1

Gel Filtration (GF)

Size Exclusion

Chromatography (SEC)

2

Gel Filtration chromatography (GF)

Principles of GF

Fractionation range

Parameters for resolution optimization

Use of GF: MW/oligomeric state – purification – buffer

exchange - QC

Examples - Troubleshooting

SEC-MALS

3

What is gel filtration?

• SEC is the most powerful chromatography technique for

obtaining reliable information about the size of

biomolecules under native conditions

• Separates molecules according to their hydrodynamic ratio

(size, conformation & oligomeric state)

• Different fractionation ranges: beads with pores of well-

defined sizes

• Mobile phase: almost all kind of buffers

4

Gel structure

AgaroseDextran

A hypothetical structure for Superdex

Different columns with beads of defined porosity: fractionation range

The degree of cross-linking determines the size of the pores and therefore the

fractionation range of the resin

SEC is not an adsorption technique (unlike all other chromatographic

procedures).

Void volume Vo

Volume of the gel matrix Vs

Pore volume Vi

How does it work?https://www.youtube.com/watch?v=oV5VB5kO3tQhttps://www.youtube.com/watch?v=E3z1wIImvHIhttps://www.youtube.com/watch?v=rPRbqYWlSEo

Smaller molecules spend longer in the

pores and elutes later

Larger molecules spend less in the pores and elutes

sooner

6

Terms and explanationsVo= Void volume: volume of the solution outside the beads, or elution from very large molecules

Ve = the volume from the time the protein is placed until it appears in the effluent

Vi = volume of the solution inside the beads = Vc - Vs - Vo

Vc = Total (geometric) volume of the column

Vt = Elution volume for very small molecules

2 3

Void volume Vo

Volume of the

gel matrix Vs

Pore volume Vi

1

Vo

Ve

Vt

Vc

7

Steric exclusion

Molecules are excluded from the gel bead to different extents according to their sizes.

Gel bead

Largest molecules - excluded from pores, travel with the mobile phase, elute rapidly from column• The volume at which large molecules elute is called the void volume, Vo (same as the volume of solution that surrounds the beads)

Smallest molecules – enter the pores of the beads, are included in the matrix and retarded in their movement, spend most of the time in the stationary phase, elute last• The volume at which small molecules elute corresponds to Vt (total volume of solution surrounding (Vo) and inside the beads, Vs) Vt = Vo + Vs

Intermediate size molecules – spend different amounts of time both inside and outside the beads (partition between the mobile and stationary phase)• The volume at which intermed.molecules elute is called the elution volume (Ve) and depends on the partition of the molecule between the Vo and Vs which is proportional to the distribution coefficient (K) Ve = Vo + KVs

8

Column calibration: MW extrapolation of unknown molecule

Kav 1

0log (Mr)

• Run MW standards and determine the elution volume for each (globular proteins)

• Calculate Kav values

• Plot log (Mr) for each standard against the calculated Kav

• Selectivity curve is usually moderately straight over the range Kav=0.1 to Kav=0.7

ot

o

VV

VVeavK

Extrapolate MW of globular proteins according to Ve

Limitations: protein is not globular; interaction with the resin

Denatured proteins

Kav 1

0.7

0.1

log (Mr)

Native proteins

Shape effects

Elution volume depends:

•Native, globular proteins

•Partially folded molecules

•Oligomeric state (monomer, dimer, trimer…soluble aggregate)

•Proteins inside detergent micelle: MW of protein + MW of micelle

10

How to choose GF type

Selectivity – fractionation range Kav 1

log (Mr)

Molecules with different shapes have different

selectivity curves

Linear polysaccharides

Globular proteins

RESIN FR Glob Prot FR Dextrans

Sephacryl S100 1-100 kDa ND

Sephacryl S200 5-250 kDa 1-80 kDa

Sephacryl S300 10-1500 kDa 2-400 kDa

Sephacryl S400 20-8000 kDa 10-2000 kDa

Protein 1: 30kDa

Protein 2: 80kDa

Vo Vt

11

Gel Filtration chromatography (GF)

Principles of GF

Fractionation range

Parameters for resolution optimization

Use of GF: Purification – buffer exchange – QC -

MW/oligomeric state – Protein/protein interaction

Examples - Troubleshooting

SEC-MALS

12

13

Efficiency

Efficiency depends on

Particle size of matrix (particle size distribution)

Packing quality of the column

Sample: volume, purity, concentration and viscosity

Flow rate (important only for big particle size)

Tubing diameter, tubing length and flow path volume

Use minimal tubing length

Shorter distance from injection valve to column

14

Peak width depends on particle size

Superdex Peptide 13-15 µm Superdex 30 prep grade 24-44

µm

0

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15

1 x Superdex® Peptide HR 10/3000 2 x Superdex® Peptide HR 10/30

Resolution depends on column length

Increasing column length increases resolution

0

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Superdex® Peptide 60 x 1.6cm ~ 120ml Superdex® Peptide 100 x 1.6cm ~ 200ml

SUMO-Atox1

SUMO-Atox1

SUMO

SUMO

Atox1

Atox1

Michal Shoshan from Edith Tshuva lab.

16

Column: Superdex® Peptide HR 10/30

Resolution depends on sample volume

25µl

0

0.02

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Retention volume (ml)200 µl

400 µl

00.010.020.030.040.050.060.07

0 10 20 30

Retention volume (ml)

00.020.040.060.080.1

0.12

0 10 20 30

Retention volume (ml)

17

Increasing resolution

Choose appropriate fractionation range

Increase column volume (Connect columns in tandem)

Reduce the flow rate

Change to a gel with smaller beads (higher efficiency)

Reduce the sample volume / protein quantity

Volume ~0.5-4% times the expected sample volume

Sample volume can be increase if resolution is OK

Check the column efficiency

Clean and/or re-pack

18

SEC Applications

Group separations: Desalting, Buffer exchange, Removing

reagents (replace dialysis)

Purification of proteins and peptides: complex samples,

monomer/dimer

QC: Size estimation. Size homogeneity: oligomeric state.

Impurities. Stability

Protein-Protein Interaction

19

HSA NaCl

volume

Desalting proteins

Desalting in a simple column

Column:

Sample:

Buffer:

PD-10

HSA, 25 mg

NaCl 0.5M

Volume for desalting: up to 25% column volume

20

Desalting / Buffer Exchange / Group separation

Adjusting pH, buffer type, salt

concentration during sample

preparation, e.g. before an assay.

Removing interfering small molecules:

EDTA, Gu.HCl, etc

Removing small reagent molecules,

e.g. fluorescent labels, radioactive

markers

Alternative to dialysis or to diafiltration

(ultrafiltration at constant retentate)

Gravity Desalting Columns

Multi Spin

Desalting Columns

FPLC Desalting Columns

Spin Desalting Columns

21

HiTrapDesalt10ml001:1_UV1_280nm HiTrapDesalt10ml001:1_Cond HiTrapDesalt10ml001:1_Fractions HiTrapDesalt10ml001:1_Inject HiTrapDesalt10ml001:1_Logbook

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HiTrapDesalt10ml002:1_UV1_280nm HiTrapDesalt10ml002:1_Cond HiTrapDesalt10ml002:1_Fractions HiTrapDesalt10ml002:1_Inject HiTrapDesalt10ml002:1_Logbook

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Waste 17 18 Waste

Desalting in the presence of buffer + 250mM NaCl

Desalting in the presence of buffer + 100mM NaCl

OD 280nm

Conductivity

Use buffer that avoid protein precipitation

Gali Prag

22

Fractionation of multiple components

Separate multiple components in a sample on the basis of differences on

their size

Best results with samples that contains few components or partially purified

samples (polishing step) : Not recommended for proteins with close MW

Limited sample volume (0.5-4% of total column volume). Not so suitable if

the sample volume is large. Volume can be increase if resolution is still OK

(scale up)

Flow-rate limitation : Time consuming

Removes higher oligomeric states and other aggregates

Protein elutes with equilibration buffer (important for storage or buffer exchange)

170726TIMPSuperdex75Prep120ml001:10_UV1_280nm 170726TIMPSuperdex75Prep120ml001:10_UV2_260nm 170726TIMPSuperdex75Prep120ml001:10_UV3_220nm 170726TIMPSuperdex75Prep120ml001:10_Cond 170726TIMPSuperdex75Prep120ml001:10_Fractions 170726TIMPSuperdex75Prep120ml001:10_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 32

Secreted Yeast TIMP9 after IMACSuperdex 75 60x1.6cm ~120ml

Jason ShiranYulia Shifman lab

Separating dimer and oligomers from monomer

24

Column size- Sample preparation

Desalting and other group separations

Column volume: four times the expected

sample volume

Column length is not so important

Preparative separation

Column volume: 0.5-4% times the expected

sample volume

Sample volume can be increase if resolution

is OK

Column length: 30-100 cm or more

(depends of the resolution: higher length

higher resolution)

How to reduce volume or concentrate your

sample

• Ultrafiltration

• Lyophilization

• Ammonium Sulfate Precipitation (or similar)

• Reverse elution (bind to absorption column

like IEX, HIC, IMAC; and reverse elution)

• Dialysis vs hygroscopic environment (glycerol, PEG, Sephadex etc)

• Others

25

Quality control: QC size estimation / oligomeric state / impurities / stability

Monitor protein prep quality

In analytical SEC, the sample volume should be approximately 0.3% of the bed

volume to achieve optimal results.

Complementary information to PAGE-SDS

Gives an estimate of molecular size in native solution

Un-native solution: Guanidine HCl, urea, detergents, etc. Precision is not so good as PAGE-SDS

For exact MW use SEC-MALS

Oligomeric state of the protein / homogeneity / complex. Aggregation profile.

Detect presence of impurities.

Identify protein interaction partners and interaction conditions

120402HLTVirE2Superose12prepEndwash001:1_UV1_280nm 120402HLTVirE2Superose12prepEndwash001:1_UV2_260nm 120402HLTVirE2Superose12prepEndwash001:1_UV3_220nm 120402HLTVirE2Superose12prepEndwash001:1_Fractions 120402HLTVirE2Superose12prepEndwash001:1_Inject 120402HLTVirE2Superose12prepEndwash001:1_Logbook

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60 80 100 120 140 160 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 29 30 31 32 33 34 35 36

HLT-VirE2Purification from 200ml cell

culture. IMAC purification and Preparative Gel Filtration

Superose 12 60x 1.6cm = 200ml

Michal Maess from Assaf Friedler lab.

SEC gives different and complementary information to PAGE-SDS

Size separation in native or un-native conditions

QC: monitoring size-homogeneity changes during storage or stress conditions

AggregationEvaluate tendency to aggregate and

quantity aggregates

DegradationEvaluate tendency to degradetion and

quantity of degraded forms

The trend is towards smaller particles of < 2 μm, with the use of

ultra high-performance liquid chromatography (UHPLC) systems for

even faster separations in high-throughput mode.

But:

• Very high back pressure

• Demands specific equipment

• Loss of resolution due to dead volumes

• Heat generation and shear stress at high flow rates could affect

proteins

A new trend: UHPLC (Ultra high-performance liquid chromatography)

29

Gel Filtration chromatography (GF)

Principles of GF

Fractionation range

Parameters for resolution optimization

Use of GF

Troubleshooting

Examples

SEC-MALS

30

Troubleshooting

Lower yield than expected

Protease degradation of the protein

Adsorption to filter, valves or top of the column

Non-specific adsorption

Sample precipitate

MW of protein is not as expected

Oligomerization state of the protein is different

Protein bounds to another protein or complex

Unfolded or naturally unfolded protein

Protein has changed during storage

Ionic or Hydrophobic interactions with the matrix

Protein precipitate

Very broad peak elution

Different oligomeric states or protein aggregation

Sticky protein

Non specific adsorption to matrix

Protein is part of complex with different sizes

Overloading

Peak of interest is poorly resolved

Sample volume is too high

Column length is not enough

Poor selectivity or efficiency of the column

Flow rate too high

Column is dirty or not well packed

Viscous sample

11/25/201931

Case study: HLT-p53CT

Capture: IMAC Affinitystart with pellet of 1.5L culture Ni-Sepharose FF 14ml

HLTp53CTNiNTA16ml004:1_UV1_280nm HLTp53CTNiNTA16ml004:1_UV2_260nm HLTp53CTNiNTA16ml004:1_Conc HLTp53CTNiNTA16ml004:1_Fractions HLTp53CTNiNTA16ml004:1_Inject HLTp53CTNiNTA16ml004:1_Logbook

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200 250 300 350 400 450 ml

F4 Waste 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

Load + 10cv 0%B + 3cv 8%B + 4cv 15%B + 4cv 100%B

POOL 17-22: 3.5OD x 35ml ~ 276mg

Ronen Gabizon from Assaf Friedler lab.

11/25/201932

Case study: HLT-p53CT

IntermediateCation Exchange

After TEV protease cleavage ON 4ºC

SP-Sepharose FF 5ml

HLTp53CTHiTrapSP5mlml005:1_UV1_280nm HLTp53CTHiTrapSP5mlml005:1_UV2_260nm HLTp53CTHiTrapSP5mlml005:1_Cond HLTp53CTHiTrapSP5mlml005:1_Conc HLTp53CTHiTrapSP5mlml005:1_Fractions HLTp53CTHiTrapSP5mlml005:1_Inject HLTp53CTHiTrapSP5mlml005:1_Logbook

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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 32 33 34 35 36 37 38 39 40 41 42 43 44

33

Case study: HLT-p53CT

Polishing SEC columnColumn: Sephacryl S100 prep. 960 x 26mm (~500ml) 6ml/fract.

Ni column – TEV protease cleavage ON – dilution – CEIX – concentration & GF

HLTp53CTSephacrylS100of500ml004:1_UV1_280nm HLTp53CTSephacrylS100of500ml004:1_UV2_260nm HLTp53CTSephacrylS100of500ml004:1_Fractions HLTp53CTSephacrylS100of500ml004:1_Inject HLTp53CTSephacrylS100of500ml004:1_Logbook

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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

Ronen Gabizon from Assaf Friedler lab.

Increasing resolution Example: Pegylated protein

120617M1605Superose12Anal001:1_UV1_280nm 120617M1605Superose12Anal001:1_UV2_260nm 120617M1605Superose12Anal001:1_Fractions 120617M1605Superose12Anal001:1_Inject 120617M1605Superose12Anal001:1_Logbook

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F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Waste

120618Superose12Prep3columns500ml001:1_UV1_280nm 120618Superose12Prep3columns500ml001:1_UV2_260nm 120618Superose12Prep3columns500ml001:1_Fractions 120618Superose12Prep3columns500ml001:1_Inject 120618Superose12Prep3columns500ml001:1_Logbook

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2 3 4 5 6 7 8 9 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93Superose 12 analytical 30 x 1cm = 23ml column

Load : ~1mg proteinSuperose 12 preparative

3 tandem columns 250 x 1.6cm = 502ml columnLoad : ~25mg protein / 5ml

Vo

123

How can we get better separation between 2 and 3 ??

Can we scale-up protein loading to separate 1 from 2 and 3 ??

35

250

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50

37

25

20

15

10

Before 6 7 M 8 9 11 13 15 17 19 21

23 25 27

Without DTT / boilingPool fractions 8-17

Results Fitting

Protein Molar Mass 1 Protein Molar Mass 2 LS UV RI

volume (mL)

9.0 10.0 11.0 12.0 13.0 14.0

Mo

lar

Mas

s (g

/mo

l)

41.0x10

51.0x10

61.0x10

160216 Darpin:10_UV1_280nm 160216 Darpin:10_UV2_260nm 160216 Darpin:10_Fractions 160216 Darpin:10_Inject 160216 Darpin:10_Logbook

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60.0 80.0 100.0 120.0 ml

cync

y to

wn

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Mer

lin 1

Cin

cy T

own

2

Waste 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Waste 32 33 34 35 36 37 38 39 40 41 42 43 Waste 45 46 47 48 49 50 51 52 53 54 55 Waste

Peak1:Total mass = 850±30 kDaProtein mass = 440±20 kDaRation = ~1:1Peak2:Total mass = 85±5 kDaProtein mass = 2.6±0.2 kDa

Protein MW (kDa) Ve (ml)

Thyroglob 669 9.76

Ferritin 440 10.95

Catalase 232 12.66

BSA 67 14.22

Chymotr 25 17.52

Peak 1 9.6

Peak 2 12.6

CMV-Viral Glycoprotein Extracellular Expression in Insect Cells

100kDa Ultrafiltration and SEC purification in tandem Superose 12 & Superdex 200 100 x 1.6cm each ~ 400ml

total SEC-MALS: Superdex 200

analytical column

36

Complex formationExample: Leptin and Leptin Receptor

Superdex75prep002:1_UV3_220nm Superdex75prep002:1_Fractions Superdex75prep002:1_Inject Superdex75prep002:1_UV3_220nm1Superdex75prep002:1_UV3_220nm2 Superdex75prep002:1_Logbook

20.0

30.0

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70.0

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90.0

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100 150 200 250 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 29 3031 32 33 34 35 Waste

Superdex 75 160x1.6cm column - Buffer: 20mMTrisHCl pH8.0 50mMNaCl 0.02%NaN3

Receptor alone

Leptin alone

Complex: Leptin + Receptor

37

Why choose gel filtration?Advantage

Separates by size

Complementary to IEX and HIC

Very gentle, high yields

Works in any buffer solution

Removes aggregates

Fast for buffer exchange

Mostly use in a final polishing step

Mandatory for QC

Complementary results than PAGE-SDS

Disadvantage

Limited sample volume

Poor resolution in a complex mixture

Flow-rate limitation – time consuming

Sample is diluted during elution

Poor selectivity compared with SDS-PAGE

Not efficient in capture or intermediate

steps

38

HTL435aaSuperdex200prep500mlB005:11_UV3_220nm HTL435aaSuperdex200prep500mlB005:11_Logbook

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Case study: Native Unfolding Protein

HTL - A natively unfolded proline-rich domain in ASPP2 that regulates its protein interactions by intramolecular binding to the Ank-SH3 domains.

Shahar Rotem et al. JBC Friedler lab

660 440 232 156 67 kDa

HTL 435aa

FoldIndex©: a simple tool to predict whether a given protein

sequence is intrinsically unfolded. Jaime Prilusky, Clifford E.

Felder, Tzviya Zeev-Ben-Mordehai, Edwin Rydberg, Orna Man,

Jacques S. Beckmann, Israel Silman, and Joel L. Sussman, 2005,

Bioinformatics.

435 aa

MW: 49.7kD

Superdex 200 prep. 100x2.6cm : trimer ??

Analytical ultracentrifugation: monomer

Size exclusion chromatograph in line with multi angle light scattering ,

added value to characterize proteins mass and shape in native solution

conditions

Calculating Mw and radius from the light scattering equations – much

more accurate.

Calculate the Mw during the elution peaks- detect homogeneity

sample.

Detect low amount of aggregation – large molecules amplify the

intensity of LS.

Useful for protein/protein or protein/ligand interaction

Size Exclusion Chromatography - Multi Angle Light Scattering

SEC-MALS

• The intensity of the radiated light depends on the magnitude of the dipole

and the macromolecule concentration.

• When a laser light hits a macromolecule, the electric field of the light

induces an oscillating dipole that re-radiates light.

Multi Angle Light Scattering (MALS)

or

Small molecule/ low concentration

Big molecule/high concentration

LS in

ten

sity

I - intensity of scattered light

c – concentration

M – mass

dn/dc = refractive index increment

2

scattered

dc

dnMcI

I

Multi Angle Light Scattering (MALS)

Quasi Elastic Light Scattering (QELS)

Dynamic Light Scattering (DLS)

• Measures by random motion of the macromolecules.

• The fluctuations are related to the rate of diffusion (D) which is related to

the radius of the molecule (R).

• Stokes-Einstein equation:

R- radiusk- Boltzmann constantT-temperatureD- diffusion coefficientη- viscosity

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

The LS detector uses the UV signal from the FPLC to

measure protein concentrations

Mini DAWN TREOS Wyatt technology

SEC-ultraviolet (UV) / LS /refractive index (RI)

approach• During elution from the column the molecules are introduced to the MALS system where

light scattering at several angles is measured, together with the dynamic light scattering and

the refractive index signals.

• The LS detector uses the UV signal from the FPLC to measure protein concentrations

• Refractive Index detector (RI ) measures the RI change of an molecule relative to the solvent

• RI gives us the possibility to calculate total concentration of all type of molecules in an

universal way, comparing it to a known reference without the need of UV

• So, we measure in this way 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

Static Light Scattering:

RMS Radius or Rg• mass averaged distance of each point in

a molecule from the molecule’s center of gravity.

• lower limit 10 nm

Dynamic Light Scattering:

Rh or Hydrodynamic Radius • radius of a sphere with the same diffusion

coefficient as “our” sample.

• lower limit ~ 0.5 nm

Rh

Radius of gyration vs. hydrodynamic radius

8 9 10 11 12 13

0.0

0.5

1.0

No

rmal

ized

ab

sorb

ance

at

28

0 n

m

Volume (ml)

10.45 ml (~45 kDa)

An intrinsically disordered protein (17 kDa)

SEC alone

Calculating mass of intrinsically disordered proteins

Predicted elution volume according to calibration curve

8 9 10 11 12 13

0.0

0.5

1.0

LS

UV

Volume (ml)

No

rmal

ized

inte

nsi

ty

17.1 0.5kDa

An intrinsically disordered protein (17 kDa)

102

103

104

105

106

Mo

lar

mas

s (D

a)

Calculating mass of intrinsically disordered proteins

Predicted elution volume according to calibration curve

Ve: 10.45 – 45kDa according to calibration curve

LS is very sensitive for aggregates

5 6 7 8 9 10 11 12

0.0

0.2

0.4

0.6

0.8

1.0 LS

UV

Volume (ml)

No

rma

lize

d in

ten

sity

102

103

104

105

106

107

108

109

1010

31.8 0.6 kDa Mo

lar

ma

ss (

Da

)

Monomer (99.7%)Aggregate (0.3%)

Protein aggregation induced by a specific molecule

8 10 12 14 16

0.0

0.5

1.0

Alone

+ M (low conc.)

+ M (high conc.)

Elution volume (ml)

No

rmal

ized

UV

inte

nsi

ty

105

106

107

108

Mass (D

a)

7 8 9 10 11

0.0

0.5

1.0 WT

L344A

Elution volume (ml)

No

rmal

ized

UV

inte

nsi

ty

P53 CTD (11 kDa) WT & L344A mutant

6050

40

30

20

Mass (kD

a)

10

P53 CTD oligomerization

WT- tetramer, L344A mutation inhibits P53 CTD oligomerization

Studying protein modifications

• Use to study protein modification such as glycosylation and pegylation.

• Can be used to characterize number of modifications.

• Can be used to study structural changes in modified proteins.

Mo

lar

mas

s

Volume Volume

Hyd

rod

ynam

ic r

adiu

s

50

51

Molar masses for two distinct ADC (antibody-drug conjugates)

formulations are determined using SEC-MALS analysisWYATT Technology

5 10 15 20 25 30

0.0

0.5

1.0

Polymer sample

LS

RI

Mass

Elution volume (ml)

No

rmal

ized

in

ten

sity

102

103

104

105

106

107

108

Mass (D

a)

Studding polymers using MALS

Downstream application in industry –measure aggregation percent

53

Preparative column:

Fast SEC-MALS experiment

5 6 7 8 9 10 11 12

0.0

0.2

0.4

0.6

0.8

1.0

Volume (ml)

No

rma

lize

d in

ten

sity

Analytical SEC-MALS :

0.01%0.5%

5%

UV

Ab

sorb

ance

Elution time (min)

Static light scattering to characterize membrane proteins in detergent solution

D.J. Slotboom et al. / Methods 46 (2008) 73–82

For the determination of the

absolute molecular mass of

membrane proteins in

protein/detergent/lipid micelles

The size exclusion column is used to physically separate

aggregates/empty micelles from the protein of interest, and

to ensure that the protein is dissolved in the correct buffer.

The elution volume is not included in the calculations.

The technique provides very similar information as

sedimentation equilibrium centrifugation, with similar

accuracy of the determined molecular masses.

Summary of SEC-MALS• SEC-MALS is a useful tool to determine protein shape and mass,

characterize oligomerization/aggregation and verify protein purity.

Very sensitive to presence of aggregates

• Additional information: modifications (glycosilation, pegylation, etc)

• Choose a good column for best separation of the sample for

achieving accurate results.

• Limitation: can detect low amount of large macromolecules but

needs high concentration of small macromolecules

• Requires longer equilibration time

56

Some molecules are highly hydrophobic, making them incompatible with fractionation via

size-exclusion chromatography (SEC).

Field flow fractionation (FFF) separates macromolecules and nanoparticles by size without

a stationary phase, eliminating most of the non-ideal surface interactions prevalent in SEC.

In an Asymmetric-Flow FFF separation channel,

macromolecules and nanoparticles are gently

pushed against a semipermeable membrane by

crossflow.

Smaller particles diffuse back up towards the

center of the channel.

Laminar channel flow induces a parabolic flow

velocity profile, causing smaller particles to elute

earlier.

Field flow fractionation (FFF)

Literature GE-Healthcare: Gel Filtration - Principles and Methodshttp://wolfson.huji.ac.il/purification/PDF/Gel_Filtration/GE_Size_Exclusion_Chromatography_Handbook.pdf

GE-HEALTHCARE Packing of Gel Filtration column and column evaluation (movies)http://wolfson.huji.ac.il/purification/Purification_Protocols.html#GF

TOSOH: GelFiltrationhttp://wolfson.huji.ac.il/purification/PDF/Gel_Filtration/TOSOH_GF.pdf

• Burgess, R.R. A brief practical review of size exclusion chromatography: Rules of thumb, limitations, and troubleshooting. Protein Expr Purif. 150, 81–85, doi: 10.1016/j.pep.2018.05.007 (2018)

• Chakrabarti, A. Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography. Antibody Engineering. doi: 10.5772/intechopen.73321 (2018).

• Mogridge, J. Using light scattering to determine the stoichiometry of protein complexes. Methods Mol Biol. 1278, 233–238, doi: 10.1007/978-1-4939-2425-7_14 (2015).

• Slotboom, D.J., Duurkens, R.H., Olieman, K., Erkens, G.B. Static light scattering to characterize membrane proteins in detergent solution. Methods. 46 (2), 73–82, doi: 10.1016/j.ymeth.2008.06.012 (2008).

• Miercke, L.J., Robbins, R.A., Stroud, R.M. Tetra detector analysis of membrane proteins. Curr Protoc Protein Sci. 77, 29.10.1-30, doi: 10.1002/0471140864.ps2910s77 (2014).

• Minton, A.P. Recent applications of light scattering measurement in the biological and biopharmaceutical sciences. Anal Biochem. 501, 4–22, doi: 10.1016/j.ab.2016.02.007 (2016).

• WYATT Technology Literature https://www.wyatt.com/library.html Bibliography https://www.wyatt.com/library/bibliography.html

Literature SEC-MALS• Some, D., Amartely, H., Tsadok, A., Lebendiker, M. Characterization of Proteins by Size-Exclusion Chromatography

Coupled to Multi-Angle Light Scattering (SEC-MALS). J. Vis. Exp.(148), e59615, doi:10.3791/59615 (2019).

• Folta-Stogniew, E., Williams, K.R. Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J Biomol Tech. 10 (2), 51–63, at <https://www.ncbi.nlm.nih.gov/pubmed/19499008> (1999)

• Kendrick, B.S., Kerwin, B.A., Chang, B.S., Philo, J.S. Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states. Anal Biochem. 299 (2), 136–146, doi: 10.1006/abio.2001.5411 (2001)

• Folta-Stogniew, E. Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. Methods Mol Biol. 328, 97–112, doi: 10.1385/1-59745-026-X:97 (2006).