Kein Folientitel

1
Fundamental concepts Charge properties of
proteins Factors influencing protein
charge Donnan effect Choice of buffer Trials to
determine pH Trials to determine salt Gradient
elution (Type I and Type II) Typical protocoll
Models describing ion-exchange process
2
Adsorption equilibrium
LINEAR
CONVEX
KONCAV
Stationäry Phase (q)
Mobile Phase
(C)
Mobile Phase
(C)
Mobile Phase
(C)
q Concentration in solid phase qm maximal
concentration in solid phase C
concentration in liquid phase K Distribution
(partition) coefficient a, b emprical
parameter
3
Mobile phase modifer and adsorption
O. Kaltenbrunner and A. Jungbauer, Adsorption
isotherms in protein chromatography Combined
influence of protein and salt concentration on
adsorption isotherm. J. Chromatogr. A 734 (1996)
183-194
4
Langmuir adsorptions isotherm
5
Scatchard plot
6
Estimation of equilibrium parameters
7
Stoffbilanz in der Säule
Unter Gleichgewichtsbedingungen
8
Ideal chromatography migration of a solute
? Phase ratio K Tangen of adsoption isotherm
9
Non-ideal/ideal chromatography
Ideal
Non-ideal
10
Models of Chromatography

• Plate model
• Martin and Synge model
• Craig model
• Mass balance
• Ideal Model
• Equilibrium-dispersive model
• Lumped kinetics model
• General rate theory

11
Plate model

• establishing a mass balance for each plate
• concept of height equivalent of a theoretical
  plate (HETP)
• originally for linear isotherms

12
Martin and Synge plate model

• Continous model
• Ratio between mobile and stationary phase volume
  are identical and constant
• Infinitestimal fraction of mobile phase volume
  passes from stage to stage

13
Martin and Synge plate model
14
Craig plate model I

• Discrete model
• Ratio between mobile and stationary phase volume
  are identical and constant
• Whole amount of mobile phase moves to the next
  stage when equilibrium is attained

15
Craig plate model II
Mass balance for the jth stage
Binomial distribution for linear
isotherm ?Gaussian distribution for high plate
numbers
16
Comparison

• Different distributions with same limit for
  increasing N
• Less peak dispersion in Craig model, related by

17
Other plate models

• Sectional model as an extension of the Craig
  model
• Tank-in-series model as an extension of the
  Martin and
• Synge model

18
Mass balance equation

• Use of partial differential equations describing
• a differential mass balance of the solute in a
  slice of column and
• its kinetics of mass transfer in the column

Ideal model Equilibrium dispersive model
Lumped-kinetic model General rate equation model
19
Mass balance
for each component in the system
20
Ideal model

• no axial dispersion nor mass transfer kinetics
• stationary phase is given by equilibrium isotherm
• focuses on influence of nonlinear thermodynamics
  of phase equilibria
• for large samples with hightly efficient columns
  there is a good agreement with the experimental
  chromatograms

21
Nonuniformity of Flow
Column inlet design (scale up) Packing
quality Column diameter Viscosity difference
between feed and eluent (concentrated samples)
viscosous fingering Velocity
22
Equilibrium dispersive model I

• When mass transfer kinetics are fast but not
  infinitely fast
• All contributions due to nonequilibrium can be
  lumped into an apparent axial dispersion term
• The apparent axial dispersion term is independent
  of the concentration of the sample components

23
Equilibrium dispersive model
with apparent dispersion coefficient
24
Band Profiles

• Relationship between equilibrium isotherm and the
  band profile for single components.

25
Mass Transfer and Kinetics of Adsorption
Biochromatography Large molecules ? slow
diffusion
26
Lumped kinetic model

• D accounts only for axial dispersion
• Also linear driving force (LDF) model
• All contributions of mechanisms involved in band
  broadening, due to their relatively slow kinetics
  are lumped in a single rate coefficient

L
27
Lumped kinetic model

• If rate of equilibrium kinetics is high, there is
  no difference between the lumped kinetic and the
  equilibrium-dispersive model
• Developed for linear isotherms, but exact for
  non-linear isotherms if external mass transfer is
  controlling
• Depending on assumptions, different models can be
  distinguished
• reaction-dispersive model
• transport-dispersive model

28
Reaction-dispersive model

• Influence of mass transfer is negligible
• Only the kinetics of adsorption and desorption
  are taken in account

29
Transport-dispersive model

• adsorption-desorption kinetics are infinitely
  fast, only mass transfer kinetics are taken in
  account

30
General rate model

• Attempts to consider silmutaneously all possible
  contributions to mass transfer kinetics
• Two mass balance equations for one solute, one
  for the mobile phase between the particles and
  one for the stagnant mobile phase inside the
  particle
• Described using the component balance equation
• Mass transfer mechanisms considered
• ? external (film) mass transfer
• ? pore diffusion
• ? solid (surface) diffusion
• ? reaction kinetics at phase boundary

31
Rate equation
32
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34
Geometry of Phases
Planar, column and capillary chromatography
35
Media for Biochromatography
36
Gel in a Shell
37
Controlled pore glass
38
Ceramic Hydroxyapatite
39
Virus purification
40
Pressure Drop of Soft Media
Rigid media Konzeny-Karman relationship
Soft media
a correlates with D/L
Joustra et al. (1967) Prodites of the Biological
Fluids 15, 57-579 Mohammad et al. 1992, (Ind.
Eng. Chem. Res.) 31-561
41
Scale up of Compressible Beds
L 20 cm (L0 24.3) T 6C
L 15 cm (L0 19.0) T 6C
42
Features of Media
43
Pore size
Particle density 0.6-0.9 g/cm3 porosity 0.4-0.6
44
Retardation Forces and Principle of Separation
45
Physicochemical Conditions In Biochromatography
46
Categories of Elution Development
(A) Elution development, (B) displacement
development and (C) frontal Analysis
47
Elution modes
Isocratic elution (A), gradient Elution (B), Step
gradient elution (C) and displacement development
(D)
48
Performance factors
Extra column effects
Efficiency (HETP)
Selectivity
Resolution
Dynamic capacity
Kinetics
Phase equilibrium
49
Height Equivalent to One Theoretical Plate
H HETP (heigth equivalent to one theoretical
plate) h Reduced plate height L Column
length N Plate count dp Particle diameter
50
Theoretical and Effective Plate Number
51
Determination of N
Peakwidth at base (w) 4 ? at point of
inflection (w) 2 ? at half heigth (w)
2.355 ?.
52
Detemination of N by Breakthrough Curves
H.P.Lettner, O. Kaltenbrunner, and A. Jungbauer,
HETP in Process Ion-Exchnage Chromatography, J.
Chromatogr. Sci. 33 (1995) 451-457 D.U. von
Rosenberg mechanics of steady state single-phase
fluid displacement from porous media, AIChJ 2
(19956) 55-61
53
Resolution (R)
54
Selectivity vs Efficiency
55
Separation Efficiency and Particle Size
56
Extra Column Effects
2
,
0
Slope e
1
,
5

l
1
,
0

m
Pulse response experiments same column diamter
different height


R
V
Intercept Ve
0
,
5
0
,
0
0
,
0
1
,
0
2
,
0
3
,
0

m
l

V

t
Kaltenbrunner, O., Jungbauer, A. and Yamamoto, S.
(1997) Prediction of the preparative
chromatography performance with a very small
column J Chromatogr A, 760, 41-53.
57
Porosity
58
Extra Column Band Spreading
0.10
1.0
0.08
0.8
0.06
0.6

2
ml2
ex
s
2
total
s
0.04
0.4
0.02
0.2
0.00
0.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0
2
4
6
8
10
2
2
V
ml

V

ml
R
t
59
Sample Loading
60
Pre- and Post Column Band Spreading
61
Influence of Extra Column Effects on Performance
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63
Das Coulombsches Gesetz
F Anziehungskraft zweier Punktladungen
N qn Ladung C r Abstand m ?0 Dielektrizitäts
konstante im Vakuum 7,854 x 10-2 C2N-1m2 ?
Dielektrizitätskonstante -
64
s Flächenladungsdichte (Anzahl der
funktionellen Gruppen, Ionisierbarkeit, Art
des Elektrolyten, Elektrolytkonzentration. ??
Potentialsprung d Dicke der Grenzschicht e1,
e0 Dielektrizitätszahl des Mediums oder Vakuum
65
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66
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67
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68
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69
Nettoladung Funktion des pH
70
Verteilung der isoelektrischen Punkte von
Proteinen
71
Säulenmethode
Wahl der Bedingungen für die Bindung eines
Proteins an einen Ionentauscher, physikochemische
Eigenschaften unbekannt
72
Zusammenhang zwischen Salzkonzentration und dem
gebundenen Protein am Beispiel des
Rinderserumalbumins
73
Wahl der Bedingungen für die Bindung eines
Proteins an einen Ionentauscher, physikochemische
Eigenschaften unbekanntTeströhrchenmethode
74
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75
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76
Donnan effect
77
Flüchtige Puffer für die Ionentauscher
Chromatographie
78
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79
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80
Tris
Phosphat
81
Tris
Phosphate
82
Die einzelnen Stufen der Ionentauscherchromatograp
hie
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84
Steric Mass Action Model
Boardmann and Partridge 1995
k relative retention b empirical
parameter a empirical parameter I ionic strenght
85
Steric Mass Action Model
Cm Protein in der mobilen Phase Cs Protein in der
stationären Phase Is Salz in der stationären
Phase Im Salz in der mobilen Phase
Kf Geichgewichtsreaktionskonstante
86
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88
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89
Absatzweise Adsorption/Desoption
f Fraktion adsorbierten Proteins q Proteinkonzentr
ation in der festen Phase C Proteinkonzentration
in der mobilen Phase ? Verteiluungskoeffizient ?
Nerntscher Verteilungskoeffizient ? q/(q
C)
Vm Volumen der flüssigen Phase,
Vs Volumen der festen Phase
90
Vs/Vm
91
Praktische Durchführung der absatzweisen
Adsorption/Desorption
92
Hydrophobic Interaction Chromatography
93
The Hydrophobic Surface
Matrix, Base, Length of ligand, Ligand density
94
Models Describing Adsorption

• Solvophobic model
• Preferential interaction analysis
• Flickering cluster model
• Random network model
• Continum model for liquid water

95
Effect of salt on protein solublity
Green, A.A. and Hughes, W.L. (1955) Methods in
Enzymology Vol I. Academic Press Inc. New York
96
Salt Promoted Adsorption

• Cavity for protein is formed
• Protein fills cavity
• (Fusion of cavities?aggregation?precipitation)
• Water and ions surround the hydrophobic surface
• Hydrophobic interaction (Van der Waals forces)
  between proteins and surface
• Structural rearrangement of protein
• Rearrangement of water and ions in the bulk
  solution

Driving force is reduction of surface area
97
The Solvophobic Model
,
Melander and Horvath 1977 Arch. Biochem.
Biophys.183 200-215
Staby and Mollerup, 1996 J. Chromatogr. A 734,
205-212
98
Solvophobic Model II
Melander et al, 1989 J. Chromatogr. 469, 3-27
Hearn, 2000 in Handbook of Bioseparations, ed.
Ahuja, Academic Press
99
lnk vs Ionic strength
100
High Selectivity
Intracellular expression of rHSA in S. cervesiae
with a FLAG-tag
kD
200
119
? rHSA
95
66
45
33
21
Yeast extract Desalted yeast extract Flow through
Anti-FLAG column Eluate Anti FLAG column
Schuster, M., Wasserbauer, E., Ortner, C.,
Graumann, K., Jungbauer, A., Hammerschmid, F. and
Werner, G. (2000) Short cut of protein
purification by integration of cell-disrupture
and affinity-extraction Bioseparation, 9, 59-67.
101
Affinity chromatography

• Selectivity of ligand ? ligand density
• Capacity of sorbent ? ligand density
• Adsorption/desorption kinetics of immobilized
  ligand
• Elution conditions
• Mass transfer properties

102
Biospecific Interactions
Selective interaction of biomolecules with
immobilzed ligands
Ligand Ligate
Substrate, Inhibitor, Cofactor Enzyme,
Receptor Antibody Antigen Lectin Glycoprote
in Nucleic acid Complementary
sequence Dye Protein Peptide Protein
103
Functional groups used for immobilization of
biomolecules
Proteins/Peptides -NH2 -SH -COOH
Nucleic acids -PO4 - pyrimidine, purine
bases
Glycoproteins - sugar moeity
Active hydrogen containing compounds
104
Activation methods
105
Restricted diffusion with large molecules
a effective molecular radius of adsorbate rrB
effectively open portion of pore
Permeability dependant on fractional saturation
and effective radius of ligate 3,4 small radius
of ligand and dsorbate 5 large radius of adsorbate
Petropoulos et al., Bioseparation 1 (1990), 69-88
106
Influence of pore size on coupling yield
Relation of pore size to surface area Quantity of
protein coupled
107
Optimization
Variables and response values
108

• Column size/shape
• height (Z)
• diameter
• height to diameter ratio height to particle
  diameter
• Mobile phase
• modifier composition
• rate of change of mobile phase modifier
  composition (gradient shape)
• Flow rate (u)
• flow rate during loading, during elution during
  regeneration
• residence time (Z/u)
• Loading
• volume
• concentration
• flow rate

109

• Physical conditions of packing
• packing quality
• packing density
• Stationary phase
• ligand type
• ligand density
• type of support
• porosity, tortuosity
• bead diameter
• beads size distribution (monodispers)
• Temperature

110
Response Values
Productivity (P) QR purity ratio C0
sample concentration VF feed volume Vt
column volume tC cycle time (process time).
tlife column life time Yield or recovery (Y)
VR product volume CR product concentration
111
DBC VBTC C0
Dynamic capacity Purity Concentration factor
(CF) C0 inital (feed) concentration, VF
feed volume CR recovered concentration VR
recovered volume Process time.
112
Dynamic and Static Binding Capacity
DBC VBTC C0
113
DBC VBTC C0
Dynamic capacity Purity CR recovered product
concentration CR,,total protein concentration in
recovered pool Concentration factor (CF) C0
inital (feed) concentration, VF feed volume
VR recovered volume Process time (tprocess)
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116
Scale Up
Z/D? constant
Z/D constant
R1 ? R2 VF1 ? VF2
117
Scale up by Constant Residence Time
H Plate height N Plate number A,B,C Parameter
in van Demter equation u Velocity of mobile
phase t0 Residence time
Consequence resolution remains
constant Everything is normalized in respect to Vt
118
DBC 0.5 cm i.d. x 10 cm
Non Agarose-based media
Agarose-based media
119
DBC versus residence time
Hahn, R., Schlegel, R. Jungbauer, A. Comparison
of Protein A affinity sorbents. J Chromatogr B
790, 35-51 (2003).
120
A ?-Lactoglobulin Separation on Source30Q
The flow rate, gradient length and load are
scaled to the column volume. The flow rate is 30
CV/h. Column dimensions are 1x10 and 2 x17.7 cm.
2
1
Scale-up 17
Flow rates 305 cm/h 531 cm/h
?t12 5.00 and 4.97 min
Scale-up experiment performed at DTU and
presented at ISPPP 2000
121
Changing Particle Diameter
Consequences Productivity ? Buffer consumption
? Process time ? Purity is constant
122
Batch operation continuous operation
Carrousell Chromatography Annular
Chromatography True Moving Bed Chromatography Simu
lated Moving Bed Chromatography
123
Bed collapse
Chaotic event not predictable
124
Flow sheeting
Choose separation process based on different
physical, chemical, or biochemical
properties. Separate the most plentiful
impurities first . Choose those processes that
will exploit the differences in the
physicochemical properties of the product and
impurities in the most efficient manner Use a
high-resolution step as soon as possible Do the
most arduous step last.
125
Factorielles Design
Follman DK, Fahrner RL Factorial screening of
antibody purification processes using three
chromatography steps without protein A. J
Chromatogr A (2004) 102479-85.