the new aggyilent 1260 infinity bio-inert hplc solution...0.010 0.015 dimer-0.005 0.000 0.005 1.00...
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The new Agilent 1260 Infinity g yBio-inert HPLC Solution
superior bioseparation tools for more confidence in large bio-molecule analytics
August 9, 20101
1260 Infinity Bio-inert quaternary LC
What is different compared to standard 1260 Infinity Quaternary LC?y y
Pump titanium basedAutosampler metal-freeC ill i d ti t l fCapillaries and connections metal-freeDetector Flow Cell metal-freepH-range 1-13, short-term pH 14Active Seal wash includedActive Seal wash included low pressure is possible
Wid t H•Widest pH range•Highest salt tolerance• Lowest surface activity• UHPLC capability• UHPLC capability
August 9, 20102
1260 Infinity Bio-inert quaternary LC
What is similar compared to standard1260 Infinity Quaternary LC?y y
Pumping performance, gradient performanceDetector, Autosampler Performance and SpecsOverall System RobustnessTCC specsTCC specs
600 bar also for fast or high gresolution Bioseparations
Modular flexibility maintainedModular flexibility maintained
August 9, 20103
Components of the 1260 BioHPLC
G5611A Bio-inertQuaternary Pump
G5667A Bio-inertHigh performance y p
Including Degasser andASW
Autosampler
all sample contact surfaces are metal-free
any G1315 and DAD or G1365 MWD with bio-inert flow cell
any G1316 TCC With bio-inert solvent heating snap in
All wetted parts are free of stainless steel and iron
flow cell
August 9, 20104
snap-in
Add-on choices
G4212 A d B DAD ith
G5664A Bio-inertAnalytical Scale Fraction Collector
G4212 A and B DAD with bio-inert Max-light flow cell
For highest sensitivity in bioanalysisFraction Collector
For small-scale prep and semi-prepPurification
G1321B FLDBio-inert flow cell
For bio inert Fl orescence Detection
G5628ABio-inertManual Injector
For large volume injectionFor bio-inert Fluorescence Detection
August 9, 20105
For large volume injection
Agilent 1260 Infinity quaternary LC New materials used for superior performanceNew materials used for superior performance
Materials used for sample flow path:
• PEEK, PEKKPEEK, PEKK• Ceramic• PTFE• PEEK capilllaries and fittings with
A „unique“ identifier to differentiate the biocompatible
p g600 bar capability
•Materials for sovent delivery: biocompatible components developed for the Bio-inert LC portfolio.
Titanium, PTFE
.
6 August 9, 2010
Target ApplicationsAnalytical workflows for therapeutic Antibodiesy p
PyroglutamateHeavy Chain
1. Physical and chemical characterization
Antigen binding Disulfide
shuffling
Pyroglutamate
2. Potency/activity
3. Product related impurities
Light Chain Fab
Hinge
Deamidation/oxidation
4. Process related impurities
F
Hinge
GlycosylationsiteTruncation
(lysine)Fc
( y )
August 9, 20107
Target ApplicationsBiologic Characterization
Reverse Phase: Separation of Intact Biologics/Variants
Ion Exchange: Separation of Isoforms
Size Exclusion: Aggregate AnalysisPeptide Mapping:
P t t l ti l difi tiSize Exclusion: Aggregate Analysis Post-translational modifications
8 August 9, 2010
NEW Size Exclusion Columns
• 5μm Particle • 3μm Particle
• Polymeric coated silica, minimizes non-specific binding
• Optimized for use at low salt concentrations (150mM Phosphate)
• Polymeric coated silica, minimizes non-specific binding
• Optimized for use at low salt concentrations (150mM Phosphate)concentrations (150mM Phosphate)
• High stability and long lifetime• Exclusion Ranges Available
– 100Å 0 1 – 100 kDa
(150mM Phosphate)• Highest resolution• Faster SEC separations• Exclusion Ranges Available100Å, 0.1 100 kDa
– 150Å, 0.5 – 150 kDa– 300Å, 5 – 1,250 kDa
500Å 15 5 000 kDa
Exclusion Ranges Available• 100Å, 0.1 – 100 kDa• 150Å, 0.5 – 150 kDa• 300Å 5 – 1 250 kDa– 500Å, 15 – 5,000 kDa
– 1000Å, 50 – 7,500 kDa– 2000Å, >10,000 kDa
• 300Å, 5 – 1,250 kDa
August 9, 20109
Agilent Bio SEC-3Monoclonal Antibody Aggregation Monitoring
0.035 Column: Bio SEC-3 300Å, 7.8x300mmMobile phase: 150 mM Phosphate, pH 7
0.020
0.025
0.030 Flow rate: 1.0 mL/minTemperature: AmbientSample: Monoclonal antibody (10 μL, 5 mg/mL)
Monomer
AU
0.010
0.015
Dimer
Monomer
-0.005
0.000
0.005
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Baseline separation of aggregates, dimer and monomer in 15 minutes. Separation run at 1 0 mL/min using 150mM phosphate pH 7 0
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
10
Separation run at 1.0 mL/min, using 150mM phosphate, pH 7.0
Agilent Bio SEC-3Improved Efficiency With Smaller Particles
Column Pressure
Å
Bio SEC-3(7.8x300mm)
1350 psi
Bio SEC-5(7.8x300mm)
650 psi Peak Protein SEC-3, 300Å (7.8x300mm)
SEC-5, 300Å (7.8x300mm)
Agilent Bio SEC-3, 300Å,7.8x300mm
1 Thyroglobulin 2460 1120
2 BSA Dimer 5100 2720
3 BSA 13090 6590
4 Ribonuclease A 22000 11160
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Agilent Bio SEC-5, 300Å,7.8x300mm
5 Uracil 38500 27860
Min0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
11 August 9, 2010
NEW Ion Exchange Columns
N PS/DVB ti l ith• Non-porous PS/DVB particles with a uniform polymeric coating
• Particle has a dense, weak cation exchange (WCX) mono layer withexchange (WCX) mono-layer with excellent selectivity for basic monoclonal antibodies
• Available in 10 µm, 5 µm, 3 µm, 1.7 µmAvailable in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes
• Small particles offering higher resolution than the commonly used 10µm particles
When to choose the Bio Mab Column?
• Want to separate charge isoforms of monoclonal antibodies
• In need of higher resolution• In need of higher resolution separations than current WCX column can offer
• Want a more reproducible selectivity from batch to batch and column to
l
12 August 9, 2010
column
Agilent Bio Mab (WCX)Separation of Monoclonal Antibody Charge Isoforms
0.55
0.60 Column: Agilent Bio MAb, NP5, 4.6mm x 250mmBuffer A: 10 mM Sodium Phosphate, pH 7.50Buffer B: A + 100 mM NaCl pH 7 50
0.40
0.45
0.50
0.55 Buffer B: A + 100 mM NaCl, pH 7.50Gradient: 15-95% B in 60 minFlow rate: 0.8 mL/min.Sample: 5 μL, 5 mg/mL, mAb
AU
0.25
0.30
0.35
Acidic Isoforms Basic Isoforms
0.10
0.15
0.20
0.00
0.05
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
August 9, 201013