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!