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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!