agilent technologies 1

April 20, 2015

Agilent Technologies

1

April 20, 2015


2

Improving HPLC Characterization of Biomolecules –Agilent Solutions to Separation Challenges

Paul Dinsmoor

Technical Specialist, Bio-

Columns

Agilent Solutions for Therapeutic Protein Characterization

3

Titer determination and purification (Affinity)

Protein A Bio-Monolith

Protein identification and impurity profiling (RP)

120A Advancebio Peptide Mapping Column and 450A RP mAb Column

Glycan analysis (HILIC)

Advancebio Glycan Mapping Column

Charge variant analysis (IEX)

Bio-IEX 1.7um Column

Aggregation analysis (SEC)

Bio-SEC 3 Column

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

April 20, 2015


4

Products in red are new!

REVERSED-PHASE BIOCHROMATOGRAPHY

April 10, 2015


5

April 20, 2015


6

April 20, 2015


7

• RP chromatography separates proteins through the interaction of the “hydrophobic foot” of the protein with a non-polar surface of the particle

• RP columns are nearly always based on silica particles

– Mechanical stability, easy to make, surface can be modified, excellent peak shape & efficiency

• Solvent

– Organic modifier: Acetonitrile, isopropanol, methanol

– Ion pair additive: Trifluoroacetic acid (TFA), formic acid

• Gradient separation

Strategy for RP-HPLC Method Development of Proteins and Peptides

April 20, 2015


8

• Start at low pH (acidic mobile phase) and choose the initial column and

conditions

• Initial selection parameters include: pore size (120A, 300A, 450A),

mobile phase, bonded phase, particle size, column length, and internal

diameter

• Obtain best resolution by optimizing: gradient steepness, bonded

phase, temperature, column configuration

• Obtain best recovery by optimizing: Bonded phase, temperature,

sample solubility

• Evaluate alternative columns/ technologies for improved selectivity

and efficiency

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

• Reduced lifetime

• Lack of analytical consistency/ method robustness

• LC system pressure limitations

• Long run times for peptide maps

April 19, 2015


9

Primary Structure Characterization Workflows - mAb

April 20, 2015


10

Improve accuracy andresolution

April 20, 2015


11

Protein/Peptide Separations by Reversed-phase

Larger Molecules = Slower Diffusion

So, a need to decrease the diffusion time for macromolecules!

To improve, we can increase the Diffusion Rate by:

elevating operating temperature decreasing solvent viscosity

and, orCCCCC..

April 20, 2015


13

April 20, 2015


14

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

April 10, 2015


15

April 20, 2015


16

Four Rapid Resolution High Definition Phases

17

C18 C8 C3 Diphenyl

Peptide Maps Increasing protein size/hydrophobicitySmall Proteins

Ligand Application

C18 Small intact proteins/peptide maps

C8 Intact proteins

C3 Larger /hydophobic proteins, including MAbs

Diphenyl Unique selectivity

ZORBAX300Å, 1.8 µm

Agilent 1290 Infinity

Family of Four Reversed-Phase Ligands

C3 versus Diphenyl

The Separation Challenge - Does Size Matter?

19

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

300SB-C18 (300Å)

SB-C18 (80Å)

PW

1/2

Proper pore size selection results in sharper peaks for large molecules

Effect of Pore Size and Molecular Size on Peak Width

0 min 12 0 min 11

NEW! AdvanceBio Peptide Mapping Column for HPLC and UHPLC:

• 2.7 µm Superficially Porous

• 120Å pore size

• 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

April 10, 2015


20

2.7 um1.7 um

0.5 um

April 20, 2015


21

Quality Assurance Testing with Agilent Peptide Mix

Peptide Mapping Standard P/N# 5190-0583

Each batch of AdvanceBio Peptide Mapping media is

tested with the Agilent Peptide Standard (PN 5190-0583)

to ensure batch to batch reproducibility

min0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

10

20

30

40

50

60

70

2.1 x 150mm AdvanceBio Peptide Mapping ColumnMobile phase: A-water (0.1%TFA), B- ACN (0.08%TFA), 40 C, flow: 0.52mL/min

Hydrophilic peptide retention

Narrow Peaks w baseline resolution

Hydrophobic peptide retention

Reduced and fast analysis time

Critical and desired peptide mapping

components to achieve fast, selective and

highly efficient peptide separations across

a wide dynamic range.

Peptide MappingAdvanceBio Peptide Mapping Column Highlights

BSA tryptic digest

Peptide Mapping by LC/MS

April 10, 2015


24

8x10

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

Native peptide

Deamidated form 2

Deamidated form 1

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

Native peptide

Deamidated form 2

Deamidated form 1

8x10

0

0.6

1.6

2.6

3.6

4.6

433 bar0.6 mL/min10-40% 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 bar0.2 mL/min10-40% B

Heavy Chain Peptide

357-366 and its two

deamidated forms

conserved

14 min. Run, 2.1 x 100 mm

Poroshell 300

• 300Å pore size

• StableBond and Extend chemistry

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

• 5 µm particle size

April 1, 2015


25

April 20, 2015


26

Column Internal DiameterPorous Particle

Flow Rate RangePoroshell

Flow Rate Range

2.1 mm 0.1 – 0.3 mL/min 0.3 – 3 mL/min

1.0 mm 30 – 60 µL/min 0.08 - 0.75 mL/min

• Very high flow rates can be used effectively with Poroshell columns

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

April 1, 2015


27

12

34

5

67

8

0 0.5 1.0Time (min)

Columns: Poroshell 300SB-C182.1 x 75 mm, 5 µm

MP: A: 0.1% TFAB: 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 II2. Neurotensin3. Rnase4. Insulin5. Lysozyme6. Myoglobin7.Carbonic Anhydrase8.Ovalbumin

Pub No# 5989-9899EN

for complete app note

High Flow Rate, High Sensitivity LCMS

min0 0.5 1 1.5 2 2.5 3 3.5

0

20000000

40000000

60000000

80000000

1E8

pmoles of protein0.5

2.5

5

0.75

1

Mobile Phase Gradient: 20 -

100% B in 5.5 min.

A: water + 0.1% formic acid

B: ACN + 0.1% formic acid

Flow Rate: 0.6 mL/min

Temperature: 80°C

Injection volume: 1 uL

LC/MS: Pos. Ion ESI

Peakwidth: 0.06 min

Sample:Mixture of insulin, lysozyme,

cytochrome C, myoglobin, BSA,

carbonic anhydrase

Column: Poroshell 300SB-C18, 1.0 x 75 mm

NEW! AdvanceBio RP mAb Column for HPLC and UHPLC

Particle

• 3.5um SP

• 450 Å pore diameter

• 600bar pressure limit

• 2um frit to reduce clogging

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

3.5 um3.0 um

0.25 um

Phases

• C4

• SB-C8

• DiphenylThe most popular phases for proteins, plus a unique selectivity

Fast Intact mAb Analysis

April 10, 2015


31

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 mmMobile 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% BSample: 5 µL injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °CDetection: UV @ 254 nm

AdvanceBio RP-mAb C4, 450Å, 3.5 µm490 bar

Competitive C4, 300Å, 1.7 µm910 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)

April 10, 2015


32

Method Parameters

Column dimensions: 2.1 x 100 mmMobile phase A: 0.1% TFA in waterMobile 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% BSample: 1 µL injection of Fc/Fab, Papain Digested Humanized Recombinant Herceptin Variant IgG1 from Creative Biolabs (2 mg/mL)Temperature: 60 °CDetection: 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

Competitor C C4-30, 300Å, 2.6 µm

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

33

Using 1.0 % B/mL gradient

RemicadeRemicade clone

Using 2.1 % B/mL gradient

RemicadeRemicade 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

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 mmMobile 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% BSample: 5 µL injection of Humanized Recombinant Herceptin IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °CDetection: UV @ 254nm

April 10, 2015

34

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 C4AdvanceBio RP-mAb SB-C8AdvanceBio RP-mAb Diphenyl

* MPA = Mobile Phase A

Large fibrous proteins

Exploiting chemical stability

PLRP-S:

Features

pH 1-14

Extreme buffer concentrations

High temperature stability

Durable and Resilient

Inherently hydrophobic so does not require a bonded alkyl chain to confer hydrophobicity

Benefits

Acid and base cleanup

Typically < 8M

200°C

Long Lifetimes

Avoids typical silica problems of silanol group

Analysis of very large biomolecules or high speed separations1000A & 4000A pores

RP Summary

Agilent Column Positioning

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

fragments

AdvanceBio Peptide Mapping

• Fast, high resolution HPLC and UHPLC analysis of peptides

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

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

fragments

PLRP-S • Polymeric for high pH stability, alternate selectivity, and large pores

April 10, 2015

39

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

April 15, 2015

40

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-STPP, 3, 5, 8 um 100, 300, 1000, and 4000A

400 bar NA

Benefits

April 20, 2015


41

Pain Features and Advantages Benefit

Insufficient resolution

Superficially porous particles and sub2um fully porous particles increase diffusion rate, mass transfer, and efficiency for biomolecules

Improved confidence in analysis results (accuracy)

Long analysis timesAnalysis time can be decreased using fasterflow rates and shorter superficially porous & sub2um fully porous particle columns without loss in efficiency

Improved throughput - reduced costs

Short column lifetimeColumn with robust Poroshell packed bed and with 2 um frit decreases chances of bed-collapse or inlet blockage

Improved resource use - reduced costs

HPLC pressure limitations

Superficially porous particles maintain compatibility with all LC instruments System flexibility

HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY(HILIC)

April 10, 2015


42

Why are glycans important to profile? Bio-Therapeutics Are Glycosylated

� Changes in glycosylation may affect drug safety and efficacy.

Reducing Risk, Variation through controlling glycosylation

� The structural characterization and quantitative estimation of glycans is highly essential in biopharmaceutical projects.

FDA Classification & Regulations

� It is tremendously challenging to comprehensively characterize glycan profiles and determine the structures of glycans.

Early stages

Glycans

Fab

Fc

Monoclonal Antibody Therapeutic

Protein

Glycans

April 20, 2015


44

Principles of HILIC

(glycans)

• Glycans are very hydrophilic

• They may be neutral or posses charges that will interact with silica and bonded phase

• Column bonded phases will greatly impact the separation performance of glycans

April 20, 2015


45

Glycan Analysis – Common Challenges

• Very long analysis times

• Instrument limitations

• Difficulty achieving reproducible results

April 19, 2015


46

N-Glycan Mapping

Glycoprotein

N-Glycan

2-AB labelled glycan

HILIC column

LC/MS

HILIC column

LC/FLD (-MS)

Majority process

April 10, 2015


47

Glycan Analysis Workflow

April 10, 2015

48

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

April 10, 2015

49

Deglycosylation

Work-up

2-AB Labelling

Work-up

HILIC FLD / MS

Glycoprotein

N-Glycans

2-AB Labelled N-glycans

Unlabelled

Standards

Labelled

Standards

April 20, 2015


51

NEW! AdvanceBio Glycan Mapping Column for HPLC and UHPLC:

Glycan Analysis by LC-FLD

April 10, 2015


53

1260 Infinity Bio-inert HPLC

AdvanceBio Glycan Mapping,

2.7 um


2.7 um+


1.7 um+

1290 Infinity UHPLC

Superficially Porous Glycan Mapping Column –High Speed and Resolution with Less Backpressure

AdvanceBio glycan column delivers faster separations with 1.8 µm particles

April 20, 2015

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

LU

0

0.2

0.4

0.6

0.8

1

1.2

FLD1 A, Ex=260, Em=430 (GLYCANAMIDE_RRHD\GLYCANS_RRHD040.D)

16

.00

01

6.0

06

17

.00

5

18

.24

01

8.2

47

18

.56

7

18

.92

6

19

.55

0

20

.58

7

23

.66

1

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

LU

0

0.2

0.4

0.6

0.8

1

1.2

FLD1 A, Ex=260, Em=430 (GLYCANAMIDE_RRHD\GLYCANS_RRHD050.D)

9.6

12

10

.53

5

11

.16

9

11

.66

8

12

.01

7

12

.34

8

12

.98

4

13

.39

0

13

.96

6

14

.37

3

17

.07

6

Column RT (min) Rs 2,1 Rs 3,2 Rs 4,3 Rs 6,5 avg. PW (min) Peak Capacity

Waters BEH Glycan 20.2 1.77 1.94 3.39 2.10 0.1085 214

AdvanceBio Glycan 12.7 2.60 2.90 5.43 2.81 0.0741 221

Agilent AdvanceBio

Glycan Mapping, 1.8 um

Waters Acquity

UPLC BEH Glycan

1.7um

Time %B ml/min

0 80 0.50

25 60 0.50

26 0 0.50

27 80 0.50

0.50mL/min, 55° C

Inj = 2uL in 70/30 ACN/water

Mobile phase A: 100mM NH4 Formate pH 4.5,

Mobile phase B: ACN

FLD: Ex=260 nm, Em=430 nm

Sample: Agilent human IgG glycan library

2

1

4

3

5

6

2

1

4

3

5

6

Same condition:• 40% faster• Better Rs• Narrower peaks• Same elution order

Time %A %B Flow rate

(mL/min)

0 25 75 1.0

12 40 60 1.0

12.15 60 40 0.5

12.5 60 40 0.5

12.9 25 75 0.5

13.05 25 75 1.0

15 25 75 1.0

Column Rs 2,1 Rs 3,2 Rs 4,3 Rs 6,5 avg. PW (min) Peak Capacity

AdvanceBio Glycan

Mapping Column, 1.8um

1.63 1.70 3.05 2.09 0.059 135

Rapid Separation of 2-AB labeled N-linked Human IgG glycan Library (Agilent):2.1 x 150mm 1.8um AdvanceBio Glycan Mapping Column

1 2 3 4 5 6 min0 1 2 3 4 5 6 7 8

9

LU

0

0.2

0.4

0.6

0.8

1

0.3

45

0.3

63

1.9

65

2.0

93

2.4

76

2.8

40

2.9

92

3.3

04

3.4

60

3.6

24

3.7

70

3.9

23

4.4

11

4.6

36

5.2

14

5.6

80

6.1

67

6.4

80

7.3

82

7.8

19

7.9

91

2-AB labelling reagent

1

2

4

3

5

10

+

Separation< 10 min

Monoclonal Antibody Glycan Mapping

April 20, 2015

56

mobile phase:A: 100mM NH4 formate

pH 4.5: B: ACNInj.: 2ul 70:30 ACN: waterTemp: 55°CFLD: Excitation 260nm

Emission 430nm

Isomer

separation

HILIC Summary

Characterization Product Features Advantage Benefit Pain Point

Glycan MappingAdvanceBio

Glycan Mapping

1.8um FPP,bonded phase

High efficiency, right selectivity

Improved accuracy and

reproducibility of data – reliable results, cost

saving

Resolution2.7um SPP,

bonded phase

1.8um FPP,bonded phase Efficiency at

higher flow rate, fast gradients

Improved analysis efficiency, cost

savingThroughput

2.7um SPP, bonded phase

FPP: Fully Porous Particle, SPP: Superficially Porous Particle

April 19, 2015


57

SIZE EXCLUSION CHROMATOGRAPHY

April 10, 2015


58

April 20, 2015


59

Aggregation Studies

• ProteinsCaggregate!

• Protein aggregates can greatly enhance the immune responses to the protein of interest

• Protein aggregates can also enhance immune responses and may cause adverse event

• These responses may impact safety and efficacy

• Protein aggregates can be induced under forced conditions and can occur simultaneously during biopharmaceutical manufacturing

April 20, 2015

60

Manufacturing ProcessesSteps and Products• Fermentation• Purification• Formulation• Storage• Shipping• Administration

Stress Conditions• Heat• Freeze-thaw• Cross-linking• Protein concentration• Formulation change – pH, salt• Chemical modification• Mechanical stress / surface

Size Exclusion Process

61

Larger molecules spend less time in the pores and elute sooner.

Smaller molecules spend longer in the pores and elute later.

Mechanism of SEC Separation – Pore Size Determines Linear Separation Range

Some General Guidelines for SEC

1. When methods are to be validated, test for ruggedness with several different column lots, mobile phase preparations, and operators.

2. As a rule of thumb, SEC will only provide baseline separation of molecules with more than a 2 fold difference in MW.

3. Sample volume should be limited to below 5% of the total column volume. Max resolution .5 – 2% CV.

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

• Consistent and robust results

• High salt conditions puts excessive wear on instrument, parts

April 19, 2015


64

Pore Choice for Antibody Analysis

65

300Å

150Å

100Å

1

2

3

4

5

1. Dimer

2. Monomer

3. Monomer Fragment

4. Azide

5. Retained Molecule

Eluent: 50mM NaH2PO4 + 0.15M NaCl, pH6.8

Columns: Agilent Bio SEC-3 ,4.6x300mm

Flow: 0.35ml/min

Detector: UV@220nm

System: Agilent 1260 Infinity Bio-Inert LC

Sample: Mouse IgG

SEC Column Choice: Resolving Ranges

April 10, 2015


66

Size Exclusion Columns

• 5µm Particle

• 100Å, 150Å, 300Å, 500Å, 1000Å, 2000Å pore sizes

• High stability and long lifetime

• Great reproducibility

• Unique, 3µm particle

• 100Å, 150Å, 300Å pore sizes

• Highest resolution

• Highest efficiency

• Faster SEC separations

Fast, High resolution SEC characterization

Step 1: Improve Resolution

Technology: Small Particle SEC ColumnsResults: Higher Resolution SEC Separations

Use Higher Resolution to Achieve Faster Separations

69

Column: Agilent Bio SEC-3, 7.8 x150mm Sample: mAb (2mg/ml)Injection: 5ulFlow rate: 1.0, 1.5 and 2ml/min (56 bar , 75 bar, 105 bar)Eluent: 150mM sodium phosphate + 100mM Na-sulfateDetection: 220nm

Flow Rate Resolution

Monomer/Dimer

Monomer

Efficiency

Percentage

Dimer

1.0ml/min 1.53 3,510 0.64

1.5ml/min 1.43 2,502 0. 47

2.0ml/min 1.13 1,917 0.64

4 Minutes

monomer

dimer

2.0ml/min 1.5ml/min 1.0 ml/min

Increase flow rate and use a shorter column for faster separations.

Technology:Small Particle SEC Columns

Results:Faster SEC separations

Step 2 – Go Faster

Fast SEC – TB5990-8613EN

April 20, 2015


70

Examples of Additives to reduce non specific interactions

• 100-150mM NaCl

• 100-150mM NaSO4

• 5-10% AcN

• 5-10% MeOH

• 50-100mM urea or Guanidine

• .1% SDS

71

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

ResolutionHigh efficiency, right MW range

3um particles High efficiencyImproved analysis

efficiency, cost saving

Throughput

April 19, 2015


72

AFFINITYCHROMATOGRAPHY

April 20, 2015


73

AFFINITYCHROMATOGRAPHY

April 19, 2015


74

April 20, 2015


75

What is a Monolith – General Monolith Characteristics

• Continuous material with porous channels that connect to other channels

• Mass transfer is convective (not diffusive) therefore faster -important for biomolecules

• Low back pressure

• High flow rates

April 20, 2015


76

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

April 20, 2015


77

2 Minute Analysis – Antibody Titer from Cell Culture Supernatant

April 20, 2015


78

min0 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 (

Abso

rban

ce (

Abso

rban

ce (

Abso

rban

ce (m

AU

mA

Um

AU

mA

Uat

280 m

m)

at 2

80 m

m)

at 2

80 m

m)

at 2

80 m

m)

Time (min)

IgG1

Column Agilent Bio-Monolith Protein ASample: Cell lysate spiked with IgG1Equilibration buffer: 50 mM NaPO4, pH 7.4Elution buffer: 0.1 M citric acid, pH 2.8Flow Rate: 1.0 mL/minDetector: UV 280nmSystem: 1200 Infinity

April 20, 2015


79

ION-EXCHANGE CHROMATOGRAPHY

April 10, 2015


80

Charge variants – What we know?

• Surface charge changed mainly by post-translational modifications- Directly, as a change in the number of charged residues

- Indirectly as a chemical or physical alteration that changes surface-charge distribution

• May result during manufacturing or under storage conditions

• Originates from- Deamidation

- Oxidation

- Glycosylation or glycation

- Proteolytic degradation

- Amino acid substitutions

- Amino acid deletions

81

Ion Exchange ChromatographyCharge Isoform Analysis of Monoclonal Antibodies

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

Column: 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 µL, 5 mg/mL, mAb

(Acidic Isoforms)weakly bound

(Basic Isoforms)Strongly bound

Ion Exchange ChromatographySeparation based on charge

Some Guidelines for IEX

1.The General Rule for choosing a Bio IEX column- Acidic proteins: SAX or WAX- Basic proteins: SCX or WCX2. 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 charge3. 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-exchange4. If your pI is unknown- Start with pH 6 for cation-exchange- Start with pH 8.0 for anion-exchange

April 19, 2015


85

Ion-exchange sorbents

Weak ion-exchange sorbents have a narrower operating pH range than strong ion-exchange sorbents

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

April 19, 2015


87

Ion Exchange – Product Families

88

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

Bio-Monolith IEX Polymer Monolith QA, DEAE, SO3 N/A N/A BioMacromoleucles

Page 46

1. Non-porous particles for high efficiency analytical separations

2. Porous particles for scale up to purification

3. Monoliths for high speed separations

Ion Exchange Columns

• Non-porous PS/DVB particles

• Uniform polymeric coating and WCX layer, specifically designed for antibody separations

• Available in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes

• Non-porous PS/DVB particles

• Uniform polymeric coating with SCX, WCX, SAX, WAX layers, designed for protein and peptide separations

• Available in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes

• High surface area

• High capacity

89

SpecificationsParameter Agilent Bio IEX

Particle Size 1.7, 3, 5, and 10um

Binding Capacity > 100 ug protein on column

pH Range 2-12

Temp Limit 80°C

Hardware/Pressure 600 bar for SS

400 bar for PEEK

Pressure Limit 10um – 275 bar (4000 psi)

5um – 413 bar (6000 psi)

3um – 551 bar (8000 psi)

1.7um - 689 bar (10000 psi)

Recommended Flow Rate: 0.1 – 1 mL/min

Mobile phase Compatibility Acetonitrile/acetone/methanol mixtures

90

Optimum Flow Rate for NP3 and NP1.7

NP1.7

Proteins are not small molecules !

Proteins are sluggish !

Proteins are complex three dimensional structures !

Higher efficiency, higher pressure

NP3 NP5 NP10

Bio-WCX Working Flow Rates/ Pressures

Column Dimension Flow Rate Agilent Bio IEX

Bio-WCX NP10 4.6x250mm .8 ml/min 50-60bar

Bio-WCX NP5 4.6x250mm .8 ml/min 150-250bar

Bio-WCX NP 3 4.6x50mm .5 ml/min 80-120 bar

Bio-WCX NP 1.7 4.6x50mm .5 ml/min 110-320 bar

92

Reasons for High Column Pressure ?

93

�� CAUTION: Risk of blockage with NP1.7 is significantly higher.

Agilent Bio IEX ColumnsComparing Separations on Each Particle Size

April 10, 2015


94

Min0 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 mmBuffer A: 20 mM PBSBuffer B: A+1.0 M NaClGradient: 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 µmSample: 1) Ribonuclease A

2) Cytochrome C3) Lysozyme

Concentration: 1.0 mg/mLDetector: 280 nm

Average N ~80,000 for WCX 1.7 µmPeak N

Peak N

Fast, High Resolution IEX Separations

95

Bio WCX NP3, 4.6 x 50 mm Bio WCX NP1.7, 4.6 x 50 mm1.0 mL/min 1.0 mL/min

Technology Used: small particle columns (3um, non porous)Result: 5 minute, high resolution IEX

Standard Protein Separation: Bio WCX

96

Conventional HPLC (400 bar)

1. Ovalbumin pI 4.52. RNase A pI 9.43. Cytochrome C pI 9.84. Lysozyme pI 11

Gradient: 20mM sodium phosphate buffer, pH 6.50-800mM NaCl (0-20 mins)

Flow rate: 1.0mL/minSample: 10µl inj.Detection: UV, 220nm

Column: Bio WCX NP3 (4.6x50mm SS)

Column: Bio WCX NP5 (4.6x250mm SS)1

2

3

4

min0

2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

100

200

300

400

500

min0

2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

50

100

150

200

250

300

350

Change a 5um standard to 3um short column:• saves time • maintains resolution

Selectivity Comparison

April 20, 2015

97

- SCX

- WCX

- MAb

1

2

3

1

2

3

1

3

Columns: Agilent Bio SCX, NP 3, 4.6x50 mm, SSAgilent Bio WCX, NP 3, 4.6x50 mm, SSAgilent Bio MAb, NP 3, 4.6x50 mm, SS

Eluent A:10 mM NaH2PO4.2H2O, pH 5.75Eluent B: A + 1 M NaCl Gradient: 0 to 100% b in 25 minsFlow rate: 0.5 mL/minTemperature : AmbientDetector: UV at 220 nm

Sample: 1. Ribonuclease2. Cytochrome C3. Lysozyme

Sample concentration: 2 mg/mLInjection volume: 2 uL

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)

April 10, 2015


98

www.agilent.com/chem/getbioguidesApplication-focused Brochures

“How To” Guides

Keys for Enabling Optimum Peptide Characterizations: A Peptide Mapping “How To” Guide5991-2348ENProtein Identification

and Impurity Profiling using Reversed-Phase HPLC/UHPLC5991-0625EN

Resolve Protein Aggregates and Degradants with Speed and Confidence5991-2898EN

Reversed-Phase

Affinity

Ion-Exchange

Characterize Charged Variants of Proteins with Speed and Confidence5991-2449EN

Size Exclusion Chromatography for Biomolecule Analysis: A “How To” Guide5991-3651EN

Selection Guide

Your Reference Guide to the Analysis of Biopharmaceuticals and Biomolecules5990-9384EN

Ion-Exchange Chromatography for Biomolecule Analysis: a “How to” Guide5991-3775EN

Characterize Charged Variants of Proteins with Speed and Confidence5991-2449EN

Size Exclusion

Resources for More Information

April 20, 2015


99

BioHPLC Columns on the Agilent Website

April 20, 2015


100

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

NEED ASSISTANCE?

LC Column help desk1-800-227-9770

• orders• customer service(option 1,1)

• technical support• applications assistance(option 3,3,2)

101

[email protected]

Thank you !

Questions?

agilent technologies 1

Documents