Chromatography Techniques For Biomolecule Purification - KMUTT - CH PDF

CHROMATOGRAPHY
TECHNIQUES FOR
BIOMOLECULE
PURIFICATION
Dr. Chester, Che-Chia Hu

July 29, 2016
Outline
Principles of chromatography
Retention mechanisms in chromatography
2 Chromatography techniques for biomolecule purification | 29.07.2016

What is biochromatography?
Purification of biological macromolecules (DNA, Proteine) with maintaining the biological function.
Aim is to obtain the target compound as pure as possible.
The separate steps are carried out predominantly (at low temperatures) in aqueous buffer systems.

What are proteins?
Proteins have a very complex three-dimensional structure, which is important for the biological function and which must
remain unchanged as far as possible during purification steps.

Amino acids I
An amino acid is the basic molecule of any protein.
Using correct terminology, it is an amino carbonic acid which contains an amino group (-NH2) and a carboxylic group (-
COOH).
Amino acids have a common basic structure, different amino acids vary from each other only by their side chain (R).
Most proteins are build from different combinations of only twenty different amino acids. This pool of twenty amino acids
can be classified according to:

Amino acids II
Nonpolar amino acids:

Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine
Polar amino acids :

Serine, Proline, Threonine, Cysteine, Asparagine, Glutamine
Aromatic amino acids:

Phenylalanine, Tryptophan, Tyrosine
Negatively charged amino acids:

Aspartate, Glutamate
Positively charged amino acids :

Lysine, Histidine, Arginine

Amino acids II
Source:
Principles of Biochemistry

Structure of proteins
Primary Secondary Tertiary Quaternary

structure structure structure structure
Amino acid α Helix Polypeptide chain Assembled

residues subunits
Source:

Structure of proteins
The characteristics imparted to the protein by the amino acid side chains serve as bases for purification process, and
include solubility, charge, hydrophobicity and intermolecular binding.

Separation methods in protein biochemistry
Chromatography
 Column liquid chromatography
 Thin layer chromatography
 Partitioning chromatography
Electrophoresis
Precipitation
Microfiltration/Dialysis
Extraction
Crystallisation
Centrifugation

Chromatography
Chromatography is the collective term ... for the separation of mixtures. It involves passing a mixture dissolved in a
mobile phase through a stationary phase, which separates the analyte to be measured from other molecules in the
mixture based on differential partitioning between the mobile and stationary phases.
Source:
Wikipedia

Chromatography
chromatogram
Chromatogram

Chromatography system
data acquisition
sample
pump detector
A B load
fraction collector

Chromatography system

Elution

Chromatography basics
+ +
+ +
++
+
+
+
+
++ +
+ ++
Equilibrate Load Wash Elute Regenerate

17
Chromatographic techniques
Adsorptive Polarity
 Ion exchange chromatography  Normal phase chromatography
 Affinity chromatography  Reversed phase chromatography
 Hydrophobic interaction
chromatography
Non-adsorptive
 Size exclusion chromatography
(Gel filtration)

Ion exchange chromatography

Ion exchange chromatography is based on the ionic attraction

between molecules of opposite electric charge.
There are two main types of resins

 anion exchange, used to separate negatively charged targets.
 cation exchange, used to separate positively charged targets.
The electrostatic interactions are short distance phenomena - and as a

result the separation is based on the total number of accessible
charges – negative or positive on their surface.
Source:
Charge of the sample may be strongly effected by the pH

 Charged amino acid side chains are weak ion exchange groups
 The charge / pH dependency is highly non-linear BUT in all cases the molecules will become more positively charged at
lower pH values and more negatively charged at higher pH values.
One important characteristic of biomolecules is the iso-electric point or pI, this is the pH at which the total number of
positive and negative charges are equal and the net charge on the molecule is zero.

Protein charge depends on pH value
COOH pI = isoelectronic point
NH3+ < pI
net charge: positive
COO-
NH3+ = pI
net charge : neutral
R
COO-
NH2 > pI
net charge: negative

Choice of the right ion exchange system
Fractogel® EMD SO3, SE Hicap, COO

Eshmuno® S, CPX
isoelectric point
of the antibody
pH
Fractogel® EMD TMAE, TMAE Hicap, DEAE, DMAE

Eshmuno® Q
charge

Classes of ion exchangers
Strong ion exchangers

 keep their charge independent of the pH value of the used buffer
Weak ion exchangers

 keep their charge only within a smaller pH range
The term „strong“ and „weak“ does not correspond to the strength of binding of proteins!

Ion exchanger binding mechanism
Matrix Ligand Protein
- -
- - - -
- + - - +- + +
+ - -
- -
Counter ion (Salt) Charged

areas

Properties of tentacle ion exchangers
Fast mass High selectivity
transport High
Flexibility DBC
tentacle resin
convent. resin

Outstanding binding capacities
Fractogel® media
Batch capacity experiments for CEX
All data from: Generic purification processes for monoclonal

35 antibodies and Fc fusion proteins; Abhinav Shukla, Peter Hinckley,
capacity (mg/ml)
Eva Gefroh, Priyanka Gupta and Brian Hubbard, Amgen, IBC

meeting 2002, San Diego, USA
25
15

Eshmuno® CPX: Superior mAb binding capacity
Static mAb07 post-protA pool binding capacities*
Eshmuno® CPX Competitor 1 (Agarose) Competitor 2 (Polymer)

12 12 12
90 50 20 80
120 130 120 60 70 50 30
130 100 80 80 60 80 70 60
70 40
11 110 80 11 11
70
130 130 90 10 50 80
10 10 90
120 100 80
Conductivity [mS/cm]
70
70 80 60
120 90 80 90
130 110
9 9 70
9
90
130
80
120 50 90
8 8 80 8
130 90 60
120 140 70 80 80 90
110 70
140 90
7 130 7 7
90
80
6 140 6 6
130 140 90
120 70 80 90 80 90
110 100 100
5 5 5
4,00 4,25 4,50 4,75 5,00 5,25 4,00 4,25 4,50 4,75 5,00 5,25 4,00 4,25 4,50 4,75 5,00 5,25
pH pH pH
* in mg/mL settled resin
Eshmuno® CPX shows highest mAb binding capacities.

Start & elution conditions in ion exchange chromatography
Binding conditions Elution conditions Cleaning step
Highest salt concentration Lowest ionic strength Higher salt concentration

at which the target protein is still which allows elution of the target than the one used for elution
well bound. protein.

Elution options in ion exchange chromatography
Increasing salt concentration

 step-wise
 as linear gradient
pH gradient (or step)

 with weak ion exchangers
(Caution: Protein must not precipitate at the IEP!)

Weak vs. strong CEX resins – mAb 1
Selectivities of different functional groups
Fractogel® EMD COO- (M) Eshmuno® CPX Fractogel® EMD SO3- (M)
Weak CEX (COO-) vs. strong CEX resins (SO3-, CPX):

Better separation of mAb aggregates from monomer
Fractogel® EMD COO- (M) > Eshmuno® CPX > Fractogel® EMD SO3-(M) media
Experimental:
Column size: 200 mm L x 5 mm i.d. (CV = 3.9 mL)
Binding capacity of Fractogel® EMD COO- (M) resin may be lower Operating buffer:
compared to strong CEX, but selectivity is often higher 50 mM acetate + 24 mM NaCl, pH 5.0 (ca. 6 mS/cm)
Linear gradient elution: 0 - 1 M NaCl in 40 CV
Load: 40 mg/mL CV
Weak vs. strong CEX resins - mAb 1
3.8% initial aggregate level
Fractogel® EMD COO- (M) resin Eshmuno CPX resin Fractogel® EMD SO3- (M) resin
6.0
mAb pool aggregate level (%)
5.0
initial mAb aggregate level: 3.8% initial mAb aggregate level: 3.9% initial mAb aggregate level: 3.8%
4.0
3.0
2.0
87% mAb yield at target ≤ 1.5% aggregates 75% mAb yield at target ≤ 1.5% aggr. 68% mAb yield at target ≤ 1.5% aggr.
1.0
0.0
0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
cumulative mAb pool yield (%) cumulative mAb pool yield (%) cumulative mAb pool yield (%)
Experimental:
Fractogel® EMD COO- (M) resin: 87% mAb yield at target ≤ 1.5% aggr. Column size: 200 mm L x 5 mm i.d. (CV = 3.9 mL)
Operating buffer: 50 mM acetate + 24 mM NaCl, pH 5.0
Eshmuno® CPX resin: 75% mAb yield at target ≤ 1.5% aggr. (ca. 6 mS/cm)
Linear gradient elution: 0 - 1 M NaCl in 40 CV
Fractogel® EMD SO3- (M) resin: 68% mAb yield at target ≤ 1.5% aggr. Flow rate: 200 cm/h
Starting level of aggregates: 3.8%
Load: 40 mg/mL CV
Comparison of elution profiles
mAb monomer
Eshmuno® CPX
competitor (agarose)
Elution peak width
%B Eshmuno® CPX: 11.4 CV
Conductivity Competitor (agarose): 14.3 CV
Conductivity at peak max.

Eshmuno® CPX: 21.4 mS/cm,
17.2% B, approx. 0.16 M NaCl
Competitor (agarose): 16.1 mS/cm
12.0% B, approx. 0.11 M NaCl
mAb aggregates
Feed: mAb06 (post-ProtA pool), aggregate level 2.9 %

Column size: 8 mm i.d. x 100 mm; Column vol. : 5.0 ml; Linear flow rate: 250 cm/h
Buffer A: 25mM sodium acetate + 50mM sodium chloride, pH 4.75, 8 mS/cm
Buffer B: 25mM sodium acetate + 1M sodium chloride, pH 4.75
Gradient: 0 to 50 %B, 20 CV; Load: 50 mg/mL
33
Ion exchange chromatography: Pros and Cons
Advantages
 High protein binding capacity
 Independent of sample volume
 Target is concentrated
 Easy scale-up
Drawbacks
 High salt concentration interfers with binding
 Eluate contains salt

Affinity chromatography

The method is based on the specific interaction between biological

counterparts (e.g. enzyme substrate).
Ligands with specific biological affinities are immobilized on Fractogel

via polymer chains. The ligand, which has a highly specific structure is
capable of binding complementary protein molecules in a "lock and
key" type mechanism. (Hydrogen bonds and polarizable molecules)
Source:
Only the protein of interest is bound to the specific ligand; other sample components are eluted from the column in the
washing buffer or during the loading.
Elution of the sample protein may either be specific (e.g. using an enzyme substrate) or non-specific using salt or a
change in pH.
Affinity chromatography is the most powerful purification technique for the isolation of proteins.

Separation mechanism of affinity chromatography
- - - - Impurities
- -
+
- - - -
Complex
Binding region
- - -
- - -
+ +
- - - -
Purified protein
Affinity molecule for elution

Ligands for affinity chromatography
Dyes
Immobilised metal ions (IMAC)
Lectins
Biospecific ligands

Protein A affinity chromatography
Protein A media Clarified feedstock from the
e.g. ProSep®, Eshmuno® A fermenter containing mAb
Clarified feedstock from the fermenter containing mAb

passed over column, product binds, impurities pass through
Intermediate wash to remove any remaining

impurities but leave mAb bound to protein A
Elution of mAb by reducing the pH of buffer

(typically pH 3.5-4.5)
Breakthrough curves at 3 min residence time
Dynamic binding capacity of protein A affinity resin
 Equilibration Buffer: 50 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.0

 Intermediate wash: 0.1 M Citric Acid, pH 5.5
 Elution buffer: 0.1 M Acetic Acid, pH 3.0
 Column dimension: 10 mm i.d. x 50 mm bed height
 Feedstream: A purified monoclonal antibody is used for this test.
Dynamic binding capacities at different residence times
 Equilibration Buffer: 50 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.0

 Elution buffer: 0.1 M Acetic Acid, pH 3.0
 Column dimension: 10 mm i.d. x 50 mm bed height
 Feedstream: A purified monoclonal antibody is used for this test.

Eshmuno® A media removes more aggregates
Eshmuno® A Media MabSelect SuRe® resin
Pre-Peak Post-Peak
Monomer in Overall mAb Total Aggregate
Resin % Aggregate % Aggregate
pool (%) Yield (%) Removed (%)
Removed Removed
Eshmuno® A Media 94.6 79.4 25.7 36.9 62.6
MabSelect SuRe® 88.9 80.3 6.1 26.1 32.2
 Equilibration Buffer: 50 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.0;

 Elution buffer: 0.1M Acetic Acid, pH 3.0;
 Column dimension: 6.6 mm i.d. x 14 cm bed height
 Feedstream: A purified monoclonal antibody is used for this test. With a loading of 12mg/ml and a max loading of
aggregate of 40mg/ml.; Initial mAb feed aggregate: 13.5 %; Gradient elution using citrate buffer, pH 6 – 3 over 20 CV
Elution & intermediate wash conditions in protein A affinity
chromatography
Intermediate Wash step Elution conditions Cleaning step
low pH, salt, solvent and lower pH acidic solution or akaline

detergent solution depends on the based
which allows elution of the mAb
matrix and ligand stability
which removes the impurities
6 M Urea, 6 M guanidine HCl,
20% ethanol/0.5 M acetic acid,
20% ethanol can be sufficient for
removing strongly bound
material

Protein A affinity chromatography: Pros and Cons
Advantages
 A powerful tool in capture step for mAb
 Very specific - high purity (> 90%) in one step
 Easy operation
Drawbacks
 Resin cost is high
 Protein A ligand leaching

Metal chelate affinity chromatography
Retention accomplished by the coordination of electron donor groups

on the protein surface with immobilised transition metal ions
IMAC is widely used for the purification of native or recombinant

proteins.
Commonly Cu (II), Ni (II), Co (II) or Zn (II) ions are immobilised onto

 Chelating ligands which are covalently attached to a support.
The binding is based on the electron-pair acceptor properties of the

 Metal ion (Lewis acid) and the electron-pair donor properties of
 Histidine containing side chains of the protein (Lewis base).
The existence of surface accessible histidine residues is necessary for

the binding of a protein.

Metal chelate affinity chromatography: PROS & CONS
Advantages
 High capacity compared to other affinity techniques
 Good selectivity
 Flexibility due to the immobilization of different metal
ions
 High stability of the ligand
 Supported by molecular techniques (expression vectors!)
 Identification of histidine residues on the protein's
surface
 Scalability/cleanability
Drawbacks
 Necessity of histidine residues on the surface
 More steps for column preparation are necessary
 Bleeding of metal ions

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography
In hydrophobic interaction chromatography binding is achieved under high salt concentration
The eluent of choice is an inverse gradient with ammonium or sodium sulphate to buffer or water
Binding is the result of permanent dipoles of water molecules conferring an ordered structure on the water and thus
increasing the association of the hydrophobic patches of the target compound and the hydrophobic regions of the
stationary phase (increased entropy in the system).

Separation mechanism of hydrophobic interaction chromatography
- -
- - - -
+ - +
-
- -
- -
„Ordered“ „Disordered“
water molecules Hydrophobic patches water molecules

Terminology
Hydrophilic = “loves water”

 Hydrophilic molecules are well water-soluble
Hydrophobic = “rejects water”

 Hydrophobic molecules behave like oil and are badly water-soluble

Start & elution conditions in hydrophobic interaction chromatography
Binding conditions Elution conditions Cleaning step
Lowest salt concentration Highest salt concentration Low salt concentration

at which the target protein is which (already) allows elution of (water or organic solvent is the
(already) well bound. the target protein. best)
 decreasing salt concentration

 linear gradient/step
 decreasing salt concentration

with simultaneously increasing
ethylene glycol concentration
(40%)
 detergents for membrane

proteins
Hydrophobic interaction chromatography: PROS & CONS
Advantages
 Sample binding occurs in the presence of salt.
 During elution salt concentration can be reduced.
 Very good separation efficiency (isoenzyme separation)
Drawbacks
 High salt load.
 Denaturation of proteins.

Size exclusion chromatography

Size exclusion chromatography
Also referred to as gel permeation chromatography or gel filtration. It is an easy

technique for the purification of proteins and other compounds according to their
molecular size and shape.
In contrast to other chromatography methods no adsorption effects should be

involved during the separation. Therefore the packing material has to be
strongly hydrophilic. To avoid hydrophobic interactions between biopolymers and
the matrix, 100 mM salt (or more) should be added to the mobile phase. Elution
of the sample can be performed under isocratic conditions without any gradient
mixer.
Since the separation mechanism is independent from the buffer, the most
suitable buffer conditions for each protein can be used.
Source:

Separation mechanism in size exclusion chromatography
Yellow = large protein

Green = medium sized protein
Red = small protein
Different migration
distances depending on the
molecule’s size and shape

Important parameters in size exclusion chromatography
Flow rate (must not be too high)
Retention time (min)

90
Sample volume
80
Appropriate salt concentration 70
60
Column dimensions (length: 40-100 cm)
50
40
Optimal column packing
30 BSA
Ovalbumin
20 Cytochrome c
10
0
0 50 100 150 200 250 300
[sodium chloride] (mM)

Separation efficiency
A280
Column: Superformance 1000-100, packed with Fractogel ® EMD BioSEC (S)

Bed height: 95 cm (7.46 Liter of packed gel)
Buffer: 20 mM Na-citrate, pH 7.4, 200 mM NaCl, 2 mM CaCl2
Flow rate: 6.2 ml/min (5 cm/h)
Sample: IgM, thyroglobulin, ovalbumin, myoglobin, vitamin B12
(each 10 mg) dissolved in 50 ml of running buffer

Size exclusion chromatography: PROS & CONS
Advantages
 No adsorptive interaction between stationary phase and
sample.
 Free choice of the buffer system (optimal conditions for
the target protein).
 Predictable elution behavior.
Drawbacks
 Loading volume is limited and large columns are
required.
 Technique is unsuitable for sample molecules of similar
size.

Biochromatography media
... an excellent investment for your process economy!
Ion exchange Affinity Size exclusion

media media media
Fractogel® EMD Eshmuno® Fractogel® EMD Fractogel® EMD

– TMAE Hicap – Q – Chelate – BioSEC
– TMAE Medcap – S
– TMAE – HCX ProSep®
– DEAE – CPX
– DMAE – ProSep Ultra Plus
– SE Hicap – ProSep-vA Ultra
– SO3- – ProSep-vA High
– COO- Capacity
Eshmuno® A
RoboColumn
RoboColumn
MiniChrom Column
MiniChrom Column
Chromabolt® Chromabolt®

Silica gel and high performance sorbent

Separation based on polarity
Normal Phase Column (Si)
N Mobile phase
P NP P POLAR SORBENT NON-POLAR
P
Like attraks like

Reversed Phase Column (RP18)
N Mobile phase
PSample
NP NON-POLAR SORBENT P POLAR
P

Analyte Stationary phase

Mobile Phase
Functional Group
polar non-polar polar
Salts Hexane
Silica
Acids
Alcohols THF
Ketones
CN
Ethers
C4
Hydrocarbons: Acetonitrile
halogenated
C8
aromatic Alcohol
aliphatic
C18
fluorinated Water
non-polar polar non-polar

-C18 low polarity
Reversed phase -C8
-C2
-Phenyl > 90% of all applications
Medium polar phase -CN
(dep. on the seperation -NH2

problematic and the
used eluent) -Diol
Normal phase -Si high polarity

Preparative chrom product range
Preparative Chromatography
PharmPrep® P spherical
Performance
LiChroprep®
irregular
Silica Gel 60 / Geduran Si 60
10 12 20 25 40 63 200 500 µm
Particle size

Influence of particle size on resolution
LiChrospher (5 µm) and LiChroprep RP-18 sorbents

Mobile phase: Buffer/ACN 68.5/31.5, flow rate 1 ml/min, UV 220 nm
Samples (1) Thymine (2) Tropic acid (3) Phenol (4) Cinnamic acid (5)
Acetophenone (6) Naphthol (7) Benzene

Silica gel and high performance sorbent
... an excellent investment for your process economy!
Silica Gel LiChroprep® PharmPrep® Others
Silica gel 60 LiChroprep® PharmPrep® P Cellulose micro-crystalline

Silica gel 60 silanised – Si 60 – Si 100 Avicel®
Geduran 60 – RP-8 – 100 RP-18e Florisil®

– RP-18 – 100 RP-8e Aluminium Oxide
– NH2 – AlOx 60, active, basic
15 - 25 μm – DIOL 10 μm – AlOx 90, active, basic
15 - 40 µm – CN 20 μm
– AlOx 90, active,
25 - 40 μm neutral
35 - 70 µm 15 - 25 μm – AlOx 90, active, acidic
40 - 63 μm 25 - 40 μm HIBAR®
– AlOx 90, st. Brockm.
63 μm 40 - 63 μm
– AlOx 150, basic
63 - 100 μm
63 - 200 μm Geduran AlOx 90
200 - 500 μm HIBAR® st. Brockm.

upgrade potential productivity
Improve your process economics
Protein A
VI UF IEX UF VRF IEX UF
Soft gel
time reduction
Protein A -44%
IEX
VI UF UF VRF IEX UF
rigid
for increased productivity rigid media are advantageous

Higher bed height = more column capacity = less re-cycling
From: An evaluation of Protein A and Non-Protein A methods for the recovery of monoclonal
antibodies and considerations for process scale-up by Martin Smith, LONZA, Presented
at“Scaling-up of Biopharmaceutical Products”, 26/27thJanuary 2004, The Grand, Amsterdam

Strictly controlled production and quality
Long term track record of successful audits by regulatory authorities
Safety stock can be held in all geographies close to customer sites
Flexible and extra manufacturing capacity supporting customer demand changes
Business contingency plans
Definition of critical product quality attributes and strict adherence in manufacturing

Strictly controlled production and quality
Rigorously controlled producton from start to finish
In accordance with cGMP
Excellent batch-to-batch reproducebility

 Ensures long-term consistency for your product quality
Outstanding regulatory support

 Assists in fullfilling regulatory requirements
Unrivalled expertise
 Over a century of experience in providing quality solutions

Chromatography training
Experiences biopharm course at Mannheim, Germany
THREE-DAY COURSE
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CHROMATOGRAPHY

Dr. Chester, Che-Chia Hu

Retention mechanisms in chromatography

2 Chromatography techniques for biomolecule purification | 29.07.2016

Aim is to obtain the target compound as pure as possible.

3 Chromatography techniques for biomolecule purification | 29.07.2016

4 Chromatography techniques for biomolecule purification | 29.07.2016

An amino acid is the basic molecule of any protein.

5 Chromatography techniques for biomolecule purification | 29.07.2016

Nonpolar amino acids:

Polar amino acids :

Aromatic amino acids:

Negatively charged amino acids:

Positively charged amino acids :

6 Chromatography techniques for biomolecule purification | 29.07.2016

7 Chromatography techniques for biomolecule purification | 29.07.2016

Primary Secondary Tertiary Quaternary

Amino acid α Helix Polypeptide chain Assembled

8 Chromatography techniques for biomolecule purification | 29.07.2016

9 Chromatography techniques for biomolecule purification | 29.07.2016

10 Chromatography techniques for biomolecule purification | 29.07.2016

11 Chromatography techniques for biomolecule purification | 29.07.2016

13 Chromatography techniques for biomolecule purification | 29.07.2016

14 Chromatography techniques for biomolecule purification | 29.07.2016

15 Chromatography techniques for biomolecule purification | 29.07.2016

16 Chromatography techniques for biomolecule purification | 29.07.2016

Equilibrate Load Wash Elute Regenerate

18 Chromatography techniques for biomolecule purification | 29.07.2016

19 Chromatography techniques for biomolecule purification | 29.07.2016

Ion exchange chromatography is based on the ionic attraction

There are two main types of resins

The electrostatic interactions are short distance phenomena - and as a

Charge of the sample may be strongly effected by the pH

21 Chromatography techniques for biomolecule purification | 29.07.2016

COOH pI = isoelectronic point

22 Chromatography techniques for biomolecule purification | 29.07.2016

Fractogel® EMD SO3, SE Hicap, COO

Fractogel® EMD TMAE, TMAE Hicap, DEAE, DMAE

23 Chromatography techniques for biomolecule purification | 29.07.2016

Strong ion exchangers

Weak ion exchangers

24 Chromatography techniques for biomolecule purification | 29.07.2016

Matrix Ligand Protein

Counter ion (Salt) Charged

25 Chromatography techniques for biomolecule purification | 29.07.2016

26 Chromatography techniques for biomolecule purification | 29.07.2016

Batch capacity experiments for CEX

All data from: Generic purification processes for monoclonal

Eva Gefroh, Priyanka Gupta and Brian Hubbard, Amgen, IBC

27 Chromatography techniques for biomolecule purification | 29.07.2016

Static mAb07 post-protA pool binding capacities*

Eshmuno® CPX Competitor 1 (Agarose) Competitor 2 (Polymer)

* in mg/mL settled resin

Eshmuno® CPX shows highest mAb binding capacities.

28 Chromatography techniques for biomolecule purification | 29.07.2016

Binding conditions Elution conditions Cleaning step

Highest salt concentration Lowest ionic strength Higher salt concentration

29 Chromatography techniques for biomolecule purification | 29.07.2016

Increasing salt concentration

pH gradient (or step)

(Caution: Protein must not precipitate at the IEP!)

30 Chromatography techniques for biomolecule purification | 29.07.2016

Weak CEX (COO-) vs. strong CEX resins (SO3-, CPX):