Logical Method Development for Peptides and Proteins … · Logical Method Development for Peptides...

Logical Method Development for Peptidesand Proteins Using RP-HPLC and SEC

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Seminar Outline

1. Gel-filtration separations by SEC

2. Developing methods for protein and peptideseparations by RP-HPLC

Mechanism of SEC Separation

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FLOW

Advantages of Size-Exclusion Chromatography

• Size-Dependent Separations

• Useable with a Wide Variety of Mobile Phases

• Samples Retain Structure and Activity

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Rapid SEC and Effect on Resolution

Rs1,2= 2.1

Rs= 2.3

Rs=1.9

Rs1,2= 1.7

Rs= 1.43.5 min

60 min

30 min

15 min

7.5 min

0.25

0.5

1.0

2.0

5.0

Run Time

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Flow mL/min

Practical Applications of SEC

• Desalting and Exchange of Sample Buffer

• Separation of Mono-, Di-, Trimer, Larger Aggregates

• Following Progress of a Reaction

• Separation of Reaction Components and Products,(esp., antibodies, fragments, and conjugates).

• Estimation of Molecular Weight

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Separation of Albumin Monomer,Dimer and Aggregate

Conditions:Column: Zorbax GF-250, 9.4 x 250mmMobile Phase: 0.2M Sodium Phosphate,

0.1% Sodium Azide, pH 7.0Flow Rate:Detection: UV 280nmTemperature:Sample: 1) Aggregate

2) Albumin dimer

1 2

DeLeenheer, A.P. et al. J. Pharm Sci., (1991) 80, 11.Reprinted with publisher’s permission

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Time Course of Antibody CleavageMonitored by SEC

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0 5 10 15 20 min

A. Undigested

B. Digested

Mab

Dimer

Fab

Mab

Conditions:Column: Zorbax GF-250, 9.4 x 250 mmMobile Phase: 100 mM phosphate, pH 7.0Flow: 1.0 mL / min.Det.: UV 225 nmTemp.: 30°CSample: A) Undigested

B) 5 mg Monoclonal IgG,antibody digested 17 hourswith pepsin at pH 3.525 µL injection volume

Separation of Plasminogen in the Presence andAbsence of 6-AHA

Conditions:Column: Zorbax GF-250, 9.4 x 250mmMobile Phase: 50mM Sodium Phosphate,

100mM KCI, pH 7.1Flow Rate: 0.7 mL / min.Detection: UV (microvolts)Temperature: 30°CSample: A) Plasminogen

B) Plasminogen + 10mM 6-AHA (aminohexanoic acid)

Dollinger, D., B. Cunico, M. Kunitani, D. Johnson and R. Jones,"Practical on-line determination of biopolymer molecular weightsby high-performance liquid chromatography with classical light-scattering detection," (1992) J. Chromatogr., 592, 215-228.Reprinted with publisher’s permission.

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Separation of Purified Serum Proteins and Total Serumby Coupled Zorbax GF-250 and GF-450 Columns

Conditions:Column: Coupled: Guard Column, Zorbax GF-450 column, 9.4 x 250mm

Zorbax GF-250 column, 9.4 x 250mmMobile Phase: 0.2M Sodium Sulfate, 0.04M sodium Phosphate, pH 6.8Flow Rate: 0.4mL / min.Detection: Differential Refractive IndexTemperature: 20°CSample: Approximately 3mg / ml for each protein

1) Human IgM (pentameric)2) Human ∝-2 macroglobulin3) Human IgA (monomeric)4) Human IgG5) Human Serum Albumin

Dotted Line: total serum

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SEC-Section Conclusions

• SEC can be used for a broad range of size-basedanalyses

• SEC often used in your samples’ native environment(both material going in and material coming out)

• A rugged SEC material should be used for stabilityand shorter run times

Break Number 1

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Seminar Outline

1. Gel-filtration separations by SEC

2. Developing methods for protein and peptideseparations by RP-HPLC

Developing methods for protein and peptideseparations by RP-HPLC

• Choosing a Silica (structure and purity)

• Pore-Size Selection (80 and 300Å)

• Consensus Mobile Phase

• Bonded-Phase Selection

• Optimize Separation (N, k’, , Column Configuration, T)

• Alternative Separation Solutions

Silica Types

XerogelRx-SIL

( Silica Sol )

STRUCTURE: UNIFORM SUB PARTICLES “SPONGE-LIKE,” POLYMERIC NETWORK

POROSITY (%):

SURFACE AREA (M 2/G):

STRENGTH:

HIGH pH RESISTANCE:

PURITY:

PORE SIZE DISTRIBUTION:

50

100Å / 150

HIGH

GOOD

HIGH

NARROW

70

100Å / 300

WEAK

POOR

LOW - HIGH

BROAD

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Chromatographic Improvement Using HighlyPurified Zorbax Rx-Sil

Original ZORBAX

ZORBAX Rx-Sil (1987)

Conditions: Flow Rate: 2.0 mL / min.Mobile Phase: 5% 2-Propanol in Heptane

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CH2 -OH.

CH2 -OH

Molecules Must Enter Pores

(C) Will be Excluded

(A,B) Enter Pores

B

C

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A

Effect of Pore Size and Molecular Size on Peak WidthGradient Separations

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)P

W 1

/2

Leu E

nkephali

n

M.W

. = 5

56

Angiote

nsin II

M.W

. = 1

046

Insu

lin B

M.W

. = 3

,496

Cyt C

M.W

. = 1

2,327

RNase

M.W

. = 1

3,684

Lysoz

yme

M.W

. = 1

3,900

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Effect of Pore Size on Sample LoadGradient Separation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0.1 1 10 100 1000

Angiotensin II (300Å)

Angiotensin II (80Å)

PW

1/2

Load (µg injected)

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Pore Size Recommendation

Use 80Å pore columns to maximizeloading capacity and retention

Use 300Å pore columns to maintainhigh efficiency. Increase columndiameter to increase loading capacity

≤ 4000 MW

4,000 to500,000 MW

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• Optimize Separation (N, k’, , Column Configuration T)


Reasons for Consensus Conditions forProtein / Peptide Mobile Phases

Acetonitrile• Low UV Cutoff, 190 nm• Volatile• Good solubilization, denaturant

TFA• Low UV Absorbance• Volatile• Good solubilization, denaturant• Acidic - improves peak shape• Mild anionic ion pair - more retention of lys, other free amines• Compatible with MS

TFA / Water : TFA / ACN mobile phase• Low pH (0.1% TFA, pH ≈ 1.9) suppresses silanol interactions• Relatively non-viscous - high efficiency, low pressure

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Column:4.6 x 150 mm 300SB-C8MobilePhase: A: 95:5, H2O : ACN, 0.1% TFA

B: 5:95, H2O : ACN, 0.085% TFAGradient: 0 - 60% B in 60 min.Flow: 1 mL / min.Temp: 35 - 40°C

Column-Technology Improvement for Low-pHSeparation

HYDROLYTICALLY UNSTABLECONVENTIONAL

HYDROLYTICALLY STABLESTERICALLY PROTECTED

Used in Zorbax StableBond columns

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Monofunctional Silanes Yield Single Step, Reproducible Reaction

End-capping groups are labile at low pH End-capping groups are not used

ZORBAX SB-C18 Shows Exceptional Stability AtLow pH - High Temperature (pH 0.8, 90°C)

Purge Solvent:50% methanol/water with

1.0% TFASolute: Toluene

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Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, 32 (1994) 473-480.

Stability of ZORBAX SB-CN at pH 2.0, 50°C

COLUMN VOLUMES

100

80

60

40

20

00 1000 2000 3000 4000 5000 6000

%k’

RE

MA

ININ

G

Si-(iPr)2PrCN

Si-(Me)2PrCNTraditional Silane

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Sterically Protected Silane

Kirkland, J.J., J.L. Glajch, and R.D. Farlee, Analytical Chemistry (1989), 61, 2.

ZORBAX StableBond® Family of Selectivities

Bulky diisobutyl (with C18) ordiisopropyl (with C8, C3, CN, phenyl)

side-chains result in stable long and short-chain

monofunctional ligands.

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SiO

SiO

NS iO

SiO

S iO

300Å / 80Å

Pore-SizeAvailability

300Å / 80Å

80Å

300Å / 80Å

300Å / 80Å

SB-CN

SB-C3

SB-phenyl

SB-C8

SB-C18

Small-Peptide Selectivity Differences onDifferent Bonded Phases

Conditions:

Columns: ZORBAX 300SB, 4.6 x 150mmMobile Phase: Gradient, 0 - 26% B in 30min. A = 0.1% TFA in Water B = 0.1% TFA in AcetonitrileTemperature: 40°CSample: 2µg of each peptideFlow Rate: 1.0 mL / min.Detection: UV-210nm

300SB-C18

300SB-C8

300SB-C3

300SB-CN

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Recovery of Polypeptides fromZORBAX 300SB Columns: Effect of Bonded Phase

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

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Effect of Bonded-Phase Ligand on Recovery of aSynthetic Lipopeptide: Effect of Bonded Phase

Conditions:15 µL ( 15 µg ) of peptide in 0.1% TFA/DMSO4.6 mm ID x 150 mm Zorbax columns1 mL/min, 60°C, 10 - 90 % B in 40 minA – 0.1% TFA/water, B – 0.1% TFA/AcN

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300SB-CN

300SB-C8

300SB-C18

Recovery 59%

Recovery 75%

Recovery 89%

Retention Time, (min.)

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How Resolution Varies with k’, N, and a

t0

Initial

Increase

k’

IncreaseN

Increaseα

time

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Increasek’

Decreasek’

Improving Resolution Using k*

Thus, all of these increase k*:

1. Longer gradient time tG 2. Shorter column Vm 3. Higher flow rate F 4. Shorter organic range ∆Φ

1 / k* = gradient steepness

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tGF

S∆Φ Vm

k* = = ∆Φ = change in volume fraction of organic S = constant F = flow rate tG = gradient time (min.) Vm = column void volume

Resolution Relationship for Gradient Elution

R ≈ √N4

α*k*

VG

Vm

Resolution Can Often Be Improved Using aShorter Column Length ( Decreasing Vm )

4.6 x 50 mm

1. GLY-TYR2. VAL-TYR-VAL3. [GLU]-ß PROTEIN

AMYLOID FRAGM 1-164. [TYR8 ] BRADYKININ5. MET-ENK6. LEU-ENK7. ANGIOTENSIN II8. KINETENSIN9. RNASE

10. INS (EQUINE)

4.6 x 150 mm

12

34

5 6 7

8 9 10

0 1284

300SB-C8, 3.5 µm Gradient: 2-75% B in 30 min.Mobile Phase: A= 5:95, ACN : H2O 0.1% TFA

B= 95:5, ACN : H2O 0.085% TFAFlow Rate: 1 mL / min.Injection: 10 µL, 2.6 µg eachTemp: 35°CUV: 215 nm

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16 min

k2=3

k2=5.6

Gradient SteepnessEffects Retention ( k*)and Resolution

0 10 20 30 40

min.

100% B

100% B

100% B

100% B

0% B

0% B

0% B

0% B

tG= 40

tG= 20

tG= 10

tG= 5

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Rapid Resolution with Reduced Particle Size

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�� 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-enkephalin 2. Leu-enkephalin 3. Angiotensin II 4. Neurotensin 5. RNase 6. Insulin (Bov) 7. Lysozyme 8. Calmodulin 9. Myoglobin10. Carbonic Anhydrase

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Mobile Phase: A: 95:5, H2O:ACN with 0.1% TFA, B: 5:95, H2O:ACN with 0.085% TFAF=1 mL/min, Det.: 215nm, Sample: 1-10µg protein (10µL inj.) in 6M Gu-HCL, pH7.0

10-60% B in 10 min.

10-60% B in 30 min.

10-60% B in 50 min.

Rapid, Reproducible Analysis of a ProteinSample -- Fast Re-equilibration

1. Met Enkephalin 2. Leu Enkephalin 3. Angiotensin II 4. Neurotensin 5. RNase 6. Insulin (BOV) 7. Cytochrome C 8. Lysozyme 9. Calmodulin10. Myoglobin11. Carbonic Anhydrase

ZORBAX 300-SB-C84.6 x 50 mm, 3.5 µm, 35°C

Inj. 1 Inj. 3Inj. 2

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Properties - 3.5 µm vs. 5 µm Particle Size

= Surface area ( m2 / g)= Pore size ( 80Å or 300Å )= Surface coverage (µM / m2)= Selectivity (α)= Retentivity (k’)

Increased Resolution (Rs)

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≥

Effect of Particle Size on Peak WidthCalmodulin Tryptic Digest

A

A

5.0 µm, 35°CP = 110 bar

3.5 µm, 35°CP = 170 bar

0 5 10 15 20 25 30 35 min.

Ab

sorb

ance

(21

0 n

m)

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Zorbax 80Å SB-C18

(4.6 x 150mm, 3.5µm)

A= 0.1% TFA in H 2O, B= 0.095% TFA in 80% ACN/ H 2O; 5-60%B / 90 minFlow=0.75 mL/min; 45 µL inj. (75 µg); 210 nm

Effect of Temperature on Peak Width

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Calmodulin Tryptic DigestA= 0.1% TFA in H2O, B= 0.095% TFA in 80% ACN/ H2O; 5-60%B / 90 minFlow=0.75 mL/min; 45 µL inj. (75 µg); 210 nm

0 5 10 15 20 25 30 35 40

000138P1.CDR

B

A

B

A

3.5 µm, 35°CP = 170 bar

3.5 µm, 85°CP = 88 bar

min.

Ab

sorb

ance

(21

0 n

m)

37 38 39

R = 1.3S

B

A

34 35 36

B

A

R = 1.5S

Zorbax 80Å SB-C18

(4.6 x 150mm, 3.5µm)

Using Higher Temperatures

Reduces Analysis Time•May Change Selectivity•May Increase Recovery

1/T (°K)001001P1.PPT

New Selectivities UsingElevated Temperatureand Stable, Short-ChainBonded PhasesZorbax 300SB-C3

Ambient

35° C

45° C

60° C

1. Leucine Enkephalin2. Angiotensin II3. RNase A4. Insulin (BOV)5. Cytochrome C6. Lysozyme7. Myoglobin8. Carbonic Anhydrase

Mobile Phase:A:5:95 ACN : Water with 0.10 % TFA (v/v%);B: 95:5 ACN : Water with 0.085% TFA (v/v%)

Gradient: 15-53 % B in 20 minutes, postime: 12 min.Flow: 1.0ml/min, Protein concentration: 2-6µg., Inj. vol.:10µL, UV/VIS=215 nm, Temp.: 35 °C

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Effect of Column Temperatureon ßAP Peptide Separation

ZORBAX 300 SB-C18 (4.6 x 150 mm)1 ml/min.; 20-45% B / 35 min.; A=0.1%TFA in H2O, B=0.09%TFA in ACN

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Boyes and Walker, (1996), Submitted

Ab

sorb

ance

(21

0nm

)

25°CßAP(1-38)

ßAP(1-43)* Recovery <10 %

40°C

60°C

0

80°C

5 10 15 20 25 30 35 40 min

Recovery >70%

10 µl injection5 µg peptide in

6M Urea/5% HOAc

Developing Methods for Protein and PeptideSeparations by RP-HPLC







Family of Bonded Phases For Best Stability,Peak Shape Across the pH Range

StableBond• Designed specifically for stability at low pH• Use appropriate buffers (e.g., Tris, Bis-Tris-Propane)• Non-endcappedEclipse XDB• Designed specifically for stability at neutral pH• Dimethyl-alkyl (C8, C18)• Proprietary double endcappedBonus-RP• Designed for stability at acidic and neutral pH• Proprietary triple endcapped• Polar alkyl phaseExtend• Designed specifically for high pH• Patented bidendate bonded phase• Endcapped

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Reasons for Separating Basic Solutes atIntermediate pH (4-8) or Higher

• Sample components are unstable at low pH

• Basic compounds elute too quickly

• Required band spacings are not found at low pH

• Near the pKi, greatest band spacing changes occur;moving out of this region makes method rugged

• At high pH, many basic compounds are not ionizedand separate without ionic interaction with silanols

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Comparison of Angiotensins Separation withTFA and NH4OH

Zorbax Extend C18(2.1 x 150 mm)

min0 2.5 5 7.5 10 12.50

1.0E6

2.0E6

3.0E6

4.0E6

5.0E6

0

1.0E7

2.0E7

3.0E7

4.0E7

5.0E7

12.

050

13.

261

min0 2.5 5 7.5 10 12.50

5.4

99

8.4

77

9.6

21

AII + AIII

AI

AI

AIIAIII

TIC (200-1500 m/z)

HP 1100 MSD: Pos. Ion ESI

Vf 70V, Vcap 4.5 KvN2=35psi, 12L/min.325°C

Gradient: 15-50%B / 15 min.0.2 mL/min

Temp: 35°CSample: 2.5 µL

(50 pmol each)Basic Conditions

A- 10 mM NH4OH in waterB- 10 mM NH4OH in 80%AcN

Acidic ConditionsA- 0.1% TFA in waterB- 0.085% TFA in 80%AcN

Separate Peptides/Proteins at High pH with Low-Noise LC/MSComparison of Angiotensin I Mass Spectra With TFA and NH4OH

Acidic Conditions A- 0.1% TFA in water B- 0.085% TFA in 80%AcN

Basic Conditions A- 10 mM NH4OH in water B- 10 mM NH4OH in 80%AcN

Conditions: 2.5 µL sample (50 pmol);Extend-C18, 2.1 x 150 mm; 0.2 mL/min;35°C; 15-50% B in 15 min.; Pos. Ion ESI-Vf 70V, Vcap 4.5 kV, N 2- 35 psi, 12 L/min.,325°Cm/z500 1000

0

20

40

60

80

100

Max: 10889

649

.5

433

.0

227

.1

659

.7

844

.8

250

.9

340

.3

127

7.9

117

9.1

102

5.2

720

.3

132

6.3

398

.0

767

.3

458

.0

146

8.8

137

3.3

+3 +2

m/z500 10000

20

40

60

80

100Max: 367225

649

.0

433

.0

659

.8

129

6.6

+1

+2

+3Note that the +1 ion is visiblewith the high-pH mobile phase,but buried in the noise with thelow-pH mobile phase.

Method Development Summary

• Method development is a logical sequence– Choose silica– Choose pore size– Use consensus mobile phase– Choose solubilization solution– Choose column chemistry for pH range– Optimize separation (k*,a *,N, column configuration, T)– Study alternative solutions

• Stable reversed-phase media offer flexibility touse temperature and short-chain bonded phasesas selectivity tools

• Use appropriate instrumentation

000963P1.PPT

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Wrap-up E-Seminar Questions

Logical Method Development for Peptides and Proteins … · Logical Method Development for Peptides...

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