66
Aggregation Insolubility Soluble aggregates Stickiness Main headache for protein purifiers
Aggregation
Insolubility
Soluble aggregates
Stickiness
Main headache for protein
purifiers
03072014 Superose12Analytic MBPAAshortNleD 48hrs concX3ish drop in conc001:10_UV1_280nm 03072014 Superose12Analytic MBPAAshortNleD 48hrs concX3ish drop in conc001:10_UV2_260nm 03072014 Superose12Analytic MBPAAshortNleD 48hrs concX3ish drop in conc001:10_UV3_220nm 03072014 Superose12Analytic MBPAAshortNleD 48hrs concX3ish drop in conc001:10_Fractions 03072014 Superose12Analytic MBPAAshortNleD 48hrs concX3ish drop in conc001:10_Inject
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01072014 MBP AA NleD short Superose 12 prep200ml001:10_UV1_280nm 01072014 MBP AA NleD short Superose 12 prep200ml001:10_UV2_260nm 01072014 MBP AA NleD short Superose 12 prep200ml001:10_UV3_220nm 01072014 MBP AA NleD short Superose 12 prep200ml001:10_Cond 01072014 MBP AA NleD short Superose 12 prep200ml001:10_Fractions 01072014 MBP AA NleD short Superose 12 prep200ml001:10_Inject
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01072014 MBP AA NleD short NiSeph4ml001:10_UV1_280nm 01072014 MBP AA NleD short NiSeph4ml001:10_UV2_260nm 01072014 MBP AA NleD short NiSeph4ml001:10_Conc 01072014 MBP AA NleD short NiSeph4ml001:10_Fractions 01072014 MBP AA NleD short NiSeph4ml001:10_Inject
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POOL 14-18
03072014 Superose12Anal MBPAAshortNleD 48hr no conc001:10_UV1_280nm 03072014 Superose12Anal MBPAAshortNleD 48hr no conc001:10_UV2_260nm 03072014 Superose12Anal MBPAAshortNleD 48hr no conc001:10_UV3_220nm 03072014 Superose12Anal MBPAAshortNleD 48hr no conc001:10_Fractions 03072014 Superose12Anal MBPAAshortNleD 48hr no conc001:10_Inject
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S P Unb W 4 16 18 28 1 11 16 17 18 19
Sup & Pellet & Frac 16 (Ni): half amount loaded.
~75
~25
~48
~35
~63
Soft aggregation: typical example
Ni column
SEC preparative Superose 12
SEC analyticalSame day
SEC analyticalAfter 48hr
Vinograd Nina/ Oded Livnah lab
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TEV protease production: buffer optimization
Capture Ni Column Polishing GF step
Increase lysis buffer volume
More DNase A in the lysis buffer
Increase [NaCl] from 0.1 to 0.5
Ronen Gabizon / Assaf Friedlerlab
Multiple pathways by which monomer protein in solution
can form aggregates
‘‘Large’’ aggregates (>10 –mer):
reversible if non-covalent
‘‘Very large’’ aggregates (50nm to
3000 nm): reversible if non-covalent
Visible particulates (‘‘snow’’):
probably irreversible
S. Webster (2013) PHARMACEUTICAL TECHNOLOGY Volume 37, Issue 11, pp. 42-48
• Solubility in a lysate extract & integrity according to PAGE-SDS, are very popular but naïve
markers of protein health. Since: is the protein correctly folded? Has the proper oligomeric
conformation? Is the protein active? Is the protein intact?
• What is the connection between insoluble expression as inclusion body and soluble
aggregates?
• Does “solubility during expression” guarantee solubility after purification and storage?
• Protein solubility and stability (in time), depends of proper conditions: buffer , concentration
• Correct conditions must be found for each protein
• Conformation and integrity can change with storage
Healthy protein: soluble, proper oligomeric conformation and more….
At what part of the process does aggregation start?
Do I have to deal with one, two or all the steps?
Expression
Cell lysis/sample
preparation/buffer
Purification
Concentration
Storage
Partial solutions can be found by optimizing each of these steps
Changing a single parameter at a time: time-consuming and often frustrating approach
How to avoid extensive and laborious screening
We need a strategy that integrates optimization of all these steps
A simple and modular toolbox
What is good expression?
Soluble or insoluble (IB) Expression
It is not enough!!!
What must be checked is the oligomerization state of the protein:Monomer – Soluble aggregate? / Folded – Unfolded?
There are soluble aggregates
Protein yield after first capture step (IMAC, etc)
It is also not enough!!!
Oligomerization state over time: Aggregation is time dependent!!!
8
Fusion proteins
Optimize purification procedure & purification strategy
Change expression system: insect, yeast, mammalian, in-vitro (RTS)
Refolding
Which domain? How many constructs to obtain
a soluble domain?
[IPTG], temperature, induction time, strain, etcChaperones: co-expression, heat shock, chemical chaperones
What can we do at the expression level?
Single target, or targets from the same familyPragmatism (HTS): random truncation
Rational domain designDomain changes
Limited proteolysis-based domain determination
MBP, GST, thioredoxin, NusA,SUMO, OthersProteins smaller than 40kDa will be attached to MBP,
whereas larger proteins will be attached to SUMO
Change expression conditions
Consider number of disulfide bridgesLarge of the protein
How to check correct folding??
• Aggregation is considered to be mediated by short “aggregation
prone” peptide segments
• Software tools for prediction of APRs: AGGRESCAN, PAGE, TANGO,
PASTA, SALSA, Zyggregator, and AMYLPRED
• PROSS (Protein Repair One-Stop Shop): algorithm to optimize
protein stability. Sarel Fleishman WIS http://pross.weizmann.ac.il/bin/steps
Aggregation prone regions (APRs) of proteins
http://pross.weizmann.ac.il/bin/steps
Pick host strain
Pick induction procedure
Test Purification conditions
Gel-filtration analysis
2 days
2-3 days
1-2 days
1-2 days
Quick expression strategy
Best host strain
with higher soluble yield
and lower IB
Best affinity binding
and lower soluble
aggregates!!!
Flowchart: Screening methodology for expressing prone-to-aggregate protein
Diagnose possible reasons for aggregation
Clone with 1-2 suitable fusion proteins containing His Tag
Screen expression conditions in auto-induced media at 25, 30 and 37°C
IMAC isolation of soluble protein: check by Coomassie staining and Western blot
Transform to 3-5 selected bacterial strains
Second round of optimization with selected strains
Scale-up selected conditions and analyze on IMAC and analytical GF
Intrinsic disorder provides several advantages:
1. Increased interaction surface area
2. Bind numerous partner ligands via the same region
3. Interaction may induce a folded conformation
4. Different ligands may induce different structures
Wright & Dyson J Mol Biol, 1999Tompa FEBS, 2005
Uversky & Dunker Science ,2008
The traditional paradigm:
“sequence → structure → function”
An updated paradigm should also include:
“sequence → disorder → function”
Van der Waals and hydrophobic attractions between side-chain and
backbone atoms
maximizing hydrogen bonding
minimizing steric clashes and energetically unfavorable bond
torsional angles
maximizing chain entropy
minimizing (maximizing) electrostatic repulsions (attractions)
minimizing unfavorable interactions between amino acids and the
solvent (water) and its co-solutes
Forces and interactions that drive foldingProtein Aggregation and Its Impact on Product Quality
Christopher J. Roberts - Curr Opin Biotechnol. 2014 December ; 0: 211–217. doi:10.1016/j.copbio.2014.08.001.
Mechanisms of Protein Aggregation Philo J.: Current Pharmac. Biotech. (2009),
10, 348-351 H.J. Lee et al. / Advanced Drug Delivery
Reviews (2011)
1) rapidly reversible non-covalent small oligomers
2) irreversible non-covalent oligomers
3) covalent oligomers(disulfide-linked)
4) Nucleation aggregation
5) Surface induce alterations
The initiation process of“non-native” aggregates
involves:
Two or more proteins “misfolding” together to a
“nuclei” (stable/irreversible)
Then grow via different mechanisms
Nucleate aggregation mechanism
Creation of nuclei as a consequence of other mechnism:
Adsorption of proteins to hydrophobic interfaces such as those between water and
air or silicone oil may promote unfolding and subsequent aggregation.
Adsorption to charged interfaces such water in contact with container materials
such as glass and stainless steel can also promote non-native aggregation
Molecular aggregates can ultimately phase separate as macroscopic particles
Nucleation-growth process
Temperature
Time Protein concentration
Buffer conditions
Reversible – Irreversible
Soluble – Insoluble
Folded – Unfolded
Covalent – Non-covalent
Aggregation: type and characteristics
Downstream aggregates are also generated via mechanical stress during filtration, high temperature holds, and excessive air-liquid or solid-liquid interfaces
H Doss et al. 2019 J. of Chromatography A -https://doi.org/10.1016/j.chroma.2019.03.044
Common types of mAbs aggregation mechanismsMechanistic and process-related drivers
Aggregation: major challenge in therapeutic protein production
The FDA generally recommends that MAb aggregates be reduced to
It is not currently possible to predict a priori when protein aggregation will occur for a given
protein, BUT, there a number of factors that influence whether and/or how quickly protein
solutions will aggregate.
The major factors include :
• solution conditions
pH,
salt concentration and which salt type,
amount and type of osmolytes
amphiphilic molecules such as surfactants;
• temperature
• pressure
• air-water interfaces
• bulk interfaces with water such as stainless steel
How can one control or mitigate aggregation?
Protein stabilizer factors
Aggregation inhibitors protein-protein interaction
reducers factors
Aggregation-prone region on thesurface of the native protein structure
Each category of additive will improve solubility of some proteins while decreasing the solubility of others
Osmolytes and kosmotropesenhance protein-protein
interactions and may increase aggregation
Chaotropes interact with aggregation-prone surface and improve solubility
+ OsmolytesOr Kosmotropes
+ Chaotropes
Chaotropes
Interacts preferentially with protein surfaces
Such interactions stabilize the exposed surface of the
protein, resulting in a decrease in the free energy of
unfolding and an increase in the free energy of aggregation
Partially unfolded protein structure exposes hydrophobic residues
Osmolytes and kosmotropeshide hydrophobic surface
and therefore may enhance folding
Chaotropes promote the unfolded (aggregation-
prone) state
+ OsmolytesOr Kosmotropes + Chaotropes
Kosmotropics
Decreased the rate of protein unfolding
from the native state
The stabilizing effects can be explained by their
preferential exclusion from protein surfaces, which
thermodynamically led to the reduction of protein
surface exposed to the solvent through unfavorable
interactions between protein surfaces and additives
Each category of additive will improve solubility of some
proteins while decreasing the solubility of others
Ideal environment Yamaguchi et al. Biotechnol. J. 2013, 8, 17–31
• Aggregation rate is decreased, and the native folded protein is stabilized
(rather than undergoing a reverse transition to intermediate states)
• Surfactants are most commonly used to protect proteins from shaking/shearing-
induced aggregation. They inhibit protein aggregation by competing with proteins
at hydrophobic surfaces.
• Polysorbate 20 and polysorbate 80 (most common) undergo auto-oxidation
yielding reactive peroxides, which cause degradation.
• Sugars protect proteins against dehydration by forming hydrogen bonds with the
protein (as water substitutes). This increase thermal unfolding temperature and
inhibits irreversible aggregation of protein molecules
• Stabilizers do not only stabilize proteins but simultaneously can enhance
aggregation. So, stabilizers can be used in combination with aggregation inhibitors
S. Kale and K. Akamanchi - Mol. Pharmaceutics 2016, 13, 4082−4093
Additive
Recommended Initial
Concentration
Recommended
Concentration Range
Sugars and Osmolytes
Glycerol 10% 0-40%
TMAO (trimethylamine N-oxide) 0.5M 0-1 M
Glucose 0.5M 0-2 M
Sucrose 0.5M 0-1 M
Trehalose 0.5M 0-1 M
Ethylene glycol 10% 0-60%
D-Sorbitol 0.5M 0.2-1M
Mannitol 2%
Xylitol 0.5M 0.2-1M
Glycine Betaine 1M
Amino acids and amino acid derivatives
Glycine 250mM 0.5-2%
Arginine L-HCl 125mM 0-2 M
Arginine Ethylester 250mM 0-500 mM
Proline 250mM 0-1 M
Potassium Glutamate 250mM 0-500 mM
Arginine L-HCl + L-glutamic acid (L-Glu) 50mM each
Non-ionic detergents
Nonidet P40 (NP40) or Triton X-100 0.01% 0-1%
Tween 80 or 20 0.1% 0-1%
DDM: n-Dodecyl β-D-maltoside 0.1% 0.01- 0.5%
Brij 56: Polyoxyethylene cetyl ether 0.05%
OG: Octyl glucoside (n-octyl-β-D-glucoside) 0.1% 0.01- 0.5%
Poloxamer 188 (BASF Pluronic® F68)
Zwiterionic detergents
NDSB: Non-detergent Sulfo Betaine 0.5M 0-1M
CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate
0.1% 0.01- 0.5%
Zwittergent 3-14 0.1% 0.001-0.2 %
LDAO: Lauryldimethylamine N-oxide 0.1% 0.01- 0.5%
Ionic detergents
CTAB: cetyltrimethylammonium bromide 0.5%
Sarkosyl : Sodium lauroyl sarcosinate 0.05% 0.01-0.5%
SDS: Sodium dodecyl sulfate Up to 0.1%
Table: Additives Used
to Stabilize Folding and to
Prevent AggregationSummary table of different
publications (De Bernardez Clark 1999, Voziyan 2000, Goloubinoff2001, Bondos 2003, Golovanov
2004, Hamada 2009, Shukla 2011, Churion 2012, Leibly 2012) and
from commercial websites (DILYX Biotechnologies OptiSol Protein
Solubility Screening Kit Application Manual, HAMPTON: Solubility &
Stability Screen)
http://www.dilyx.com/protein_solubility_screen_home2http://hamptonresearch.com/default.aspx
Mild chaotrope agents and chaotrope salts
Urea 0.5M 0-2M
Guanidine HCl 0.5M 0-2M
N-Methylurea 250mM up to 2.5M
N-Ethylurea 100mM up to 2M
N-Methylformamide 3-15%
NaI 0.2M 0-0.4 M
CaCl2 10-50 mM 0-0.2M
MgCl2 10-50 mM 0-0.2M
Mild and strong kosmotrope salts
NaCl (weak) 300mM 0-1 M
KCl (weak) 200mM 0-1 M
MgSO4 (strong) 100mM 0-0.4 M
(NH4)2SO4 (strong) 50mM 0-0.2M
Na2SO4 (strong) 500mM 0-0.2M
Cs2SO4 (strong) 50mM 0-0.2M
Potassium citrate 100mM
Citric Acid 50mM
Alcohols, Polyols, Polymers, Polyamines, and others
Ethanol 5-10% Up to 25%
n-Penthanol 1 to 10mM
n-Hexanol 0.1 to 10mM
Cyclohexanol 0.01 to 10mM
Polyethylene glycol (PEG 3350) 0.3-1.5% 0.1-0.4 g/L
Polyvinylpyrrolidone 40 (PVP40) 0.05-4%
Alpha-Cyclodextrin 8-40mM
Beta-Cyclodextrin 1-5mM
Putrescine, spermidine, and spermine 0.1M
Formamide 0.1%
Reducing Agents
β-mercaptoethanol (BME) 2 to 5mM 1 to 10mM
Dithiothreitol (DTT) 1mM 0.1 to 10mM
tris(2-carboxyethyl)phosphine (TCEP) 1 to 5mM 1 to 50mM
Table: Additives Used
to Stabilize Folding and to
Prevent AggregationSummary table of different
publications (De Bernardez Clark 1999, Voziyan 2000, Goloubinoff2001, Bondos 2003, Golovanov
2004, Hamada 2009, Shukla 2011, Churion 2012, Leibly 2012) and
from commercial websites (DILYX Biotechnologies OptiSol Protein
Solubility Screening Kit Application Manual, HAMPTON: Solubility &
Stability Screen)
http://en.wikipedia.org/wiki/TCEPhttp://www.dilyx.com/protein_solubility_screen_home2http://hamptonresearch.com/default.aspx
Flowchart: Screening methodology showing the best additives for purifying prone-to-aggregate proteins
Parallel bacterial lysis with different families of additives
Small-scale IMAC purification in the presence of different additives
Expand the screening of selected families of additives
Final optimization step: minimize the additive concentration Starting from the lysis
Starting from the wash & elution
Non-ionic detergent (0.5%)Zwittergent (0.5%) Osmolyte (0.5M) Urea or GuHCl (1M)Add 0.5M Arg or 50mM Glu/Arg
only in the elution bufferControl
Analysis of partially purified protein after overnight incubation at 4°C
Buffer with high salt (0.5M) + 10% glycerol +/- reducing agents
a) Supernatant b) pellet c) unbound wash to IMAC and d) eluted protein
Analytical SEC of the best conditions
Visual selection of non-turbid
samples
Short spin and PAGE-
SDS analysis
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Or use a combination of families
Best conditions during purification are not necessarily the same for
other steps like protein concentration or long term stability storage
Stable conditions after ON incubation: best monomeric / soluble aggregation ratio
Final Goal of the screening for best additives
Optimal IMAC conditions:least protein in the unbound fraction and
higher elution yield
Selection of optimal lysis conditions: least protein in pellets
Example of screening
methodology
Ronen Gabizon / Assaf Friedler lab
un
b
Guanidine
E1 E2 un
b
E2E1
Urea
un
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Tween
un
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E2E1Trehalose
un
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E2E1
L-Arg
Additives
Example of screening methodology: different additives
Additives
HMTV
HMTV
Ihab abd-Elrahman / Ben Yehuda Dina lab
un
bGuanidine-HCl
E1 E2
HMTV
HMTV
E1 E2 E1 E2
0.2 0.05 0.001
Tween20 0.05 0.005 0.0001
E1 E2 E1 E2 E1 E2un
bExample of screening methodology: additive concentration & time
Aggregation screening after 24hs 4C
HMTV
HMTV
0.2 0.05 0.001
Tween20
0.05 0.005 0.0001
P S P S P S
P S P S P S
Aggregation screening: additive concentration
Guanidine -HCl
MTVHMTV
• O.N Dialysis with 10mM imidazole and 0.02 M Guanidine and with TEV
After Negative Ni
Before TEV
Example of screening methodology: cut with TEV
1M Guanidine HCl 1M Urea 0.5% Tween 20 0.5% Zwitergent (3-14)
500mM L-ArgHCl 50mM L-ArgHCl + 50Mm GlutAcid Guy Mayer / Assaf Friedler lab
Example of screening methodology: different
additives. 1st optimization
Example of screening methodology: different additives. 2nd optimization
Guy Mayer / Assaf Friedler lab
Lysis, load, wash and elution in the presence of 1M GuHCl
Lysis & load in the presence of 1M GuHCl , wash & elution, add 50mM Arg & Glu after elution
HLT-VIP1 purification: Buffer screen
50mM MES pH 6.0 50mM Hepes pH 8.0
200mM 500mM 200mM 500mM
Elution buffer
no additives
Arg 500mM
Arg + Glu 50 + 50mM
Tween20 1%
Tween80 1%
Zwittergent 1%
Trehalose 1M
TMAO 1M
LDAO 0.1%
CHAPS 0.1%
NDSB-201 0.5M
Urea 1M
GndChl 1M
120102HLTVIP1Superose12prepEndwash001:1_UV1_280nm 120102HLTVIP1Superose12prepEndwash001:1_UV2_260nm 120102HLTVIP1Superose12prepEndwash001:1_UV3_220nm 120102HLTVIP1Superose12prepEndwash001:1_Fractions 120102HLTVIP1Superose12prepEndwash001:1_Inject 120102HLTVIP1Superose12prepEndwash001:1_Logbook
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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
Lysisbuffer50mM Hepes pH 8.0500mM NaCl10% glycerol1M urea
NiNTA Batch binding
Superose 12 prep SEC: same buffer without Urea
Michal Maes / Assaf Friedler lab
Lack of reducing agents
Ohad Solomon/ Assaf Friedler lab
Lysis and IMAC purification in buffer without reducing agents and 0.3M NaCl
Overnight dialysis with low conductivity (50mM NaCl) and AEIX in the presence of 1mM DTT
Lysis and IMAC purification in buffer with reducing agents and 0.3M NaCl.Add to peak 10mMDTT 50mMArg/Gluand load immediately on SEC in the presence of 1mM DTT & 0.3M NaCl
Integrase purification: Buffer screen
pHis2IN2mut(1-288)
Grow conditions: Rosetta ON IPTG indiction at 22C
Batch binding, and wash 20% buffer B
Buffers A IMAC: HEPES 20 mM pH7.5, 1M NaCl, 10%
glycerol, 25 mM Imidazole, 2 mM βME (added fresh)
B: A + 500 mM Imidazole
Add 2mM EDTA to avoid cloudiness
Concentrate and add TEV protease 1:10 because of
the high NaCl under dial buffer A + 50mM Imid
Negative IMAC
Gel filtration buffer: HEPES 20 mM pH 7.5, 1 M NaCl,
10% glycerol, 0.1 mM EDTA, 1 mM DTT
Concentrate and aliquot. Keep -80C
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260 nm
IN(1-288)
Tzvika Ayhuka and Ronen Gabizon / Assaf Friedler lab
Conclusion: Important factors to consider during purification of prone to aggregate proteins
Protein specific issues: additives, conductivity, buffer, others.
General Issues as:
Purification time and Low temperature
Protein concentration in each step (ratio lysis buffer / cell or
overloading column, etc)
Rational selection and arrangements of methods and columns
Prevention of mechanical or not mechanical stresses to the protein
such as: freezing / thawing
exposure to air
interactions with metal surfaces
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TEV protease production: buffer optimization
Capture Ni Column Polishing GF step
Increase lysis buffer volume
More DNase A in the lysis buffer
Increase [NaCl] from 0.1 to 0.5
Ronen Gabizon / Assaf Friedler lab
Strategy: Effect of concentration and time on protein aggregation
Sil5NiSeph4ml002:1_UV1_280nm Sil5NiSeph4ml002:1_UV2_260nm Sil5NiSeph4ml002:1_Conc Sil5NiSeph4ml002:1_Fractions Sil5NiSeph4ml002:1_Inject Sil5NiSeph4ml002:1_Logbook
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F3 F4 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19
pool 13-15 (12ml) was loaded on sephacryl S200
next time: longer steps
Sil6Superdex75prep001:1_UV1_280nm Sil6Superdex75prep001:1_UV2_260nm Sil6Superdex75prep001:1_Fractions Sil6Superdex75prep001:1_Inject Sil6Superdex75prep001:1_Logbook
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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
97ml = 160kDa
Next time: Superose 12 or Sephacryl S200
Sil6SephS200prep001:1_UV1_280nm Sil6SephS200prep001:1_UV2_260nm Sil6SephS200prep001:1_Fractions Sil6SephS200prep001:1_Logbook
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264ml =50kDa
Ni column after lysis. Buffers with glycerol and high [NaCl]
Sephacryl S200 100x2.6cm ~500ml column inmediately after Ni purification
Superdex 75 100x1.6cm ~200ml column after Ni purification and several hours of
concentration
Processing time is critical!!!!Protein concentration between
IMAC and GF sometimes is problematic
Michal Maes / Assaf Friedler lab
HLTiASPPprolRichSephacrylS200Prep001:1_UV1_280nm HLTiASPPprolRichSephacrylS200Prep001:1_UV2_260nm HLTiASPPprolRichSephacrylS200Prep001:1_Inject HLTiASPPprolRichSephacrylS200Prep001:1_Logbook
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HLTiASPPprolRichSephacrylS200Prep001:1_UV1_280nm HLTiASPPprolRichSephacrylS200Prep001:1_UV2_260nm HLTiASPPprolRichSephacrylS200Prep001:1_UV3_220nm HLTiASPPprolRichSephacrylS200Prep001:1_Logbook
-50
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HLTiASPPAnkSH3NiSepharFF16ml001:1_UV1_280nm HLTiASPPAnkSH3NiSepharFF16ml001:1_UV2_260nm HLTiASPPAnkSH3NiSepharFF16ml001:1_Conc HLTiASPPAnkSH3NiSepharFF16ml001:1_Inject HLTiASPPAnkSH3NiSepharFF16ml001:1_Logbook
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Two big changes:
1) Use X 2 more IMAC resin
2) Separate aggregates immediately after IMAC
Strategy: Avoid column overloading & speed process
IMAC captureSephacryl S200 100x2.6cm ~500ml
For prone to aggregation proteins sometimes is better to avoid column overloading
Michal Maes / Assaf Friedler lab
DefrozeniASSPAnkSH3Superose12 Anal002:1_UV1_280nm DefrozeniASSPAnkSH3Superose12 Anal002:1_UV2_260nm DefrozeniASSPAnkSH3Superose12 Anal002:1_UV3_220nm DefrozeniASSPAnkSH3Superose12 Anal002:1_Fractions DefrozeniASSPAnkSH3Superose12 Anal002:1_Inject DefrozeniASSPAnkSH3Superose12 Anal002:1_Logbook
-5.0
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25.0
mAU
6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Waste
GF of Defrozen iASSP Ank SH3
Strategy: Should TEV protease be cleavage before or after GF separation of soluble aggregates?
Try to eliminate your soluble aggregates as soon as possible!!!
Only monomer undergoes TEV protease cleavage(aggregates can not be cleaved)
HLTiASPPAnkSH3Superose12Prep001:1_UV1_280nm HLTiASPPAnkSH3Superose12Prep001:1_Inject HLTiASPPAnkSH3Superose12PrepDelay002:1_UV1_280nm HLTiASPPAnkSH3Superose12Prep001:1_Logbook
0
20
40
60
80
100
mAU
60 80 100 120 140 ml
Load inmediately after affinity
Load 12hours after affinity
After TEV cleavage, monomers are stable
Load immediately after Ni affinity
Load 12h after Ni affinity
+-
Anat Iosub / Assaf Friedler lab
Column selection: develop the best strategy to avoid further aggregation
Ni column
TEV protease + dialysis
Negative Ni column
GF
Concentration
Ni column
GF immediately
TEV protease
Negative Ni column
and/or Ion exchange
REVIEW Nature Methods - 5, 135 - 146 (2008)Protein production and purification
Structural Genomics Consortium
chelating2mlAir Imp7Beta001 18 12 12001:1_UV1_280nm chelating2mlAir Imp7Beta001 18 12 12001:1_UV2_260nm chelating2mlAir Imp7Beta001 18 12 12001:1_Conc chelating2mlAir Imp7Beta001 18 12 12001:1_Fractions chelating2mlAir Imp7Beta001 18 12 12001:1_Logbook
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%B
120.0 130.0 140.0 150.0 160.0 170.0 180.0 190.0 ml
F4 1 F2 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3132
Fractions 16-22 Peak-833 mAU
100%B
20%B
15%B
Chelating2mlAir, Imp7598 ImpBeta1-442 from 1L 17C ON 19.12.12
Imp7BetaSuperose12prepar200ml 18 12 12001:1_UV1_280nm Imp7BetaSuperose12prepar200ml 18 12 12001:1_UV2_260nm Imp7BetaSuperose12prepar200ml 18 12 12001:1_Fractions Imp7BetaSuperose12prepar200ml 18 12 12001:1_Inject Imp7BetaSuperose12prepar200ml 18 12 12001:1_Logbook
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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
Extra ImpBeta Peak - 106ml
Complex Peak - 87ml
Aggregate Peak - 65ml
Superose12prepar200ml Imp7 598 ImpBeta1-442 + Tween20 0.01% after Ni 20/12/12 Imp7BetaSuperdex75prep320ml 25 04 12001:1_UV1_280nm Imp7BetaSuperdex75prep320ml 25 04 12001:1_UV2_260nm Imp7BetaSuperdex75prep320ml 25 04 12001:1_Fractions Imp7BetaSuperdex75prep320ml 25 04 12001:1_Logbook
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80 100 120 140 160 180 200 ml
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
Peak2: 182 ml
Peak1: 137 mlPeak agregate: 115 ml
Imp7 (598-C) + Imp Beta (1-442) after Ni and conc - Superdex75prep320ml 25.04.12
Optimization of Complex Formation
Co-Purification + Buffer optimization
Capture: IMAC column
Superose 12 GF
Komornik Nadav / Oded Livnah lab
Conclusions
A simple and modular toolbox for designing a minimized
expression and purification screen
Protein production in E.coli-based systems following these
hierarchic rules is more efficient, simpler, and less costly
Minimized hierarchical screening platform
Combination of most beneficial approaches:
rational domain design possible fusion partners
suitable bacterial hosts optimal induction conditions
Conclusions
Main goal at the expression level is to obtain maximal yield of the correct
oligomeric conformation along with the minimal presence of insoluble proteins,
or soluble aggregates, in the shortest possible time.
Selection of optimal lysis conditions: the least protein in pellets
Reduce screen to one additive of each family
Optimal IMAC conditions: least protein in the unbound fraction and a higher
elution yield
Best monomeric/soluble aggregation ratio after overnight incubation
Consider and optimize all the factors that can affect solubility during the
processing before scale-up: chromatography, concentration and storage
It looks difficult!!!But it works….
Review: Production of prone-to-aggregate proteins
Mario Lebendiker , Tsafi Danieli
FEBS Lett. (2014), http://dx.doi.org/10.1016/j.febslet.2013.10.044
Chapter: A screening methodology for purifying proteins with aggregation
problems
Mario Lebendiker, Michal Maess and Assaf Friedler (2015)
Methods in Molecular Biology (Springer)"Insoluble Proteins“ Vol.1258, pp 261
http://dx.doi.org/10.1016/j.febslet.2013.10.044
Monodispersity, aggregation state
SEC
SEC-MALS
Native PAGE
Ultrafiltration assays
Dynamic Light Scattering
Analytical Ultracentrifugation
How to check aggregation and stability
Biological and/or specific activity is extremely important, but, it does not always reflect all the picture
Stability
Differential Scanning
Calorimetry
Circular Dichroism
Termal Shift Assay
Dynamic Light Scattering
DSF: Differential Scanning Fluorimetry
Screens in 24 or 96 wells (or microarrays), covering a range of conditions to be
tested (pH, salts, additives)
Protein is mixed with each solution and then screened visually, under a
microscope or by different analytical methods
Methods for Screening Solubility Conditions
PAGE-SDS
Western blot
Intrinsic protein fluorescence
Visual screen: from clear soluble drop to light precipitation, heavy
aggregation, phase separation (like oil in water), or small crystals
Light Scattering
Biological Activity
Others Circular Dichroism (CD)
Differential Scanning CalorimetryStability
Self-Interaction Chromatography (SIC) HSC Technologies Soluble Therapeutics
Native PAGE
Spectrophotometer
Extrinsic dye fluorescence
Monitor over time or different temperatures or with chemicals (H2O2, urea, etc)
Native PAGE
Like regular SDS-PAGE, but samples are not denatured
Proteins separate based on size and shape
Aggregation appears as streaks instead of a discrete band
Not easy. Not always work. Artifacts
Thermal Shift Assay
• Can be high-throughput
• Monitor by light scattering or fluorescence
• Fluorescence: – Upon denaturation, dye binds
exposed hydrophobic portions of the protein
– Can use real time PCR machine
• Can be adapted to search for ligands that bind
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/real-time-pcr/real-time-pcr-applications/real-time-pcr-protein-analysis/protein-thermal-shift.html
A Novel Protein Aggregation Assay for Biologics Formulation Studies and Production
ENZO-BioTek: QA/QC - ProteoStat® Fluorescent Reagent for Microplate-based
Aggregate Quantitation
The filter assay detects both soluble and insoluble aggregates
1. Dilute protein
1:5 or 1:10 into
test buffer, final
volume ~100 µl.
2. Incubate at
room tempera-
ture 1 hour.
3. Apply protein
to top of ultrafilter
unit, spin at 16,000g
for 15 minutes.
4. Resuspend re-
tained aggregates
in 30 µl H2O.
5. Detect protein
in filtrate (Sol) and
retentate (Agg) by
SDS-PAGE.
Bondos & Bicknell (2003) Anal. Biochem. 316 223-231
Churion & Bondos (2011) Methods Mol. Biol. 896, 415-427
OptiSol™ Protein Solubility Screening Kit
• Filtration assay from Dilyx Biotechnologies
Optim 1000 from Avacta
• Uses an instrument that combines, the powerful analytical capabilities of fluorescence and static light scattering technologies
• High throughput
• Measures unfolding temperature and aggregation temperature
• When laser light hits a macromolecule, the oscillating electric field of the light induces an oscillating dipole that re-radiates light.
• The intensity of the radiated light depends on the magnitude of the dipole.
• The intensity of the scattered light is proportional to the macromolecule concentration.
• When monomers form a dimer, the intensity of the scattered light increases.
• By increasing the mass, while keeping the concentration the same, the intensity of the scattered light increases.
MALS principles(Multi Angle Light Scattering)
55
LS- intensity of scattered light
c – concentration
K – a constant for a specific solute in a solution
Static & Dynamic Light Scattering (LS and DLS) non-invasive measurement for mass and radius
Light hitting small (
Hydrodynamic radius vs Mw Sabine Supman / Max Plank / P4EU course
PeakRadius (nm)
Mw-R (kDa) %Pd %Intensity %Mass %Number
Peak 1 (True) 8.5 509.2 19.3 21.7 39.7 100.0
Peak 2 (True) 45.3 25296.0 22.9 55.6 1.4 0.0
Peak 3 (True) 5546.2
1938105428.8 28.0 22.7 58.9 0.0
MBP: highly concentrated sample frozen , unfiltered in 50% glycerol
DLS of fresh vs frozen sample Sabine Supman / Max Plank / P4EU course
Radius (nm) %Pd Mw-R (kDa) %Intensity %Mass
3.9 18.9 78.9 4.0 91.5
25.0 49.0 6309.9 96.0 8.5
MBP: highly concentrated sample before freezing
Size Exclusion Chromatography (SEC)Size, oligomeric conformation, aggregations, & purity.
Particles separated by size
Smaller particles trap in beads and elute later
Larger particles avoid beads and elute first
Mini analytical columns for fast results
Resolution to discriminate monomers from oligomers
Underestimation of aggregates due to Removal of aggregates by frits and column filtration
Dilution of sample and thus potential dissociation of reversibly aggregated species
Non-specific interaction of unfolded, exposed hydrophobic surfaces with the matrix
Stickyness of properly folded proteins (e.g ß-sheet)
01072014 MBP AA NleD short Superose 12 prep200ml001:10_UV1_280nm 01072014 MBP AA NleD short Superose 12 prep200ml001:10_UV2_260nm 01072014 MBP AA NleD short Superose 12 prep200ml001:10_UV3_220nm 01072014 MBP AA NleD short Superose 12 prep200ml001:10_Cond 01072014 MBP AA NleD short Superose 12 prep200ml001:10_Fractions 01072014 MBP AA NleD short Superose 12 prep200ml001:10_Inject
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F3 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
HLTp53CTSephacrylS100of500ml004:1_UV1_280nm HLTp53CTSephacrylS100of500ml004:1_UV2_260nm HLTp53CTSephacrylS100of500ml004:1_Fractions HLTp53CTSephacrylS100of500ml004:1_Inject HLTp53CTSephacrylS100of500ml004:1_Logbook
0
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mAU
150 200 250 300 350 400 ml
F3 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 41
Fractions around 23 and 32 are higher MW impurities
POOL 7-14 Difficult HTS!!Reliable QC for low to relatively high MW aggregates
DSF: Differential Scanning FluorimetryNanoTemper
Method for measuring ultra-high resolution protein stability using
intrinsic tryptophan or tyrosin fluorescence.
Applications include antibody engineering, membrane protein research,
formulation and quality control.
Protein solutions can be analyzed independent of buffer compositions
(concentration range from more than 200 mg/ml down to 5 µg/ml)
Methods to quantify the structural stability of a
protein: thermal and chemical unfolding
Thermal unfolding experiments use a constantly
increasing temperature to monitor protein
conformational changes over time
Chemical unfolding experiments employ
denaturing agents to unfold proteins
Thermal unfolding occurs over a narrow
temperature range
Transition from folded to unfolded - referred to
as 'melting temperature' or 'Tm' - serves as a
measure for protein stability
DSF: Differential Scanning FluorimetryNanoTemper
DSF: Differential Scanning FluorimetryNanoTemper
Fluorescence of the tryptophans (and
tyrosins) in a protein is strongly dependent on
their close surroundings
Changes in protein structure affect both the
intensity and the emission wavelength of Trp
fluorescence
Tm is calculated from the
changes in tryptophan
fluorescence intensity, or from
the ratio of tryptophan
emission at 330 and 350 nm
A) Establishing a protein unfolding standard
B) Long-term storage of membrane protein
C) Forced-degradation stress-test on MEK1
A) Unfolded IgG at different concentrations was mixed with folded IgG and subject to thermal unfolding. The percentage of unfolded IgG in the solution was quantified based on the F350/F330 ratio meassured at 25 °C.
B) Membrane protein HiTeha were stored at 4 °C and at RT, respectively, and thermal unfolding curves were measured over a time period of 34 days. %unfolded protein was calculated based on the F350/F330 ratio
C) MEK1 protein was subject to the indicated stresses, and the fraction of unfolded protein was calculated based on the F350/F330 ratio at 25 °C
Exploring Protein Stability by nanoDSF - Prometheus NT.48
Melanie Maschberger et al. 2015 - NanoTemper Technologies GmbH, Munich, Germany
Type of Stressor Example for Experimental Stress
Elevated Temperature Incubate 24 hours at 37°C
Long-Term Storage Store 2 weeks at room temperature
Ice/Liquid Transition Freeze and thaw 20 times
Shear Force Force material 20 x through narrow syringe need
Intense Light Expose samples to direct sunlight or UV light for 1 h
Chemical Compatibility Add 10 mM of caustic reagent (i.e. heavy metal)
Surface Exposure Add 5 uL of 10 um diameter glass beads
Air Oxidation Bubble 10 ml of air through sample
Chemical Oxidation Add Hydrogen Peroxide
Experimental stressesOptiSol™ - Protein Solubility Screening Kit Application Manual http://www.dilyx.com/protein_solubility_screen_home2
http://www.dilyx.com/protein_solubility_screen_home2
rAlbumin (15 mg/ml)
Glycine (20 mg/ml)
PEG 400 (1 mg/ml)
Ethylene glycol (10%)
Polysorbate 80 - Tween-80 (0.82 mg/ml to 8.2 mg/ml) oxidation problems
Arginine (400mM)
Histidine (400mM)
Mannitol (10%)
Trehalose (500mM)
TMAO (1.0M)
Arg/Glu: each amino acid is 50mM
Excipients applied in the industry to enhance protein stability or avoid aggregation
Potential Stabilizers of Influenza Virus-Like ParticlesAggregation
Kissmann J. et al. Journal of Pharmaceutical Sciences, Vol. X, 1–12 (2010)
Ascorbic acid 0.15 M
Aspartic acid 0.075 M
Lactic acid 0.15 M
Malic acid 0.15 M
Arginine 0.3 M
Diethanolamine 0.3 M
Guanidine HCl 0.3 M
Histidine 0.3 M
Lysine 0.3 M
Proline 0.3 M
Glycine 0.3 M
Brij 35 0.01%
Brij 35 0.05%
Brij 35 0.10%
Tween 20 0.01%
Tween 20 0.05%
Tween 20 0.10%
Tween 80 0.01%
Tween 80 0.05%
Tween 80 0.10%
Pluronic F-68 0.01% Pluronic F-68 0.05% Pluronic F-68 0.10%Albumin (human) 1% Albumin (human) 2.5%Albumin (human) 5% Gelatin (porcine) 2.5% Gelatin (porcine) 5% Lactose 10% Lactose 15%Lactose 20%Trehalose 10%Trehalose 15%Trehalose 20%Sucrose 10%Sucrose 20%
Dextrose 10%Dextrose 15%Dextrose 20% Mannitol 10%Sorbitol 10%Sorbitol 15%Sorbitol 20%Glycerol 5%Glycerol 10%Glycerol 15%Glycerol 20%α-Cyclodextrin 2.5%2-OH propyl β-CD 5%2-OH propyl β -CD 10%2-OH propyl γ-CD 5%2-OH propyl γ-CD 10%
