU

UltiMate 3000 BioRS

Vanquish Flex, Forced Air

NMB = 12,950

αDMP-MP = 1.17

αDMP-MP = 1.17

NMB = 12,450

RDMP-MP = 1.58

RDMP-MP = 1.53

29






0

200

450 1

2 3

4

min

mAU

0

200

450 1 2 3

4

min

mAU

UltiMate 3000 BioRS


NMB = 12,950

αDMP-MP = 1.17

αDMP-MP = 1.17

NMB = 12,450

RDMP-MP = 1.58

RDMP-MP = 1.53

0 0.5 1.0 1.5 2.0

0

200

450 1 2 3

4

min

mAU

Vanquish Flex, Still Air

αDMP-MP = 1.12

NMB = 14,000

RDMP-MP = 1.16

30






0

200

450 1

2 3

4

min

mAU

0

200

450 1 2 3

4

min

mAU

UltiMate 3000 BioRS


NMB = 12,950

αDMP-MP = 1.17

αDMP-MP = 1.17

NMB = 12,450

RDMP-MP = 1.58

RDMP-MP = 1.53

0 0.5 1.0 1.5 2.0

0

200

450 1 2 3

4

min

mAU


αDMP-MP = 1.12

NMB = 14,000

RDMP-MP = 1.16 Excellent efficiency Poor

selectivity

31






0

200

450 1

2 3

4

min

mAU

0

200

450 1 2 3

4

min

mAU

UltiMate 3000 BioRS


NMB = 12,950

αDMP-MP = 1.17

αDMP-MP = 1.17

NMB = 12,450

RDMP-MP = 1.58

RDMP-MP = 1.53

0 0.5 1.0 1.5 2.0

0

200

450 1 2 3

4

min

mAU


αDMP-MP = 1.12

NMB = 14,000

RDMP-MP = 1.16

Retention and selectivity very sensitive to temperature with this application

Minor changes in eluent pre-heating and frictional heat dissipation can matter

Excellent efficiency Poor

selectivity

32

Correlation of retention factors of origin UltiMate 3000 BioRS system ( Forced Air principle) and target Vanquish Flex system ( Still Air mode) by reducing incoming eluent temperature enabled by the active pre-heater

Setting the Eluent Pre-heater Temperature Different to the Column Compartment

33



0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]

Temparture of active pre-heater [°C]

Dimethylphthalate

Temperature of active pre-heater [°C]

34



0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


35



0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


36

k ≈

0.68

5 @

30.5

°C



0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


37

k ≈

0.68

5 @

30.5

°C



0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


0,70

0,73

0,76

0,79

0,82

0,85

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylparabene


38

k ≈

0.68

5 @

30.5

°C



k ≈

0.80

5 @

34

°C

0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


0,70

0,73

0,76

0,79

0,82

0,85

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylparabene


39

k ≈

0.68

5 @

30.5

°C



k ≈

0.80

5 @

34

°C

0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


0,70

0,73

0,76

0,79

0,82

0,85

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylparabene


1,42

1,44

1,46

1,48

1,50

1,52

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylbenzoate


40

k ≈

0.68

5 @

30.5

°C



k ≈

0.80

5 @

34

°C

k ≈

1.49

@

32

°C

0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


0,70

0,73

0,76

0,79

0,82

0,85

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylparabene


1,42

1,44

1,46

1,48

1,50

1,52

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylbenzoate


41

k ≈

0.68

5 @

30.5

°C



In compromise an averaged incoming eluent temperature of 32 °C should deliver best matching retention factors for all 3 substances in Still Air mode

k ≈

0.80

5 @

34

°C

k ≈

1.49

@

32

°C

0,65

0,66

0,67

0,68

0,69

0,70

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Dimethylphthalate


0,70

0,73

0,76

0,79

0,82

0,85

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylparabene


1,42

1,44

1,46

1,48

1,50

1,52

29 30 31 32 33 34 35 36 37 38 39 40

Rete

ntio

n fa

ctor

k' [

-]


Methylbenzoate


42

• Why using Still Air and reducing incoming eluent temperature when coming from Forced Air?

Setting the Eluent Pre-heater Temperature different to the Column Compartment

43



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]


Efficiency of Methylbenzoate

Vanquish Flex in Still Air Mode

UltiMate 3000 BioRS with Forced Air principle and passive pre-heating

44



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





45



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





46



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

47



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%

48



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%

49



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%

10% better efficiency

50



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%


But do we still meet the resolution?

51



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%

1,00

1,20

1,40

1,60

1,80

2,00

2,20

29 30 31 32 33 34 35 36 37 38 39 40

Reso

lutio

n [-]


Resolution critical peak pair





52



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%

1,00

1,20

1,40

1,60

1,80

2,00

2,20

29 30 31 32 33 34 35 36 37 38 39 40

Reso

lutio

n [-]







53



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%

1,00

1,20

1,40

1,60

1,80

2,00

2,20

29 30 31 32 33 34 35 36 37 38 39 40

Reso

lutio

n [-]







we exceed!

54



12500

13000

13500

14000

14500

29 30 31 32 33 34 35 36 37 38 39 40

Plat

es N

[-]





8%

10%

1,00

1,20

1,40

1,60

1,80

2,00

2,20

29 30 31 32 33 34 35 36 37 38 39 40

Reso

lutio

n [-]







Still Air mode + reduced pre-heater temperature deliver better efficiency + better resolution

we exceed!

55

Method Transfer with Improved Resolution by Unique Thermosttating Features

0 0.50 1.00 1.50 2.00

0

100

200

300

400

500

600

700

800

1 2

3

4

min

mAU

UltiMate 3000 BioRS Forced Air, 40 °C Passive pre-heater

Vanquish Flex Still Air, 40 °C Active pre-heater = 32 °C

Original System

Target System

56


0 0.50 1.00 1.50 2.00

0

100

200

300

400

500

600

700

800

1 2

3

4

min

mAU

RDMP-MP = 1.58 αDMP-MP = 1.17



NMB = 12,950

Original System

Target System

57


0 0.50 1.00 1.50 2.00

0

100

200

300

400

500

600

700

800

1 2

3

4

min

mAU





NMB = 12,950

NMB = 14,450

Original System

Target System

58


0 0.50 1.00 1.50 2.00

0

100

200

300

400

500

600

700

800

1 2

3

4

min

mAU





NMB = 12,950

NMB = 14,450

Match of retention factors and improved resolution

Successful method transfer with increased performance (resolution and plates) through unique thermostatting features of Vanquish platform

Original System

Target System

59

Method transfer

• Gradient delay volume (GDV) adaption • Adjustable metering device idle volume

• Mixer and Viper capillaries

• Delayed injection

• Match the thermostatting mode • Forced and still air mode give different results

• Mimic thermal conditions used in existing systems

• UltiMate 3000 TCC or column compartments of competitors

Vanquish

Vanquish Flex

UltiMate 3000 (Bio)RS

60

Increasing peak capacity by smart temperature control

61

Method transfer with MS Method transfer from UltiMate 3000 RS HPG to Vanquish

62

What is the best practice?

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• Consider changes of prescribed method parameters vs. hardware adaptations

• Consider physico-chemical effects of gradient generation principle (HPG vs. LPG)

• Consider the advantage of seamless GDV adaptation by the adjustable metering device in Vanquish autosamplers

• Consider the influence of column and eluent thermostatting specifics and the unique thermostatting features that the Vanquish systems offer

Method Transfer Best Practice Summary

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Method Transfer Best Practice - Thermo Fisher Scientific · Method Transfer Best Practice . 2 ... cylinder) 0.9 0.8 . 0.6 . 0.7 0.5 0.4 ... Fine-tuning of GDV by the autosampler metering