iex-mals: applications in process developing and protein
TRANSCRIPT
IEX-MALS: Applications in process
developing and protein characterization
Mario Lebendiker
The Protein Purification Facility
Wolfson Centre for Applied Structural Biology
Hebrew University of Jerusalem
vP4EU meeting November 16th 2020
Size Exclusion Chromatography (SEC)
• A chromatographic method for protein separation by their size in the
native form
• Calculates Mw based on calibration curve – inaccurate in many cases
High mass• SEC-MALS: calculate the mass
of individual proteins in the
sample using light scattering
equations
High mass
Low mass
SEC-MALS • SEC-MALS is a powerful tool to study protein structure (shape and
mass), characterize oligomerization/aggregation and verify protein
purity. Percentage of glycosilations, nucleic acid, etc.
• A good column should be used for best separation of the sample for
achieving accurate results.
• Limitation: - needs relatively high concentration of a protein (low MW)
- presence of high oligomers can interfere with calculations
- resolution is very poor
- column separates only molecules with different size
- long equilibration time
• Choose appropriate fractionation range
• Increase column volume (Connect columns in tandem)
• Increase efficiency by reducing flow rate or smaller bead resin
• Reduce sample volume and/or protein quantity
• Change column supplier
• But any of this parameters are able to solve situations where
presence of higher order aggregates are interfering with
calculations for lower oligomeric species
SEC: Parameters to increase resolution
Schematic illustration of IEX-MALS method
• IEX chromatography separates molecules by charge. High resolution.
• Proteins with same mass but different charge elute differently.
• Elution is performed with salt (NaCl) or pH gradients.
IEX-MALS: calculate
the mass of
individual proteins in
the sample using
light scattering
equationsAmartely H. et al. Scientific Reports 2018
Mini DAWN TREOS Wyatt technology
Triple-angle MALS, 60mW Laser Mass up to 100,000 kDa
In line with refractive index detector:
measuring concentration in a universal way.
Comparing it to a known reference
(no need to aromatic residues)
The LS detector uses the UV signal from the AKTA Pure M - FPLC to
measure protein concentrations
Amartely H. et al. Scientific Reports 2018
We measure concentration according to UV and according to RI
This allows calculations of the molar masses and hydrodynamic radii for each peak eluted during chromatography
Moreover, we can extrapolate protein mass from the total mass of the molecule (glycoproteins, proteins inside micelles)
SEC-MALS and AIEX-MALS of BSA
Amartely H. et al. Scientific Reports 2018
IEX-MALS requires RI baseline subtraction
Amartely H. et al. Scientific Reports 2018
Mass calculations according to RI
requires RI baseline subtraction
• Shape and slope of the gradient or steps
• Flexibility of changing gradient shape and slope during the run
• Flow rate
• Different pH or pH gradient
• Different salts
• Use of additives (detergents, ligands, co-factors, etc)
• Temperature
• Column length (volume)
• Type of ligand: cation or anion – strong or weak - mixed mode
• Particle size of matrix (efficiency) and Degree of substitution (ligand)
• Supplier
• Injected volume: as much as you need (column capacity is very high)
• Equilibration takes minutes instead of hours as SEC
IEX: Parameters to increase resolution
SEC-MALS and AIEX-MALS of hoefavidinvariant
Orly Avraham - Department of Biological Chemistry – HUJI – Prof Oded Livnah
Amartely H. et al. Scientific Reports 2018
Low MW oligomers elutes before and allow calculations
Better resolution Huge optimization possibilities
Molar Mass 1
Molar Mass 2
LS
UV
RI
Results Fitting
volume (mL)
5.0 10.0 15.0
Mo
lar
Mass
(g
/mo
l)
1000.0
41.0x10
51.0x10
61.0x10
71.0x10
81.0x10
Other examples of SEC-MALS vs AIEX-MALS
Shimon Bershtein, BSU
UV 280nmUV 260nm UV 220nm
UV 280nmMALS
RI
NgMetK-SEC-MALS
Molar Mass 1
Molar Mass 3
Molar Mass 4
LS
UV
RI
Results Fitting
volume (mL)
30.0 35.0 40.0 45.0
Mo
lar
Mass
(g
/mo
l)
1000.0
41.0x10
51.0x10
61.0x10
71.0x10
81.0x10
91.0x10
101.0x10
UV 280nmUV 260nm UV 220nm
Conductivity%B
UV 280nmMALS
RI
NgMetK-AEIX-MALS
Molar Mass 1
Molar Mass 2
Molar Mass 3
LS
UV
RI
Results Fitting
volume (mL)
35.0 40.0 45.0
Mo
lar
Mass
(g
/mo
l)
1000.0
41.0x10
51.0x10
61.0x10
71.0x10
81.0x10
UV 280nmUV 260nm UV 220nm
Conductivity%B
UV 280nmMALS
RI
BsMetK-SEC-MALS BsMetK-AEIX-MALS
Molar Mass 1
Molar Mass 2
Molar Mass 3
LS
UV
RI
Results Fitting
volume (mL)
10.0 11.0 12.0 13.0 14.0 15.0 16.0
Mo
lar
Mass
(g
/mo
l)
41.0x10
51.0x10
61.0x10
71.0x10
UV 280nmUV 260nm UV 220nm
UV 280nmMALS
RI
Low MW oligomers elutes before and allow better calculations
SEC-MALS and AIEX-MALS of SARM1, octamer in crystal and in solution
Sporny M. et al. Journal Molecular Biology 2019 Opatowsky Y. lab in BIU
Results Fitting
Molar Mass 1 LS UV RI
volume (mL)
12.4 12.5 12.6 12.7 12.8
Mo
lar
Mass
(g
/mo
l)
45.0x10
46.0x10
47.0x10
48.0x10
49.0x10
51.0x10
Molar Mass 1
Molar Mass 2
LS
UV
RI
Results Fitting
volume (mL)
55.0 60.0 65.0
Mo
lar
Mass
(g
/mo
l)
41.0x10
51.0x10
61.0x10
71.0x10
UV 280nmUV 260nm UV 220nm
Conductivity%B
UV 280nmMALS
RIUV 280nmUV 260nm UV 220nm
UV 280nmMALS
RI
Mass according to UV peak 1 (30.87mS): 6.866×104
Mass according to UV peak 2 (33.48mS): 6.695×104
Mass according to UV 6.896×104
Same mass but different conformation
SEC-MALS and CIEX-MALS of HumiraTherapeutic protein
Separation of charge variants
Same mass but different charge
IEX-MALS of a peptide (predicted mass 2026 Da)
Amartely H. et al. Scientific Reports 2018
Samples with very low signal in
SEC-MALS, like peptides or
prone to aggregate proteins
(difficult to concentrate) can be
loaded in high quantities (no
volume restriction) in order to
get enough LS signal
Loading volume: no restrictionLoad as much protein as you need in order to get
good calculations
IEX-MALS of a detergent soluble peptide
Ariella Shalev MSc thesis (2020)Y. Arkin lab. (HUJI)
SEC-MALS
AEIX-MALS
Tetramer
IEX-MALS advantages for membrane proteins
• Separate free micelles from membrane proteins
(do not bind to column)
• Very useful for low MW membrane proteins
• Aggregates elutes after target or do not bind at all
• Good separation from correct oligomeric
conformations
• Purification
• Sample concentration
• Sample can be easily reload (after dilution to low salt)
• Detergent exchange is possible
IEX-MALS of a VLP VaccineProtein Molar Mass 1
Protein Molar Mass 2
Protein Molar Mass 3
LS
UV
RI
Results Fitting
volume (mL)
30.0 31.0 32.0 33.0 34.0 35.0
Mo
lar
Mass
(g
/mo
l)
59.0x10
61.0x10
62.0x10
63.0x10
64.0x10
Protein molar mass: 2.809×106 (±0.736%) daltonModifier molar mass: 2.142×106 (±1.846%) daltonTotal Molar mass: 4.955×106 (±0.735%) dalton
Protein molar mass: 3.522×106 (±0.566%) daltonModifier molar mass : 2.930×106 (±1.294%) daltonTotal Molar mass: 6.510×106 (±0.488%) dalton
Rh(Q)(avg) 14.209 (±0.187%)
SEC Column: X Bridge BEH from WatersResults Fitting
Protein Molar Mass 1 Protein Molar Mass 2 LS UV RI
volume (mL)
15.5 16.0 16.5 17.0 17.5
Mo
lar
Mass
(g
/mo
l)
71.0x10
81.0x10
UV 280nmMALS
RIMolar Mass
UV 280nmUV 260nm UV 220nm
UV 280nmUV 260nm UV 220nm
Conductivity%B
Mono Q - MALSPossible option to check vaccine QC
IEX-MALS of neg charge HMW Polysacharides
UV 280nmUV 220nm UV 215nm
Conductivity%B
BLANK
Results Fitting
Molar Mass 1 LS RI
volume (mL)
36.0 38.0 40.0 42.0 44.0 46.0
Mo
lar
Mass
(g
/mo
l)
1000.0
41.0x10
51.0x10
MALSRI
Mass (avg): 4.147×104 (±0.028%)
Sample: Sodium Alginate MW ~12,000 – 40,000 from SIGMA
Detect presence of non-protein molecules (without aromatic residues): polymers, micelles,
charge polysaccharides, others
Algin, is a polysaccharide distributed widely in the cell walls of brown algae Significant component of the biofilms produced by the bacterium Pseudomonas aeruginosa. Na Alginate is used in many industries including food, animal food, fertilizers, textile printing, dental and pharmaceuticals.
IEX-MALS of charge polysachridesMolar Mass vs Charge
LS RI
Mass
Na Alginate ~12-40kDa
CMC ~90kDa
CMC ~250kDa
CMC (carboxymethyl cellulose): Pure CMC used for food, pharmaceutical, and tooth-paste applications.
Characterized only by Rahman -FTRI
CMC 90000
CMC 250000
Na Alginate
UV 280nmMALS
RIMass
Differential weight distribution
IEX-MALS of charge polysachridesDifferential weight distribution
Na Alginate ~12-40kDa
CMC ~90kDa
CMC ~250kDa
IEX-MALS of charge polysachridesDifferential weight distribution vs Radius (nm)
For NaAlginate and the 90K CMC it seems that there is a significant fraction that has increased charge per unit molar mass, as you might expect, and then some highly-charged fraction that elutes later with a different dependence, perhaps due to a different molecular conformation.
For 250K CMC it appears that the main fraction is either very uniform or is not separating well, and it has the same highly-charged, late-eluting fraction.
Differential weight distributionvs Radius (nm)
CMC ~90kDa
CMC ~250kDa
Calculate charge per unit molar mass
IEX-MALS of charge polysachridesRadius (nm) vs Molar Mass in a short portion of
CMC 90,000
The central portion of the 90K CMC peak has a conformation that is clearly quite extended (slope of 1 in the conformation plot), which you would expect from a charged polymer
LS 2
UV
dRI
QELS
aRI
Strip Chart: 200706 Shai 5 2020 2MNaCl ULVAN MonoQMALS
volume (mL)
0.0 20.0 40.0 60.0 80.0
dete
cto
r vo
ltag
e (
V)
0.05
0.10
0.15
diffe
ren
tial re
fractiv
e in
dex (R
IU)
0.0
2.0x1080
4.0x1080
6.0x1080
8.0x1080
1.0x1081
IEX-MALS of Charge HMW Sulpho Polysacharides
UV 280nmUV 220nm UV 215nm
Conductivity%B
MALS280nmQUELS
Sample: Inject 0.5ml of 10mg/ml Algae extract - Gradient 0 – 2MNaCl - buffer pH 5.0
Detect presence of non-protein molecules (without aromatic residues)
polymers, micelles, charge polysaccharides, others
Rh(Q)(avg): 19.327 nm (±0.167%)
Method parameters SEC-MALS IEX-MALS
Principle of separation Hydrodynamic Size Charge
Parameters that increase
selectivity and resolution
RestrictedDifferent columns can be used with different
fractionation range, resin particle size, matrix
or different column length
VariedDifferent steps/gradient running programs, gradient slope, pH or
salt gradient, type of salts, type of buffer, resin particle size,
different matrixes, type of column (CIEX/AIEX) and column length
Injected volume Limited Unlimited
Sample concentration Concentrated sample Diluted or concentrated sample
Sample buffer As desired Conditions that allow binding
Equilibration time Long Short
Flexibility of changing
parameters during the run Not flexible Flexible
Conjugate analysis
(for modified proteins)
achieved by using RI signal
Easy to performMore laborious
RI signal changes during salt or pH gradients. Requires high sample
concentration
Analysis of low masses Not recommended Possible
Analysis of mixtures of
proteins with similar sizeNot recommended Recommended
Complexity of experiment Easy and intuitive Requires prior optimization or knowledge of conditions
Advantages and limitations of SEC-MALS vs. IEX-MALS
Conclusion I
• A new application of Multi Angle Light Scattering coupled to Ion Exchange chromatography
(IEX-MALS) for protein characterization
• The limited resolution of SEC restricts in some instances the accurate analysis that can be
accomplished by MALS
• IEX-MALS allows more precise analyses of samples that cannot be resolved by SEC-MALS
• Main advantages:
Different principle of separation (Analysis of mixtures of proteins with similar size)
Therefore it can be used for analysis of mix proteins samples (not only pure samples)
Huge variety of parameters that increase selectivity and resolution
Elution of high order oligomers after monomers, dimers, etc
Unlimited injection volume (Analysis of low masses, peptides, IDP, others)
Conclusion II
• We conclude that IEX-MALS is a valuable and complementary
method for protein characterization for Academy and Pharm:
therapeutic proteins, biosimilars, VLPs, AAV, vaccines, others
• Characterization of charge polysaccharides/polymers
• At Academic level can be use on-line during last purification steps
to quickly discriminate between our target to impurities, non-
protein impurities, and aggregates
• Possible use in Industry: online detection of aggregates elution at
the end of main peak
Kibutz Magal – Old Olive treesIsrael – February 2020
Hadar Amartely
Dan Some - Wyatt Technology
Danyel Biotech (GE in Israel)
All collaborators
Thank you!