Bull Vet Inst Pulawy 48, 321-327, 2004
ISOLATION AND PURIFICATION OF POLYCLONAL IgG
ANTIBODIES FROM BOVINE SERUM BY HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY
JAN STEC, LEOKADIA BICKA AND JACEK KUŹMAK
Department of Biochemistry, National Veterinary Research Institute
24-100 Puławy, Poland
e-mail: [email protected]
Received for publication March 08, 2004.
Abstract
Three
different
high-performance
liquid
chromatographic (HPLC) techniques, i.e. affinity, ion
exchange, and gel filtration chromatography, have been used
to purify polyclonal antibodies from bovine serum. Polyclonal
antibodies were obtained from animals infected with bovine
leukaemia virus, which was confirmed by AGID and ELISA
tests. Precipitation of the antibodies by ammonium sulphate
prior to HPLC made possible to purify the antibodies in one
chromatographic step. However, if the highest purity is
required and separation IgG1 from IgG2, it should be used one
and/or two additional columns. In sodium dodecyl sulphate
polyacrylamide gel electrophoresis, the purest preparation
revealed only one band without tailing or any additional extra
peaks. Attempts were also made to purify the antibodies
without prior ammonium sulphate precipitation. But the best
results in immunoglobulins preparation were obtained in a
combination of affinity, ion-exchange and gel filtration
chromatography, and sample preparation by ammonium
sulphate precipitation and delipidation by n-hexane. These
preparations are comparable in purity to those commercially
available immunoglobulin standards. The rapid HPLC
techniques were found to be very useful for the purification of
polyclonal antibodies on a preparative scale, where sample
loading of up to 25 mg of serum protein could be fully
resolved in satisfactory yields.
Key words: cows, serum, IgG, HPLC,
purification.
Purification of immunoglobulins (Ig) is
required for many applications in numerous field of
science and technology. A growing list of purification
procedures has emerged, reflecting the heterogens nature
of this group of molecules and also different researchers,
demands for varying levels of purity. Most papers deal
with monoclonal antibodies from mouse ascites as a
starting material (3, 4, 7), and for the isolation of murine
monoclonal IgG antibodies, affinity chromatography on
immobilized protein A or G is probably the most
commonly used technique (11). However, considerable
attention has been paid to alternative methods, for
instance, when a special procedure is required in field of
veterinary or human medicine (6). General adsorption
technique,
using
high
performance
liquid
chromatography (HPLC), is attractive in this respect.
Recent works have shown that high performance ion
exchange and high performance size exclusion have the
potential for rapid isolation and/or purification of
monoclonal and polyclonal antibodies from different
range of biological material (2, 5). The classical
protocols for the isolation and purification of IgG
antibodies were frequently long and tedious, and often
did not result in a high degree of the purity. For
example, the purification of antibodies by conventional
precipitation with ammonium sulphate followed by
DEAE-cellulose chromatography which has been used
to isolate polyclonal and monoclonal antibodies from
mouse ascites fluid and animal serum requires several
hours to complete and is often limited by poor recovery
(11). Affinity chromatography using protein A has been
successfully employed for the purification of mouse and
human IgG, but one subclass (IgG1) was bound only
weakly, and another ( IgG3) did not bound at all (12).
Hydroxyapatite adsorption chromatography is very
useful for simple and rapid fractionation of proteins, but
has intrinsic limitations for routine use, and is difficult
to scale-up for IgG purification from serum of animal
species (8, 9). Some authors have demonstrated that IgG
from human serum could be partially purified on an
anion-exchange Mono Q column followed by gel
filtration on Superose 6 column (6, 10). Recently, a
mixed mode ion-exchange chromatography matrix,
utilizing silica gel as the support, was used for the rapid
purification of immunoglobulins. This chromatographic
matrix demonstrated little or no affinity for albumin,
transferrins, proteases or pH indicator dyes from tissue
culture media (7).
Separation and recovery of proteins from ionexchange chromatographic media are affected by factors
such as buffer type and pH, length of gradient, flow rate
of the mobile phase, ionic strength and nature of counter
322
ion, and characteristic of the proteins (13). The selection
of ideal conditions for protein purification involves
changing some or all of these parameters. Recent
development
of
high-performance
liquid
chromatography (HPLC) and fast performance liquid
chromatography (FPLC), using combination of several
columns, anion exchange, immunoaffinity, size
exclusion, gel filtration and hydrophobic interaction,
have opened up new possibilities for separation of
polyclonal immunoglobulins from animal sera.
In the present study, we have performed a
comprehensive evaluation of high performance anion
and cation exchange chromatography, immunoaffinity
chromatography, and high performance size exclusion
separation as a single-step technique or combine
protocol for purification of polyclonal IgG antibodies
from bovine serum. The samples were pretreated
similarly to allow a direct comparison between different
techniques. Ammonium sulphate precipitation and nhexane delipidation of serum prior to chromatography
separation in relation to the final purity of polyclonal
antibodies were also examined. Based on these findings,
we propose a strategy for the purification of polyclonal
IgG antibodies from bovine serum by high performance
adsorption chromatography techniques.
Material and Methods
Samples. Blood samples were collected from
cows infected with bovine leukaemia virus (BLV) by
jugular venipuncture and serum was prepared in the
usual manner, clarified by centrifugation (1000 g, 15
min) and prior to preparation of IgG diluted 1:1 with
sodium phosphtate buffer at pH 7.2. The presence of
polyclonal antibodies (pAb) against BLV was confirmed
serologically by AGID and ELISA.
Ammonium sulphate precipitation.
The
precipitation was performed at 40C. Equal volumes of
diluted serum and saturated ammonium sulphate were
mixed by slow addition of the ammonium sulphate
solution during gentle stirring. On the next day this
material was centrifuged (10 000 g for 20 min) and
washed twice with 50% saturated ammonium sulphate
solution. The precipitate was dissolved in distilled water
and dialyzed against phosphate buffer (10 mM Naphosphate + 0.15 M NaCl, pH 7.2). The precipitate
solution was mixed with equal volume of n-hexane and
centrifuged (20 000 g, 40C for 30 min) to remove lipids.
The final solution was filtered through a Millipore filter
(0.22 µm), and clear supernatant was loaded into the
column.
Instruments.
Whole
experiment
was
performed on HPLC instrumentation Akta Explorer 10 S
(Pharmacia Biotech, Uppsala, Sweden) consisting of a
solvent pump system model 900 and 910, gradient
module M-925, detection system UV 280 nm, pH/C-900
monitor, fraction collector Frac-900, recorder HP-890.
The HPLC system was controlled by software Unicorn
ver. 3.0 with a Compaq computer. The fractions were
collected every 0.5 - 2.0 min in volume program mode,
depending on the used method.
Ion exchange chromatography. Ion exchange
chromatography was performed on a Mono Q HR 5/5
anion exchange column (50 mm x 5 mm I.D., Phamacia
Biotech, Uppsala, Sweden) with starting buffer A, 20
mM bisTris propane (pH 9.5), and final buffer B, 20
mM bisTris propane containing 1 M NaCl (pH 7.5). The
gradient was generated over 20 min at a flow rate of 1
ml/min. The sample load was between 0.5 – 10 mg
protein injected with 0.1 – 2.0 ml sample loop.
Affinity
chromatography.
Affinity
chromatography was performed on a Protein G
Sepharose 4 (fast flow) 1 and 5 ml columns
(Pharmacia). Equilibration buffer was 0.02 M Naphosphate, pH 7.2, and eluting buffer was 0.1 M glycine
at pH 2.5. The column was washed with 0.1 M glycine
buffer at pH 2.0, following immediately with
equilibration buffer. The flow-rate used, at all stages
was 1 ml/min. Samples were applied on the column by
sample loop: 0.5 ml or 2.0 ml, and protein load was
between 0.1 – 25.0 mg for analytical and preparative
scale, respectively. The collected fraction of 1 ml
volume were immediately treated with neutralizing 1 M
Tris-HCl at pH 9.0.
Gel filtration. Gel filtration was carried out on
Superdex 200 Prep grade 10 x 300 columns
(Pharmacia). The column was equilibrated with 0.05 M
Na-phosphate buffer + 0.015 M NaCl, adjusted to pH
7.4. The samples (sample volume as rated in the manual
from the column supplier) were subjected to
chromatography at flow-rates of 0.5 ml/min. In order to
determine the molecular weight of all sample
components, a molecular weight calibration curve was
set up.
Buffer exchange. Buffer exchange or desalting
of collected fractions, were carried out on a Fast
Desalting HR 10/10 column packed with Sephadex G 25
Fine (Pharmacia). The samples were applied in 1.0 ml
volume by sample loop and subjected to
chromatography with buffer, into which the sample was
to be transferred, at a flow-rate of 2 ml/min.
SDS-PAGE electrophoresis. The purity of
various IgG preparations was checked by sodium
dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) of reduced and non reduced samples. The
total polyacrylamide concentration in the separating gel
was 8 or 12%. Coomassie brilliant blue R-250 was used
to visualize the protein bands. As a standard, the lowmolecular-weight (LMW) references from MBI
Fermentas (Vilnus, Lithuania) was used: β-galactosidase
MW 116 000; albumin MW 66 000; ovoalbumin MW
45 000; lactate dehydrogenase MW 35 000; restriction
endonuclease Mw 25 000; β-lactoglobulin MW 16 000;
lysozyme MW 14 000.
All samples and buffers used in this study were
filtered through 0.22 µm Millipore filters to remove
small particles, and buffers were additionally degassed
prior to use. After ten chromatographic separations on
each column the clean in place (CIP) was carried out,
according to Pharmacia protocols, to prevent loss in
performance of the columns. Protein determination was
323
performed by the Bradford method (1), using bovine
serum albumin as a standard.
Results
In the first set of the experiments, bovine serum
containing pAb against bovine leukaemia virus was
subjected to chromatography. Before chromatography,
ammonium sulphate precipitation and n-hexane
delipidation was used as a general purification step. The
result for the three consecutive columns are shown in
Figs. 1 A, B, and 2 C. The main peak of IgG, after
ammonium sulphate precipitation, was exclusively
isolated by affinity chromatography on Protein G
Sepharose column with a 0.1 M glycine eluting buffer at
pH 2.5, but the remaining ballast proteins were removed
with Na-phosphate starting buffer (Fig. 1 A). The
protein G is covalently bound to Sepharose, and is able
to trap IgG subclasses eg. IgG1, IgG2 and IgG3. The
most of the ballast proteins were eliminated in the flow
of starting Na-phosphate buffer at pH 7.2. Separation of
IgG fractions to the subclasses was not successful on
Protein G Sepharose column with stepwise elution
gradient of pH ranged from 3.5 to 2.2 and cannot be
recommended. When the purity of IgG fraction is not
very important, the ammonium sulphate precipitation
and protein G affinity chromatography can be used for
processing of the isolation and purification of bovine
polyclonal antibodies.
The affinity Protein G column served very well
on analytical scale with the protein load less than 0.5 mg
and also can satisfactory work on small-preparative
scale with the protein loading on the column up to 25
mg (Fig. 2 D). The chromatographic profile of this
separation gave very broad and flattened peaks,
however, the both peaks, the ballast protein and IgG
fraction were separated very well.
The second step of the purification of bovine
pAb consisted in chromatography by anion exchange
technique on Mono Q HR 5/5 column. This column
was used for separation of the IgG peak obtained from
the affinity column. The best results were obtained when
elution of the proteins was performed with 20 mM bisTris propane buffer at pH starting from 9.5 and ending
with the 7.5, applied with the linear gradient of the salt
from 0 to 1 M NaCl concentration over 20 min at a flow
rate of 1 ml/min. The resulting chromatogram has been
shown on Fig. 1 B. The elution profile gave at least three
main peaks. The first one was unidentified proteins as
the remaining portion of ballast materials and this
amount of waste proteins were removed in void volume
of the column. The next two peaks had retention volume
of buffer: 7.62 ml and 12.46 ml, and they completely
distinguished one from another. According to retention
volume of standard immunoglobulins, those two peaks
were identified as IgG1 and IgG2. In a similar
experiment, commercially available pAb from bovine
serum was tested and the obtained results of affinity
chromatography and anion exchange chromatography
were very close to those from the previous preparation.
When a diluted bovine serum was run by ion
exchange technique (Fig. 3 E) on Mono Q HR 5/5
column, the peak of IgG has been separated. According
to the chromatogram profile, there were many different
proteins accompanying the IgG fraction. The identity of
IgG peak was confirmed by the standard sample of
bovine IgG. However, the precipitation step removed
more than 50% of serum proteins, the majority of
proteins were the ballast proteins, among those the peak
of IgG made up only few percent of the total protein
content. The anion exchange column, Mono Q HR 5/5
was also used as the first column for the isolation of IgG
fractions from ammonium sulphate precipitation. The
results were satisfactory and there were obtained two
well separated peaks identified as IgG1 and IgG2.
However, the Mono Q HR column performance was
disturbed after the several consecutive runs of
ammonium sulphate preparation, and the routine
cleaning up procedure was necessary to restore
efficiency of the column. After cleaning in place (CIP),
as recommended by the manufacturer, the column was
tested for efficiency, to check column performance, by a
standard mixture of two proteins: β-lactoglobulin and
ovoalbumin at concentration of 2 mg/ml each (result not
shown). Even so, as the column was used for the next
series of chromatographic runs, the live time of the
columns have been shortened, and they should be
withdrawn, when the cleaning procedure did not restore
their performance. Because of high price of a Mono Q
column and reasonably lower an affinity column, this
was in support to used sequence of the two columns, the
first Protein G Sepharose followed by Mono Q.
Gel filtration chromatography on HP Superdex
200, 10/300 column was used as a final “polishing” step
to obtain IgG1 and IgG2 fractions of very high purity.
The column was loaded with 1 to 5 mg per ml of the
individual IgG subfractions or both together and the best
performance was achieved at a flow rate of 0.5 ml/min.
This step allowed to cut of the immunoglobulin with
molecular weight (MW) lower than 150 000 kD from
any other proteins, or the immunoglobulins partially
degraded during the all protocol of preparation. The
chromatogram profile of the fine purity IgG1 fraction on
the Superdex column is presented on Fig 2 C. The large
peak represents IgG1 fractions (retention volume 11.52
ml) separated from the small one (retention volume
10.79 ml) which contained the rest of impurities.
The purity of the various chromatographic fractions was
analysed by SDS-PAGE (Fig. 4). Lane a-1 contains the
pooled fractions from Fig. 1-A. There are present some
protein tailing bands from the bovine serum. The peaks
eluted from Mono Q column (Lane a-2) or Mono Q and
Superdex 200 (Lane a-3) show the highest purity. Lane
MW contains molecular weight marker proteins. SDS
PAGE with reducing condition is presented on Fig. 4-b.
The appropriate lanes: 5, 6, 7, and 8 correspond to the
same samples that in Fig. 4-a, 1, 2, 3, and 4,
respectively. On this figure, both subunits show
molecular weights about 50 kD, which correspond to
subunits of bovine IgG with broken - S - S - bonds.
324
mAU
A
:
pH
7
80
6
1200
waste
100
0
0
800
4
waste
5
10
15
60
IgG2
5
200
mS/cm
IgG1
1600
300
IgG
B
mAU
3
400
2
0
40
20
ml
0
10
15
20
25
ml
Fig. 1. A - Isolation of bovine IgG on Protein G Sepharose column. Low protein load (0.5 mg). Elution with
0.1 M glycine buffer at pH 2.5.
B - Chromatographic pattern for bovine polyclonal antibodies obtained on Mono Q HR column with
20 mM bis Tris propane buffer (pH 9.5→7.5) and (0→1 M) NaCl gradient elution.
D
C
pH
mAU
mAU
7
800
4000
600
3000
400
2000
4
200
1000
3
0
0
2
0
10
20
30
ml
waste
6
IgG
0
5
10
5
15
ml
Fig. 2. C - Gel filtration chromatography of pAb on Superdex 200 HR column. The large peak represents
IgG1. Sample load of 1 mg protein. Elution with 50 mM sodium phosphate buffer at pH 7.2.
D - Purification of bovine IgG on Protein G Sepharose column. High protein load (25 mg). Elution with
0.1 M glycine buffer at pH 2.5.
325
E
mS/cm
mAU
500
400
IgG
300
200
100
F
mS/cm
mAU
5.0
80
2500
60
2000
4.0
40
1500
3.0
20
1000
10
500
2.0
1.0
0
10
20
30
ml
0
0
5
10 15
ml
Fig. 3. E - Chromatographic separation of diluted bovine serum obtained on Mono Q HR 5/5 column. Sample
load was 2 mg protein. The elution with 20 mM bisTris propane buffer at 9.5→7.5 pH. The gradient
of 1 M NaCl 0→100% was generated over 20 min.
F - Chromatograpic pattern of buffer exchange on Sephadex G 25 Fine 10/10 column. The sample load
was 25 mg of protein after affinity chromatography. Isocratic elution with 20 mM bis-Tris propane
buffer at the flow rate of 2 ml/min.
a
MW
1
2
b
3
4
MW 5
116 kD
66
116 kD
45
66
6
7
8
35
25
18
45
35
Fig. 4. SDS-PAGE non-reduced (a) and 2-mercaptoethanol-reduced (b) samples from various purification steps.
MW - molecular weight reference, a-1 – sample used in Fig. 1 A (affinity), a-2 – sample used in Fig. 1 B
(anion exchange), a-3 – sample used in Fig. 2 B (gel filtration), a-4 – sample used in Fig. 3 F (buffer
exchange). Samples on gel b correspond to samples from gel a.
326
The analysis confirmed the high purity of IgG1 and
IgG2 fractions obtained by the three steps
purification procedure. From these results it is
obvious that the ammonium sulphate step gives a
final pAb preparation of higher purity than that
obtained if it is omitted. The yield of pAb after two
step procedure (ammonium sulphate precipitation
and Protein G Sepharose chromatography) was
found to be about 75%. From various experiments
with the Mono Q column followed by Superdex 200,
the protein recoveries in our experiments were
estimated to be about 50%.
During all experiments, samples to be
loaded into columns were applied in a starting
buffer. The buffer exchange and/or desalting after
gradient was performed by gel filtration
chromatography on Sephadex G 25 Fine column
(Fast Desalting HR 10/10). The flow rate of 2
ml/min and collected fractions of 1 ml allowed for
completely separation of a protein peak from salt of
a previous buffer. As an example, the typical
chromatogram profile of buffer exchange is showed
on Fig. 3-F. For example, the fraction of IgG, after
affinity column step on Protein G Sepharose, in 0.1
M glycine buffer pH 2.5 neutralized with 1 M TrisHCL to pH 7, was separated from IgG fraction,
removed and changed with 20 mM bis-Tris propane,
pH 9.5 as the starting buffer for the Mono Q HR
column. The protein peak was well separated from
the salt peak exactly at the bottom of both peaks, and
only such separation was acceptable.
Discussion
In this study, we have obtained highly
purified polyclonal IgG antibodies, of G1 and G2
subclasses from bovine serum in combination of
affinity,
ion-exchange
and
gel
filtration
chromatography using HPLC system. While the
present work deals with small scale purification, both
the affinity and ion exchange used here can easily be
scaled up.
Affinity chromatography with Protein G
Sepharose at pH 2.5 was shown to be a powerful
method for the isolation and purification of IgG
antibodies from bovine serum. This method is
attractive since mild condition is used and,
furthermore, since several of the proteins normally
found in serum do not bind to the column. The
capacity of the immunoaffinity bead is thereby used
for binding the protein of interest. Generally,
immunoaffinity column with protein G appears to be
particularly suited for the isolation of IgG molecules
from bovine serum. The affinity chromatography on
Protein A Sepharose did not bind strongly bovine
IgG and the performed experiment showed high
losses of IgG which could not be accepted. However,
this column is routinely used for the isolation and
purificaton of IgG fractions from human and some
animal species sera: mouse, rabbit, guinea pig, dog,
pig (9).
By anion exchange with Mono Q column at
pH 7.5 – 9.5, two IgG antibodies (subclasses IgG1
and IgG2) with isoelectric points above neutral, were
purified almost to homogeneity. The high pH of
starting buffer appears not to affect the
immunoglobulin molecules. Similar results were
obtained by anion exchange with Mono Q in other
studies (11). The capacity of this technique is
potentially lower than that of affinity with protein G
when the bovine serum precipitate is used as a
sample, since essentially all components in this fluid
are adsorbed to the column. Furthermore, extremely
tight binding of certain molecules may cause an
increase in column backpressure which necessitates
extensive cleaning procedure between experiments.
However, pretreatment of the sample by ammonium
sulphate precipitation or a similar method, may
eliminate this problems (12). Such pretreatment may
be particularly relevant when large sample volumes
are used.
Attempts were also made to purify
polyclonal antibodies by high performance gel
filtration on a Superdex 200 column. Evidently
immunoglobulins generally have the molecular
weight 150 kD compared to the other proteins in
serum. This technique has been successfully used for
the separation of IgG from ascites fluid and serum
(5). The IgG fractions, which we obtained by
additional purification on gel filtration using very
fine molecular sieving column, were completely
resolved from the proteins with molecular weight
other than 150 kD. The antibodies used in this study
were purified to near homogeneity in a combine
method by these techniques.
In conclusion, the following strategy for
combine HPLC purification of bovine IgG antibodies
from serum can tentatively be suggested, in cases
when other columns are considered less suitable: (i)
affinity chromatography on immobilized protein G at
pH 2.5 is the first choice for antibodies due to mild
condition
employed;
(ii)
anion
exchange
chromatography for antibodies with high isoelectric
points (>7.2), as the second column for further
purification and separation of IgG1 from IgG2: (iii)
gel filtration as the final “polishing” step to obtain
very pure individual classes of IgG antibodies. If
very high purification is not necessary, the
ammonium sulphate precipitation and affinity
chromatography on immobilized protein G should be
satisfactory, but for obtaining a completely
homogenous product, at least the second
chromatographic step should be included. The last
column optionally may be used for very fine
purification of obtained bovine IgG subclasses.
However, when ammonium sulphate precipitation
must be avoided, diluted bovine serum can be
subjected to chromatography directly on the anion
exchange Mono Q column 5/5, as a starting material.
327
Our results show that the HPLC techniques are
efficient for the purification of pAb and that the
analytical columns used in this study can be utilized
for preparative scale purification. The use of larger
columns will, of course, improve the capacity.
7.
8.
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