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4/4/2013
1
Peptide Mapping
Hardware and Column Optimization
BioLC Master Class: Peptides
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Peptide Map: definition
Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASSELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases.
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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ICH Guidelines for Peptide Map
Peptide map
Selective fragmentation of the product into discrete peptides is performed using suitable enzymes or chemicals and the resultingperformed using suitable enzymes or chemicals and the resulting peptide fragments are analyzed by HPLC or other appropriate analytical procedure. The peptide fragments should be identified to the extent possible using techniques such as amino acid compositional analysis, N-terminal sequencing, or mass spectrometry. Peptide mapping of the drug substance or drug product using an appropriately validated procedure is a method that is frequently used t fi d i d d t t t f l t l
BioLC Master Class: PeptidesBioLC Master Class: Peptides
to confirm desired product structure for lot release purposes.
Source: http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
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Innovation: 1290 Infinity Quaternary PumpQuaternary Pump with Binary Pump-like Performance
1290 Infinity Quaternary Pump
• Boost Performance
Highest accuracy and precision for composition and flow, with exceptionally low delay volume
• Save Time
Accelerates transfer of existing HPLC methods to UHPLC
BioLC Master Class: PeptidesBioLC Master Class: Peptides
UHPLC
• Reduce Costs
Outstanding UHPLC performance at HPLC-like costs of ownership
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Power Range
For any kind of analysis
Composition Accuracy and Precision
Agilent 1290 Infinity Quaternary PumpSpecifications & Benefits
Composition Accuracy and Precision
< 0.15 % RSD or 0.02 min SD
± 0.4 % (1-99 % Composition B)
High RT precision in gradient runs
Flow Accuracy and Precision
< 0.07 % RSD or 0.01 min SD
± 1.0 % or 10 µL
Composition Range
1-99 %
BioLC Master Class: Peptides
0 % o 0 µ
High RT precision in isocratic runs
Wide analytical range
Delay Volume
< 350 µL
For fast quaternary gradients
1290 Quaternary Pump Flow Diagram
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Different mixtures (H2O, MeOH, ACN) at different pressures and different flow rates
Full application range!
Composition Accuracy and PrecisionStep Gradient Analysis with Tracer
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Multipurpose valve functions for highest comfortExtra Mixing Volume for lowest baseline ripple (TFA applications)
BioLC Master Class: Peptides
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Why is delay/dwell volume important
1. Different dwell volumes result in a RT time shift(the time for the mobile phase to reach the column head)
2. Different dwell volume could effect resolution(peaks spends different time under isocratic/gradient(peaks spends different time under isocratic/gradient conditions)
3. additionally, the dwell volume effects the gradient shape (dispersion effects, flush out behavior => the programmed gradient becomes deteriorated)
4. Therefore even with the same “geometrical” delay volume the chromatograms could look different on different systems
BioLC Master Class: PeptidesBioLC Master Class: Peptides
5. The dwell volume has an big impact for narrow bore applications, especially combined with fast gradient
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Dwell volume determination
Programmed
BioLC Master Class: Peptides
W. Dolan LCGC Vol 24, No 5, 458-466
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1290 Quaternary Pump with J380 Mixer and bypass J380 mixer
Note different transition volumes
J380 Mixer 480 uL delay
Bypass Mixer335 uL delay
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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TFA Mixing Noise:Effect of JetWeaver Mixer
Without Mixer
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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With Mixer
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1290 Infinity Quaternary Pump - BlendAssist
Simple tool for online-dilution of modifiers and gradient set-up
You need different concentrations of modifiers in your analysis, would like to have just one stock-solution and do online dilution to profite from the quaternary mixing capability of your pump? Here is a simple tool – BlendAssist!
Water Water 1% TFA
Desired method conditions - example: 1. 5 to 95% gradient of ACN with 0.1% TFA in
Water and 0.08% TFA in ACN2. 20 – 80% gradient of ACN with 0.5% TFA in
Water and 0.4% TFA in ACN
Without BlendAssist you need to either pre-mix the required solvents or by using stock-solutions of TFA in Water and ACN to program complex
BioLC Master Class: Peptides
ACNACN
1% TFA
TFA in Water and ACN to program complex gradients (%A, B, C, D).
With BlendAssist: just program your binary organic/aqueous gradient and define the dilution factor!
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1290 Infinity Quaternary Pump - BlendAssistSimple tool for online-dilution of modifiers and gradient set-up
BioLC Master Class: Peptides
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Norm.
20
25
30
Analysis of Glucocorticoids - Comparing dynamically mixed mobile phase vs. user influence
A:B=70:30
Norm.
35
40
6 dynamically mixed mobile phases
min1 2 3 4
0
5
10
15
A:B=70:30
BioLC Master Class: PeptidesBioLC Master Class: Peptides
min1 2 3 4
0
5
10
15
20
25
30
6 premixed mobile phases (same user)
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1290 Infinity Diode-Array Detector1290 Infinity Diode-Array Detectory yy y
BioLC Master Class: Peptides
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Dispersion volume- Why it makes no sense to specify a geometrical volume
LFr
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Volumetric peak varianceF: Flow rateDm: Molecular diffusion coefficient
Dispersion:
Simple example: disperision in a straight tube
mD24Dm: Molecular diffusion coefficientL: Capillary length
Derived from Aris, Taylor and Golay equation
Same geometrical volume (V1 = V2), but totally different dispersion volumes
BioLC Master Class: Peptides
R. Tijssen, “Mechanism and Importance of Zone-Spreading, in Handbook of HPLC, Vol 78, 1998, Marcel Dekker, NY
V1 V2
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Why do we need a new cell technology?10 mm pathlength
13 µl volumeShort 2.1 mm ID column
How to achieve smaller cell
+ =
How to achieve smaller cell volume?
short path-length (3 mm, 2 µl)
High light transmission =>
low noise
Low S/N Low light transmission=> high noise
long path-length (10 mm, 0.5 µl)
Low S/N
BioLC Master Class: Peptides
low noise
Optofluidic Waveguides- Long path length- Small cell volume- High light transmission
Highest S/N
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Max-Light Cartridge Cell- Optofluidic waveguides
Non-coated fiber (fused silica)
High Light Transmission due to Total-Internal Reflection (TIR) principle (~ 100 % Light efficiency)
Benefits: Highest Sensitivity (S/N) with small cell volumes (dispersion effects) More reliable and robust peak integration (automated) due to
BioLC Master Class: Peptides
More reliable and robust peak integration (automated) due to nearly no Refractive Index and thermal effects (solvent temperature)
Coating free fused silica (no special care instructions or smiling baseline effects) Easy cell selection (one cell for all major applications) Cartridge design for ease of use
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Reversed-Phase Separation – brief overview
Reversed phase HPLC is used for the analysis of complex biologics (protein therapeutics, cell lysates, serum, etc)intact proteinspeptide mapping amino acid analysisamino acid analysis
Detailed information about the primary structureIncluding disulphide shifting
Mass spec compatible (no salt buffer)
The pore size of the media must be matched to the size of the molecule
Intact Protein Analysis 300Å
AdvanceBio Peptide Mapping 120
BioLC Master Class: Peptides
tact ote a ys s 300Peptide Mapping 120Å
Confirm protein identity (primary structure analysis) Look for impuritiesDevelop impurity profilesPeptide mappingAmino acid analysis
ZORBAX RRHD 300SB-C18
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Reversed-Phase – Product Families Particle Porosity Functionalities Particle
SizesPore Size Application
ZORBAX Stable Bond Silica Fully Porous C18, C8, C3, CN 3.5um, 5um 300A Low pH
ZORBAX 300Extend Silica Fully Porous Extend-C18 3.5um, 5um 300A High pH
ZORBAX RRHD Silica Fully Porous SB-C18 SB-C8 1 8um 300A High speedZORBAX RRHD Silica Fully Porous SB-C18, SB-C8, SB-C3, Diphenyl
1.8um 300A High speedHigh resolution
ZORBAX Eclipse AAA Silica Fully Porous C18 3.5um, 5um 80A Amino Acids
AdvanceBio PeptideMapping
Silica Superficially EC-C18 2.7um 120A Peptide Mapping
Poroshell 300 Silica Superficially SB-C18, SB-C8, SB-C3, Extend-C18
5um 300A High speedHigh resolution
PLRP-S PS/DVB Fully Porous 3um, 5um, 8um, 10um,12um, 18um
100A, 300A Peptides, oligos, proteins
BioLC Master Class: Peptides
PLRP-S PS/DVB Fully Porous 5um, 8um, 10um, 30um
1000A, 4000A
Proteins and DNA
VariTide RPC PS/DVB Fully Porous 6um 200A Syntheticpeptides
Page 17Emphasized column technology
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Protein/Peptide Separations by Reversed-phase
Larger Molecules = Slower Diffusion
So, a need to decrease the diffusion time for macromolecules!
To improve, we can increase the Diffusion Rate by:
elevating operating temperature decreasing solvent viscosity
BioLC Master Class: Peptides
elevating operating temperature decreasing solvent viscosity
and, or……………..
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Decrease the Diffusion Distance!
1. Rapid Resolution High Definition (RRHD) 300 Family (C3, C8, C18, diphenyl)
Developed very small (totally porous) particles (<2 um)
RRHD 1.8um, UHPLCSuperior efficiency, Superior efficiencyVery narrow bandsUltra high resolutionFast flowsShort column lengths (pressure
constraint) 2. Poroshell 300 and AdvanceBio120 Peptide Mapping
Limit diffusion distance into a particle (shell particle) HPLCS perior efficienc
BioLC Master Class: Peptides
Superior efficiencyVery narrow bandsUltra high resolutionFast flowsLong column lengths (no pressure
constraint)
5um Poroshell 3002.7um AdvanceBio120 Peptide
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1. ZORBAX RRHD 300Å 1.8 µm Columns
Application Objectives:
• The ZORBAX RRHD 300Å 1.8 µm columns are designed for primary structure analysis - confirming protein identity, quantifying post translational modifications and fingerprinting impurity profiles by reversed-phase UHPLC.
• With UHPLC, 1200 bar stability, faster separations are achieved to improve productivity but with no compromise in data accuracy.
• Primary benefit of these Agilent RRHD reversed-phase columns for separations of proteins is increased resolution with shorter analysis times.
BioLC Master Class: Peptides
• Resolution is critical in these separations due to the small differences in structure between the candidate protein and the “impurities”.
• RRHD provides HPLC and UHPLC level productivity for large molecules.
5990-8124EN
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Four Rapid Resolution High Definition Phases
ZORBAX300Å, 1.8 µm
Family of Four Reversed-Phase Ligands
C18 C8 C3 Diphenyl
Lysates Increasing protein size/hydrophobicitySmall Proteins
Ligand Application
C18 Small intact proteins/peptide maps/ cell lysates
BioLC Master Class: Peptides
C8 Intact proteins
C3 Larger /hydophobic proteins, including MAbs
Diphenyl Unique selectivityAgilent 1290 Infinity
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2. 5um Poroshell 300 and 2.7um AdvanceBio 120
5 04 5
0.25 um
Poroshell 300 AdvanceBio Peptide Mapping120
0.50 um
5.0 um4.5 um
Use AdvanceBio Peptide Mapping 120 for peptide maps containing small proteins and peptides
Peptide digests of proteins or monoclonal antibodies
Highly compatible with TFA and formic acid mobile phases for efficient LC/MS analysis
2.70 um1.70 um
BioLC Master Class: Peptides
Highly compatible with TFA and formic acid mobile phases for efficient LC/MS analysis
Use Poroshell 300 for intact protein analysis and large peptide fragments
Does the sample contain all polypeptides or larger?
Poroshell 300 can be used with very large proteins/monoclonal antibodies, complex biologicals
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Reversed-Phase – Intact Protein Separations
RRHD 300High Efficiency – ultra fast protein separations
RRHD 300 columns for the separation of large peptides, proteins and antibodies.
RRHD 300 particles provide HPLC and UHPLC level productivity for large molecules
1.8 um
BioLC Master Class: Peptides
productivity for large molecules
Sharper peaks and fast, efficient separations with large proteins and peptides
Stable Bond phase technology
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mAU
17.5 48 4
1.51
2
mAU
80
Heat- degraded CharacterizationSeparation of IsoformsA
B
Intact protein Analysis - Fast Separation of Insulin
RRHD 300 SB-C18
0
2.5
5
7.5
10
12.5
15
0.14
1.38
4
1.64
8
0
10
20
30
40
50
60
70Heat-treated insulin (55C for 24 h)
Forced degradation products
1.8 min.2.5min.
Oxidized insulinChain B
Oxidized insulinChain A
insulin
Subspecies of oxidized Insulin chain B
Injection 2ul, flow: 1.0mL/minPress: 680 bar
Gradient 35-50%B, 0-4 min.33%B, 4-5min.
Injection 4ulflow:1.0mL/minPress: 680 barGradient 35-50%B, 0-5 min.
BioLC Master Class: PeptidesBioLC Master Class: Peptides
min0 0.5 1 1.5 2 2.5 min0 1 2 3 4 5
Insulin, a small protein biotherapeutic, was used in this study together with its isoforms and degradation products todemonstrate utility for rapid separation profiling and characterization of a biopharmaceutical API. Using a ZORBAX RRHD300SB-C18 column (2.1 x 50mm) and optimized chromatographic condition for ultra fast analysis, chromatograms in A and B showexcellent resolution and selectivity for resolving insulin and its products in run times under 3 minutes. Additionally, low TFAaddition (0.01% ) in ACN (see Experimental) make the separations highly transferable to LC/MS analysis.
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Reversed-Phase – Peptide Separations
AdvanceBio Peptide Mapping 120
SOLID CORE
Porous Shell0.5um
2.7 um
AdvanceBio Peptide Mapping 120 columns for the separation of small proteins, polypeptides and peptides.
AdvanceBio Peptide Mapping 120 particles have a solid silica core with a superficially porous surface with a 120Å pore size
BioLC Master Class: Peptides
CORE1.7um
This results in more efficient mass transfer, sharper peaks and fast, efficient separations with small proteins and peptides
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What Parts of the Knox/VanDeemter Equation Are Affected by the Column Particle
A term – eddy diffusion and flow distribution• Impacted by the column particle p y p• Column particle size and column packing quality impact this• A tight particle size distribution improves the A term
B term – longitudinal diffusion • Impacted by column particle• This is impacted by column pore size
C term – mass transfer component• Impacted by the column particle and its properties
BioLC Master Class: PeptidesBioLC Master Class: Peptides
• Mass transfer is improved by using smaller particles with shorter diffusion paths• Improved with a superficially porous particle• Allows high efficiency at high flow rates
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BioLC Master Class: Peptides
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Comparison of Three ColumnsTryptic Digest
Zorbax 1.8um, 2.1mm x 100mm
PoroShell 300
AdvanceBio Peptide Mapping 120
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Premixed versus Blend Assist;0.05%TFA-Water/0.04%TFA-ACN
Premixed
Note Fine Structure
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Blend Assist
Duplicate Runs Premixed0.05%TFA-Water/0.04%TFA-ACN
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Triplicate Runs Blend Assist: Stock 1%TFA; 0.05%TFA Water/0.04%TFA ACN
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Overlay of Blend Assist TFA percent Differences:Blue: 0.05%TFA-Water/0.04%TFA-ACNRed: 0.1%TFA-Water/0.08%TFA-ACN
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Maximum Peaks Plotted for Three Columns
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Lab Session Goals
Setup 1290-quaternary system to run tryptic digest gradient on AdvanceBio Peptide Mapping 120 column
U Bl d A i t t t G di t Use Blend Assist to create Gradient
Use Openlab Chemstation to create virtual sequence set of data to compare columns
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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Questions
BioLC Master Class: PeptidesBioLC Master Class: Peptides
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