Increased Protein/Peptide Mass Transfer Rates for Improved ... · Sample: 3 mg each protein 1. RNase 6. CDR 2. Insulin 7. Myoglobin 3. Cytochrome C 8. Carbonic Anhydrase 4. Lysozyme

Increased Protein/Peptide Mass Transfer Rates for Improved Resolution and Faster LC

Page 2

• Proteomics studies • LC/MS studies• Combinatorial chemistry analyses• Rapid monitoring for process control• 2D-methods

Applications for Fast HPLC of Large Molecules and Proteins

Page 3

First, A Few Basics on Bio Molecule Separations

• Factors That Optimize Bio Separations• Sample Solubility• Appropriate Pore Size• Bonded Phase• Low pH Mobile Phase• High pH Mobile Phase• Temperature• Gradient

Page 4

Solvent Application Comments 0.05-5% TFA in Water General Effective solubilization of many

samples 6 M Guanidine, buffered at pH 6-8

General Very good for many proteins and peptides

5-80% Acetic Acid or Formic Acid; 0.1-0.5M Perchloric Acid

Peptides

Frequently used to extract peptides from tissues, precipitating many proteins and cellular debris

6 M Urea/ 5% Acetic aid Hydrophobic Peptides, Proteins

Useful for membrane proteins, fragments, aggregating systems

Water-Miscible Organic Solvents: Acetonitrile, Methanol, THF, Dioxane, DMSO; +/- TFA; +/- Water

Hydrophobic Peptides, Polypeptides

Limit injection volume to avoid problems; add water, as possible, to improve volume tolerance; acidify with TFA as required

Optimize SolubilitySelected Sample Solvents and Application for Protein and Peptides

Page 5

Improved Peak Shape for Large Molecules in Solution

Columns: 4.6 x 150 mm, 5 mm Mobile Phase: 60% MeOH: 40% 0.1% TFA Flow Rate: 0.75 mL/min Temperature: RT Detection: UV 282 nm Sample: Tylosin (MW 916)

SB-C3 (80Å) 300SB-C3 (300Å)

• The size of a molecule in solution determines which pore size column is best.• The narrower peak width indicates unrestricted access to the pores.

Pw1/2 = 0.442 Pw1/2 =0.125

O

O

NHOHO

CHO

HO

OHOH

O

OOO

O

O O

O

CH3

CH3CH3

OCH3OCH3

CH3

CH2

CH2

CH2

CH3

CH3

CH3

H3CH3C

H3C

0 5 10Time (min)

0 5 10Time (min)

Page 6

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

300SB-C18 (300Å)SB-C18 (80Å)

PW 1/2

LeuEnke

phalin

M.W. =

556

Angiotensin

II

M.W. =

1046

Insulin

B

M.W. =

3,49

6

CytC

M.W. =

12,32

7

RNase

M.W. =

13,68

4Lys

ozyme

M.W. =

13,90

0

Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations

Wide Pore (300Å) Columns are Needed for Proteins and Polypeptides

•Proper pore size selection results in sharper peaks for large molecules

Page 7

50%

60%

70%

80%

90%

100%

110%

300SB-C8 300SB-C3 300SB-CN

ParvalbuminMyoglobinRNase AInsulinLysozymeCarbonic AnhydraseCalmodulin

Columns: 4.6 x 150 mmMobile Phase: 5 - 40% B in 20 min.

A: 0.1% TFA / WaterB: 0.1% TFA / ACN

Flow Rate: 1 mL / min.Temperature: 60°CSample: 4 µg each protein

25 µL injection

Rel

ativ

e R

ecov

ery

to 3

00S

B-C

18

•Recovery may vary depending on bonded phase.• All 300SB bonded phase generally provide good recovery.

Recovery of Polypeptides from ZORBAX 300SB Columns

Page 8

12,3

4 56

7 89

10

1 24 5

6,7

89

103

1 24 5

68

9 103

7

Ret ent ion Time (min)0 5 10 15 20 25 30 35 40

1 24 5

68

910

37

Comparison Separation of Large Polypeptides on 300SB Bonded Phases

Columns: ZORBAX StableBond 300SB4.6 x 150 mm, 5 mm

Mobile Phase: Linear Gradient, 25- 70% B in 40 minA: 0.1% TFA in WaterB: 0.09% TFA in 80% ACN/20% water

Flow Rate: 1.0 mL/minTemperature: 60°CSample: 3 mg each protein

1. RNase 6. CDR2. Insulin 7. Myoglobin3. Cytochrome C 8. Carbonic Anhydrase4. Lysozyme 9. S-100β5. Parvalbumin 10. S-100α

300SB-C18

300SB-C8

300SB-C3

300SB-CN

• Four different 300SB bonded phases allow selectivity optimization of proteins.

Page 9

Conditions:Column: ZORBAX 300SB, 4.6 x 150 mm, 5 mmMobile Phase: A – 0.1% TFA/water, B – 0.1% TFA/canGradient: 10 - 90 % B in 40 minFlow Rate: 1 mL/minTemperature: 60°C, Sample: 15 µL ( 15 µg ) of peptide in 0.1% TFA/DMSO

300SB-CN

300SB-C8

300SB-C18

Recovery 59%

Recovery 75%

Recovery 89%

Retention Time, (min.)

• Recovery on each bonded phase is not always predictable so evaluate different bonded phases.

Effect of Bonded-Phase Ligand on Recovery of a Synthetic Lipopeptide

Page 10

ZORBAX 300SB HPLC Column Developed Expressly for Bio Separations

Page 11

000336P1.PPT

Column: ZORBAX 300SB-C8, 4.6 x 150mm; Mobile Phase Gradient 0 - 60% in 120 min.A= 0.1% TFA in Water, B= 0.086% TFA in ACN Temp.: 40°C Flow Rate: 1mL/min. Det. UV 210nm Sample: 50 µg of rhGH Tryptic Digest

After 495 mL

After 13680 mL

ZORBAX 300SB Designed for Long Life in Low pH Bio Separations

rhGH Tryptic Digest

Reference 3

Page 12

High pH Can be Used for Separating Hydrophobic or Other Low-Solubility Peptides

Comparison of Aß Peptide RP-HPLC Separations at Low and High pH

Column: ZORBAX 300Extend C18 2.1 x 150 mm, 5 mm

Flow Rate: 0.25 mL/min Sample: 5 µL sample (100 pmol each)

Amyloid ß Sequences:Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys Leu17 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly AlaIle Ile Gly Leu Met Val Gly Gly38 Val Val40 Ile Ala42 Thr43-COOH

Abs

orba

nce

(210

nm

)TFA Conditions, 25°C

A- 0.1% TFA in waterB- 0.085% TFA in 80%AcN33-45%B in 30 min.

TFA Conditions, 80°CA- 0.1% TFA in waterB- 0.085% TFA in 80%AcN29-41%B in 30 min

.

Aβ(1-38)Aβ(1-40)

Aβ(1-42)Aβ(1-43)

Aβ(1-38) Aβ(1-40)

Aβ(1-43)

Aβ(1-42)

Aβ(1-42/3)Aβ(1-40)

Aβ(1-38)

NH4OH Conditions, 25°CA- 20 mM NH4OH in waterB- 20 mM NH4OH in 80%AcN26-38%B in 30 min.

Reference 1

• High pH and room temperature improve peak shape, recovery and change selectivity.

Page 13

pH Can Change Selectivity in Bio SeparationsComparison of TFA and NH4OH For Peptide RP-HPLC \ ESI-MS

Column: ZORBAX Extend C18, 2.1 x 150 mmFlow rate: 0.25mL/minTemp: 25°C Gradient: 5-60% B in 20 min; LC/MS: Pos. Ion ESI- Vf 70V, Vcap 4.5 kV,

N2- 35 psi, 12L/min, 300°C4 µL (50 ng each peptide);

0

.5E7

1E7

1.5E7

2E7

2.5E7

3E7

m in0 2 4 6 8 10 12 14 16 180

.5E7

1E7

1.5E7

2E7

2.5E7

3E7

TFA Conditions:A- 0.1% TFA in waterB- 0.085% TFA in 80% AcN

TIC

(150

-150

0 m

/z)

0.1% TFA

20mM NH4OH

LLG LHL

LLL-NH2LLL

NH4OH Conditions:A- 20 mM NH4OH in waterB- 20 mM NH4OH in 80% AcN

LLVF

LLVF

LLL-NH2

LLG

LHL

LLL

Page 14

Peptide RP-HPLC/ESI-MS NH4OH Mobile Phase Yields Positive and Negative Ion Spectra

Column: ZORBAX Extend C18 2.1 x 150 mm, 5 mm

Flow rate: 0.25mL/min Temp: 25°C Gradient: 5-60% B in 20 minLC/MS: Pos. Ion ESI- Vf 70V, Vcap 4.5 kV,

N2- 35 psi, 12L/min, 300°CSample: 4µL (50 ng each peptide)

0

.5E7

1E7

1.5E7

2E7

2.5E7

3E7

min0 2 4 6 8 10 12 14 16 180

.5E7

1E7

1.5E7

2E7

2.5E7

3E7

TIC

(150

-150

0 m

/z)

Positive Ions

LHL

LLG

LLL

LLVF

LLL-NH2

Negative Ions

LHL

LLG LLL

LLVFLLL-NH2

• The Extend-C18 column is ideal for LC/MS of proteins and peptides at high pH• Both positive and negative ion MS are possible with NH4OH mobile phase.

Page 15

LC/MS at Low pH on Extend-C18

Column: ZORBAX Extend-C18, 2.1 x 150 mm, 5 mm Flow Rate: 0.2 mL/min Temperature: 35°CMobile Phase Gradient: 15-50% B in 15 min. A: 0.1% TFA in water B: 0.085% TFA in 80% ACN

LC/MS: Pos. Ion ESI – Vf 70V, Vcap 4.5 kV, N2-35 psi, 12L/min., 325°C Sample: Angiotensin I, II, III 2.5 mL (50 pmol each)

• Acidic conditions can not resolve all three Angiotensins.

min0 2.5 5 7.5 10 12.50

1.0E6

2.0E6

3.0E6

4.0E6

5.0E6

12.

050

13.

261

AII+ AIII

AITIC

(200

-150

0 m/

z)

m/z500 1000

0

20

40

60

80

100Max: 10889

649

.5

433

.0

227.1

659.7

844.8

250.9

340.3

1277.

9

1179.

1

1025.

2

720.3

1326.

3

398.0

767.3

458.0

1468.

8

1373.

3

+ 3 + 2

Page 16

LC/MS at High pH on Extend-C18

• At high pH all 3 Angiotensins are resolved and the mass spectrum shows improved spectral clarity.

Column: ZORBAX Extend-C18, 2.1 x 150 mm, 5 mm Flow Rate: 0.2 mL/min Temperature: 35°C Mobile Phase Gradient: 15-50% B in 15 min. A: 10 mM NH4OH in water B: 10 mM NH4OH in 80% ACN

LC/MS: Pos. Ion ESI – Vf 70V, Vcap 4.5 kV, N2-35 psi, 12L/min., 325°C Sample: Angiotensin I, II, III, 2.5 mL (50 pmol each)

Signal x10

m/z500 10000

20

40

60

80

100Max: 367225 6

49.0

433

.0

659

.8

129

6.6 + 1

+ 2

+ 3

min0 2.5 5 7.5 10 12.50

1.0E7

2.0E7

3.0E7

4.0E7

5.0E75.

499 8.

477AII

AITIC

(200

-150

0 m/z)

AIII9.

621

Page 17

Column: ZORBAX 300SB-C18, 4.6 x 150 mm, 5 mm Mobile Phase: A- 0.1%TFA in water, B- 0.09%TFA in acetonitrileGradient: 20-45% B in 35 min Flow Rate: 1 mL/min Sample: 10 µl injection of 5 µg peptide in 6M Urea/5% HOAc

Abs

orba

nce

(210

nm

)

25°CßAP(1-38)

ßAP(1-43)* Recovery <10%

40°C

60°C

Time (min.)0

80°C

5 10 15 20 25 30 35 40

Recovery >70%

Optimize Recovery by Changing TemperatureßAP Peptide Separation

Page 18

Protein and Peptide Retention by RP-HPLC –High Sensitivity to Changes in % Organic:

0.14 0.18 0.22 0.26 0.30 0.34 0.38 0.42

100

10

1

k«

φ

Leucine Enkephalins = 11

Lysozymes = 40

Benzenes = 2.7

log k

log k = log k0 - Sφ

φ = W ater/Organic

Large proteins like Lysozyme (14,000 MW) are more sensitive to changes in mobile phase conc of organic modifier (15X) than small molecules like benzene (78 MW) and 4X more sensitive than leucine enkephalin (600 MW).

Page 19

Longitudinal diffusion

Smaller Particles Improve Mass Transfer Effect on Column Dispersion

Plat

e H

eigh

t H

Linear Velocity u

Eddy Diffusion

Sum Curve: van-Deemter

Resistance to Mass Transfer

H = A + B/u + C uLarge Particle

SmallParticle

The smaller the plate height, the higher the plate number and the greater the chromatographic resolution

u opt

H min

Page 20

Improved Efficiency Over Wide Flow Range with Smaller Particles

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0μL Octanophenone in

Eluent

Volumetric Flow Rate (mL/min)

HETP (cm

)

5.0μm3.5μm1.8μm

H = A + B/u + Cu

Smaller particle sizes yield flatter curves, minima shift to higher flow rates

Title of Presentation Agilent Restricted

0 10 20 30 40

Changing Gradient Time to AffectRetention (k*) and Resolution

Time (min)

100% B

100% B

100% B

100% B

tg= 40

tg= 20

tg= 10

tg= 5

000995P1.PPT

tg F

S Δ%B Vm

k* ∝

DF = change in volume fraction of B solventS = constantF = flow rate (mL/min.)tg = gradient time (min.)

Vm = column void volume (mL)

0% B

0% B

0% B

0% B

• S ≈ 4–5 for small molecules

• 10 < S < 1000 for peptides and proteins

Page 22

0 5 10 15 20 25 30 35 40 45 50

0 2 4 6 8 10 12 14 16 18 20 24 26 28 30

12.

16 22

0 1 2 4 5 6 8 9 1073

1 23 4 5 6 7

89 10

ZORBAX 300SB-C8 4.6 x 250 mm, 5 µm

Rapid Resolution 4.6 x 150 mm, 3.5 µm

Rapid Resolution 4.6 x 50 mm, 3.5 µm

40 min.

24 min.

9 min.

1. Met-enkephalin2. Leu-enkephalin3. Angiotensin II4. Neurotensin5. RNase6. Insulin (Bov)7. Lysozyme8. Calmodulin9. Myoglobin

10. CarbonicAnhydrase

Conditions:

Mobile Phase: A: 95:5, Water : ACN with 0.1% TFAB: 5:95, Water : ACN with 0.085% TFA

Flow: 1.0 mL/minDetection: 215nmSample: 1-10µg protein (10µL inj.) in

6M Guanidine HCL, pH7.010-60% B in 10 min.

10-60% B in 30 min.

10-60% B in 50 min.

Optimize Resolution: Column Length, Particle Size and Gradient Time

Page 23

300SB-C8, 4.6 x 150 mm, 5µm 300SB-C8, 4.6 x 50 mm, 3.5µm

Mobile Phase: A: 95% Water : 5 % ACN, 0.1% TFAB: 5% Water : 95% ACN, 0.085% TFAGradient: 10-60% B in 30 min.

Flow Rate: 1.0 mL / min.Temperature: Ambient

Sample: 1. Gly-Tyr 6. Leu-Enk2. Val-Tyr-Val 7. Angiotensin II3. [Gln11] Amyloid-β- 8. Kinetensin

Protein Fragm 1-16 9. RNase4. (TYR8) Bradykinin 10. Insulin (Eq.)5. Met-Enk

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16

1

2

3

4 56

7 8

9

101

23

45

6

7 8

9

10

000997P1.PPT

Improving Resolution Using ShortColumn Length ( Vm ) and Particle Size (N)

Page 24

Gradient Resolution and Shorter ColumnsFaster Analyses for Complex Samples: HSA Tryptic Digest

min15 20 25 30 35 40 45 50 55

min3 4 5 6 7 8 9 10

min5 10 15 20 25

2.1x150mm, 5μmP/N 883700-92270 min gradient0.2 mL/min

2.1x50mm, 1.8μmP/N 822700-90210 min gradient0.5mL/min

2.1x50mm, 1.8μmP/N 822700-90230 min gradient0.5mL/min

120 peaks60 mins

125 peaks10 mins!

156 peaks!25 mins

Conditions: M obile Phase A: W ater w/ 0.1% TFA, B: ACN w/0.1% TFA, Gradient 2%B to 50%B, Temp: 50°C UV 214 nm

Page 25

Particle Size - More Peak Capacity with 1.8 um RRHT Columns - Peptide Map of BSA

673

502

Peak CapacityColumn used

SB-C18, 2.1x150mm, 3.5µ

SB-C18, 2.1x150mm, 1.8µ

min14.5 15 15.5 16 16.5 17 17.5 18 18.5 19

mAU

-10

0

10

20

30

40

50

min15 16 17 18 19

mAU

-20

0

20

40

60

Less peak capacity, less resolution

35% More peak capacity, more resolution

Conditions: Columns: as listed, Mobile Phase: A:0.1% TFA in Water B:0.08% TFA in ACN Gradient: 5% B to 60%B in 25 min. Temperature: 80°C Sample: BSA tryptic digest

StartingPressure380 bar

StartingPressure105 barPeak Capacity

Smaller particle size = sharper peaks = greater peak capacity

Page 26

What Makes High Speed HPLC Possible?

• Small particle sizes (< 5 mm)• Short columns (10 cm or less)• Low viscosity mobile phases• Optimized HPLC – minimal extra column volume, low

volume flow cell, minimal delay volume for gradients• Detector set to fast response time• High diffusion coefficient of the sample in the mobile

phase • Rapid diffusion = less band broadening = higher

linear velocities with efficient peaks • But large molecules have small diffusion

coefficients!!

Page 27

PorousShell

SolidCore

• Increase the Diffusion Rate • Elevate operating temperature -- Need stable bonded

phase• Decrease solvent viscosity -- Helps but changes elution

• Decrease the Diffusion Distance• Develop very small particles (<2-um) • Limit diffusion distance into a particle!

Slower Diffusion of Large Molecules Limits Speed and Resolution

So, decrease the diffusion time for macromolecules!

Page 28

A solid silica core with a superficially porous surface of smaller silica sol particles derivatized with bonded phase.

Scanning Electron Micrograph of Poroshell Particles

Scale: = 1 µm

What is Poroshell 300SB?

• Poroshell 300SB superficially-porous particles, reduce diffusion distance for proteins for fast HPLC.

• A RP-HPLC phase such as C18, C8 or C3 are bound to sol particles in the porous shell.

Page 29

0.25 µm

5 µm Totally Porous Particle

2.5 µmRequired diffusion distance

for a macromoleculereduced 10 fold !

5 µm Superficially Porous Particle

Comparison of Diffusion DistanceTotally porous silica vs. superficially porous silica

Page 30

Column: Poroshell 300SB-C18, 5 μm Mobile phase: Urease, 44.7% ACN / 55.2% 0.1% TFA, Insulin1, 32.6% ACN / 67.3% 0.1% TFA Insulin 2, 28% ACN / 72% 0.1% TFA Detection:UV 214 nm Samples: in 6 M guanidine, pH 6.8 phosphate buffer Temperature: 60°C

Mobile Phase Velocity, cm/sec0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Pla

te H

eigh

t, cm

0.000

0.005

0.010

0.015

0.020

Data fitted to Knox equation

Urease; MW=83,000(sub-unit); column: 3 x 100 mm

Insulin1; MW=5,7003 x 100 mm column

Insulin2; MW=5,7002.1 x 75 mm column

• Slow increase in plate height with velocity means high flow rates can be used for fast separation with modest loss in resolution

• Steeper plot for urease reflects slower diffusion of much larger protein

Maintain Efficiency at High Velocity with Poroshell 300SB-C18

Van Deemter Plot for Urease (83,000D) and Insulin (5,700D)

Page 31

How Do We Use Poroshell Columns?

• Faster separations• High flow rates• High flow rates for fast analysis with high resolution in

comparison to totally porous silica• Polypeptides• Large proteins

• High recovery of large proteins• Elevated temperature

• High resolution• High resolution of protein digests• High resolution of protein impurities• High resolution for LC/MS of proteins

• Change selectivity• Different bonded phases• Comparison to totally porous 300SB materials

Page 32

High Flow Rates with 2.1 mm ID Poroshell for High Resolution and Fast Separations

12

34

5

67

8

0 0.5 1.0Time (min)

Columns: Poroshell 300SB-C182.1 x 75 mm, 5 mm

Mobile Phase:A: 0.1% TFAB: 0.07% TFA in ACN

Gradient: 5 – 100% B in 1.0 min.Flow Rate: 3.0 mL/min.Temperature: 70°CPressure: 250 barDetection: UV 215 nm

Sample:1. Angiotensin II2. Neurotensin3. Rnase4. Insulin5. Lysozyme6. Myoglobin7.Carbonic Anhydrase8.Ovalbumin

• Only Poroshell can provide high efficiency at higher flow rates for extremelyrapid separations of proteins and peptides.

• This is due to more rapid mass transfer of the superficially porous particle

Page 33

Flow Rates for Poroshell Columns

Column Internal Diameter

Porous Particle Flow Rate Range

Poroshell Flow Rate Range

2.1 mm 0.1 – 0.3 mL/min 0.3 – 3 mL/min

1.0 mm 30 – 60 mL/min 0.08 - 0.75 mL/min

•Very high flow rates can be used effectively with Poroshell columns

Page 34

min0 2 4 6 8 10 12 14 16 18

mAU

-1000

100200300400500

min*0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

mAU

-1000

100200300400500

(Poroshell chromatogram - expanded)

min*0 2 4 6 8 10 12 14 16 18

mAU

-1000

100200300400500

1\DATA\PORSHL3\PrSHL010.d\ßamy-ovr.win

Poroshell 300SB-C182.1x75 mm, 5 µm

Zorbax 300SB-C18 4.6x150 mm, 5 µm

F =1 mL/min, 5-100 %B / 20 min

ß-amylase

ß-amylase

ß-amylase

Poroshell chromatogramexpanded

•Poroshell technology facilitates ultra-fast HPLC analysis of proteins Note the similar separation of minor impurities in the 2 and 20 min

separations.

12 min

2min

12min

0 1 1.4 1.80.6 0.80.40.2 1.2 1.6

Poroshell for Fast Analysis of High MW Proteins - ß-amylase (200kdal)

Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Column: as shown Gradient: as shown Mobile Phase: A= 95% H2O, 5% ACN with 0.1%TFA; B= 5% H2O, 95% ACN, with 0.07%TFA Flow Rate: as shown Temp.: 70°C, Detection.: UV 215 nm

F =2 mL/min, 5-100 %B / 1 min

Page 35

Poroshell Has Different Selectivity from Totally Porous 300SB

min0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

mAU

0

200

400

600

800

min0.10

0

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

mAU

0

200

400

600

800

Agilent 1100 WPS with AutoBypass Column: 300SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase: A= 95% H2O, 5% ACN with 0.1%TFA B= 5% H2O, 95% ACN, with 0.07%TFA Gradient: 5 –100%B in 0.67 min Flow Rate: 3 mL/min Piston Stroke: 20 µL Temp.: 70°C Det.: UV 215 nm

300SB-C18

Poroshell 300SB-C18

1,2 43

32

1

4

•Poroshell and 300SB-C18 can have different selectivity due to the different ratios of bonded phase on the surface.

Page 36

5 min

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

Totally Porous2.1x75mm, 5µm3 mL/min

0-100%B in 0.67 min

1. Neurotensin2. Rnase A3. Lysozyme4. Myoglobin

1 2 3 4

2 mL/min0-100%B in 1 min

1 mL/min0-100%B in 2 min

0.5 mL/min0-100%B in 4 min

Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Temp.: 70°C, Det.: 215 nmMobile Phase: A= 95% H2O, 5% AcN with 0.1%TFA; B= 5% H2O, 95% AcN, with 0.07%TFA

•Increased flow rate decreases run time, but, decreases resolution in protein analysis when using totally porous particles!

Effect of Increasing Flow Rate in Protein Analysis with use of Totally Porous Silica

Page 37

5 min

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

200

400

600

800

1000

Poroshell 300SB-C182.1x75mm, 5µm

3 mL/min0-100%B0.67 min

1. Neurotensin2. Rnase A3. Lysozyme4. Myoglobin

12 3 4

Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Temp.: 70°C, Det.: 215 nmMobile Phase: A= 95% H2O, 5% ACN with 0.1%TFA; B= 5% H2O, 95% ACN, with 0.07%TFA

2 mL/min0-100%B1 min


0.5 mL/min0-100%B4 min

Sustained Efficiency and Resolution

• As flow rate increases, protein/peptide analysis time is dramatically reduced when using superficially porous particles!

Effect of Increasing Flow Rate in Protein Analysis with Poroshell

Page 38

min0 2 4 6 8 10 12 14 16 18

mAU

-1000

100200300400500

ß-amylase

A=0.1%TFA, H2O; B=0.07%TFA, ACN

min*0 2 4 6 8 10 12 14 16 18

mAU

-1000

100200300400500

*DAD1 A, Sig=215,16 Ref=310,10

ß-amylase(Poroshell chromatogram - expanded)

min*0 2 4 6 8 10 12 14 16 18

mAU

-1000

100200300400500

*DAD1 A, Sig=215,16 Ref=310,10

ß-amylase

1\DATA\PORSHL3\PrSHL010.d\ßamy-ovr.win


Temp: 70ºC

F =2 mL/min, 5-100 %B / 1 min

ZORBAX 300SB-C18 4.6x150 mm, 5 µm

Temp: 70ºC

F =1 mL/min, 5-100 %B / 20 min

ß-amylase

ß-amylase

ß-amylase


• Poroshell technology facilitates ultra-fast HPLC analysis of proteins• Note the similar separation of minor impurities in the 2 and 20 min separations.

12 min

2min

12min

0 1 1.4 1.80.6 0.80.40.2 1.2 1.6


Poroshell for Fast Analysis of High-Molecular Weight Proteins

ß-amylase (200kdal)

Page 39

min0 2 4 6 8 10 12 14 16 18

mAU

-50

0

50

100

150

200

250

DAD1 A, Sig=215,16 Ref=310,10 (PORSHL3\PRSHL010.D)

Poroshell 300SB-C18, 2.1x75mm, 5µm

F=2, 5-100%B/1 min, 70°C


Thyroglobulin subunits

min*0 2 4 6 8 10 12 14 16 18

mAU

-50

0

50

100

150

200

250

*DAD1 A, Sig=215,16 Ref=310,10 (PORSHL3\PRSHL010.D)

(Poroshell chromatogram - expanded)


min*0 2 4 6 8 10 12 14 16 18

mAU

-50

0

50

100

150

200

250

*DAD1 A, Sig=215,16 Ref=310,10 (PORSHL3\PRSHL042.D)

1\DATA\PORSHL3\PrSHL042.d\Thyr-ovr.win


Zorbax 300SB-C18, 4.6x150mm, 5µm

F=1, 5-100%B/20 min, 70°C


F =2 mL/min, 5-100 %B / 1 min

F =1 mL/min, 5-100 %B / 20 min

Thyroglobulinsubunits


• Poroshell technology facilitates ultra-fast HPLC analysis of very large protein subunits.• Note the improved peak shape and recovery on Poroshell, in <2 min.



12 min

2min

12min

Zorbax 300SB-C18 4.6x150 mm, 5 µm

Good Peak Shape and Recovery with PoroshellAnalysis of High-Molecular Weight Proteins - Thyroglobulin 660 kDa

Page 40

Aligned

min0 1 2 3 4 5 6 7

mAU

0

50

100

150

200

min*0 1 2 3 4 5 6 7

mAU

0

50

100

150

200


BSA tryptic digest15hrs., 70 pmol

Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Temp.: 70°C, Det.: 215 nmMobile Phase: A= 95% H2O, 5% AcN with 0.1%TFA; B= 5% H2O, 95% AcN, with 0.07%TFA


0.208 mL/min0-100%B120 min

Zorbax 300SB-C182.1x150 mm, 5 µm

6min

60min

• Extremely fast (6 min), high, resolution of BSA tryptic digest, using Poroshell 300SB-C18. • Under typical conditions, a comparable run takes 60min on a totally porous particle.

High Resolution and Fast AnalysisSeparation of a Protein Digest on Poroshell 300SB-C18

Page 41

UltraHigh Speed HPLC Peptide M aps of a M onoclonal Antibody on Several Zorbax Poroshell Phases

Aligned

min1 2 3 4 5

min1 2 3 4 5

min1 2 3 4 5

mAU

0

20406080

100

mAU

0255075

100125150175

mAU

0

50

100

150

200

Aligned

min1 2 3 4 5

min1 2 3 4 5

min1 2 3 4 5

mAU

0

20406080

100

mAU

0255075

100125150175

mAU

0

50

100

150

200

min1 2 3 4 5

min1 2 3 4 5

min1 2 3 4 5

min1 2 3 4 5

min1 2 3 4 5

min1 2 3 4 5

mAU

0

20406080

100

mAU

0255075

100125150175

mAU

0

50

100

150

200

mAU

0

20406080

100

mAU

0255075

100125150175

mAU

0

50

100

150

200

Conditions: M obile phase A = 0.1% TFA in water M obile phase B = 0.1% TFA in ACN; Temperature: 70°C; Detection: VW D, 210nm; Injection: 10 µl Lys-C digest of Human M onoclonal Antibody; Flow: 1.0 ml/min; Gradient: 0 min, 0% B; 5.5 min, 55% B; 5.6 min, 55% B; 7.0 min, 0% B

Zorbax Poroshell 300SB-C18, 2.1 x 75mm, 5µ



•Zorbax Poroshell technology facilitates ultra-fast HPLC analysis of peptides

Original method –120 min tG using a C18 4.6 x 250 mm –57 peaks detected

For more details see 5989-0590EN

46 to 48 peaks, 1/20 analysis time

Data courtesy of:Novartis Pharma, Biotechnology, Basel Dr. Kurt ForrerPatrikRoethlisberger

Page 42

LC/MS of Proteins with PoroshellFormic acid Provides High Sensitivity

m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

0

10000

20000

30000

40000

50000

60000

1254.4

1187.3

1231.2

1146.4

1303.6

1278.7

1166.5

1356.7

1208.8

1108.2

1126.9

1329.7

1090.0

1189.6

1211.9

1445.2

1128.7

1257.3

1385.5

1233.2

1414.4

1149.1

1306.8

1168.4

1331.8

1110.3

1360.0

1510.9

1545.8

1477.2

1448.7

1055.5

1282.0

1621.3

1075.2

1582.6

1661.8

1704.1

1749.1

1023.3

1796.7

m/z1000 1200 1400 1600 1800 2000

0

500

1000

1500

2000

2500

3000

3500

4000

1385.2

1796.5

1513.7

1704.2

1955.1

1624.3

1749.1

1303.6

1546.2

1414.4

1665.6

1480.6

1621.8

1586.3

1388.2

1359.6

1705.9

2004.7

1510.7

1801.0

1904.5

2144.6

2171.8

2076.9

1848.8

1734.8

1254.4

min0.5 1 1.5 2 2.5 3 3.5

0

5000000

10000000

15000000

20000000

25000000

Abundance

Formic Acid

TFA

TIC

Column: Poroshell 300SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase Gradient: 20-100% B in 5.5 min. A: water + 0.1% TFA or FA B: ACN + 0.1% FA or TFA Flow Rate: 500 mL/min Temperature: 60°C Injector: 1 mL Sample: 10 pmol of BSA Electrospray ionization: positive ion Vcap:6000V Drying Gas: 12L/min 350ºC Nebulizer: 45 psi Scan: 600-2500 amu Step size:0.15 amu Peak width:0.06 min

Page 43

High Flow Rates and High Sensitivity LC/MSUsing 1.0 mm ID Poroshell

min0 0.5 1 1.5 2 2.5 3 3.5

0

20000000

40000000

60000000

80000000

1E8

pmoles of protein on column0.5

2.55

0.751

•These TIC’s show good sensitivity with only 0.5 pmoles on column.

Column: Poroshell 300SB-C18, 1.0 x 75 mm, 5 mm Mobile Phase Gradient: 20-100% B in 5.5 min. A: water + 0.1% formic acid B: ACN + 0.1% formic acid Flow Rate: 600 mL/min Temperature: 80°C Injection volume: 1 mL Sample: insulin, lysozyme, cytochrome C, myoglobin, BSA, carbonic anhydraseLC/MS: Pos. Ion ESI –, Vcap 6000 V, Drying gas flow: 12 l/min Drying gas temperature: 350°C Nebulizer: 45 psi, Fragmentor volatage: 140 V Scan: 600 –2500 Stepsize: 0.15 amu Peakwidth: 0.06 min

Page 44

min0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75

mAU

10

20

30

40

50

min0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75

mAU

10

20

30

40

50

Untreated

Fast Analysis of Insulin (5.7 kDa) Impurities using Poroshell 300SB-C18

Column: Poroshell 300SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase Gradient: 5-100% B in 5 min. A: water + 0.1% TFA B: ACN + 0.0.07% TFA Flow Rate: 2 mL/min Temperature: 70°C Sample: Bovine pancreas insulin

INSULIN - bovine pancreas

27 hrs. at 55°C

Page 45

8 min

min0 1 2 3 4 5 6 7

mAU

0200400600

min0 1 2 3 4 5 6 7

mAU

0200400

min0 1 2 3 4 5 6 7

mAU

0100200300

.

min0 1 2 3 4 5 6 7

mAU

0

100

min0 1 2 3 4 5 6 7

mAU

-500

50100 Impurities CA

CA

CA

CA

CA

•Poroshell technology is compatible with a wide range of gradients that result in a corresponding range of resolution and run times.

•Note the consistent separation of minor impurities even in the 1 min run at top.

tG = 2 min

tG = 4 min

tG = 1 min

tG = 8 min

tG = 16 min

Column: Poroshell 300SB-C18, 2.1 x 75 mm, 5 mm Mobile Phase Gradient: 5-100% B in tG min. A: water + 0.1% TFA B: ACN + 0.0.07% TFA Flow Rate: 2 mL/min Temperature: 70°C Sample: Carbonic Anhydrase

Increasing Gradient Times on PoroshellRapid Analysis of Fresh Carbonic Anhydrase

Page 46

4 min

0 0.5 1 1.5 2 2.5 3 3.5

mAU

0

100

200

300Carbonic Anhydrase Poroshell 300SB-C18 (2.1x75mm)

5 hrs. Incubation at 55°C

Flow=2mL/min, 5-100%B/4 min, 70°C

0 0.5 1 1.5 2 2.5 3 3.5

mAU

0

100

200

300

1 hrs. Incubation at 55°C

0 0.5 1 1.5 2 2.5 3 3.5

mAU

0

100

200

300FRESH

2


F=2mL/min, 5-100%B/4 min, 70°CA=0.1%TFA in H2O, B=0.07%TFA in ACN

Carbonic Anhydrase (CA)

5 hrs. incubation at 55°C

1 hr. incubation at 55°C

FRESH

CA

CA

CA

•Rapid HPLC analysis of carbonic anhydrase and its aging products is achieved in 4 minutes.

Impurities

Rapid Analysis of Carbonic Anhydrase Aging RP-HPLC on Poroshell 300SB-C18

Page 47

• Extremely fast resolution of carbonic anhydrase aging products using Poroshell under four different gradient times. High resolution is obtained in 2 or 3 min.

0 0.5 1 1.5 2 2.5 3 3.5

mAU

50100150200250

0 0.5 1 1.5 2 2.5 3 3.5

mAU

5075

100125150175200

0 0.5 1 1.5 2 2.5 3 3.5

mAU

020406080

100

min0 0.5 1 1.5 2 2.5 3 3.5

mAU

020406080

100

Carbonic Anhydrase (CA)Several M onths at 4°C

1 min

2min

3 min

Fast Separation of Aged Carbonic AnhydraseRP-HPLC on Poroshell 300SB-C18

Column: Poroshell 300SB-C18, 2.1 x 75 mm, 5 μm M obile Phase Gradient: 5-100% B in tG min. A: water + 0.1% TFA B: ACN + 0.0.07% TFA Flow Rate: 2 mL/min Temperature: 70°C Sample: Carbonic Anhydrase1 mg/mL Injection Volume: 2 μL

tG = 2 min

tG = 4 min

tG = 8 min

tG = 1 min

Page 48

More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution

min0 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

Poroshell SB-C18, 2.1 x 75 mm

1

2 3 4

5, 6

7

8

9 0.9

76

1.0

77

1.2

89

1.3

95

1.4

63

1.5

23

1.7

41

1.8

60

min0 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

512

34

6

7

8

9

Poroshell SB-C3, 2.1 x 75 mm

0.9

01

1.0

36 1.2

64

1.3

54 1.4

30 1

.449

1.4

97

1.7

39

1.8

37

Column: Agilent Poroshell (2.1 x 75 mm); Temp.: 70 0C; Flow: 0.5 mL/min; Det: UV 215 nmMobile Phase: A= 0.1% TFA/H2O, B= 0.07% TFA/ACN; Gradient: 5-100% B in 3.0 min

1.5

5

4 6

7

1.5

5

4 6

7

1.5

4

5, 6

7

1.5

4

5, 6

7

Samples:1. Angiotensin II2. Neurotensin3. RNase A4. Insulin B Chain5. Insulin6. Cytochrome C7. Lysozyme8. Myoglobin9. Carbonic

Anhydrase

Poroshell 300SB-C182.1 x 75 mm

Poroshell 300SB-C32.1 x 75 mm

•Changing from SB-C18 to SB-C3, within the Poroshell family results in resolution of peaks 5 and 6, still in 3 min!

Page 49

Summary

• Fundamentals of Bio Separations apply whether using conventional columns or columns that optimize mass transfer

• These fundamentals can be used to optimize bio separations

• 1.8 µm particles can be used for much faster gradients peptide maps

• Poroshell columns have greatly reduced surface area and markedly reduce resistance to large molecule mass transfer

• This allows for fast analysis of proteins – including very large proteins with high recovery.

• Optimized bonded phases for low and high pH offer more options for RPLC and Mass Spec assays

Page 50

Column Description

Monolith stationary phase.

Page 51

Column Description

• Currently there are 4 Bio-Monolith Products:

• Bio-Monolith QA (strong anion-exchanger)

• Bio-Monolith DEAE (weak anion-exchanger)

• Bio-Monolith SO3 (strong cation-exchanger)

• Bio-Monolith Protein A (immunoaffinity)

Further products to expand the product portfolio (CM: weak cation-exchanger; Protein G (?), Specialty columns (pDNA analytics, virus analytics).

Page 52

Catalog number 5069-36395

Column chemistry QA: Strong anion exchanger

Matrix Poly(glycidyl methacrylate – ethylene dimethacrylate)

Ligand density 2.0 ± 0.2 mmol/g

Matrix dimensions Diameter: 5.2 mm; Length: 4.95 mm;

Bed volume: 0.1 mLDynamic binding capacity > 25 mg BSA/ml

Maximum loading capacity 1.2 to 1.5 mg

Working flow rates 0.2 to 2 ml/min (max. 3 mL/min)

Maximum allowed pressure over the column 150 bar

Temperature stability 4 to 40 °C

Recommended pH 2 to 13 working

1 to 14 CIP

Bio-Monolith Columns: Data SheetsBasic characteristics

Page 53

Column Column LLengthength

Bio-Monolith Columns are characterized by a high resolving power at extremely short column lengths – only 5 milimeter.

10 ml/min = 4500 cm/h

= 360 CV/min (res. time: 0,1 s)

Page 54

Different Loadings on Bio-Monolith QA

10uL 50uL 100uL Gradient

Page 55

Consecutive injections Bio Monolith QAAnalytical Column

Page 56

Dynamic Binding Capacity for DNA

Dynamic Capacities

1% 5% 10%

1%5%

10%

1% 5% 10%

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

DBC

mg

DNA

QFF – porous particles Q nano - membrane QA - monolith

Note 50 times higher dynamic binding capacity than particle based resinwhile operating at 4-fold higher flow rate!

Courtesy of Pete Gagnon, for details visit validated.com

http://www.validated.com/revalbio/pdffiles/PUR07a.pdf

Page 57

Bio Monoliths

• Monolithic supports are highly porous rigid polymers with:

• High porosity (over 60 %)

• Flow-through channels (“pores”) having large diameter (1.5 µm)

• Uniform channel connectivity in 3D (homogeneous structure).

10 100 1000 10000

Pore diameter [nm]

Documents

Increased Protein/Peptide Mass Transfer Rates for Improved ... · Sample: 3 mg each protein 1. RNase 6. CDR 2. Insulin 7. Myoglobin 3. Cytochrome C 8. Carbonic Anhydrase 4. Lysozyme