agilent technologies april 7, 2015 1 · peptide mapping standard april 7, 2015 agilent technologies...

April 7, 2015

Agilent Technologies

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April 7, 2015


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Improving your laboratory HPLC analysis workflow with new Agilent BioHPLC Columns

Paul Dinsmoor

Technical Specialist, Bio-Columns

April 7, 2015


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Tools for Therapeutic Protein Characterization

Size Exclusion Chromatography

• Aggregates and fragment analysis

• Molecular size analysis

Ion-Exchange Chromatography

• Charge variant analysis

2- 1-

+

Reversed-Phase Chromatography

• Primary structure analysis

• Post-translational modifications/ degradations

• Peptide mapping • Amino acid

analysis

Affinity Chromatography

• Titer and protein quantitation

Hydrophilic Interaction Chromatography (HILIC)

• Glycan mapping • Glycopeptide analysis H20

H20 H20

H20

H20

H20

H20 H20

Agilent Bio-LC Column Portfolio

Agilent Bio-LC Columns

Affinity

Bio-Monolith Protein A

Multiple Affinity Removal System

Reversed Phase

AdvanceBio Peptide Mapping

ZORBAX RRHD 300A 1.8um

Poroshell 300

AdvanceBio RP mAb

ZORBAX 300SB

ZORBAX Amino Acid Analysis

PLRP-S

HILIC

AdvanceBio Glycan Mapping

ZORBAX RRHD 300-HILIC

Size Exclusion

Bio SEC-3

Bio SEC-5

ProSEC 300S

ZORBAX GF-250

ZORBAX GF-450

Ion Exchange

Bio-Monolith (QA, DEAE, SO3)

Bio mAb

Bio IEX (SAX, SCX, WAX, WCX)

PL SAX

PL SCX

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Products in red are new!

AFFINITY CHROMATOGRAPHY

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Analytical Bio-Monolith Protein A Columns

Used for: • Fast screening of harvest cell culture

samples for IgG – process optimization

• Accurate analysis of mAb quantities to determine protein harvest

• Capture and purification of protein for further characterization

Features and benefits: • Bio-Monolith Protein A

(immunoaffinity) • Monolith type material for fast, flow

rate independent separations • Monolith material does not clog easily

with cell debris • Attaches easily to all LCs with

standard fittings

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2 Minute Analysis – Antibody Titer from Cell Culture Supernatant

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min 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

mAU

0

50

100

150

200

250

Lysate proteins containing mAb

wash elute re-equilibrate

IgG1 (2.5 µg) Flow through (8 µg)

Abso

rban

ce (m

AU

at 280 m

m)

Time (min)

IgG1

Column Agilent Bio-Monolith Protein A Sample: Cell lysate spiked with IgG1 Equilibration buffer: 50 mM NaPO4, pH 7.4 Elution buffer: 0.1 M citric acid, pH 2.8 Flow Rate: 1.0 mL/min Detector: UV 280nm System: 1200 Infinity

REVERSED-PHASE BIOCHROMATOGRAPHY

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Characterization of Primary Structure – RP LC

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Levels of Characterization

Improve accuracy and resolution

Intact mAb

Reduction/Alkylation

Light and Heavy Chain

Enzymatic Digestion

Peptide Map

Common Challenges with RP Bioseparations

• Poor characterization in the separation results in poor identification - Need best stationary phase to perfect the separation - Need high resolution/efficiency

• Lack of analytical consistency

• Method robustness

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Poroshell 300

• 300Å pore size

• StableBond and Extend chemistry

• Available in SB-C3, SB-C8, SB-C18, and Extend-C18

• 5 µm particle size

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High Flow Rates with 2.1 mm id Poroshell for High Resolution and Fast Separations

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12

34

5

67

8

0 0.5 1.0Time (min)

Columns: Poroshell 300SB-C18 2.1 x 75 mm, 5 µm MP: A: 0.1% TFA B: 0.07% TFA in ACN Gradient: 5 – 100% B in 1.0 min. Flow Rate: 3.0 mL/min. Temperature: 70 °C Pressure: 250 bar Detection: UV 215 nm

Sample: 1. Angiotensin II 2. Neurotensin 3. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.Ovalbumin

Pub No# 5989-9899EN for complete app note

ZORBAX 300SB RRHD for Proteins

• Stablebond 300 silica/bonding

• C18, C8, C3, and Diphenyl bonded phase

• 1.8 µm particle size for high resolution

• 1200 Bar pressure limit for UHPLC

• 2.1 x 50 mm and 2.1 x 100 mm

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C18 C8 C3 Diphenyl

Increasing protein size and hydrophobicity

UHPLC Columns Increase Resolution and Increase Speed

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Column Length (mm)

Resolving Power

N(5 µm)

Resolving Power

N(3.5 µm)

Resolving Power

N(1.8µm)

150 12,500 21,000 32,500

100 8,500 14,000 24,000

75 6000 10,500 17,000

50 4,200 7,000 12,000

30 N.A. 4,200 6,500

15 N.A. 2,100 2,500

Analysis Time

Peak Volume

Analysis Time*

-33%

-50%

-67%

-80%

-90% Solvent Usage

* Reduction in analysis time compared to 150 mm column

Comparison C3 and Diphenyl Phases

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min 1 1.2 1.4 1.6 1.8 2 2.2 2.4

mAU

2

4

6

8

10

12

1.3

37

1.5

87

1.6

66

1.7

54

1.8

11

min 1 1.2 1.4 1.6 1.8 2 2.2 2.4

mAU

2

4

6

8

10

12 1.3

45

1.5

64

1.6

46

1.7

87

1.8

46

ZORBAX RRHD 300SB-C3, 1.8 µm

ZORBAX RRHD 300-Diphenyl, 1.8 µm

Ribonuclease A, Cytochrome C and Lysozyme (3 mg/mL)

Time (min)

%B

0 10

2.5 70

Arrows indicate better separation resolution of ZORBAX RRHD 300-Diphenyl

Pub No# 5990-9668EN for complete application note.

Peptide Mapping: Common Challenges

• Analytical consistency

• Long analysis times to get full resolution

• Insufficient resolution and sensitivity

• Method robustness

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AdvanceBio Peptide Mapping Column for HPLC and UHPLC: • Peptide Maps in < 15 minutes • 2.7 µm Superficially Porous • 120Å pore size • C18 functionality • 600 bar pressure limit • 2 µm frit to reduce clogging

Greater analytical confidence: Each batch is tested with a rigorous peptide mix to ensure suitability and reproducibility Save Time: 2 to 3 times faster than fully porous particles Increased Flexibility: Highly compatible with TFA and formic acid mobile phases for efficient LC UV and LC/MS analysis

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Superficially Porous Particle Technology

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Decrease the diffusion time for macromolecules and limit the diffusion path!

The particle has a solid core (1.7 µm) and porous outer layer with a 0.5 µm diffusion path Reduces secondary interactions and enables selective separation for a wide range of peptides.

Peptide Mapping Standard

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Peak # /Peptide MW 1 Bradykin frag (1-7) 756.85 2 Bradykin Acetate 1045.53 3 Angiotensin II 1060.21 4 Neurotensin 1296.48 5 Angiotensin I 1672.92 6 Renin 1759.01 7 {Ace-F-3,-2H-1] Angiotens-

inogen (1-14) 2231.61 8 Ser/Thr Protein Phosphotase

(15-31) 1952.39 9 [F14] Ser/Thr Protein Phosphotase

(15-31) 2099 10 Mellitin (Honey

bee venom) 2846.46

Each batch of AdvanceBio Peptide Mapping media is tested with the Agilent Peptide Standard (PN 5190-0583) to ensure batch to batch reproducibility

Peptide Mapping by LC/MS

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8 x10

0

0.75

1.75

2.75

3.75

+ESI TIC Scan Frag=200.0V igg023.d

Counts vs. Acquisition Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

40 min. Run, 2.1 x 100 mm 6x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

Cpd 154: B(357-366): + ECC Scan igg036-Agilent long run-peptides in -20.d

Cpd 154: B(357-366)

Counts vs. Acquisition Time (min)

18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5

Native peptide

Deamidated form 2

Deamidated form 1

Critical Post Translational Modifications (PTM) Identified in Fast and Slow Analyses

7x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

Cpd 132: B(357-366): + ECC Scan igg035-Agilent short run-peptides in -20.d

Cpd 132: B(357-366)

Counts vs. Acquisition Time (min)

6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2 8.3 8.4 8.5

Native peptide

Deamidated form 2

Deamidated form 1

8 x10

0

0.6

1.6

2.6

3.6

4.6

433 bar 0.6 mL/min 10-40% B

+ESI TIC Scan Frag=200.0V igg011.d

Counts vs. Acquisition Time (min) 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13

140 bar 0.2 mL/min 10-40% B

Heavy Chain Peptide 357-366 and its two deamidated forms

conserved

14 min. Run, 2.1 x 100 mm

New AdvanceBio RP mAb

Particle

• 3.5 μm SP particle

• 0.25 μm porous layer depth

• ρ value of 0.86

• 450 Å pore diameter

The optimum large molecule resolution for use with both HPLC and UHPLC systems

April 7, 2015

Agilent Confidential

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3.5 um 3.0 um

0.25 um

Phases

• C4

• SB-C8

• Diphenyl The most popular phases for proteins, plus a unique selectivity

New

New

Fast Intact mAb Analysis

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AdvanceBio RP-mAb provides superior peak shape at a lower pressure and resolves more fine detail than a UHPLC protein column from competitor

Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water:IPA (98:2) Mobile phase B: IPA:acetonitrile:MPA* (70:20:10) Flow rate: 1.0 mL/min

Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 5 μL injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °C Detection: UV @ 254 nm

AdvanceBio RP-mAb C4, 450Å, 3.5 μm 490 bar

Competitive C4, 300Å, 1.7 μm 910 bar

* MPA = Mobile Phase A

Fast, High Resolution mAb Fragment Analysis

min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200

DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...C_MD\AEM_PS450_FAB-FC_MD_4 2014-09-10 09-02-53\1443508-69-0005.D)

min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200

DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-11 13-54-40\USRIT001297-003.D)

min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200

DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-11 14-37-19\706785-1-000003.D)

min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

200

DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-10 11-45-32\CD4F123-0000003.D)

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Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water Mobile phase B: n-propanol/acetonitrile/MPA (80/10/10) Flow rate: 0.8 mL/min

Gradient: 5-40% B in 5 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 1 μL injection of Fc/Fab, Papain Digested Humanized Recombinant Herceptin Variant IgG1 from Creative Biolabs (2 mg/mL) Temperature: 60 °C Detection: UV @ 220nm

AdvanceBio RP-mAb provides superior peak shape and resolution than other columns designed for protein separations

AdvanceBio RP-mAb C4, 450Å, 3.5 μm

Competitor A Protein C4, 400Å, 3.4 μm

Competitor B WIDEPORE C4, 200Å, 3.6 μm

Competitior C C4-30, 300Å, 2.6 μm

Fast Intact mAb Analysis AdvanceBio RP-mAb Diphenyl resolves additional fine detail - the Diphenyl phase is unique to Agilent

Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water/IPA (98/2) Mobile phase B: IPA/acetonitrile/MPA* (70/20/10) Flow rate: 1.0 mL/min

Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 5 μL injection of Humanized Recombinant Herceptin IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °C Detection: UV @ 254nm

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min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

mAU

0

20

40

60

80

100

120

140

DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)

min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

mAU

-4

-2

0

2

4

6

8

10

12

14

DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)

AdvanceBio RP-mAb C4 AdvanceBio RP-mAb SB-C8 AdvanceBio RP-mAb Diphenyl


Fast, High Resolution mAb Fragment Analysis

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Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water Mobile phase B: n-propanol/acetonitrile/MPA* (80/10/10) Flow rate: 0.8 mL/min

Gradient: 5-40% B in 5 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 1 μL injection of Fc/Fab, Papain Digested Humanized Recombinant Herceptin IgG1 from Creative Biolabs (2 mg/mL) Temperature: 60 °C Detection: UV @ 220nm

min2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25

mAU

0

100

200

300

400

DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...C_MD\AEM_PS450_FAB-FC_MD_4 2014-09-10 09-02-53\1443508-69-0005.D)

AdvanceBio RP-mAb provides sharp peaks and good resolution in less than 5-minutes

AdvanceBio RP-mAb C4


AdvanceBio RP-mAb C4 Separtes Proteins with small differences: Biosimilars in development

Agilent-RIC collaboration biopharma - Nov 2014

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Using 1.0 % B/mL gradient

Remicade Remicade clone

Using 2.1 % B/mL gradient

Remicade Remicade clone

Shift of Fc

Annotated shift of the Fc part implicates differences in hydrophobicity of the Fc part due to a 2-point mutation in the AA sequence of the biosimilar compared to the originator. The shift is observed with either a fast or slow gradient.

Shift of Fc

RP Summary

Agilent Column Positioning

AdvanceBio RP-mAb • Designed for mAb separations • Fast, high resolution HPLC and UHPLC analysis of intact mAbs and mAb

fragments

ZORBAX RRHD 300SB • High resolution UHPLC analysis of proteins, including intact mAbs, and protein fragments

Poroshell 300 • Fast, HPLC analysis of large intact proteins, including intact mAbs

ZORBAX 300SB • High resolution HPLC analysis of proteins, including intact mAbs, and protein fragments

PLRP-S • Polymeric for high pH stability and alternate selectivity.

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AdvanceBio RP-mAb is an addition to the market leading Agilent reversed-phase bio-column portfolio and complements existing columns

How the New Columns Fit: Agilent Positioning

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Match The Column To System Pressure Capabilities

Agilent Column Particle Pressure Rating Phases

AdvanceBio RP-mAb SPP, 3.5 μm, 450Å 600 bar SB-C8, C4, Diphenyl

ZORBAX RRHD 300SB TPP, 1.8 μm, 300Å 1200 bar SB-C18, SB-C8, SB-C3, Diphenyl

Poroshell 300 SPP, 5 μm, 300Å 400 bar SB-C18, SB-C8, SB-C3, Extend-C18

ZORBAX 300SB TPP, 3.5 & 5 μm, 300Å 400 bar SB-C18, SB-C8, SB-C3, SB-CN

PLRP-S TPP, 3, 5, 8 um 100, 300, 1000, and 4000A 400 bar NA

HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY(HILIC)

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Glycan Analysis – Common Challenges

• Very long analysis times

• Instrument limitations

• Difficulty achieving reproducible results

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N-Glycan Mapping

Glycoprotein

N-Glycan

2-AB labelled glycan

HILIC column LC/MS

HILIC column LC/FLD (-MS)

Majority process

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Glycan Analysis Workflow

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Glycoprotein

N-Glycans

2-AB Labelled N-glycans

Deglycosylation

Work-up

2-AB Labelling Work-up

HILIC FLD / MS

Deglycosylation Kit

Description (24 or 96 samples)

Reaction buffer 5X

Denaturant

Detergent

PNGase F

Deglycosylation Work-up


SPE cartridges

2-AB Labeling Kit


2-AB solution

Reductant solution

2-AB Labeling Work-up


SPE cartridges

Glycan Analysis Workflow

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Deglycosylation

Work-up

2-AB Labelling Work-up

HILIC FLD / MS

Glycoprotein

N-Glycans

2-AB Labelled N-glycans

Unlabelled Standards

Labelled Standards

AdvanceBio Glycan Mapping Column

• Super-fast glycan analysis

• 1.8 µm fully porous for highest performance

• 2.7 µm superficially porous for lower pressures

• Unique hydrophilic bonding

High-speed, high-resolution performance Ideal for all UHPLC/HPLC instruments Batch tested with the Agilent IgG glycan standard to ensure reproducibility

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Glycan Analysis by LC-FLD

1260 Infinity Bio-inert HPLC

AdvanceBio Glycan Mapping,

2.7 um +

AdvanceBio Glycan Mapping,

1.7 um +

1290 Infinity UHPLC

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HILIC Summary

Characterization Product Features Advantage Benefit Key Challenge

Glycan Mapping AdvanceBio Glycan Mapping

1.8um FPP, bonded phase

High efficiency, right selectivity

Improved accuracy and

reproducibility of data – reliable results, cost

saving

Resolution 2.7um SPP,

bonded phase

1.8um FPP, bonded phase Efficiency at

higher flow rate, fast gradients

Improved analysis efficiency, cost

saving Throughput

2.7um SPP, bonded phase

FPP: Fully Porous Particle, SPP: Superficially Porous Particle

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SIZE EXCLUSION CHROMATOGRAPHY

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Common SEC challenges

• Insufficient/incorrect pore sizes can reduce resolution

• Non-specific interactions contribute to loss of sample, lead to inconsistent results, rework

• SEC is typically slow

• Poor pressure stability creates rework and increased cost

• Consistent and robust results

• High salt conditions puts excessive wear on instrument, parts

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SEC Column Choice: Resolving Ranges

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Pore Size Comparison

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Eluent: 50 mM NaH2PO4 + 0.15M NaCl, pH6.8 Columns: Agilent Bio SEC-3, 4.6 x 300 mm Flow: 0.35 mL/min Detector: UV @ 220 nm System: Agilent 1260 Infinity Bio-inert LC System Sample: BioRad Gel Filtration Standards Mix

1. Thyroglobulin Aggregates 2. Thyroglobulin 3. IgA 4. γ-globulin 5. Ovalbumin 6. Myoglobin 7. Vitamin B12

300Å

150Å

100Å

1

2

3

4 5 6

7 Best starting point for mAbs

Improved Efficiency With Smaller Particles

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Column: Bio SEC-3 300Å and Bio SEC-5 300Å Buffer: 150 mM Phosphate buffer, pH 7 Flow rate: 1.0 mL/min for 7.8 x 300 mm Temperature: Ambient (~23 °C) Detection: UV 214 nm Injection: 10 µL (3 µL for 4.6 x 300 mm) Sample: 1) Thyroglobulin (1.0 mg/mL), 670 kD; 2) BSA dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD; 4) Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil (2.5 µg/mL), 120 D.

Min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Agilent Bio SEC-3, 300Å, 7.8 x 300 mm 93 bar

Agilent Bio SEC-5, 300Å, 7.8 x 300 mm 45 bar

Peak Protein Efficiency Gain

SEC-3, 300Å (7.8x300mm)

SEC-5, 300Å (7.8x300mm)

1 Thyroglobulin 2.2X 2460 1120

2 BSA Dimer 1.9X 5100 2720

3 BSA 2.0X 13090 6590

4 Ribonuclease A 2.0X 22000 11160

5 Uracil 1.4X 38500 27860

Fast monomer/dimer separation using the Agilent Bio SEC-3 300Å 7.8 x 150 mm column

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Flow Rate Resolution Monomer/Dimer

Monomer Efficiency

Percentage Dimer

1.0 mL/min 1.53 3,510 0.64

1.5 mL/min 1.43 2,502 0. 47

2.0 mL/min 1.13 1,917 0.64

monomer

2.0 1.5 1.0 mL/min mL/min mL/min

dimer

Size Exclusion Summary

Characterization Product Features Advantage Benefit Key Pain Point

Aggregation Bio SEC

3um particles, porosities

High efficiency, right selectivity

Improved accuracy and

reproducibility of data – reliable results, cost

saving

Resolution High efficiency, right MW range

3um particles High efficiency Improved analysis

efficiency, cost saving

Throughput

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ION-EXCHANGE CHROMATOGRAPHY

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Charge Variant Analysis

Common challenges

• Resolution can be limited and inconsistent – can require troubleshooting and rework

- Need capability to handle complex analyses consistently - mAbs present special challenges, due to their complexity

• Column contamination can lead to early column failure and produce incomplete sample recovery

• Method development is time-consuming - Need capability for faster, systematic method development with

different buffer strengths

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Some Guidelines for IEX

1.The General Rule for choosing a Bio IEX column - Acidic proteins: SAX or WAX - Basic proteins: SCX or WCX 2. Consider the isoelectric point (pI) of your protein when choosing the pH

of your mobile phase: - If pH>pI, your protein will have a net negative charge - If pH<pI, your protein will have a net positive charge 3. The pH of your starting buffer should be 0.5 to 1 pH unit from your pI - Above pI for anion-exchange - Below pI for cation-exchange

4. If your pI is unknown - Start with pH 6 for cation-exchange - Start with pH 8.0 for anion-exchange

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Ion Exchange – Product Families

47

Particle Porosity Functionalities Particle Sizes Pore Size Application

Agilent Bio-IEX Polymer Non-porous SAX, WAX, SCX, WCX

1.7um, 3um, 5um 10um

N/A Peptides, proteins

Agilent Bio MAb Polymer

Non-Porous WCX 1.7um, 3um, 5um 10um

N/A IgG

PL-SAX PS/DVB Fully Porous SAX 5um, 8m, 10um, 30um

1000A, 4000A Peptides, oligos, proteins. Larger column sizes

PL-SCX PS/DVB Fully Porous SCX 5um, 8m, 10um ,30um

1000A, 4000A Peptides, proteins. Larger column sizes

Page 46

1. Non-porous particles for high efficiency analytical separations

2. Porous particles for scale up to purification

Ion-Exchange Columns

• Non-porous PS/DVB particles (polystyrene divinylbenzene) • Uniform polymeric coating with SCX, WCX, SAX, WAX layers, designed

for protein and peptide separations • Available in 10, 5, 3, 1.7 µm particle sizes • High surface area • High capacity

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Agilent Bio IEX Columns Comparing Separations on Each Particle Size

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Min 0 2 4 6 8 10 12 14 16 18 20 22

1.7 µm

3 µm

5 µm

10 µm

Column: Bio WCX, 4.6 x 50 mm Buffer A: 20 mM PBS Buffer B: A+1.0 M NaCl Gradient: 0-100%B (20 min) Flow rate: 1.0 mL/min for 10 µm, 5 µm, 3 µm 0.75 mL/min for 1.7 µm Sample: 1) Ribonuclease A 2) Cytochrome C 3) Lysozyme Concentration: 1.0 mg/mL Detector: 280 nm Average N ~80,000 for WCX 1.7 µm

Peak N

Peak N

Ion-Exchange Columns – Special for mAbs

• Non-porous PS/DVB particles with a uniform polymeric coating • Particle has a dense, weak cation-exchange (WCX) mono-layer with excellent

selectivity for basic monoclonal antibodies • Available in 10, 5, 3, 1.7 µm particle sizes • Small particles offering higher resolution than the commonly used 10 µm

particles

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Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies

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Min 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

A

B

C

D

E

Optimization of method conditions for the isoform characterization of a monoclonal antibody. Changes in the buffer conditions, pH and gradient

conditions sharpen peaks and increase resolution of acidic and basic isoforms.

Columns: Agilent Bio MAb, 10 µm, 4.6 x 250 mm Mobile phase: A, 10 mM phosphate, pH 7.5 B, A + 0.1 M NaCl Gradient: A) 15-75% B in 30 min B) 15-65% B in 30 min C) 15-55% B in 30 min D) 15-47.5% B in 30 min E) 15-40% B in 30 min Flow rate: 0.8 mL/min Sample: Monoclonal Antibody Injection: 10 µL (1.5 mg/mL) Temperature: 25 oC Detection: UV 214 nm

Analytical Bio-Monolith Ion Exchange Columns

• Polymer based monolithic discs • Fast, high resolution ion-exchange • Key applications are for large proteins and

biomolecules (virus particles, pDNA, antibodies [IgG and IgM])

• Agilent Bio-Monolith QA (strong anion-exchanger) • Agilent Bio-Monolith DEAE (weak anion-exchanger) • Agilent Bio-Monolith SO3 (strong cation-exchanger)

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www.agilent.com/chem/getbioguides Application-focused Brochures

“How To” Guides

Keys for Enabling Optimum Peptide Characterizations: A Peptide Mapping “How To” Guide 5991-2348EN Protein Identification

and Impurity Profiling using Reversed-Phase HPLC/UHPLC 5991-0625EN

Resolve Protein Aggregates and Degradants with Speed and Confidence 5991-2898EN

Reversed-Phase

Affinity

Ion-Exchange

Characterize Charged Variants of Proteins with Speed and Confidence 5991-2449EN

Size Exclusion Chromatography for Biomolecule Analysis: A “How To” Guide 5991-3651EN

Selection Guide

Your Reference Guide to the Analysis of Biopharmaceuticals and Biomolecules 5990-9384EN

Ion-Exchange Chromatography for Biomolecule Analysis: a “How to” Guide 5991-3775EN

Characterize Charged Variants of Proteins with Speed and Confidence 5991-2449EN

Size Exclusion

Resources for More Information

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http://www.chem.agilent.com/Library/selectionguide/Public/5991-2348EN%20Peptide%20Mapping%20Guide.pdf

http://www.chem.agilent.com/Library/brochures/5991-0625EN_Rev%20Phase%20Brochure.pdf



http://www.chem.agilent.com/Library/brochures/BioSEC%20Brochure.pdf

http://www.chem.agilent.com/Library/brochures/5991-2449EN_BioIEXBrochure.pdf



http://www.chem.agilent.com/Library/primers/Public/5991-3651EN_LR.pdf

http://www.chem.agilent.com/Library/selectionguide/Public/5990-9384EN.pdf



http://www.chem.agilent.com/Library/primers/Public/5991-3775EN_BioIEX_HowTo_LR.pdf




BioHPLC Columns on the Agilent Website

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To learn more and order online visit www.agilent.com/chem/biocolumns

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Thank You Any Questions?

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