Peptide Mapping Hardware and Column Optimization...Peptide Mapping Hardware and Column Optimization...

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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


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


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 %


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


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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


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Multipurpose valve functions for highest comfortExtra Mixing Volume for lowest baseline ripple (TFA applications)


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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


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


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


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TFA Mixing Noise:Effect of JetWeaver Mixer

Without Mixer


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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


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


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Norm.

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25

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Analysis of Glucocorticoids - Comparing dynamically mixed mobile phase vs. user influence

A:B=70:30

Norm.

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6 dynamically mixed mobile phases

min1 2 3 4

0

5

10

15

A:B=70:30


min1 2 3 4

0

5

10

15

20

25

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6 premixed mobile phases (same user)

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1290 Infinity Diode-Array Detector1290 Infinity Diode-Array Detectory yy y


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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


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


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


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


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


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


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


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.


• 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


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


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


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

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Heat- degraded CharacterizationSeparation of IsoformsA

B

Intact protein Analysis - Fast Separation of Insulin

RRHD 300 SB-C18

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2.5

5

7.5

10

12.5

15

0.14

1.38

4

1.64

8

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10

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30

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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.


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


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


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


• 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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Comparison of Three ColumnsTryptic Digest

Zorbax 1.8um, 2.1mm x 100mm

PoroShell 300



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Premixed versus Blend Assist;0.05%TFA-Water/0.04%TFA-ACN

Premixed

Note Fine Structure


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Blend Assist

Duplicate Runs Premixed0.05%TFA-Water/0.04%TFA-ACN


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Triplicate Runs Blend Assist: Stock 1%TFA; 0.05%TFA Water/0.04%TFA ACN


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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


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Maximum Peaks Plotted for Three Columns


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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


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Questions


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Peptide Mapping Hardware and Column Optimization...Peptide Mapping Hardware and Column Optimization...

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