Talk3 Hardware and Software · 2016. 9. 4. · Agilent Technologies Hardware and Software 1. BioLC Master Class: Peptides Topics 1290 Quaternary Pumping System for Peptide Mapping

BioLC Master Class: Peptides

Enhancing Productivity in Peptide Mapping, Ion Exchange Chromatography and Size Exclusion Chromatography with

Agilent Technologies Hardware and Software

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BioLC Master Class: PeptidesBioLC Master Class: Peptides

Topics

� 1290 Quaternary Pumping System for Peptide

Mapping

� 1260 BioInert HPLC System with Buffer Advisor for

Ion Exchange Chromatography

� 1260 BioInert Multi-Detector System for Size

Exclusion Chromatograpy

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Peptide Mapping

Hardware Optimization

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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 and/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.

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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. The resulting peptide

fragments are analyzed by HPLC or other appropriate analytical

procedures. 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 a peptide-based drug substance or drug product, using an

appropriately validated procedure, is a method that is frequently used

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

• 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

< 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

� High RT precision in isocratic runs

Agilent 1290 Infinity Quaternary PumpSpecifications & Benefits

Composition Range

1-99 %

� 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

Composition Accuracy and PrecisionStep Gradient Analysis with Tracer

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Multipurpose ValveOptional Extra Mixing Volume for lowest baseline ripple (TFA applications)

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TFA Mixing Noise:Effect of JetWeaver Mixer: Lower Short Term Noise

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With Mixer

Without MixerWithout Mixer


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 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

W. Dolan LCGC Vol 24, No 5, 458-466

Programmed

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1290 Quaternary Pump with J380 Mixer and bypass J380 mixer

J380 Mixer 480 uL delay

Bypass Mixer335 uL delay �

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1290 Quaternary Pump with J380 Mixer and bypass J380 mixer

J380 Mixer 480 uL delay

Bypass Mixer335 uL delay �

Note different transition volumes

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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

ACN

ACN

1% TFA

Desired method conditions - example: 1. 5 to 95% gradient of ACN with 0.1% TFA in

Water and 0.08% TFA in ACN

2. 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

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 - BlendAssist

Simple tool for online-dilution of modifiers and gradient set-up

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1290 Infinity Diode-Array Detector1290 Infinity Diode-Array Detector

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Dispersion volume- Why it makes no sense to specify a geometrical volume

mD

LFr

⋅

⋅⋅⋅=

24

4

2 πσ

Volumetric peak varianceF: Flow rateDm: Molecular diffusion coefficientL: Capillary length

Derived from Aris, Taylor and Golay equation

Dispersion:

Simple example: dispersion in a straight tube

R. Tijssen, “Mechanism and Importance of Zone-Spreading, in Handbook of HPLC, Vol 78, 1998, Marcel Dekker, NY

Same geometrical volume (V1 = V2), but totally different dispersion

volumes

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 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

Optofluidic Waveguides- Long path length

- Small cell volume

- High light transmission

Highest S/N

+ =

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Max-Light Cartridge Cell- Optofluidic waveguides

High Light Transmission due to Total-Internal Reflection (TIR) principle (~ 100 % Light efficiency)

Non-coated fiber (fused silica)

Benefits:� Highest Sensitivity (S/N) with small cell volumes (dispersion effects)� 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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Comparison of Three ColumnsTryptic Digest

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Zorbax 1.8um, 2.1mm x 100mm

PoroShell 300

AdvanceBio Peptide Mapping 120


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

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Premixed

Blend Assist

Note Fine Structure


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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Agilent TOF Schematic

Pulser Detector

Vacuum ~1*10-7 Torr

Internal support structure with low coefficient of thermal expansion

1 meter flight tube, 2 meter flight path

Reflectron (ion mirror) with self-compensating thermal design

Optics for beam shaping and conditioning

Flight acceleration orthogonal to ion introduction

Turbopumps


A Time-of-Flight Scan

Pulser Detector

1. Pulse ions every 100 microseconds

2. Measure at detector each nanosecond (1 GHz)

3. 100,000 data points in each transient

4. Sum ~10000 transients into one scan (1 scan/second operation)

5. Produces spectra with excellent ion statistics

20 µsec – m/z 118

46 µsec – m/z 622

90 µsec – m/z 2421


“Traditional” Peptide Map

Column: 4.6 X 250 MMC18 stationary phase5 micron particle size,Mobile Phase: A= 0.1%TFA in water, B=0.1%TFA in CANGradient: 10% B to 90 B in 100 minutesRuntime: 115 minutesRe-equilibration time: 20 minutes


1290 Infinity Diode-Array Chromatogram Lysate Digest

LC Conditions: Peptide Mapping Column, Flow Rate: 2.75 ml/min; Split 3:1 ;

A=Water+0.1% TFA /B= ACN + 0.1% TFA, Gradient 5% B � 95% B in 10 minutes,

Column Temp. 40 deg. C., Inj Vol 2.0 uL; Detector rate: 80 Hz


Analysis of Peptide Map with Diode-Array Detector


Time of Flight Total Ion Chromatogram Lysate Digest

LC Conditions: Peptide Mapping Column 3.0mm x 150 mm, Flow Rate: 2.75 ml/min; Split

3:1A=Water+0.1% TFA /B= ACN + 0.1% TFA, Gradient 5% B � 95% B in 10 minutes,

Column Temp. 40 deg. C., Inj Vol 2.0 uL


Analysis of Peptide Map with Time-of-Flight MS Detection


Individual Compound Report from Time of Flight


Compound List

RT Label Mass Ions

1.101 Cpd 1: 1.101 446.2486 3

1.102 Cpd 2: 1.102 347.1794 3

1.134 Cpd 3: 1.134 360.2 3

1.141 Cpd 4: 1.141 329.1001 3

1.141 Cpd 5: 1.141 327.1033 2

1.146 Cpd 6: 1.146 389.1282 2

1.396 Cpd 7: 1.396 492.2358 3

1.507 Cpd 8: 1.507 372.1016 2

1.508 Cpd 9: 1.508 431.1501 2

1.546 Cpd 10: 1.546 617.3481 6

2.106 Cpd 11: 2.106 479.0991 2

2.151 Cpd 12: 2.151 328.0876 2

2.556 Cpd 13: 2.556 438.2107 3

2.627 Cpd 14: 2.627 300.0267 2

2.629 Cpd 15: 2.629 322.0082 2

2.822 Cpd 16: 2.822 315.0805 2

2.822 Cpd 17: 2.822 337.0622 2

3.224 Cpd 18: 3.224 447.2687 4

3.707 Cpd 19: 3.707 624.2751 7

3.876 Cpd 20: 3.876 685.4113 8

4.048 Cpd 21: 4.048 442.0331 2

4.049 Cpd 22: 4.049 444.0296 2


The Agilent 1260 Infinity Bio-inert LC


The Agilent 1260 Infinity bio-inert quaternary LCThe New Standard in Bioanalysis

100% bio-inert � Precious sample never touch metal surfaces

� Extended pH range 1-13 (shortterm 14)

� High salt tolerance: 2M salt, 8 M urea

� No stainless steel in mobile phase flow path

� New capillary technology

UHPLC capability� 600 bar

Superior Ease of Use and Robustness� Active seal wash

� Quaternary buffer mixing

� Superior Bio-HPLC columns for biotherapeutic

characterization

� Column compartement for up to 30 cm columns

The choice for both, bioanalytical and biopurification up to 10 ml/min


New Capillary Design

Metal cladded PEEK capillary

Newest fitting design: Hybrid technique

New capillary technolgy enables

600 bar AND is completely

metal-free!!


Applications

Agilent has a complete solution for bioanalytical workflows including LC-Instrument, column and applications

Typical applications for the 1260 bio-inert LC in Biopharma: 1. Aggregate analysis of mABs with SEC2. Charge variant analysis with WCX3. Peptide Mapping with RP4. Glycan analysis with HILIC, RP or AEX5. Structural analysis with HIC


G1315 DAD or MWD with bio-inert Detector Flow-cell (#028)

G5611 A Bio-inertQuaternary Pump

G5667A Bio-inert High Performance Autosampler

G1316 C Column Compartment with Bio-inert Solvent Heater (#019)

Bio-inert materials used:

PEEK, PEKK, ceramic, titanium (only in solvent delivery), gold, saphire,

PTFE

Components of the 1260 Infinity bio-inert HPLC typical configuration


G5664A Bio-inertFraction Collector

For small-scale prep and semi-prepPurification (up to 10 ml/min)

G5628A Bio-inertManual Injector

For large volume injection

Bio-inert flow cell for fluorescence detector G1321B # 028

For bio-inert fluorescence detection

Modular Flexibility - More bio-inert choices


Fraction Trigger Mode and Peak detection

May 5, Confidentiality 43


Adapting Peak DetectionWithin the method setup of Chemstation

May 5, Confidentiality 44


Bio-inert valves for G1316C #20 or for G1170A

+ =

Valve head Valve drive

2pos / 6port G5631A4-column selector G5639A

12/13 Solvent selection G1160A2 pos/10port valve

• Full flexibility for easy and fast valve exchange• 600 bar capability. • Available in the G1316C or • in the universal actuator G1170A


Agilent Buffer Advisor

G5611A: Agilent Buffer Advisor (with 1260 Bio-inert LC)G5617AA: Agilent Buffer Advisor (standalone)


Instead of setting up different bottles with different pH…

BufferAdvisor - Saving Time and Resources….


What is BufferAdvisor?

• BufferAdvisor is an independent utility software to calculate pump time tables for ion-exchange chromatography with 1260 Infinity Bio-inert Quaternary Pump

• Provides the foundation for automated mixing of acid, base, water and salt in IEX

• Is capable to calculate the selected buffer concentration and pH and high accuracy

• Suggests for the most suitable stock solutions and provides recipes

• Has included more than 50 different buffers and more than 20 were experimentally validated

• Is capable to correct automatically for pH shifts during salt gradients

• Provides the option to include user defined buffers can be included

• Generates an xml file as output which can be imported into the method of the 1260 Infinity Bio-inert Quaternary Pump by a button implemented in the driver


What Applications can be done with Buffer Advisor?

Ease of use for Ion- Exchange Chromatography• Anion-exchange, • Cation-exchange • Composite buffers• with either salt gradient or pH gradient

Customer Benefit:• Enables automated pH scouting with only 4 stock solvents e.g. phosphate buffer,

buffering range 6.1- 7.2, use buffers at 6.2, 6.4, 6.6, 6.8,7.0 would require to titrate 10 solvent bottles independently. With buffer advisor its only H20, salt, acid stock and base stock (4 solutions, no titration)

• Provides recipes and amount to weigh in for most suitable stock solutions• Corrects for pH shifts through salt by insertion of additional time points• Provides additional time points for more precise pH gradients

Buffer Advisor is a calculator software

Buffer Advisor will export the optimized Timetable


How it works

In the end it defines four stock solutions for your quaternary LC

� Line A: Water� Line B: NaCl (Salt)� Line C: Acidic buffer component� Line D: Basic buffer component


How to use Buffer Advisor Step by Step

� Select the buffer of choice

� Define concentrations for buffers, salt start and end concentration and gradient time

� Check whether the proposed buffer stock solutions are suitable for the separation task

� Press optimize gradient tab to compensate for pH shifts

� Export xml pump time table

� Import xml pump time table into OpenLab method file


Software view

Area 1Select Buffer and Gradient Solution. Here you can choose between single buffer mode, usually applied for salt gradients and Composite buffer mode for pH gradients

Area 2Enter Gradient Time TableHere you define the gradient , eg time, maximum salt concentration, pH and buffer concentration

Area 3Review Composition of Stock Solutions and AdjustConcentrations for stock solutions are shown and the recipe button shows amounts of chemical to be added to water to create the respective components.

Area 4Gradient Display Section: Process Gradient and Review Optimized Gradient with Compensation Steps


Software view

Single buffer is a buffer consisting of an acidic and basic component. For salt gradients, NaCl can be added in increasing concentrations.

Composite buffer is a buffer consisting of multiple buffering components. The composite buffer can be used over a wide pH range without compromising the buffer capacity.


Select Optimum Buffer System with applicable pKa

in Range of PI


Software view

The Recipe options are available in two locations

Recipe provides the amount of chemicals needed to be weighed in to prepare the stock solutions


Software view

The gradient time table can be saved as *.xml file format.OpenLAB CDS Chemstation C.01.04 or OpenLAB CDS Ezchrom A.01.03 can be used to IMPORT the gradient time table.

Note.xml will export the time table.asb will export the time table,the signal curves of present/actual ph and the Ionic strength(importing the .asb gradient file into OpenLAB will not import the signal curves)


How to use: example Single Buffer

Select Buffer & Gradient Mode:- Single Buffer- Cation Exhange

- Select Buffer from drop down menu

Define Gradient Table:Time, Salt, pH, Buffer as the picture

Compose Stock Solution:Press SetThen select Recipe


How to use: process

Pressing the

button calculates the

percentages of stock solutions

for each channel for of the

quaternary pump.

Furthermore it calculates

whether the pH range you

entered is suitable for the

buffer system you selected.


How to use: optimize

Optimize Gradient will adjust

the Result Pump Gradient

Table adding or correcting the

lines.

This compensation points are

added to the Timetable such

that the pH is kept constant

while the ionic strength is

changing.


How to use: export

Export Gradient Timetable will help you to Export the

Timetable from Buffer Advisor

to OpenLAB.


How to use: pump method

OpenLAB Pump Method (Default)


How to use: importing

OpenLAB Import Solvent Blending


How to use: importing

OpenLAB Timetable

Buffer Advisor Timetable


Real-time Monitoring of pH and Conductivity


Characterization of proteins and biomolecules using

Size Exclusion Chromatography

Agilent 1260 Infinity

Multi-detector

Bio-SEC Solution

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Agilent 1260 Infinity Bio-SEC Solution What is it?

• The 1260 Infinity Multi-Detector Bio-SEC is a solution for sizing and aggregation studies of proteins and other large biomolecules using Size Exclusion Chromatography

• The Agilent 1260 Infinity Bio-inert LC System combines true bio-inert sample flow path with UHPLC power range. The sample never touches metal surfaces since there is no stainless steel in the mobile phase flow path.

• The combined Static and Dynamic Light Scattering Detector simultaneously determines the absolute Molecular Weight (Mw) as well as the size of the molecule (Rh).

• Agilent Bio-SEC Software is a dedicated Chromatography Data System for an easy, yet powerful, workflow based solution

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Why Use SEC/GFC to characterize proteins?

• Separation and isolationSEC is an ideal tool to separate proteins of interest from other species

Allows further analysis, required for crystallisation and clinical applications

• Powerful analytical toolAllows determination of protein molecular weight, size and conformation

Can be used to investigate oligomerisation and aggregation phenomenon

Studies protein conformation and behavior in solution

Monomer

DimerTrimerTetramer

Aggregates

UV detection

Light scattering detection

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1260 Infinity Bio-SEC MDS System Light scattering detection

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� Reproducible, accurate molecular weights

and size information

� Sensitive detection of aggregates with market-

leading, low cell volume, light scattering detection

� Easy to use software simplifies workflow with

routine and expert mode

� Excellent repeatability and transferability minimizes

effort on data review and potential rework

� High system uptime due to robustness of a fully

tested single vendor solution

� Metal-free sample flow path for lowest surface

activity & high salt tolerance coupled with sub-

ambient thermostatting

� High Efficiency Bio-SEC columns providing

maximum resolution


Agilent 1260 Infinity Bio-SEC SolutionHow does it work?

2.9 kDa to 780 kDafor Globular Proteins

Molecular weight range kDa

1 150,000 600,000300,000

RH range nm1 102 3 4 5 6 7 8 9

Static Light Scattering determines an accurate molecular weight.Dynamic Light Scattering determines size across the molecular weight distribution.

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50,000


• In static light scattering, coherent incident laser radiation interacts with components of the biomolecule in a small scattering volume

• Excitation of the biomolecule results in re-emission of the radiation at the same wavelength and frequency but at variable intensity

• Measurement of the intensity of the scattered radiation allows the calculation of molecular weight and the chain dimensions

Static Light scattering from biomolecules

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Dynamic Light scattering from biomolecules

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Dynamic light scattering is used to measure the intensity fluctuation from the particles which are in Brownian motion.

Based on the

number of

photons

detected, DLS

provides

diffusion

coefficients

which are related

to size and Rh.

Auto correlation function

Large Molecules Small Molecules

The fluctuation

observed depends

on the size of the

biomolecule


Dual Angle Light Scattering DetectionIs it an advantage to use more angles?

• Generally, using multiple angles does not improve the data unless themolecule is very large, i.e. a molecular weight of several million daltons and aradius of gyration of greater than 10nm, which is not the case for proteins.

• Because proteins are relatively small there is no angular dependence so it isn’t possible to measure Rg, but better to use DLS to accurately determine Rh.

• The Agilent light scattering detector delivers :-• Low volume flow cell of 10ul to minimize band broadening effects• Stable baselines and highest sensitivity• Data acquisition and processing through user friendly, easy to use

software• No manipulation of raw data to make instrument related corrections

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Agilent 1260 Infinity Bio-SEC SolutionThe application - SEC analysis of biomolecules

Monomer

Dimer Fragments

Higher order oligomers

Aggregates

Sensitive to aggregation Size in solution

UV (280nm)

LS (90°)Rh (nm)

Linear response to all species

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A Monoclonal Antibody (MAB)

Documents

Talk3 Hardware and Software · 2016. 9. 4. · Agilent Technologies Hardware and Software 1. BioLC Master Class: Peptides Topics 1290 Quaternary Pumping System for Peptide Mapping