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Ion Exchange Chromatography

Optimisation and Robustness Analysis Using Design of Experiments
Emelie Stenborg
Department of Chemical Engineering, Lund Institute of Technology
Abstract
Proteins are often separated using ion-exchange chromatography. This method can however
show poor resolution if not carefully optimised. This work aims to find all the parameters that
affect the separation as well as determining which are critical. By using Design of Experiment the
optimum separation is found and a robustness analysis is conducted. Polyclonal antibody
Immunoglobulin G (IgG) and Bovine Serum Albumin (BSA) are separated on a strong anion-
exchange column. The parameters focused on are the elution gradient, pH, sample concentration,
flow rate and sample size.
Keywords: Ion Exchange Chromatography, Design of Experiments, Optimisation, Robustness analysis
Introduction
The purification and separation of
antibodies is an important chromatographic
procedure and ion exchange chromatography,
which uses a charged ligand bound to the
stationary phase is often used. The proteins
are sent through the column and depending
on their charge, adjusted by pH, they interact
differently to the stationary phase. By
changing the mobile phase so that more
counter ions are present the proteins elute in
order of increasing interactions with the
stationary phase [1] [2].
The benefits of ion exchange
chromatography are that it has a high
throughput, the column has a long lifespan
and it is a well-known method. However it is a
labour intense procedure since the
optimisation is time consuming and involves
several parameters. In a process, the most
important parameters to optimise towards are
recovery and speed [2].
This work tries to find a way of
developing an efficient procedure by using
design of experiments in order to find more
information from fewer experiments.
Immunoglobulin G (IgG) and Bovine serum
albumin (BSA) are separated on a strong
anion exchanger using a linear salt gradient
[3]. The optimisation aims mainly towards
finding a good degree of separation but also
the efficiency of the column is investigated.
Theory
The parameters that are the most
important for separation was first found and a
favourable range was defined, by looking at
the effect of the parameters one by one. This
is the traditional method for optimisation but
here it is only used as a screening method [4].
The need for this step can be disputed but if
the range investigated by design of
experiments is to big no new information will
be gained and the linearity of the model will
be questionable. Also the aim was to reduce
parameters. The parameters investigated were
pH, gradient length (column volume, CV),
flow rate (ml/min), sample concentration
(mg/ml), sample volume (ml) and
temperature (C) [4].
In the optimisation step a range of the
four most important parameters, pH, gradient
slope, sample concentration and sample size
was defined and experiments performed at all
the combinations of their high respectively
low level. Together with three experiments in
the mid point, for precision, nineteen
experiments were run [4].
The software, MODDE 7.0, used
statistical methods to evaluate the data and
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found an equation that describes the optimum
separation. It also randomised the
experiments in a full factor two level test [6].
The separation was optimised towards the
following responses.
L HETP
2
2

o
= (1)
HETP (height equivalent to a theoretical
plate) describes with what efficiency the
separation is made where L is the length of
the column, is the weighted retention time
and the deviation [3].
300 2
) ( ) ( w A separation A - = (2)
The term (2) is a way to describe the degree of
separation (fig 2). It takes into account the
retention times for IgG and BSA as well as
the gap between the peaks at an absorbance
of 300 mAU. The two responses are
maximised on the behalf of the other but are
used to evaluate if it is possible to maximise
both and if not which one should be used.
A
2
w

Fig 1. Schematic figure of the

equation, separation.
IgG is due to multiple peaks considered to
be non-Gaussian while the monomer of BSA
is considered to be a Gaussian peak. This is
confirmed by the responses where different
models are examined. The first peak in the
chromatogram is IgG and the second is BSA.
Experiments
Material
All chromatographic separations were
performed using an KTA-purifier with a
strong anion-exchange column Q Sepharose
High Performance, 1 ml (Amersham
Pharmacia). Detection was performed with an
online conductivity meter and an UV detector.
The pH was set using the pH meter in the
KTA system. The software controlling and
partly evaluating the chromatography was
Unicorn 4.11 and MODDE 7.0 was used to
design and evaluate the design of experiments.
Chemicals
The buffers used for loading and elution
where Trizma Base, C
4
H
11
NO
3
, Sigma
Chemical Co., St Louis, USA, titration grade
with the addition of NaCl, Merck,
Darmstadt, Germany p.a for the eluting
buffer. The pH of the buffers where set
using HCl and NaOH both Merck, p.a. The
sample solution consisted of the proteins
Bovine Serum Albumin (BSA), Sigma
Chemical Co., >97% and Immunoglobulin G
(IgG), 157 mg/ml, kindly given by
Biovitrum. Sodium azide, NaN
3
, BDH
Laboratory Supplies, Poole, England, p.a.
where used to prevent bacterial growth in
the sample solution. The Milli Pore water
used was prepared in-house.
Screening
The parameters investigated are found
below (table 1 and 2).
Table 1. Experiments performed.
pH Gradient length (CV)
8 10, 16, 20, 24
8,5 10, 12, 16, 18, 20, 22, 24
9 10, 16, 20, 24
9,5 10, 16, 20, 24
Table 2. Experiments performed
flow (ml/min) 0,5 0,7 1 1,2 1,5 2
sample conc.
(mg/ml)
0,5 1 2 3 5
sample size (ml) 2 4 6 8 14
temp. (C)
5 15 23 30
300
mAU
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The experimental conditions were loading
buffer 20 mM TRIS-HCl, eluting buffer 20
mM TRIS-HCl with 1 M NaCl. The pH was
8,5, the flow 1 ml/min, the concentration 0,5
mg/ml, the sample size 2 ml and the
temperature was ambient to start with but
changed to the values in bold italic (see table 1
and 2) during the course of the screening
experiments.
Optimisation
The parameters chosen where pH,
gradient slope, sample concentration and
sample size. The range for respectively
parameters are pH 8,75-9,25, slope 12-16 CV,
sample concentration 2-4 mg/ml and sample
volume 2-4 ml. The mid separation was pH
9,0, slope 14 CV, protein concentration 3
mg/ml and sample volume 3 ml.
Robustness analysis
Here a smaller range, when possible,
than for the optimisation was used to evaluate
the robustness of the model. The range of the
parameters was pH 8,50-9,00, slope 15-17 CV,
sample concentration 3,5-4,5 mg/ml and
sample volume 1,66-2,66 ml. The only
difference from the optimum point chosen by
MODDE was that a higher protein
concentration seemed to have very little effect
on the separation and hence a higher
concentration was used. The mid point was
pH 8,75, slope 16 CV, sample concentration 4
mg/ml and sample volume 2,06 ml.
Validation
Three points were chosen to represent
optimisation towards maximum efficiency,
maximum separation and a combination of
both. These where run to investigate the
validity of the model.
Results and Discussion
Screening
This step was done to eliminate
parameters and to define a range for the
optimisation for the remaining parameters.
The temperature could not be controlled
in the apparatus since the eluate held the same
temperature no matter what the ingoing
buffer temperature was. The flow rate had no
influence on the separation between the peaks
but within the same peak the subclasses and
dimers where slightly better separated. The
flow rate was hence chosen to be as high as
possible within the columns limitations.
Already in this step a decision had to be
done regarding what is desired from the
separation and what parameters are most
important. Industrially the optimisation is
often done towards speed and recovery, with
a given purity. The main criteria here however
was to achieve the best degree of separation as
possible within a reasonable time. This means
that the remaining parameters where to be
optimised within the range in table 3.
The pH is chosen to be 9 since it gives a
better efficiency of the separation and because
the dimer of BSA is more prominent and this
means that the assumption that the monomer
of BSA is Gaussian is more valid. Selecting
the gradient slope is a compromise between
speed and resolution. The real interesting
feature appears when the sample
concentration is high and a reasonably small
sample size is chosen. The front gets sharp
and still a Gaussian shape is maintained which
means low sample loss (fig 3).
0
500
1000
1500
0 5 10 15 20
m
A
U
ml
Fig 3. PH 9 CV16 3mg/ml 2 ml
Optimisation
The aim of the procedure is to find a
local minima or maxima within or outside of
the experimental volume to find the best
separation possible. But the model is linear for
almost all parameters in the range and this
means that no minima or maxima will be
found. As follows there has to be knowledge
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that the experiments are done in the right area
and also a further definition of towards what
direction optimisation is done needs to be
established. The later decision has to be made
since the responses efficiency and separation
are optimised in opposite ways.
Table 3. The parameters and range
used for optimisation
parameter range
pH 8,75-9,25
Slope (CV) 12-16
Sample conc. (mg/ml) 2-4
Sample size (ml) 2-4
MODDE evaluated the results and the
model received was valid with a good ability
to predict data and a reasonable ability to
predict variation. The different responses
where dependent on the parameters as seen in
fig 4.
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
Efficiency IgG Separation
pH
grad
conc
size
pH*grad
pH*conc
conc*size
Fig 4. The normalised coefficients for
the responses in the
optimisation step.
The two responses where combined to
give the three validation experiments seen in
table 4.
Table 4. The three optimised separations.
optimised
response
pH CV sample
size
conc.
efficiency 9,20 12 2,84 4
separation 8,75 16 2,06 2
efficiency,
separation
9,20 16 2 2
Robustness
The robustness analysis was made in a
small range around the optimum point chosen
by the validation experiments. The results
show a dependence of less parameters as well
as a greater dependence of the most
important ones. A non-optimised pH can be
compensated with a correct gradient length.
Even if the sample concentration shows a
linear relationship with the separation it can
still be fairly high without loss of separation as
long as the sample size is kept low. All
together this means that the parameters that
are the most difficult to control can be
manageable through the other parameters that
are easy to set.
Hence the model is robust but since the
response is still linear it means that it is also
sensitive enough to provide correct
information.
-0.60
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
efficiency separation
pH
grad
conc
size
pH*size
Fig 6. The normalised coefficients for
the responses in the robustness
analysis.
Validation
As previous mentioned three optimum
separations where chosen and the validation
was made to see if MODDE predicted the
result correctly and if what response ought to
be used for optimisation.
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Table 5 The predicted and real
responses.
efficiency
(10
3
mm)
separation
Fig MODDE exp MODDE exp
7
-0,362,8
2,6
0,930,56
1,2
8
203,8
38
5,80,76
7,0
9
8,03,5
6,1
4,90,71
5,8
As seen above the prediction for
efficiency was good when it was optimised
but the separation tends to be more difficult
to predict. The reason for this could be that
the difference in actual figures are more
prominent then the difference in the real
separation.
Optimisation towards the response
separation was chosen for the robustness
analysis since a high efficiency leads to
extensive tailing and therefore product loss
(Fig. 6).
0
500
1000
1500
2000
2500
3000
3500
0 5 10 15 20
m
A
U
ml
Fig 7. Optimised efficiency. pH 9,20
CV12 4mg/ml 2,83 ml
Trying to optimise both separation and
efficiency leads to a very sharp front but the
actual separation is visually overestimated due
to the displacement of the weighted retention
time (fig 7).
0
500
1000
1500
2000
2500
0 5 10 15 20
m
A
U
ml
Fig 8. Optimised efficiency and
separation. pH 9,20 CV16
2mg/ml 2 ml
Optimising against optimum separation
leads to an early eluting peak of Gaussian
shape and hence little product loss (fig 8).
0
200
400
600
800
1000
0 5 10 15 20
m
A
U
ml
Fig 9. Optimised separation. pH
8,75 CV16 2mg/ml 2,06 ml
Conclusion
In order to get Gaussian peaks
optimisation towards efficiency should be
avoided, since it results in tailing of the IgG
peak and hence less recovery. The most
limiting parameter for a good separation is the
sample size. A large sample size tends to fill
the whole column with protein and the
separation is worsened. A non-optimised pH
can be compensated with the gradient. The
sample concentration can be high but the
sample size has to be kept low for a good
separation.
Design of experiments is a good way of
performing an optimisation and a robustness
analysis. It takes less experiments and
information about correlating factors is
achieved. An interesting fact is that the best
separation from MODDE also is the best
separation visually. The model is also robust
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as well as sensitive enough to provide accurate
results.
References
[1] Amersham Biosciences, Ion Exchange
Chromatography, Principles and methods,
Amersham Pharmacia Biotech, Bjrkgatan 30,
SE-751 84 Uppsala, Sweden, 2002
[2] Amersham Biosciences, Protein
Purification - Handbook, Amersham
Pharmacia Biotech, Bjrkgatan 30, SE-751 84
Uppsala, Sweden, 2002
[3] C F Poole, The Essence of
Chromatography, Elsevier, Amsterdam, 2003,
ISBN 0444501991
[4] Y-B Yang, K Harrison, J. Chromatogr. A,
743 (1996) 171-180
[5] R G Brereton, Chemometrics, Data
Analysis for the Laboratory and Chemical
Plant, John Wiley & Sons, Ltd, Chichester,
2003
[6] L Eriksson, E Johansson, N Kettaneh-
Wold, C Wikstrm, S Wold, Design of
Experiments - Principles and Applications,
Learnways AB, Stockholm, 2000
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