Optimization of Affinity Chromatography in Purification of

Monoclonal Antibodies (IgG)

Nirmal Kumar Ganesan, Nurul Mufitdhah and Malin Wollens
Downstream Processing-Protein Factory
Flensburg University of Applied Sciences

Abstract: optimization of buffer and running condtion is

required in order to seperate specific type of
Affinity separation is the most selective IgG1.
type of chromatography used in purification
technology due to its high resolution. To
Keywords: Affinity chromatography,
optimize the protein purification process of a
technical mouse IgG, Protein A column,
monoclonal antibody, the affinity
Protein G column, Elution buffer,
chromatography column with the best
Equlilbration buffer.
suitable ligands and suitable buffers for
equilibration and elution should be selected. Introduction:
Binding capacities of Sartobind Protein A
membrane and HiTrap Protein G HP column Monoclonal antibodies (mAbs) are highly
against technical grade mouse IgG was
specific antibodies produced using
evaluated. Eluting capacity of five different
elution buffers such as Glycine Hcl, Glycine hybridoma cells. Each monoclonal
NaCl, Sodium citrate, Citric acid and acetic
antibody will only recognize one epitope
acid where tested against both columns.
Resulting fractions were analyzed by on an antigen, therefore they have a high
Bradford assay and ELISA assay. In terms of specificity that has significant
total protein elution acetic acid is the best
elution buffer for both columns. If a mixture advantages, particularly in therapeutic
of different IgGs are to be separated, protein applications [1]. Current major
G column with sodium phosphate as running
therapeutic applications of monoclonal
buffer and acetic acid as elution buffer is the
most suitable. For a product containing only antibodies include cancer, chronic
IgG1, protein A is most appropriate. Further inflammatory disease, and infection and
they constitute the largest and fastest immunoglobulins (Ig) [5]. Protein G is a
growing sector of the biological bacterial cell wall protein expressed in
pharmaceutical industry [2]. Group C and G streptococci and type III
Fc-receptor binding to all mouse IgG
For therapeutic applications, monoclonal subclasses. Its binds preferentially to the
antibodies should be produced in very Fc portion of IgG, but can also bind to
high quality. In the pharmaceutical the Fab region, making it useful for
industry, chromatography is a critical and purification of F(ab)2 fragments of IgG1.
widely used separation and purification Some species and IgG subtypes bind
technology due to its high resolution [3]. differentially to Protein A or Protein G [6].
Affinity separation is the most selective
type of chromatography used in In the previous study of the purification
biotechnology. It separates proteins process to obtain the anti-CD45 antibody
based on a reversible interaction from hybridoma cell line CF-10H5, there
between a protein and a specific ligand has been a problem in the affinity
coupled to a chromatography matrix. The chromatography step. One possible
technique is a commonly used step in reason might be that the elution time was
purification processes [4]. too short and and the elution volume was
too small; therefore some IgGs were still
An efficient way of purifying mAbs is by bound to the column matrix [7]. Another
using high affinity ligands as protein A possibility is that the buffer system was
and G. The high affinity of protein G and not effective or the protein binding to the
protein A for the Fc region of polyclonal column was too weak. Therefore, it is
and monoclonal immunoglobulin G (IgG)- necessary to optimize the buffer system.
type antibodies forms the basis for Appropriate buffer conditions for binding
purification of IgG, IgG fragments and elution steps in affinity purification
containing the Fc region, and IgG are as varying as the types of molecules
subclasses [4]. Protein A is a component applied and their chemical binding
of the cell wall of the bacterium properties.
Staphylococcus aureus and specifically
binds to the Fc-region of various
Unique binding or elution conditions exist of the product, several methods of
for certain affinity systems. However, analysis are also needed. This analysis
conditions for binding and elution of such as ELISA for IgG determination,
antibody-antigen affinity interactions and Bradford test for measuring the total
(immunoaffinity) are more predictable. protein concentration is necessary from
Antibody-antigen binding usually is most the start, and than after each and every
efficient in aqueous buffers at single step during the whole downstream
physiological pH and ionic strength. process.
Elution often can be accomplished by Materials and methods:
raising or lowering the pH or altering the
Optimization of affinity chromatography
ionic state to disrupt the binding
in purification of monoclonal antibody
interaction [8].Which type of elution
includes selection of suitable binding
condition (pH, ionic strength, chaotrope,
ligands for IgG and strong elution buffer
or denaturant) is most effective for a
(see Figure 1). 100 g of Technical
particular antibody-antigen interaction
grade IgG from mouse serum with 80%
depends on the specific composition of
purity diluted with PBS buffer was used
ionic, hydrophobic and hydrogen bonds
for each run of the optimization process.
involved. The ideal elution buffer
Analytics were done before and after the
effectively releases the antibody or
affinity chromatography.
antigen without irreversibly denaturing or
inactivating them [8].
Technical mouse IgG
In this study the optimization process
includes the selection of the column with
Analytics Bradford assay and ELISA assay
the best suitable ligands (protein A or G)
and suitable buffers for equilibration and
Affinity Chromatography
elution. This can be achieved by
repeating the experiment in small scale
with both columns using different buffers Protein A Protein G
for the elution with the same
equilibration. In order to monitor the Elution buffer optimization

development and know the current status Analytics Bradford assay and ELISA assay

Result
 purification of desired IgG type, usage of
suitable Fc receptor column is required.
Figure 1: Process flow diagram.
The suitable methods for the
Affinity Chromatography:
chromatographic experiment were
The NGC Biorad HPLC device was used created and running conditions were
for the affinity chromatographic adjusted corresponding to the protein
purification process. Bacterial Fc columns, by the software Chromlab from
rceptors are the important tools in the Biorad (see Table 2 and 3).
affinity purification of immunoglobulins
The pumps were primed with the
[9].
respective buffers by using a 10 ml
Table 1: Binding of mouse IgGs to Protein A and G [12][13] syringe. Flow rates were adjusted to 2

HiTrap ml/min and 1.5 ml/min for protein A and

Sartobind
Protein G column, respectively. Maximum
Species Subclass Protein A
G HP pressure was set to 44 psi. Sartobind
membrane
column
Protein A membrane (2 ml) from
Mouse IgG1 + ++++ Sartorius Stedim Biotech GmbH and
HiTrap Protein G HP column (1 ml) from
IgG2a ++ ++++
GE Healthcare were used to compare
IgG2b +++ +++
the binding capacity of Protein A and G
IgG3 ++ +++ regarding IgG. 1 ml Sample containing
100 g of technical IgG was injected by
+ means weak binding and ++++ the strongest binding
using a sample injection loop.

The best studied Fc receptors are Table 2: Running condition for affinity chromatography
Protein A column [12]
protein A from Staphylococcus aureus
and protein G from Streptococcus Flow rate 2 ml/min
species, which can bind with IgG from
Sample volume 1.0 ml
different sources [10,11]. The binding
Max. pressure 44 psi
capacities of both ligands for different
mouse IgG types are shown below (see Wavelengths 210, 254, 280, 495 nm

table 1). To obtain the efficient

Sartobind Protein A membrane was of elution buffers. The collected fractions
connected to the column port number. were analysed using Bradford assay and
0.1 M PBS buffer was used as ELISA assay.
equilibration buffer.

Table 3: Running condition for affinity chromatography Table 4: Titer analysis for neutralization buffer
Protein G column [13]
Amount of Tris HCl
Flow rate 1.5 ml/min Elution buffer
(pH 9.0)

Sample volume 1.0 ml

0.1 M Glycine Hcl (pH 450 l

Max. pressure 44 psi 2.8)

Wavelengths 210, 254, 280, 495 nm 0.1 M Glycine NaCl 350 l

(pH 2.8)

0.1 M Sodium citrate 2.1 ml

The elution buffer was optimized by (pH 3.8)

repeating the experiment with various 0.1 M Citric acid (pH 2 ml

elution buffers in the following order: 0.1
M Glycine HCl (pH 2.8), 0.1 M Glycine
with 2M NaCl (pH 2.8), 0.1 M Sodium
citrate (pH 3.8), 0.1 M Citric acid (pH 3)
and 1 M Acetic acid (pH 3). To attain the
pH of 7.2, Tris HCl (pH 9.0) was used as
a neutralization buffer. The amount of
neutralization buffer to be added was
determined by using titre analysis.
The eluted sample with a volume of 10
ml was collected in a tube containing 1M
Tris HCl (pH 9.0) with an volume
according to the elution buffer (see Table
4). The same experiment was repeated
with 0.02 M sodium phosphate (pH 7.2)
as equilibration buffer with the same set
3)
1 M acetic acid (pH 3) 8 ml
Analytical methods:
For analytics 96-well-plates Microlon 96K
f-form cap from Greiner Bio-One were
used for Bradford and ELISA.
Bradford assay [14]:
The total protein content was determined
using Bradford assay. The Bradford
reagent containing 0.01% (w/v) comassie
blue G250, 4.7% (w/v) ethanol and 8.5%
(w/v) phosphoric acid was prepared and
 plate was incubated for 4.5 mins. 200 l
Total
Conce Bradfor
protein
of 0.9% NaCl was used as blank.
Volum ntratio d
concent Table 5: Bradford assay overview
e (l) n reagent
ration
(l/ml) (l)
(l/ml)
The plate was measured in a
BSA - 25 200 175 200
spectrophotometer at 595 nm after the
Dilu 1
incubation and the concentration was
BSA - 25 100 175 100 calculated.
Dilu 2
ELISA Assay [15]:
BSA - 25 50 175 50
Dilu 3 [delete, is double] Plates were coated
BSA - 25 25 175 25 with 100 l anti-mouse IgG coating
Dilu 4 antibody and incubated over night at

BSA - 25 12.5 175 12.5

room temperature. Unbound antibodies
Dilu 5 are rinsed two times with washing buffer
containing PBS and 0.05% (v/v)
BSA - 25 6.25 175 6.25
Dilu 6 Tween20 and blocking buffer containing
1% (w/v) BSA and PBS was added.
BSA - 25 3.125 175 3.125
Plates were incubated at 37C for one
Dilu 7
hour with constant shaking at 60rpm.
Sampl 25 Unkno 175 Unknow
The plates were washed thrice with 200
e- wn n
l of washing buffer. The samples to be
the extension value was evaluated. The measured and antibody standard mouse-
reagent was filtered using filter paper IgG was diluted serially with dilution
before it was used for the assay. Before buffer containing 0.1% (w/v) BSA in
measuring the samples they were PBS-buffer. 100 l of diluted standard
centrifuged at 10.000 g for 2 mins. A and diluted samples were added.[Make
standard curve was created by using more sentences it out of it and be more
BSA standard. The unknown samples specific with the added volumes] 100 l
and the BSA standard were diluted with of dilution buffer was used as blank. The
0.9% NaCl (see Table 5). Finally, capped plates were incubated at 37C
Bradford reagent was added and the for one hour. After incubation plates were
washed thrice with 200 l of washing Figure 1 and Error! Reference source not
buffer. Then 100 l of freshly mixed found. show the amount of protein in
detection antibodies containing percentages for the two different
IgG+alkaline- columns, the bars grouped after the
different elution buffers. We could see
phosphatase-conjugate [Sigma-A-5153]
that there are huge differences in the
were added to each well followed by an
amount of protein in the fraction of the
incubation of 1 hour. Substrate solution
sample application. That is an expected
was added to each well (100 l/well) and
result due to the fact that the used
the first well was monitored for the
elution buffers are picked out from
reading of the absorption at 410 nm.
different sources [4] and compared in
Once the monitored wells attain
order to find the best one. We applied
absorption of 410 nm the enzyme
100g/mL of sample in a volume of 1 mL
substrate reaction was stopped by
on each column in each run. The
adding 100 l NaOH and the plates were
maximum loading capacity of Sartobind
read again. IgG -concentration can be
Protein A membrane and HiTrap Protein
calculated by the normalized extinction
G HP column are 5mg/mL [13] and
values from the absorption values. By
11mg/mL [12]. Therefore, we can
using the logit values and respecting the
exclude overloaded columns as
dilution factor the antibody concentration
explanation for the binding behaviour. A
is calculated.
possible reason is the specificity of the
Results & Discussion columns.

Protein A column
100.0
Protein amount [%]

80.0
60.0
40.0
20.0
0.0
Glycine Glycine Citric Sodium Acetic
HCl NaCl acid citrate acid

Sample application Column wash Elution

Figure 1: Protein amount for protein A column given for all

fractions of the chromatography (sample application, column
wash and elution) for each of the five elution buffers (Glycine Buffers column column
HCl, Glycine NaCl, Citric acid, sodium citrate and acetic acid)
in percentages.
Glycine HCl 48,3 40,1

Protein G column Glycine NaCl 31,1 69,2

100.0
Protein amount [%]

80.0
Citric acid 46,8 59,5
60.0
40.0
Sodium 39,3 29,6
20.0
0.0 citrate
Glycine Glycine Citric Sodium Acetic
HCl NaCl acid citrate acid
Acetic acid 22,8 3,4
Sample application Column wash Elution

Figure 2: Protein amount for protein G column given for all

One aim was to reduce the overall loss
fractions of the chromatography (sample application, column
wash and elution) for each of the five elution buffers (Glycine during the affinity chromatography. The
HCl, Glycine NaCl, Citric acid, sodium citrate and acetic acid)
in percentages. loss in percentage is given in

When protein G columns is binding with Protein A Protein G

Elution
a broad affinity and protein G column Buffers column column
much more specific only particular types
Glycine HCl 48,3 40,1
of IgG, our finding could be explained.
Glycine NaCl 31,1 69,2
Our applied sample is technical IgG
containing different types of IgG and not Citric acid 46,8 59,5
one purified type. Therefore, differences
Sodium 39,3 29,6
in the affinity of the columns is citrate
reasonable.
Acetic acid 22,8 3,4
This finding allows to work according to
, showing that the loss for acetic acid is
the purpose. when all available mouse
the the lowest with only 3,4% in the
IgG should be bound then protein G
protein G column and 22,8% in protein A
column is the best choice, binding a
column. This is a quite good result for the
specific IgG is not possible with this
most successful purification step in the
column but rather with protein A column.
downstream process. The highest loss
Table 6: Loss of protein in percentage according to the has glycine NaCl after protein G column
different elution buffers listed for both columns.
with 69,2%, which is still better then the
Elution Protein A Protein G
result from the last years group In terms
of the best elution buffer acetic acid is
doing the best job and Glycine HCl is Protein A column
another good possibility for both. Since 100.0

IgG amount [%]

80.0
the aim is to elute antibodies and not 60.0
only proteins, a more specific test i.e 40.0
20.0
ELiSA is performed in order to detect 0.0
Glycine Glycine Citric Sodium Acetic
and quantify IgG. But the process is HCl NaCl acid citrate acid
aiming for this and furthermore can Non-binding Binding

proteins still be detected if antibodies are

Figure 4: IgG content of protein A column in percentages,
not working anymore or degraded. In
sorted after the different elution buffers into columns,
order to be able to compare both tests showing the non-bonding and binding amount of IgG

the same results as above are displayed

Protein G column
differently below.
180.0
IgG amount [%]
150.0
The performed ELiSA is a Sandwich 120.0
90.0
ELiSA detecting all types of mouse IgG. 60.0
30.0
Only the standard used is only containing 0.0
Glycine Glycine Citric Sodium Acetic
IgG1. Again the applied sample is HCl NaCl acid citrate acid
measured and the fraction of the flow Non-binding Binding
through called non-binding and the
elution in the following binding. Figure 4 Figure 5: IgG content of protein A column in percentages,
sorted after the different elution buffers into columns,
and Figure 65 show the IgG content of showing the non-bonding and binding amount of IgG

both columns, but in order to compare

both figures there has to be paid some
attention to the y-axis. For protein A
column the axis goes up to 100%
whereas for protein G column the axis
ends at 180%. Apparently, there can be
nearly nothing found for the non-binding
fraction for both columns. Only very low
results distinctly below 5%, nearly visible
in the figures.
The first viewpoint lies on the very small
bar for the elution with acetic acid. For
both columns nearly nothing can be
found although the protein content in the
analysis before was quite high. This can
be easily explained with the harsh
conditions the antibody is kept in with an
aggressive elution buffer in relatively
high molarity of 1M and low pH of 3.0. The exact values of the loss of IgG in
This might simply have degraded the percentages for each elution buffer and
antibodies but not harming the protein, column can be find in Table 7.
the basic component of each antibody.
Table 7: Loss of IgG in percentages grouped for the columns
Therefore, the high protein and very low after the different elution buffers, a plus in front of the number
meaning no loss but an increase of the IgG
IgG amount are reasonable. Same holds
Protein A Protein G
true for the citric acid in the protein G Elution buffer
column column
column.
Glycine HCl 28,3 +55,1

Secondly, it is quite astonishing that the Glycine NaCl 34,8 +66,8

results for protein G column with the Citric acid 66,4 99,9
buffers glycine HCl and glycine NaCl are
Sodium citrate 28,9 59,2
far above 100%. How can more then the
Acetic acid 93,2 96,7
applied IgG be washed off the column ?
It seems that there are old samples still
bound on the column and washed off For all experiments a potential error
with a stronger elution buffer as both source might be the multiple use of each
glycine containing buffers seem to be. column without cleaning after every step.
Therefore, for further experiments, for
For protein A column there are three
more precise results the column should
really good working buffers all reaching a
be cleaned after every run. Otherwise a
yield of approximately 70%. Glycine HCl
phenomenon like the one for the IgG
and Glycine NaCl are eluting a lot of the
content of protein G column for the
IgG and this results are consistent with
glycine-containing buffers might occur.
the findings of the protein amount.
Therefore, there seems to be no Conclusion
degradation, so the buffer is gentle
Regarding the purpose of the affinity
enough with our antibody and eluting
chromatography the column should be
only in a range which fits to the applied
chosen very specifically. If the product
sample.
contains all IgGs in the end, no matter
which species, then protein G column
should be used with sodium citrate or
testing other buffers due to the high loss process development for monoclonal
that should be expected. antibody production. Landes
Bioscience mAbs 2:5, 480-499.
If the product contains only one specific
5. GE Healthcare. 2016. Affinity
IgG as IgG1, then protein A column
Chromatography Handbook, Vol. 1:
should be used with either glycine HCl or
Antibodies
glycine NaCl with no risk of a very high
6. Bjrck L. and Kronvall G. 1984.
loss or degradation of the antibody.
Purification and some properties of
Acknowledgements streptococcal protein G, a novel IgG-
binding reagent. J. Immunol. 133:
The authors thank the University of Applied
969-974.
Sciences Flensburg for providing the
7. GE Healthcare. 2016. Affinity
laboratory equipment and rooms. Special
Chromatography Handbook, Vol. 1:
thanks go to Prof. Dr Antje Labes, Prof. Dr.
Antibodies
Hans-Udo Peters and Dipl.-Ing. Leif Tretbar
8. Thermo Fisher Scientific Inc. 2009.
for the support and collaboration.
Optimize elution conditions for
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