Bio-inert UHPLCThe 1260 Infinity Bio-inert LC System

Sebastian KraheProduktspezialistAgilent Technologies

1

Good Reasons for Having a Dedicated Bio-inert HPLC

Protein Analysis:

• Unspecific surface binding

• Low throughput, low resolution

• Peak tailing for critical proteins

• Corrosion and pH issues with standard LC

• Decreased column lifetime and chromatographic performance

Other Applications:

• metal-free chromatography

• high pH applications

• Phosphorylated compounds, oligonucleotides

2

1260 Infinity Bio-inert HPLC

What is similar to standard 1260 Infinity?

Based on proven technologySystem performance specificationSystem Robustness

600 bar (UHPLC) for fast or high resolution bio-separations without any

compromises!

3

1260 Infinity Bio-inert HPLCWhat´s new ?

No corrosion due to high saltSample management completely metal-freeNo usage of MP35N capillaries with cobalt,

nickel, chromium…pH-range 1-13, shortterm pH 14low and high pressure possiblelarge volume injections

• Widest pH range• High salt tolerance• Lowest surface activity

no passivation needed !!

4

New Capillary Design Ensures Bio-inertnessCapillaries

Metal cladded PEEK capillary

Newest fitting design: Hybrid technique

New capillary technolgy enables 600 bar

AND is completely metal free !!

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Proof of concept: surface activityATP Analysis – 1260 Infinity standard LC system

min0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

500

600

0 %

MeO

H

10 %

MeO

H

50 %

MeO

H

70 %

MeO

H

90 %

MeO

H

Chromatographic conditionsFlow rate: 0.5 mL/minIsocratic run with buffer A, B, C, D or EStop time: 5 minutesInjection volume: 0.2 μLTemperature TCC: 40 °CDiode array detector: 254 nmNo column, PEEK restrictionSolventsBuffer A: 10 mM ammonium acetateBuffer B: 10 mM ammonium acetate +10% methanolBuffer C: 10 mM ammonium acetate +50% methanolBuffer D: 10 mM ammonium acetate +70% methanolBuffer E: 10 mM ammonium acetate +90% methanol

Proof of concept: low surface activityATP Analysis – 1260 Infinity Bio-inert LC

min0 1 2 3 4 5 6

mAU

0

100

200

300

400

500

600

700 0 %

MeO

H

10 %

MeO

H

50 %

MeO

H

70 %

MeO

H

90 %

MeO

H

Chromatographic conditionsFlow rate: 0.5 mL/minIsocratic run with buffer A, B, C, D or EStop time: 5 minutesInjection volume: 0.2 μLTemperature TCC: 40 °CDiode array detector: 254 nmNo column, PEEK restrictionSolventsBuffer A: 10 mM ammonium acetateBuffer B: 10 mM ammonium acetate +10% methanolBuffer C: 10 mM ammonium acetate +50% methanolBuffer D: 10 mM ammonium acetate +70% methanolBuffer E: 10 mM ammonium acetate +90% methanol

Quaternary Buffer Mixing withBuffer Advisor and 1260 Infinity Bio-inert LC System

8

…a Quat LC and Buffer Advisor enables Automation….

Na2HPO4 NaH2PO4

Water NaCl

Mix 2 components directly to getdesired pH

Dilute online or add salt for saltgradients

9

Instead of setting up different bottles with different pH…

…you use a quaternary Bio-inert LC with BufferAdvisor software toinstantly run salt- and pH gradients

…Saving Time and Resources….

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Simplifies Ion Exchange Workflows

Buffer Advisor SW Customer benefits:Ease of use• Automates preparation of separation buffers for IEX from stock solutions• Eliminates the need to prepare multiple buffer solutions • Provides recipes for buffer preparationIncrease of productivity• Simplifies pH scouting studies • Simplifies pH gradient setupIncrease of Robustness and run to run repeatibility• Compensates for pH shifts in salt gradients• Calculates optimized gradient steps

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Buffer Advisor: How does it work?

Area 1: Select Buffer and Gradient Solution

Area 2: Enter Gradient Time Table

Area 3: Review Composition of Stock Solutions and Adjust

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

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Step 1: Select Optimum Buffer System with applicable pKas in Range of PI

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Step 2: Enter your time table for the salt or pH Gradient: Retrieve Stock Solution information and Recipes

14

Step 3: Press Process and obtain your new optimized gradient table and the corresponding displayExport the Gradient Timetable as xml file

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Step 4: Import the Gradient timetable into OpenLAB CDS ChemStation

1. Go to the OpenLABCDS ChemStationand set up and save a new method,

2 . Go to the QuatPump tab and click Import Solvent Blending File

3.Select the file that you have generated in the Buffer Advisor software

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Determination of Low Metal Release in the 1260 Infinity Bio-inert LC

The Agilent 1260 Infinity Bio-inert Quaternary LC system releases less metals using acidic, basic, and salt containing buffers compared to

• the Agilent 1260 Infinity LC stainless steel system and

• to the bio-inert LC system from another vendor

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Agilent 1260 Infinity LC Bio-inert systemfrom other vendor

Agilent 1260 Infinity Bio-inert LC

H2Odd

ACN/ H2Odd

0.1 % FA

0.1 % TFA

100 mM NaOH

Buffer(150 mM NaPO4,150 mM NaCl)

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

< 10 µg/L > 10 µg/L< 1 µg/L

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

MoTi Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

Mo

Ti Cr Mn Fe

Zr

Cu ZnNiCo

MoTi Cr Mn Fe

Zr

Cu ZnNiCo

Mo

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

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Characterization of therapeutic proteins and monoclonal Antibodies drugs

1. Physical and chemical characterization (product characterization)

2. Potency/activity

3. Product related impurities

4. Process related impurities

Light Chain

Fc

Fab

Antigen binding

Hinge

GlycosylationsiteTruncation

(lysine)

Disulfide shuffling

Pyroglutamate

Deamidation/oxidation

Heavy Chain

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Monitoring Aggregation and Impurities by SEC with the 1260 Infinity bio-inert LC

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

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

Column: Agilent Bio SEC-3 300Å, 7.8x300mmMobile phase: 150 mM Phosphate, pH 7Flow rate: 1.0 mL/minTemperature: AmbientSample: Monoclonal antibody (10 μL, 5 mg/mL)

Buffer/ExcipientsMAb

DimerMAb

Aggregates

Low Molecular Weight Impurities

Detection: UV (diode array detector)

min5 10

mAU

0

20

40

60

80

100

120

140A

min5 10

mAU

0

50

100

150

200

250

300B

Agg

rega

te

Agg

rega

te

DAD1 A, Sig=220 DAD1 A, Sig=220

Aggregation induced through pH change(Sample: pharmaceutical IgG1)

Sample after pH induced aggregation

IgG before and during aggregationexperiment

Column: Agilent Bio SEC-3, 300Å, 7.8 x 300 mm, 3 μm

Parameters ConditionsMobile phase A

150 mM sodium phosphate (pH = 7.0) containing 150 mMsodium chloride

TCC Temper-ature

Ambient

Isocratic run Mobile phase AInjection volume

5µl

Flow rate 0.8mL/minUV detection 220 and 280 nm

HPLC Analysis of Charged Isoforms

Column: Agilent Bio MAb 5um, 4.6x250mm PEEKEluent A: 10 mM Na phosphate buffer, pH 5.5Eluent B: A + 0.5 M NaClFlow rate: 0.85 ml/min Gradient: 5 – 30%B in 25 minutesVolume: 5 ul of 1mg/ml Detection: UV 225nmInstrument: Agilent 1260 Infinity Bio-inert system

min5 7.5 10 12.5 15 17.5 20 22.5 25

mAU

0

5

10

15

20

25Lys-1

Lys-2

Lys-0Basic Charged Variants

K K K

Charge VariantsCharge Isoform Analysis of Monoclonal Antibodies

‐ a typical example how it should look like ‐

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

Minutes0.00 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

Agilent 1260 Bio –inert LC,  Agilent Bio MAb, NP5, 4.6mm x 250mmBuffer A: 10 mM Sodium Phosphate, pH 7.50Buffer B: A + 100 mM NaCl, pH 7.50Gradient: 15‐95% B in 60 minFlow rate: 0.8 mL/min.Sample: 5 ul 5 mg/mL, mAbPEEK column hardware

Acidic Isoforms Basic Isoforms

RP Characterization: Peptide mapping for sequence confirmation of mABs

High peak capacity is crucial for sufficient coverage

• Approach: stationary phases with small particles 1.8 um totally porous or 2.7 um coreshell, long or coupled columns, 1200 bar UHPLC

• Advantages: Increased resolution at similar time as larger particle columns or increased analysis time at similar resolution as larger particle columns

Peptide map map of IgG1 min10 20 30 40 50

mAU

-10

0

20

40

6070

DAD A, Sig=214

Pressure: ~ 402 bar Instrument: 1290 Infinity binary LCColumn: Agilent RRHD SB C18, 2.1 x 150 mm, 1.8 µm column) Flow rate: 0.25mL/minMobile phase: A = Water + 0.1 % TFA, B = Acetonitrile+ 0.09 % TFA

Time (min)

Solution B (%)

0 215 2050 4055 6060 9061 2

Post time 10 min

Gradient:

How to further increase peak capacity in peptide mapping for mABs?

Coupled Column Approach:

DAD A, Sig=214

A

min20 40 60 80 100

mAU

-10

0

10

20

30

40

50 Pressure: ~ 598 bar

Peptide map map of IgG1 (A) on 1290 Infinity LC using coupled columns

Instrumentation Platform

Column Peak Capa-city

Agilent 1260 Bio-inert LC

Poroshell 120 SB C18 4.6 x 150 mm,

2.7 µm

292

Agilent 1290 Infinity LC

Single RRHD SB C18, 2.1 x 150 mm,

1.8 µm

313

Agilent 1290 Infinity LC

Coupled RRHD SB C18, 2.1 x 150 mm,

1.8 µm

406

Comparison on Peak capacity with different columns and instruments

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