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