Lars Haneskog, PhD GE Healthcare Life Sciences · Lars Haneskog, PhD GE Healthcare Life Sciences. 2 Protein aggregation ... Additive Proposed mode of action Glycerol (5 - 40%)

Protein aggregationDetect, prevent, remove

Lars Haneskog, PhDGE Healthcare Life Sciences

2Protein aggregation

9/24/2009

Contents

Introduction

Detect

Prevent- mechanisms for aggregate formation- preventive actions and condition screening

Remove


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Protein aggregates can be…

• covalent (caused by S-S bridges) or non-covalent

• irreversible or reversible

• large (>100µm) or small (oligomeric; ~20nm)

• assembled from non-native or native protein

”There is no single protein aggregation pathway but a variety of pathwayswhich may differ between proteins and may result in different end states.”

Mahler, H.-C. et al. (2008) J. Pharm. Sci. Wiley InterScience DOI 10.1002/jps.21566


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When does aggregation occur?

Expression

Chromatographicpurification

Storage

End application

Samplepreparation

Inclusion body formation

Proteins stuck on cell debris

Mechanical stress

Low pH

Freeze-thawing

High/low ionic strength

High protein concentrations


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Introduction

Detect


Remove


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Techniques for aggregate detection

10nm 100nm 1µm 10µm 100µm 1mm

Native gel electrophoresis

Dimers/oligomers

Gel filtration

Absorbance

Visual


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0

0,5

1

Absorbance

Optical density measurement at a wavelength where the protein does not absorb (e.g., 340, 490 or 600 nm)

OD

490

pHAdditive

Additive screening in microtiter plate


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6%24%70%

Native gel electrophoresis

• Mobility depends on protein charge and hydrodynamic size

• Straightforward to try: Omit SDS and reducing agent from a standard SDS-PAGE protocol

• May require optimization

Sample: Purified IgG Mab, 90µgGel: 4-12% Tris-glycineStaining: Deep PurpleAnalysis: ImageQuantTM TL


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Rapid gel filtration

SuperdexTM 200 5/150 GLSuperdex 75 5/150 GL

• Sufficient resolution for detection of dimers and oligomers

• Separation time only ~10 min

• Sample volumes 4 – 50 µl


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Rapid gel filtration for semi-quantitativeaggregate detectionSample: Purified IgG MabColumn: SuperdexTM 200 5/150 GLSystem: ÄKTAexplorerTM 10Sample volume: 50 µlSeparation time: 10 min

A280


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Gel filtration and multi-angle laser light scattering (GF-MALLS)

• Light scattering (LS) measurements allow direct calculationof the average molecular weight

• Gel filtration separates the sample into size homogeneousfractions. The molecular weight can then accurately be determined by LS

• Read-out is proportional to the mass: highly sensitive to aggregates


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Degassernoise reduction

Buffer filternoise reduction

A/D 900 converterimports the scattering signal into Unicorn™

Wyatt miniDAWN* Treos*

3 angle light scattering detector

Wyatt Optilab rEX*

refractometer

GF-MALLS with ÄKTAmicro™ and miniDAWN

*Trademarks owned by Wyatt Technology Corporation


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Mw determination of chymotrypsinogenusing GF-MALLS

• The UV monitor in ÄKTA™ can be used for determination of protein concentration, if the extinction coefficient, ε, is known

Mw dn/dc ε

Refractometer 25,300 0.185 n.a.

UV 25,220 n.a. 1906*

* Theoretical calculation, assuming all cys are half-cystins

Expected Mw: 25,755


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Analysis of ovalbumin aggregation

2x Superdex 200 5/150 GL

Peak 1: 107,600 (trimer/dimer)Peak 2: 42,710 (monomer)

(Mw from sequence is 42,750)

1

2 Gel filtration - light scattering

Gel filtration – A280Sample: Ovalbumin2x Superdex™ 200 5/150 GL


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Introduction

Detect


Remove


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Solvated phase Less solvated phaseAdsorptionCrystallisationAggregation

Change in:TemperatureSolventIonic strengthpH…


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Proposed mechanisms

Native protein

Largeraggregates

Partial unfolding Oligomers of non-native protein

Largeraggregates

Oligomers of native protein


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Protein aggregation can be minimized by

• stabilizing the native state

• reducing protein-protein interactions

• avoiding

- mechanical stress- repeated freeze-thawing- high protein concentrations


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Protein stability assessment- differential scanning calorimetry (DSC)

Measures the temperature associated with a conformational change of a protein

Tm is the thermal transition midpoint:50% native / 50% unfolded

Tm is an indicator of stability


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A comparison of protein stability windowsobtained by DSC and gel filtration

Gel filtration(aggregation)

DSC(Tm)


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Additives proposed to stabilize the native state

Hydration shell is strengthened by kosmotropes(”water structure makers”)

Glycerol SucroseSorbitol Glycine


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Additives proposed to reduce protein-protein interactions

Potential protein-proteininteraction sites are shielded

+-

Hydration shell is weakenedby chaotropes (”water structure breakers”)

Polyethylene glycolNonionic detergents

Citrate

UreaArginine


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Commonly used additives to minimizeprotein aggregation

Additive Proposed mode of action

Glycerol (5 - 40%)Sucrose (10 - 40%)Glycine (0.02 – 0.5 M)Sorbitol (5 - 40%)

Polyethylene glycol (1 - 15%)Nonionic detergents (below cmc)

Citrate (0.02 - 0.4 M) Shields surface exposed charged sites(reduces protein-protein interactions)

Urea (< 2 M)Arginine (< 2 M)

Reduces protein-protein interactions

Dithiothreitol, DTT (0.1 – 1 mM) Prevents formation of intermolecular S-S bonds

Stabilizes native, intramolecular protein interactions

Shields surface exposed hydrophobic sites(reduces protein-protein interactions)


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Screening of elution conditions for antibodypurification

Superdex™ 200 5/150 GL

PreDictor™ MabSelect SuRe™, 20 µl

Fractions collected into neutral buffer

IgG Mab in cell culture supernatant

Elution (different conditions)

Aggregate analysis (gel filtration)

Adsorption to protein A (multiplexed)

Recovery(A280)


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Elution condition screening – parameter space

Buffersubstance

Acetate

Citrate

AdditiveArginine Sucrose Urea

pH3 -

5

NaCl concentration0.1 – 0.5 M


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Elution condition screening:IgG recovery versus monomer content

0.1 M NaCl0.3 M NaCl0.5 M NaCl

0

20

40

60

80

100

3 3,5 4 4,5 5pH

Recovery

Monomer content%

Sample: IgG Mab, eluted from MabSelect SuRe™ under different conditionsAnalysis: A280 (for recovery)

Superdex™ 200 5/150 GL (for monomer content)


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Elution condition screening:Effect of additives

pH

0

5

10

15

20

25

3 3,5 4 4,5 5

% aggregates1 M arginine1 M urea0.1 M glycineReference (20mM citrate)0.1 M sucrose

Sample: IgG Mab, eluted from MabSelect SuRe™ under different conditionsAnalysis: Superdex™ 200 5/150 GL


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Screening of storage conditions

PD MultiTrap™ G-25

Superdex™ 200 5/150 GL

Purified monoclonal antibody

Storage

Absorbance measurement

Buffer exchange in 96 well plate

Gel filtration

(if low A490)


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Results

Sample: MabX, filteredColumn: SuperdexTM 200 5/150 GLSystem: ÄKTAexplorerTM 10Buffer: 50 mM Sodium phosphate,

150 mM NaCl, pH 7.5Flow rate: 0.3 ml/min

0

50

100

150

200

A215(mAU)

0.0 0.5 1.0 1.5 2.00

50

100

150

200

A215(mAU)

0.0 0.5 1.0 1.5 2.0 ml

Day 1Day 10

0.2 M glycine

0

50

100

150

200

A215(mAU)

0.0 0.5 1.0 1.5 2.00

50

100

150

200

A215(mAU)

0.0 0.5 1.0 1.5 2.0 ml

Day 1Day 10

2 M arginine

0

50

100

150

200

A215(mAU )

0.0 0.5 1.0 1.5 2.0 ml0

50

100

150

200

A215(mAU )

0.0 0.5 1.0 1.5 2.0 ml

Day 1Day 10

0.25 M sorbitol


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Introduction

Detect


Remove


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Filtration

• Removal of insoluble aggregates

• Low protein adsorption: e.g., cellulose acetate or polyvinylidene fluoride (PVDF) filters

• 0.22 µm nominal pore size


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Gel filtration

Sample: Purified IgG monoclonal antibodyColumn: SuperdexTM 200 10/300 GL

Monomer

Dimer

Larger aggregates

A280

0.15

0.10

0.05

00 5 10 15 20 25 ml


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Multimodal ion exchange chromatography

O O N+

OH

OH OH

O O N+

OH

OH OH

Process step Yield(%)

Dimers / aggregates

(%)

HCP(ppm)

Start material spiked with host cell protein (HCP)

100 130000

MabSelect SuReTM 95 0.7 55

CaptoTM adhere 95 < 0.1 7.5

IgG MabDimers,

aggregates


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%

36.86

46.04

57.19

IMACRemoval of aggregates of histidine tagged proteins

Sample: E. coli lysate with SLK kinase-his6Column: HisTrapTM HP, 1 ml

Sample: Fractions from HisTrapColumn: SuperdexTM 75 5/150 GL

00 0 5 1 0 15 20 25 l

00 0 5 1 0 15 20 25 l

Monomer

0 10 20 30 40 50V (ml)

A280


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Summary

DetectGel filtration for detection and quantitation of dimersand oligomers in the presence of large amounts of monomeric protein

PreventpH/salt/additive screen in multiplexed format to identifybest conditions

RemoveFiltration for insoluble aggregatesLiquid chromatography for soluble aggregates


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Documents

Lars Haneskog, PhD GE Healthcare Life Sciences · Lars Haneskog, PhD GE Healthcare Life Sciences. 2 Protein aggregation ... Additive Proposed mode of action Glycerol (5 - 40%)