Post on 17-May-2018
2Protein aggregation
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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
Prevent- mechanisms for aggregate formation- preventive actions and condition screening
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
Prevent- mechanisms for aggregate formation- preventive actions and condition screening
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
Prevent- mechanisms for aggregate formation- preventive actions and condition screening
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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