Simplifying Sample Prep

Desalt/
Starting Fractionate
Sample
Deplete Purify
Concentrate Remove Analyze
Detergent
 Novel methods for Protein Purification
using Pall Life Sciences
Chromatography resins.

Monica Isaacs
Technical 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
Sample
Procurement
Experimental Design
• plasma, serum
• lysates Sample • LC-MS/MS
Processing • MALDI, SELDI
• 2D gel
• fractionate
• deplete Data
• digest…
Collection

Data
Design decisions should be
made based on sample Analysis
• univariant
limitations, experimental • multivariant
goals, and resources • supervised or no?
 Typical Proteomics
Process Flow

Sample for Analysis

• Optional Clean Up

Tag based method Intact protein analysis

• Tag (ICAT) Global digest
• Deplete
• Digest • Deplete
• Fractionate
• Fractionate • Fractionate
• Optional sample
• Affinity capture • Digest
cleanup
• Optional sample • Optional sample
cleanup cleanup
Analysis method
• 2D gel electrophoresis
Analysis method Analysis method • FTMS, ETD/ CID
• LC-MS • LC-MS • LC-MS/MS
• MS/MS • MS/MS (peptidomics)
• MALDI • MALDI • MALDI/SELDI

Complexity reduction = depletion, fractionation, tagging

MS analytical ‘aids’ = digestion
 Depletion Followed by
Ion Exchange

Serum or Plasma Sample

Optional Clean Up (filtration)

Optional Denaturation /
Depletion of Fractionate with
Protein interaction
IgG & HSA IEX (strong anion)
disruption

pH elution

Analysis method Optional

• 2D gel electrophoresis digestion
• FTMS, ETD/ CID Optional sample
• LC-MS/MS cleanup
(peptidomics)
• MALDI/SELDI
Methods of Chromatography

Ion Exchange (IEX)

Affinity

Mixed Mode Chromatography

Size Exclusion (Gel Filtration)

Pall Specialty Sorbents
 Pall Chromatography Range

DEAE, CM, SP Trisacryl® M/LS

DEAE, SP Spherodex® LS
Ion Exchange
QMA Spherosil® LS

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

Q and CM HyperZ

Heparin HyperD®

Lysine HyperD®
Affinity
Protein A Ceramic
® Hyper D

Blue Trisacryl M Affinity

IMACTM HyperCel

Methyl HyperD®
HIC
SDR HyperD®
And
HA Ultrogel
Specialty
MEP Hypercel

HEA/ PPA HyperCel

Ultrogel AcA
Size

Trisacryl GF
Exclusion

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

“a gel in a shell”

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

Monomer intrusion

In situ polymerization
 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
ave bead
Resin 1 ml/min 4 ml/min
size
Slurry Volume % 10% 50% 10% 50%
Q HyperD F1 50 µ 125 129 114 122
Q HyperD 20 20 µ 139 134 130 129
Q (competitor A) 30 51 13 22
Q (competitor B) 15 µ 75 76 66 40
Q (competitor C) 42 62 15 33
DEAE HyperD F1 50 µ 121 121 112 118
DEAE (competitor A) 52 81 27 42
DEAE (competitor C) 28 26 13 20

• Anion Exchange: 5mg/ml BSA in 25 mM Tris pH 8.5, conductivity = 4-5 mS

• ~0.85 ml column, run on Akta Explorer
NEW Membrane-based Chromatography at Pall

Mustang ion exchange

96-well format

Acrodisc format

Larger devices
Fractionation of Human Serum in Mustang Q Anion
Exchange Membrane in 96-Well Plate Format
Effect of Loading pH and NaCl

Compare flow through and pH 7.0 7.0 10.0 10.0
[NaCl] mM 150 0 150 0
eluate by 1D SDS-PAGE,
reduced MW - FT E FT E FT E FT E FT E
kD

Consistent with expected IEX 250
behavior, at pH 7, very few 150
proteins captured, at pH 10 100
most proteins capture
75

The presence of 150 mM
NaCl in buffer reduces 50
number of bound proteins
37

Easier then using beads

Binding capacity relative to 25
20
membrane bed volume. 15
10
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
Enchant™ Kits – Abundant Protein
Depletion

• Albumin Depletion
• Albumin Fractionation
• Albumin & IgG Depletion
– Affinity Ligand based kit
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
Ligand : CIBACRON BLUE (Blue TRISACRYL M)
• Vaccines BASILEN BLUE ( Blue TRISACRYL PLUS LS)
• Interferons.
• Purification of growth factors.
• Isolation of DNA-dependent enzymes.
• Purification of coagulation factors.
• Purification of lipoproteins.
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

Adsorption at near-neutral pH

N
Hydrophobic
pKa = 4.8 interaction

Desorption
Desorptionat
atpH
pH44.0 – 5.8
+
S + pH
% in (+)
Form
+
N
+
4.8 50%
H +
5.8 10%
Electrostatic +
Repulsion
 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:
Ligand is tridentate IDA
Cu(II), Ni(II), Zn(II), Co(II),
(Iminodiacetic acid) immobilised
on HyperCel base sorbent
Ag(I), Ga(III), Zr(IIII) and
Fe(III)

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 Line
BioSepra 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
 Desalting in Gravity Flow Column

AcA202 Resin GF05M

0.50 30 0.35 40

0.45
35
0.3
25
0.40
30
0.35
0.25
Absorbance @ 280 nm

20

Conductivity mS/cm
25
0.30
0.2

0.25 15 20

Conductivity (mS/cm)
0.15

Absorbance @280 nm
0.20
15
10
0.15
0.1
10
0.10
5
0.05
0.05
5

0.00 0 0 0

0 5 10 15 20 25
0 5 10 15 20 25

Fraction number
Fraction number
A280 nm NaCl

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 Nanosep Spin Column

Resin Volume recovered Conductivity

(ml)1 (mS/cm)2
GF-05M 0.12 0.45

AcA 202 0.10 0.021

Competitive
0.095 1.05
agarose

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
 Specialty sorbents

SDR HyperD

Unique to Pall Life Sciences
SDR Mechanism of Detergent Binding

10 kD
Silica Surface exclusion
limit

Hydrophobic Polymer

SDR HyperD Chemistry

SDR Mechanism of Detergent Binding

10 kD
exclusion
Triton limit

TnBP

SDR HyperD Chemistry

 Dynamic Binding Capacity Determination

ASB-14 Removal on SDR HyperD

5 mg/ml HSA in 1%
detergent onto 1 ml 1.40 0.35
packed column 1.20 0.30
After loading, 1%

Absorbance @
Absorbance @

1.00 0.25
detergent in buffer was

280 nm
595 nm

0.80 0.20
added until break 0.60 0.15
through was seen 0.40 0.10

Break through 0.20 0.05
determined based on 0.00 0.00
inhibition of dye 0 10 20 30 40
Fraction number
binding assay at OD
595 nm A 595 nm, detergent A 280 nm, HSA
SDR HyperD Detergent Removal
Examples of Binding Capacity

5 mg/ml HSA in 1%
Detergent SDR HyperD
detergent onto 1 ml
ASB-14 60.0 mg/ml packed column

After loading, 1%
ASB-14 + 6M Urea / 2M detergent in buffer was
70.0
Thiourea added until break
through was seen
CHAPS 75.0

Break through
SDS 15.0 determined based on
inhibition of dye binding
SDS + 0.1 M NaCl 28.0 assay at OD 595 nm
Removal of Detergents from Protein Solutions

Detergent Protein solutions

IgG AT-III Bovine Serum

Triton (DBC = 60-80 mg/ml)
Initial Conc. (ppm 10,000 10,000 10,000
Final Conc. (ppm) <10 <10 340
Removal efficiency >99.9% >99.9% 95.2%

TnBP (DBC = 40-60 mg/ml)

Initial Conc. (ppm 5,000 5,000 5,000
Final Conc. (ppm) <0.4 <0.4 3.8
Removal efficiency >99.99% >99.99% 99.92%
 Sample prep tools for protein purification
Enrichment & Prefractionation Detergent/ Solvent Removal Desalting

IEX AFFINITY
MIXED MODE GEL FILTRATION

Mustang Resin Recombinant IMAC

Protein SDR HyperCel Trisacryl
Blue tris acryl Ultrogel

Mustang Q Protein A Ceramic HD F SDR Hyper D

Mustang S Heparin HD M
Lysine HD Trisacryl GF05M
Blue Trisacryl M Ultrogel AcA 202

Q Ceramic HD F IMAC Hypercel

S Ceramic HD F
DEAE Ceramic HD F
CM Ceramic HD F IgG, Albumin Solvent &Detergent
Purification / Depletion Removal
Desalting &
Protein & Monoclonal Ab Coagulation factors Small molecule
Peptide Recombinant Lipoproteins, GH Tagged Biomolecule removal
Enrichment proteins Glycoproteins Purification
 Summary of Resins
At this stage you are pulling
out what you want and
and/ Ion eliminating the junk.
Affinity or Exchange
Then, you may wish to
fractionate what is left based
on size – resolution.

Size
Exclusion

Once you have isolated the right Polishing

fraction, if using size exclusion you affinity
will need to concentrate the
product using ultrafiltration or a and/
polishing affinity resin step. Concentrate or
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
Protein Purification – Scouting Experiment
An example of superb well-to-well reliability using an
AcroPrep™ 96 filter plate with hydrophillic media as
chromatography minicolumns
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 AcroPrep
filter 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.
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
Spin Filter Columns – Single Sample Format
• 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
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.
Making Protein Sample
Preparation and Analysis Easy

• 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
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!