logical method development for peptides and proteins … · logical method development for peptides...
TRANSCRIPT
Slide 4
Logical Method Development for Peptidesand Proteins Using RP-HPLC and SEC
001284P1.PPT
Slide 5
Seminar Outline
1. Gel-filtration separations by SEC
2. Developing methods for protein and peptideseparations by RP-HPLC
Slide 6
Mechanism of SEC Separation
000855P1.PPT
FLOW
Slide 7
Advantages of Size-Exclusion Chromatography
• Size-Dependent Separations
• Useable with a Wide Variety of Mobile Phases
• Samples Retain Structure and Activity
000831P1.PPT
Slide 8
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
000845P2.PPT
Flow mL/min
Slide 9
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
000831P1.PPT
Slide 10
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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Slide 11
Time Course of Antibody CleavageMonitored by SEC
001486P1.PPT
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
Slide 12
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.
001477P1.PPT
Slide 13
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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Slide 14
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
Slide 15
Break Number 1
• For Questions and Answers
• Press *1 on Your Phone to
• Ask a Question
Slide 16
Seminar Outline
1. Gel-filtration separations by SEC
2. Developing methods for protein and peptideseparations by RP-HPLC
Slide 17
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
Slide 18
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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Slide 19
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
000945P1.PPT
CH2 -OH.
CH2 -OH
Slide 20
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
Slide 21
Molecules Must Enter Pores
(C) Will be Excluded
(A,B) Enter Pores
B
C
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A
Slide 22
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
000970P2.PPT
Slide 23
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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Slide 24
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
000973P1.PPT
Slide 25
Break Number 2
• For Questions and Answers
• Press *1 on Your Phone to
• Ask a Question
Slide 26
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
Slide 27
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
Slide 28
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
Slide 29
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
Slide 30
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.
Slide 31
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.
Slide 32
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.
000949P1.PPT
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
Slide 33
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
000979P1.PPT
Slide 34
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
001007P1.PPT
Slide 35
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.)
Slide 36
Break Number 3
• For Questions and Answers
• Press *1 on Your Phone to
• Ask a Question
Slide 37
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
Slide 38
How Resolution Varies with k’, N, and a
t0
Initial
Increase
k’
IncreaseN
Increaseα
time
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Increasek’
Decreasek’
Slide 39
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
Slide 40
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
Slide 41
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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Slide 42
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
000976P1.PPT
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.
Slide 43
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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Slide 44
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)
001032P1.PPT
≥
Slide 45
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
Slide 46
Effect of Temperature on Peak Width
001004P1.PPT
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)
Slide 47
Using Higher Temperatures
Reduces Analysis Time•May Change Selectivity•May Increase Recovery
1/T (°K)001001P1.PPT
Slide 48
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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Slide 49
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
000028P2.PPT
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
Slide 50
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
Slide 51
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
000000P1.PPT
Slide 52
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
000917P1.PPT
Slide 53
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
Slide 54
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.
Slide 55
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
Slide 56
Break Number 4
• For Questions and Answers
• Press *1 on Your Phone to
• Ask a Question
Slide 57
Wrap-up E-Seminar Questions