Starting Sample

DepleteFractionate

PurifyConcentrate

Desalt/ Remove

DetergentAnalyze

Simplifying Sample Prep

Novel methods for Protein Purification using Pall Life Sciences Chromatography resins.

Monica IsaacsTechnical Marketing Manager

Pall Biosciences - ASIA

Agenda

� Recommended strategies for Sample Preparation� Chromatography Methods

� Special feature resins� Platforms for Chromatography� Ultrafiltration� Western Blotting� Protecting your column and samples� Summary� Questions

Proteomics Experimental Design

Sample Procurement

• plasma, serum• lysates Sample

Processing

• fractionate• deplete• digest…

Data Collection

• LC-MS/MS• MALDI, SELDI• 2D gel

Data Analysis

• univariant• multivariant• supervised or no?

Design decisions should be made based on sample limitations, experimental goals, and resources

Sample for Analysis• Optional Clean Up

Tag based method• Tag (ICAT)• Digest • Fractionate• Affinity capture• Optional sample

cleanup

Global digest

• Deplete • Fractionate• Digest• Optional sample

cleanup

Intact protein analysis• Deplete• Fractionate• Optional sample

cleanup

Analysis method

• LC-MS• MS/MS• MALDI

Analysis method• LC-MS• MS/MS• MALDI

Analysis method• 2D gel electrophoresis• FTMS, ETD/ CID• LC-MS/MS

(peptidomics)• MALDI/SELDI

Typical Proteomics Process Flow

� Complexity reduction = depletion, fractionation, tagging� MS analytical ‘aids’ = digestion

Serum or Plasma Sample Optional Clean Up (filtration)

Depletion of

IgG & HSA

Optional sample cleanup

Fractionate with

IEX (strong anion)

Analysis method

• 2D gel electrophoresis• FTMS, ETD/ CID• LC-MS/MS

(peptidomics)• MALDI/SELDI

Depletion Followed by Ion Exchange

Optional Denaturation / Protein interaction disruption

pH elution

Optional

digestion

� Ion Exchange (IEX)

� Affinity

� Mixed Mode Chromatography

� Size Exclusion (Gel Filtration)

� Pall Specialty Sorbents

Methods of Chromatography

Pall Chromatography Range

Ion Exchange

SizeExclusion

HIC

AndSpecialty

�DEAE, CM, SP Trisacryl® M/LS

�DEAE, SP Spherodex® LS

�QMA Spherosil® LS

�Q, S, DEAE, CM Ceramic HyperD® 20 and F

�Q and CM HyperZ

Affinity

�Heparin HyperD®

� Lysine HyperD®

�Protein A Ceramic Hyper D�Blue Trisacryl

®

M Affinity� IMACTM HyperCel

�Methyl HyperD®

�SDR HyperD®

�HA Ultrogel

�MEP Hypercel

�HEA/ PPA HyperCel

�Ultrogel AcA

�Trisacryl GF

� Ion Exchange Chromatography

� Q/ S/ DEAE/ CM Ceramic HyperD resins� Mustang Q/ S membranes

� Q (Quarternary Ammonium) = Strong Anion Exchanger

� S (Sulfonic Acid) = Strong Cation Exchanger� DEAE (Dextran) = Weak Anion Exchanger� CM (Carboxymethyl) = Weak Cation Exchanger

Ceramic HYPERD sorbents

In situ polymerization

Monomer intrusion

� Porous, non-compressible ceramic bead

� >0.2 µm (2000 Å) ‘pores’� In situ polymerization to form hydrogel

bead, containing the functional groups � CM, Q, S, DEAE IEX sorbents

“a gel in a shell”

Q/S/DEAE/CM Ceramic HyperD

� Features:� High Dynamic binding capacity at high flow rates� High-efficiency capture from dilute feedstock� Rigid, non compressible sorbent – easy to pack� Easy cleaning with NaOH� High speed, high capacity affinity preparative resins

for the purification of biological molecules by charge

Anion Exchange Dynamic Binding Capacity

• Anion Exchange: 5mg/ml BSA in 25 mM Tris pH 8.5, conductivity = 4-5 mS• ~0.85 ml column, run on Akta Explorer

Resinave bead

size

Slurry Volume % 10% 50% 10% 50%

Q HyperD F1 50 µ 125 129 114 122Q HyperD 20 20 µ 139 134 130 129Q (competitor A) 30 51 13 22Q (competitor B) 15 µ 75 76 66 40Q (competitor C) 42 62 15 33DEAE HyperD F1 50 µ 121 121 112 118DEAE (competitor A) 52 81 27 42DEAE (competitor C) 28 26 13 20

1 ml/min 4 ml/min

� Mustang ion exchange� 96-well format� Acrodisc format� Larger devices

NEW Membrane-based Chromatography at Pall

Fractionation of Human Serum in Mustang Q Anion Exchange Membrane in 96-Well Plate Format

� Compare flow through and eluate by 1D SDS-PAGE, reduced

� Consistent with expected IEX behavior, at pH 7, very few proteins captured, at pH 10 most proteins capture

� The presence of 150 mMNaCl in buffer reduces number of bound proteins

� Easier then using beads� Binding capacity relative to

membrane bed volume.

Effect of Loading pH and NaCl

pH 7.0 7.0 10.0 10.0 [NaCl] mM 150 0 150 0

250

150

100

75

50

37

25

15

20

10

kD

MW - FT E FT E FT E FT E FT E

Affinity Chromatography

� Separation using specific ligands� Reversible binding (buffer/ salt/ pH)

� Enchant Protein Depletion Kits� Blue Trisacryl M� Protein A Ceramic HyperD� IMAC Hypercel� Heparin HyperD� Lysine HyperD

• Albumin Depletion

• Albumin Fractionation

• Albumin & IgG Depletion

– Affinity Ligand based kit

Enchant™ Kits – Abundant Protein

Depletion

Abundant Proteins

� There are six abundant proteins that researchers often want to remove� Albumin

� IgG

� IgA� Transferrin� Anti-trypsin� Haptoglobin

Why Deplete High Abundant Proteins?

� Starting sample contains a mixture of both high abundance and low abundance proteins

� Proteins researchers are interested in are present in a complex sample

� Need to reduce the complexity of the sample� Proteins of interest are low molecular weight, low

abundant proteins

� First step in complexity reduction is the removal of high abundant proteins

� Without removing high abundant proteins, studying/identifying proteins of interest is like looking for a needle in a haystack

2DGE Plasma

Blue Trisacryl®M Affinity Sorbent

Structure of Blue Trisacryl M.

Typical applications:

• Albumin

• Vaccines• Interferons.• Purification of growth factors.• Isolation of DNA-dependent enzymes.• Purification of coagulation factors.• Purification of lipoproteins.

Ligand : CIBACRON BLUE (Blue TRISACRYL M)BASILEN BLUE ( Blue TRISACRYL PLUS LS)

Protein A Ceramic Hyper D

Protein A Sorbents : introduction

� Protein A is the industry standard for the capture step of antibodies, both in a laboratory and large-scale production.

� Generic, simple, very selective, allows direct recovery with minimal pre-treatment

� Several FDA approved Mabs processes for therapeutic usage refer to protein A sorbents.

� Typically, Protein A capture is followed by two « orthogonal » steps (ion exchange or HIC), to get rid of contaminants and remove traces of protein A.

Limitations of Protein A

� Leaching of Protein A� Cost� Variable binding capacity for different IgGs

� Species� Class� Sub-class� The need for Protein G

� Loss of protein activity over time due to regeneration in harsh conditions

Pall offer another alternative to Protein A and G

MEP HyperCel™

MEP chemistry

� Hydrophobic charge induction mechanism

Interaction of 4-MEP Ligand with Antibody

S

N

pKa = 4.8Hydrophobicinteraction

Adsorption at near-neutral pH

Desorption at pH 4

H

S

N

+++++++++

++++

++++

++++

Electrostatic Repulsion

pH% in (+)Form

4.8 50%

5.8 10%

Desorption at pH 4.0 – 5.8

IMAC HyperCel Resin

� For immobilised metal ion affinity chromatography

� Pre-fractionation method to increase resolution

� purify and collect tagged proteins ie His-tag, antibodies or phosphorylated proteins

� Can be charged with: Cu(II), Ni(II), Zn(II), Co(II), Ag(I), Ga(III), Zr(IIII) and Fe(III)

Ligand is tridentate IDA (Iminodiacetic acid) immobilisedon HyperCel base sorbent

� Size Exclusion Chromatography

� Also called gel filtration or gel permeation chromatography� Separates molecules according to size� Simple to use, non-denaturating method� Separates monomers from aggregates (i.e. IgG)� Desalting (buffer exchange) � Time consuming, low flow rates� LIMITATION: Final product is diluted

� Alternative is MWCO separation using ultrafiltration membrane

Ultrogel® AcA Product LineBioSepra Frac. range Excl. limit

sorbent (dalton) (dalton)

Ultrogel AcA202 1,000-15,000 22,000

Ultrogel AcA54 5,000-70,000 90,000

Ultrogel AcA44 10,000-130,000 200,000

Ultrogel AcA34 20,000-350,000 750,000

Ultrogel AcA22 100,000-1,200,000 3,000,000

Application of Pall Size Exclusion Sorbent

Sample Cleanup (Desalting)

� AcA 202 and GF05 can be used for desalting:

� very low non-specific interactions� ready to use, no gel swelling required� better resolution

� Desalting is one of the most commonly performed size exclusion steps

� Goal – remove small molecules (salt, free label)

� Using a spin device or filter plates avoids that common problem of sample dilution

� 5 mg/ml HSA in 1M NaCl onto 10 ml column, 1 ml fractions collected

� A280 measured the HSA coming through the column

� Conductivity was used to measure the removal of NaCl

Desalting in Gravity Flow Column

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0 5 10 15 20 25

Fraction number

Ab

so

rban

ce @

280 n

m

0

5

10

15

20

25

30

Co

nd

ucti

vit

y m

S/c

m

A280 nm NaCl

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20 25

Fraction number

Ab

so

rba

nc

e @

28

0 n

m0

5

10

15

20

25

30

35

40

Co

nd

uc

tivit

y (

mS

/cm

)

AcA202 Resin GF05M

� 0.1 ml of 5 mg/ml HSA in 1M NaCl was loaded onto nanosep column (0.2ml resin)

� Minimal sample dilution is seen when a centrifugation protocol is used

� Measured conductivity indicates very efficient removal of NaCl

Desalting in Nanosep Spin Column

1.050.095Competitive

agarose

0.0210.10AcA 202

0.450.12GF-05M

Conductivity

(mS/cm)2

Volume recovered

(ml)1

Resin

Specialty sorbents

�SDR HyperD�Unique to Pall Life Sciences

Silica SurfaceSilica SurfaceSilica SurfaceSilica Surface

Hydrophobic PolymerHydrophobic PolymerHydrophobic PolymerHydrophobic Polymer

SDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD Chemistry

SDR Mechanism of Detergent Binding

10 kDexclusion limit

Triton

TnBP

SDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD Chemistry

SDR Mechanism of Detergent Binding

10 kDexclusion limit

ASB-14 Removal on SDR HyperD

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 10 20 30 40Fraction number

Ab

so

rban

ce @

595 n

m

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Ab

so

rban

ce @

280 n

m

A 595 nm, detergent A 280 nm, HSA

� 5 mg/ml HSA in 1% detergent onto 1 ml packed column

� After loading, 1% detergent in buffer was added until break through was seen

� Break through determined based on inhibition of dye binding assay at OD 595 nm

Dynamic Binding Capacity Determination

SDR HyperD Detergent Removal

Examples of Binding Capacity

28.0SDS + 0.1 M NaCl

15.0SDS

75.0CHAPS

70.0ASB-14 + 6M Urea / 2M Thiourea

60.0 mg/mlASB-14

SDR HyperDDetergent � 5 mg/ml HSA in 1% detergent onto 1 ml packed column

� After loading, 1% detergent in buffer was added until break through was seen

� Break through determined based on inhibition of dye binding assay at OD 595 nm

Removal of Detergents from Protein Solutions

Triton (DBC = 60-80 mg/ml)

Initial Conc. (ppm

Final Conc. (ppm)

Removal efficiency

10,000

<10

>99.9%

10,000

<10

>99.9%

10,000

340

95.2%

Detergent Protein solutions

AT-IIIIgG Bovine Serum

TnBP (DBC = 40-60 mg/ml)

Initial Conc. (ppm

Final Conc. (ppm)

Removal efficiency

5,000

<0.4

>99.99%

5,000

<0.4

>99.99%

5,000

3.8

99.92%

Enrichment & Prefractionation

Mustang Resin Recombinant

Protein

Blue tris acryl

IMAC

IEX AFFINITY

DesaltingDetergent/ Solvent Removal

GEL FILTRATIONMIXED MODE

Trisacryl

UltrogelSDR HyperCel

Protein &

Peptide

Enrichment

Mustang Q

Mustang S

Q Ceramic HD F

S Ceramic HD F

DEAE Ceramic HD F

CM Ceramic HD F

Protein A Ceramic HD F

Heparin HD M

Lysine HD

Blue Trisacryl M

IMAC Hypercel

SDR Hyper D

Trisacryl GF05M

Ultrogel AcA 202

Monoclonal Ab

Recombinant

proteins

IgG, Albumin

Purification / Depletion

Coagulation factors

Lipoproteins, GH

Glycoproteins

Tagged Biomolecule

Purification

Solvent &Detergent

RemovalDesalting &

Small molecule

removal

Sample prep tools for protein purification

AffinityIon

Exchange

Size Exclusion

Concentrate

Polishingaffinity

and/

or

and/ or

Summary of Resins

At this stage you are pulling out what you want and eliminating the junk.

Then, you may wish to fractionate what is left based on size – resolution.

Once you have isolated the right fraction, if using size exclusion you will need to concentrate the product using ultrafiltration or a polishing affinity resin step.

Useful Chromatography Formats for Biology

� 96 well plates� Medium through put small scale fractionation

for proteomics or purification� Small scale purification without expensive

chromatography systems or expertise� Purification development – scouting� Disposable – no chance for cross contamination

� Centrifugal devices (spin columns)� Small scale purification without expensive

chromatography systems or expertise� Disposable – no cross contamination

� Small to very large traditional column chromatography

High throughput sample processing in proteomic research requires the protein recovery be consistent from well to well in the filter plates.

As shown, the elution from 96 identical samples processed by minicolumns in a single AcroPrepfilter plate with 0.45 µm GHP membrane (PN 5030) was consistent from well to well as judged by the intensity of protein bands in SDS PAGE gels. In addition, the protein concentration of each eluted sample was quantified by BCA assay, giving a CV of 9.3%.

This result indicated superb well-to-well reliability of the AcroPrep 96 filter plate in processing multiple samples.

Protein Purification – Scouting Experiment

An example of superb well-to-well reliability using an

AcroPrep™ 96 filter plate with hydrophillic media as chromatography minicolumns

Uses of UltraFiltration Devices

� Desalting & size exclusion with MWCO� Omega UF membranes� Spin devices – Nanosep, Microsep, Macrosep,

Jumbosep� TFF – also for sample concentration- Minimate

� Limited to 2 fractions (retentate and filtrate)� Some proteins can be lost on/in the membrane

� Might be faster then bead based separations� Less likely to see sample dilution, possibly combine

with a concentration step

• Small scale purification without expensive

chromatography systems or expertise

– Varying Sizes • Nanosep®: less than 0.5 mL• Microsep™: 0.5 – 3.5 mL• Macrosep®: 3 –15 mL• Jumbosep™: 15 – 60 mL

– Ultrafiltration Devices• Desalting• Concentration• Buffer Exchange

– Microfiltration Devices

• Batch mode chromatography• Small, fast protein preps

Spin Filter Columns – Single Sample Format

Western Blotting

� What? When? Where?� Nitrocellulose

� BioTrace NT – pure unsupported Nitrocellulose � 0.45um PVDF

� BioTrace PVDF – strong hydrophobic interaction� 0.2um PVDF – increased sensitivity, lower burn-

through� FluoroTrans – N-terminal sequencing� FluoroTrans W – optimised for Western blots

� 96-well plate Acrowell – ELISPOT assays� BioTrace PVDF� BioTrace NT

Western Blotting - Troubleshooting

� Before you blame the membrane…� Has your protein transferred properly?� Have you checked your blocking solution?� Have you checked your primary antibody?� Have you checked your conjugate?� Do your detection reagents give ideal results?� Is your water actually ultrapure 18.2MOhm?� All this should be run with known working

standards.

• Biosepra® Chromatography Resins

• Enchant™ Protein Purification/Depletion/Fractionation Kits

• Nanosep®, Microsep™, Macrosep®, &Jumbosep™ Centrifugal Spin Filters

• AcroWell™ & AcroPrep™ Multi-well Filter Plates

• Minimate™ TFF Systems

• Acrodisc® Syringe Filter Columns

• BioTrace™ & FluoroTrans®

Blotting Membranes

Making Protein Sample

Preparation and Analysis Easy

LC Sample and Mobile Phase Filtration

� Traditional options� Hydrophilic Nylon/ PVDF – Aqueous/ some organics� Hydrophobic PTFE – Solvent/ Aggressive organics

� 0.45um standard� 0.2um recommended for smaller bead sizes� Introducing GHP

� Universal HPLC Membrane� Hydrophilic Polypropylene� Aqueous and Aggressive solvents� Low Protein binding

HPLC: Why should I filter?

� What Pore size?� 0.2um – Beads 3um and smaller� 0.45um – larger than 3um

� Mobile Phase/ Sample: Removing particulate/ bacteria from your column will extend the life of your column, saving you money

� Features to consider:� Accuracy of pore size� Extractables� Biomolecule binding

Thank You for

Your Attention!