Study of imidazole performance as pseudo-affinity ligand in the purification of IgG from bovine milk

Article abstract of DOI:10.1016/j.ab.2020.113693

The spherical sepharose CL-6B beads were activated by epichlorohydrin in different epoxy contents (80, 120 and 160 μmol_epoxide/mL_gel) and, L-histidine and imidazole as pseudo-affinity ligands were covalently immobilized to them. Some linkers with different length, (1,2-ethanediol diglycidyl ether and 1,4-butanediol diglycidyl ether) were synthesized for activation of sepharose and the activated sepharose beads modified with imidazole and the performance of these adsorbents in the purification of immunoglobulin G from bovine milk were evaluated. Among the L-histidine bearing adsorbents, higher adsorption of IgG (0.28 mg/mL) was obtained by adsorbent with the lower concentration of L-histidine. The highest amount of IgG adsorption (0.53 mg/mL) was obtained by imidazole bearing adsorbent with the highest amount of imidazole and Among the adsorbents with synthesized linkers, the adsorbent with 1,2-ethanediol diglycidyl ether showed better performance and was able to purify 0.25 mg/mL IgG with high purity. The synthesized pseudo-affinity adsorbents represented the abbility to purify immunoglobulin G in one-step process with high purity and efficiency.

Full text of DOI:10.1016/j.ab.2020.113693

AnalyticalBiochemistry597(2020)113693
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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Study of imidazole performance as pseudo-affinity ligand in the purification
of IgG from bovine milk
Pariya Pourrostam-Ravadanaq, Kazem D. Safa∗, Hassan Abbasi
Department of Organic Chemistry and Biochemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
The spherical sepharose CL-6B beads were activated by epichlorohydrin in different epoxy contents (80, 120 and
160 μmolepoxide/mLgel) and, L-histidine and imidazole as pseudo-affinity ligands were covalently immobilized to
them. Some linkers with different length, (1,2-ethanediol diglycidyl ether and 1,4-butanediol diglycidyl ether)
were synthesized for activation of sepharose and the activated sepharose beads modified with imidazole and the
performance of these adsorbents in the purification of immunoglobulin G from bovine milk were evaluated.
Among the L-histidine bearing adsorbents, higher adsorption of IgG (0.28 mg/mL) was obtained by adsorbent
with the lower concentration of L-histidine. The highest amount of IgG adsorption (0.53 mg/mL) was obtained by
imidazole bearing adsorbent with the highest amount of imidazole and Among the adsorbents with synthesized
linkers, the adsorbent with 1,2-ethanediol diglycidyl ether showed better performance and was able to purify
0.25 mg/mL IgG with high purity. The synthesized pseudo-affinity adsorbents represented the abbility to purify
immunoglobulin G in one-step process with high purity and efficiency.
Immunoglobulin G
Affinity chromatography
Pseudo-affinity ligands
Histidine
Imidazole
Bovine milk
1. Introduction
reversible interactions between a protein and related ligand to isolate
the target protein [25–29]. These binding properties that offered by
Because of the vital role of antibodies in the biotechnology, medi-
cine, pharmaceutical, and food industries, isolation and purification of
antibodies are very important and it has increased dramatically over
the past decades [1–5]. Antibodies or immunoglobulins (Ig), are Y-
shaped, unique and soluble glycoproteins that are secreted by lym-
phocyte B cells in the immune system to counteract foreign antigens.
These specific antibody-antigen interactions have led to extensive ap-
plications of antibodies in various fields [5–9]. Immunoglobulin G (IgG)
is one of the important and interesting antibodies of bovine milk and
purification of this bioactive protein from bovine milk has attracted the
researcher's attention during the last years [10–14]. It is the most
common group of antibodies which is the smallest antibody among the
other immunoglobulins and it can be stable throughout the purification
process [15,16]. According to the important role of IgG for im-
munoaffinity chromatography, immunotherapy, drug delivery, and
treatment of immune disorders like alloimmunization, rheumatoid ar-
thritis, and cancer, it is very essential to isolate and purify it [17–22]. A
large number of methods, including precipitation, electrophoresis, fil-
tration, ion exchange, size exclusion, and affinity chromatography have
been used for purification of antibodies [23,24]. Affinity chromato-
graphy is a kind of separation technique that employs the unique and
affinity ligand are used for selective adsorption of target protein from a
protein mixture [30,31]. Recently for simplifying the purification pro-
cess and providing high selectivity, efficiency and easy recovery of
protein, considerable efforts have been made. To recompensate these
requirements, novel affinity methods have been developed by identi-
fying and designing new ligands and matrices. The matrices can be
employed in the affinity chromatography are divided into three groups:
natural (Agarose, dextrose and cellulose beads), synthetic (acrylamide,
polystyrene and polymethacralate derivatives) and inorganic type
(porous silica and glass) [1,32–34]. Also, the most bispecific ligands
that are extensively used for affinity separation of IgG, include protein
A and G, textile dyes, histidine, thiophilic ligands and chelated metals
[34–38]. Protein A chromatography is the most widely used method for
purification of immunoglobulins, which has some disadvantages like
high cost, ligand leakage, low stability, prone to degradation and loss of
antibody activity under severe elution conditions [39–45]. On the other
hand, pseudo-specific affinity chromatography can be extensively used
to purify biomolecules, which at the same time compensates for the
defects of protein A chromatography. In pseudo-specific affinity chro-
matography, the interactions among the immobilized ligand with the
target protein, depending on the complementarity of their shape,
∗ Corresponding author.
E-mail address: dsafa@tabrizu.ac.ir (K.D. Safa).
https://doi.org/10.1016/j.ab.2020.113693
Received 20 January 2020; Received in revised form 13 March 2020; Accepted 18 March 2020
Availableonline19March2020
0003-2697/©2020ElsevierInc.Allrightsreserved.

P. Pourrostam-Ravadanaq, et al.
AnalyticalBiochemistry597(2020)113693
charge and hydrophobicity [46–49]. The benefits and particular prop-
erties of histidine and imidazole as pseudo-affinity ligands have made
them unique. Imidazole can be found in the structures of important
biomolecules, including histidine, which has an important role in the
location of protein binding [50–52]. Furthermore, they possess some
specific properties such as good stability, mild hydrophobicity, a wide
range of pKa values, the asymmetric carbon atom and weak charge
transfer due to the presence of imidazole moiety [46,53–57].
Herein we report the design and preparation of new adsorbents
based on sepharose for purification of immunoglobulin G from bovine
milk in one step and with high purity and efficiency. The sepharose
beads activated by 1,2-ethanediol diglycidyl ether, 1,4-butanediol di-
glycidyl ether, and epichlorohydrin in different binding capacities and
L-histidine and imidazole ligands were covalently attached to them. The
performance of modified beads and effects of the binding capacity, li-
gand type and linker length on the purification of immunoglobulin G
from bovine milk were evaluated.
2.3.1. 1,2-Ethanediol diglycidyl ether (EDDGE)
1H NMR (400 MHz, CDCl3): δ 3.59–3.71 (m, 4H, OCH2CH2),
3.77–3.80 (dd, 2H, OCH2), 3.38–3.42 (dd, 2H, OCH2), 3.13–3.16 (m,
2H, CH2(O)CH), 2.58–2.60 (dd, 2H, CH(O)CH2), 2.76–2.78 (dd, 2H, CH
(O)CH2).
2.3.2. 1,4-Butanediol diglycidyl ether (BDDGE)
1H NMR (400 MHz, CDCl3): δ 1.62–1.63 (m, 4H, –CH2-), 3.45–3.50
(m, 4H, OCH2CH2), 2.55–2.56 (dd, 2H, CH(O)CH2), 2.73–2.75 (dd, 2H,
CH(O)CH2), 3.09–3.11 (m, 2H, CH2(O)CH), 3.30–3.35 (dd, 2H, OCH2),
3.65–3.68 (dd, 2H, OCH2).
2.4. Epoxy activation of sepharose
The sepharose CL-6B was activated with epichlorohydrin, 1,2-
ethanediol diglycidyl ether, and 1,4-butanediol diglycidyl ether by
procedures as previously described elsewhere with some modifications
[58,59].
2. Experimental
2.4.1. Activation of sepharose with epichlorohydrin (Sep-Epch)
In a round-bottom flask equipped with a magnetic stirrer, 1 g of
washed and suction dried sepharose CL-6B was added into the aqueous
solutions of 2 mL NaOH (0.5 M, 1 M, 2 M) containing 5 mg sodium
borohydride. Epichlorohydrin (0.075, 0.15, 0.25 mL) was added to the
mixture and shaken at 45 °C for 6 h. The activated gels were washed
with an excess of distilled water and dried under vacuum conditions.
Finally, epoxy activated sepharose gels with various amounts of ep-
oxide groups (containing around 80, 120, 160 μmol of epoxide groups/
mL gel) were obtained.
2.1. Materials
Sepharose CL-6B was obtained from Arg. Biotech. Co. (Tabriz, Iran).
L-histidine (98%, Sigma-Aldrich), imidazole (99%, Sigma-Aldrich), 1,2-
ethanediol (99%, Sigma-Aldrich), 1,4-butanediol (99%, Sigma-Aldrich),
sodium borohydride (98%, Sigma-Aldrich), sodium sulfate (99%,
Sigma-Aldrich), tetrabutylammonium chloride (97%, Sigma-Aldrich),
acetic acid (99%, Sigma-Aldrich), sodium acetate (99%, Sigma-Aldrich)
and sodium carbonate (99%, Dae-Jung) were used as received without
further purifications. Sodium hydroxide (97%) and epichlorohydrin
(99%) were obtained either from Merck (Darmstadt, Germany) and
used as received. All the commercial solvents such as CH2Cl2 were
distilled before use.
2.4.2. Activation of sepharose with EDDGE and BDDGE (Sep-EDDGE and
Sep-BDDGE)
In a round-bottom flask equipped with a magnetic stirrer, 1 g of
washed and suction dried sepharose CL-6B was added into the aqueous
solution of 1 mL NaOH 1 M containing 2 mg sodium borohydride. Then
1 mL EDDGE or BDDGE was added to the reaction mixture and in-
cubated overnight on a shaking at room temperature. The activated gels
were washed with an excess of distilled water and vacuum dried.
2.2. Instrumentation
The 1H NMR spectra of synthesized compounds were recorded using
a Bruker FT-400 MHz (Bruker Co., United States) spectrometer in
deuterated chloroform (CDCl3) as solvent at room temperature. The FT-
IR spectra of synthesized compounds were recorded in/on pressed KBr
plate on a Bruker-Tensor 270 (Bruker Co., United States) spectrometer.
The morphology, microstructures and particle size distribution of the
samples were analyzed by scanning electron microscopy (SEM) and
Energy-dispersive X-ray spectroscopy (EDX) using an MIRA3 FE-SEM
(TESCAN, Czech Republic) instrument. Fast protein liquid chromato-
graphy (GE healthcare Amersham AKTA FPLC UPC-900 P-920 INV-
907 M-925 system) was used for purification of target protein from
protein mixture. The electrophoresis analysis was carried out on an
SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
page by a Mini-PROTEAN® 3 (Bio-Rad Co., United States) cell.
2.5. Determination of epoxy groups
The determination of epoxy groups on the activated sepharose gels
was carried out according to the method described by Sundberg and
Porath [60]. 150 mg of modified gel, was added into the 15 mL of 1.3 M
sodium thiosulfate solution and titrated with 0.1 M hydrochloric acid.
2.6. Immobilization of L-histidine and imidazole on epoxy activated
sepharose (Sep-Epch-His and Sep-Epch-Im, Sep-EDDGE-Im and Sep-
BDDGE-Im)
1 g of suction-dried epoxy activated sepharose with different epoxy
content (80, 120 and 160 μmolepoxide/mLgel), EDDGE or BDDGE acti-
vated Sepharose, was transferred to 25 mL shaking flask containing
200–500 mg L-histidine or imidazole in 2 mL sodium carbonate
(Na2CO3) 2 M and 5 mg sodium borohydride. The reaction mixture was
stirred at 50 °C for 18 h. In the end, the modified sepharose gel was
washed with plenty of distilled water and vacuum dried.
2.3. General method for synthesis of diglycidyl ether intermediates
Diglycidyl ether intermediates were prepared under phase-transfer
catalytic condition: To the vigorously stirred mixture of epi-
chlorohydrin (9.34 mL, 120 mmol), sodium hydroxide pellets (4.8 g,
120 mmol), water (0.5 mL, 28 mmol), and tetrabutylammonium
chloride (0.28 g, 1 mmol), cooled Glycol (20 mmol, 1,2-ethanediol or
1,4-butanediol) was added dropwise. After the completion of the ad-
dition, the mixture was stirred for 45 min at 40 °C. The solid products
that were formed alongside the reaction removed by filtration and
washed with CH2Cl2 (30 mL×3). The combined organic layers were
dried over anhydrous Na2SO4 and additional dichloromethane and
excess epichlorohydrin evaporated off by distillation to give a yellow
oil. The resulting oil was purified by distillation under reduced pressure
to give a pure and colorless product (89% yields).
2.7. Chromatographic separation of IgG from skim milk
For preparation of the skim milk, the fresh bovine milk was cen-
trifuged at 6000 rpm for 15 min at room temperature to remove the fat
in the form of the upper cream layer. The pH of milk was adjusted in 4.6
with HCl 1 N and centrifuged again at 6000 rpm for 15 min to pre-
cipitate the casein. Afterward, the clear skim milk was collected and the
pH raised to 7 by 1 N NaOH to protect immunoglobulins from
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P. Pourrostam-Ravadanaq, et al.
AnalyticalBiochemistry597(2020)113693
denaturation. The targeting antibody was purified from the pre-
prapared skim milk by using an automated chromatography system (GE
healthcare Amersham AKTA FPLC) and the column with the dimension
of (6 cm × 1 cm i.d.) to give a bed volume of approximately 2 mL. The
outlet of the column was connected to a UV monitor (UV monitor UPC-
900 P-920) and to a fraction collector (Frac-950 fraction collector). The
whole process of chromatography was performed at the flow rate of
1 mL/min at room temperature. The resulted modified sepharose gels
were packed into the column and it was equilibrated with 25 mM
acetate buffer, pH = 5 (equilibration buffer). 50 mL of skim milk
containing 30 mg IgG was injected into the column and washed with
equilibration buffer until any protein wasn't detected by absorption at
280 nm. Elution was carried out with the same buffer +0.2 M NaCl and
absorption measured at 280 nm. For regeneration of the column, the
50 mM NaOH solution, water, and equilibration buffer were used.
Determination of Protein concentration was performed by Bradford's
method [61].
imidazole ligand was immobilized onto the surface of activated se-
pharose beads (Fig. 1).
The two diglycidyl ether linkers, 1,2-ethanediol diglycidyl ether
(EDDGE) and 1,4-butanediol diglycidyl ether (BDDGE), with different
length were also synthesized by the reaction of 1,2-ethanediol and 1,4-
butanediol with epichlorohydrin in the presence of sodium hydroxide
and tetrabutylammonium chloride as a phase-transfer catalyst at room
temperature (Fig. 2).
The sepharose beads were activated with synthesized diglycidyl
ethers (EDDGE and BDDGE) to obtain Sep-EDDGE and Sep-BDDGE gels
respectively. The imidazole was then immobilized on the activated
sepharose beads surface (Sep-EDDGE and Sep-BDDGE) by the ring-
opening base reaction to prepare Sep-EDDGE-Im and Sep-BDDGE-Im
respectively (Fig. 3). The synthesized adsorbents are summarized in
Table 1.
3.1. FT-IR study of adsorbents
2.8. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE)
The FT-IR spectrum of adsorbents Sep-Epch-His with binding ca-
pacities of 80 and 120 μmolepoxide/mLgel is depicted in Fig. 4. As can be
seen in Fig. 4A, the characteristic peak of O–H and N–H stretching vi-
brations are appeared at 3436 cm−1 as a strong and broad peak. The
vibration peak at 2918 cm−1 is assigned to the aliphatic C–H. The
characteristic peaks of C]C bond can be observed at 1465 cm−1. The
absorption peaks that are appeared at 1039 cm−1 and 1186 cm−1 as-
sociated to the C–N and C–O vibrations.
The obtained elution fractions from chromatographic runs were
analyzed by SDS-PAGE to determine the purity of aim antibody using a
Mini-PROTEAN® 3 system (Bio-Rad, USA) as described by Laemmli
[62]. Acrylamide was used at 10.0% in the running gel and 4% in the
stacking gel. Samples with pre-prepared sample buffer (5 μL of sample
buffer +15 μL of the sample) were placed in a boiling water bath at
95 °C for 5 min to denature the proteins. The samples were loaded onto
a gel and run at a constant voltage of 30 V. Finally, the protein bands
were stained with the Coomassie Brilliant Blue R-250 for 30 min and
destained for 3–4 h [63].
In the FT-IR spectrum of Sep-Epch-His (120 μmolepoxide/mLgel
(Fig. 4B), the vibration peaks of the O–H and N–H are appeared at
3434 cm−1. The C–H aliphatic vibration gives a peak at 2921 cm−1
)
.
The vibration peak of the carbonyl group of acid (COOH) due to the low
concentration of the ligand compared to the sepharose appears as a very
small peak at 1710 cm−1. The vibration peak assigned to the C]C bond
can be observed at 1466 and around 1519 cm−1. The peaks are ap-
peared at 1051 and 1178 cm−1 represent the C–O and C–N bonds.
3. Results and discussions
The pseudo-specific affinity adsorbents with a different type of
linkers and ligands were used for the isolation and purification of im-
munoglobulin G from bovine milk. to prepare these pseudo-specific
affinity adsorbents, the surface of the sepharose beads were activated in
two different binding capacities (80 and 120 μmolepoxide/mLgel) using
epichlorohydrin by means of nucleophilic substitution reaction. The L-
histidine ligand was attached to the surface of activated sepharose
beads via its amine group by the ring-opening base reaction. In the
following, the surface of the sepharose beads was coated by epi-
chlorohydrin using nucleophilic substitution reactions with three dif-
ferent binding capacities (80, 120 and 160 μmolepoxide/mLgel) and then
The FT-IR spectra of Sep-Epch-Im (80, 120, 160 μmolepoxide/mLgel)
are represented in Fig. 5. Fig. 5C is depicted the FT-IR spectrum of Sep-
Epch-Im (80 μmolepoxide/mLgel). The peaks assigned to the stretching
vibration of O–H and C–H aliphatic bonds are observed at 3432 cm−1
and 2901 cm−1 respectively.
The peak centered around 1500 cm−1 is represented the C]C bond
and the peaks appeared at 1070 cm−1 and 1175 cm−1 are associated
with vibrations of C–O and C–N bonds. The FT-IR spectrum is re-
presented in Fig. 5D is belonged to the Sep-Epch-Im (120 μmolepoxide
/
mLgel). The observed peak at 3437 cm−1 and 2918 cm−1 are related to
the O–H and C–H aliphatic bonds respectively. The characteristic peaks
Fig. 1. Synthesis of (a) Sep-Epch-His and (b) Sep-Epch-Im.
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P. Pourrostam-Ravadanaq, et al.
AnalyticalBiochemistry597(2020)113693
Fig. 2. Synthesis of (a) EDDGE and (b) BDDGE
Fig. 3. Synthesis of (a) Sep-EDDGE-Im and (b) Sep-BDDGE-Im.
Table 1
Synthesized adsorbents with different linkers, ligands, and binding capacities.
Entry
Adsorbent
Binding capacity (μmolepoxide/mLgel)
1
2
3
4
5
6
7
Sep-Epch-His
Sep-Epch-His
Sep- Epch-Im
Sep- Epch-Im
Sep- Epch-Im
Sep-EDDGE-Im
Sep-BDDGE-Im
A
B
C
D
E
F
80
120
80
120
160
–
G
–
of C]C bond are appeared at 1463 and 1516 cm−1 and the vibrations
of C–O and C–N bonds appeared at 1048 and 1183 cm−1. The broad
and sharp peak in comparison with spectrum 5C indicates a large
amount of epoxy and loading of ligand groups in the Sep-Epch-Im (120
μmolepoxide/mLgel).
The FT-IR spectrum of 5E belongs to Sep-Epch-Im (160 μmolepoxide
/
mLgel). The O–H vibration appears at 3438 cm−1, which in comparison
to the spectra 5C and 5D has a wide and strong peak. The absorption
peak at 2923 cm−1 represents C–H aliphatic and two peaks at 1464 and
1522 cm−1 represent C]C bond. The vibrations of C–O and C–N are
observed at 1044 and 1187 cm−1. Due to the high content of the epoxy
groups and ligand, the peaks were appeared stronger and sharper than
others (5C and 5D).
Fig. 4. FT-IR spectra of A) Sep-Epch-His (80 μmolepoxide/mLgel) and B) Sep-
Epch-His (120 μmolepoxide/mLgel).
vibrations of C]C and the appeared peaks at 1053 and 1170 cm−1
associated to the vibrations of C–O and C–N bonds respectively.
Fig. 6 represents the FT-IR spectra Sep-EDDGE-Im (6F) and Sep-
BDDGE-Im (6G). The stretching vibrations of O–H and C–H aliphatic
peak are observed at 3562 cm−1 and 2926 cm−1 respectively. The peak
is related to the C]C bond observed at 1518 cm−1 and the peaks at
1030 and 1180 cm−1 represent the C–O and C–N bonds.
In Fig. 6G the stretching vibration of O–H and C–H aliphatic bonds
appear at 3436 cm−1 and 2926 cm−1 respectively. The absorption
peaks are appeared a 1463 cm−1 and 1516 cm−1 associated to the
3.2. Energy dispersive X-ray spectroscopy (EDX)
For further investigation of the chemical composition of prepared
pseudo-affinity adsorbents Energy-Dispersive X-ray (EDX) spectroscopy
was carried out and shown in Fig. 7(a–h). The EDX analysis of se-
pharose shows only oxygen (O) and carbon (C) related to the chemical
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P. Pourrostam-Ravadanaq, et al.
AnalyticalBiochemistry597(2020)113693
adsorbent has approximately dimension with an average size of
44–177 μm, which in comparison with the size of the initial sepharose
CL-6B particles there is no significant change in the particle size and
distribution and there is no accumulation of particles after the surface
modification.
3.5. IgG adsorption studies
The chromatographic results (Fig. 10-a) showed that both Sep-Epch-
His adsorbents with a binding capacity of 80 (A) and 120 (B) μmo-
l
epoxide/mLgel have the capability to attach to IgG and isolate it from
bovine milk. Adsorbent A demonstrated more IgG adsorption efficiency
in comparison with adsorbent B. It is due to the high amount of epoxy
groups and the high concentrations of L-histidine ligand on the surface
of the adsorbent B. It seems that there is a repulsive force between the
free COOH groups and proteins, that leads to a reduction in the anti-
body recovery. The results of the chromatography and SDS-PAGE of
adsorbents A and B are shown in Figure (10-I and 11). According to the
obtained data, adsorbent A was able to purify target antibody with a
yield of 46.66% and purity of > 80% from bovine milk. The results of
the purification process are represented in Table 2.
Fig. 5. FT-IR spectra of Sep-Epch-Im C) 80, D) 120 and E)160 μmolepoxide
/
mLgel
.
The results obtained from Figure (10-II, 11) showed that all three
adsorbents bearing imidazole (C, D, and E) have the capability to ad-
sorb IgG from bovine milk with different efficiency and adsorption
capacity. As can be seen, IgG with a molecular weight of 25 KDa (light
chain) and 50 KDa (heavy chain) is observed as single bands in SDS-
PAGE analysis with negligible impurities (Fig. 11). The adsorbent E
demonstrated the best performance in adsorbing IgG due to the highest
content of epoxy groups (160 μmolepoxide/mLgel) and the highest con-
centration of imidazole ligands (Fig. 10). According to obtained data
adsorbent E purified IgG from the bovine milk with a yield of 88.33%
and purity of > 85%. The results are summarized in Table 2.
To evaluate the linker length in the purification process, the se-
pharose activated by the synthesized linkers (EDDGE and BDDGE). The
activated sepharose was modified with imidazole by the means of ring-
opening base reaction to form adsorbent Sep-EDDGE-Im and Sep-
BDDGE-Im. The obtained results from chromatography (Fig. 10-III)
revealed that both adsorbents Sep-EDDGE-Im and Sep-BDDGE-Im had
the ability to separate IgG from bovine milk. The interaction of both
adsorbents was specific to IgG. It can be seen in the SDS-PAGE analysis
that IgG was detected as a single bond without any further impurities
(Fig. 11).
The adsorbent F shows better performance in adsorption of IgG than
adsorbents G and it was able to purify IgG from the bovine milk with
the yield of 41.66% and the purity of > 90% (Table 2).
Fig. 6. FT-IR spectra of F) Sep-EDDGE-Im, G) Sep-BDDGE-Im.
Histidine is one of the amino acids that could be hold a positive
charged in the side chain. Histidine has an isoelectric point (pI) of 7.59
and if the pH falls to 5, it can take a positive charge. The bovine im-
munoglobulin with pI value between 5.5 and 8.3 can became positively
charged at a pH of 5. It seems that positive charge of ligand and bio-
molecule, resulted a repulsive force leads to the separation of IgG from
the ligand [56].
composition of sepharose. Existence of the nitrogen (N), in Fig. 7(b–h)
confirmed the immobilization of histidine and imidazole by means of
ring-opening reaction on the sepharose surface.
3.3. Microscopic images
Microscopic images of the prepared adsorbents were performed by
using an optical microscope and results shown in Fig. 8. As could be
seen in Fig. 8(a–e), the spherical structures of particles are in micro size.
The structure of the adsorbent particles after activating their surface
with the linkers and ligands has remained spherical and no accumula-
tion and aggregation detected in the structure of particles.
3.6. Comparison with the related literature
There are a lot of reported articles about the different adsorbents
with the various ligands and binding capacities for affinity purification
of IgG in the literatures [47,64]. Premysl et al. [65] reported the pur-
ification of IgG using thiophilic ligands attached to crosslinked agarose
bead with the yield of 0.29–0.32 mg IgG/mL and the purity of 74% and
Protein G-Sepharose 4FF with binding capacity of 0.38–0.42 mg IgG/
mL and purity of 81% from bovine milk. Denizli and Pishkin reported
adsorption capacity of 24.0 mg HIgG/g with protein A-immobilized-
poly(2-hydroxyethylmethacrylate) [66]. Muller-Shulte et al. [67] used
several polymeric adsorbents made of different polymers and histidine
as the ligand and obtained adsorption values of 0.05–0.23 mg IgG1/mL
3.4. Scanning electron microscopy (SEM)
The morphologies of the prepared adsorbents and sepharose CL-6B
were evaluated by scanning electron microscopy (SEM) with different
magnification and depicted in Fig. 9. As could be seen in Fig. 9a, the
sepharose has a spherical shape with a dimension of 44–177 μm. Ac-
cording to SEM images, the particle size and distribution in each
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P. Pourrostam-Ravadanaq, et al.
AnalyticalBiochemistry597(2020)113693
Fig. 7. EDX spectra of a) sepharose CL-6B b) Sep-Epch-His (80 μmolepoxide/mLgel) c) Sep-Epi-His (120 μmolepoxide/mLgel) d) Sep-Epch-Im (80 μmolepoxide/mLgel) e)
Sep-Epch-Im (120 μmolepoxide/mLgel) f) Sep-Epch-Im (160 μmolepoxide/mLgel) g) Sep-EDDGE-Im h) Sep-BDDGE-Im.
of sorbent. The different commercial protein A affinity chromatography
matrices including Affi-Gel, Eupergit, Ultrogel, Sepharose series, and
Prosep A with adsorption capacities of 0.7–20.1 mg IgG3/g were re-
ported by Fuglistaller [45]. D. Muller-Schulte reported the purification
of IgG with sepharose adsorbent and histidine ligand about 0.2 mg/mL
[68]. Cu (II) metal ion affinity adsorbents were applied for purification
of IgG with the yield of 97.4% with an 8-fold purification by Sulakshana
Jain and Munishwar N. Gupta [37]. Stefano Menegatti et al. [35] were
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P. Pourrostam-Ravadanaq, et al.
AnalyticalBiochemistry597(2020)113693
Fig. 8. Microscopic images of adsorbents: a) sepharose Cl–6B, b) Sep-Epch-His, c) Sep-Epch-Im, d) Sep-EDDGE-Im, e) Sep-BDDGE-Im.
recovered IgG from skim milk with 74% yield and 92% purity using a
hexamer peptide ligand.
sepharose through ring-opening reaction. The adsorption behaviors of
IgG on adsorbents bearing L-histidine demonstrated that the adsorbent
with lower content of epoxy groups and ligand showed better perfor-
mance with adsorption of 0.28 mg/mL IgG from bovine milk. A sig-
nificant increase in IgG adsorption capacity was achieved by imidazole
bearing adsorbent with the highest epoxy groups and ligand content up
to 0.53 mg/mL. Besides, adsorbents with imidazole ligand separate
immunoglobulin G with higher purity than histidine. The result re-
presents that among the adsorbents with different linkers such as
EDDGE and BDDGE, EDDGE showed better performance in adsorption
of IgG. It purified the IgG about 0.25 mg/mL without any impurities.
The results obtained from chromatography and SDS-PAGE analysis
4. Conclusion
In summary, we designed and synthesized novel adsorbents based
on sepharose with various content of epoxy groups and binding capa-
cities, ligand type and linker length that showed good potential to
purify immunoglobulin G from bovine milk. The sepharose surface has
been activated by epichlorohydrin, 1,2-ethanediol diglycidyl ether and
1,4-butanediol diglycidyl ether. L-histidine and imidazole as pseudo-
affinity ligands were then covalently immobilized on activated
Fig. 9. SEM image of adsorbents: a, b) sepharose CL-6B c) Sep-Epch-His d) Sep-Epch-Im e) Sep-EDDGE-Im f) Sep-BDDGE-Im.
7

P. Pourrostam-Ravadanaq, et al.
AnalyticalBiochemistry597(2020)113693
Fig. 10. Effect of the different ligands, linkers and binding capacities on the adsorption and elution of bovine IgG by affinity chromatography on adsorbents (A–G).
conditions: bed volume, 2 mL; injection volume, 50 mL of skim milk containing 30 mg IgG; flow rate, 1 mL/min washing was carried out using 25 mM acetate buffer,
pH = 5. Elution was carried out using 25 mM acetate buffer +0.2 M NaCl, pH = 5. Column regeneration was done with the 50 mM NaOH solution, water, and
equilibration buffer.
Table 2
Results of IgG adsorption from bovine milk by adsorbents A-G.
Entry
adsorbent
Adsorbed IgG (mg/mL)
Yield (%)
1
2
3
4
5
6
7
A
B
C
D
E
F
0.28
0.15
0.17
0.19
0.53
0.25
0.16
46.66
25.00
28.33
31.66
88.33
41.66
26.66
G
Abbasi: Writing - review & editing, Software.
Acknowledgements
Financial support provided by University of Tabriz, Iran, is grate-
fully acknowledged. Also we would like to express our gratitude to Prof.
Siavoush Dastmalchi for his kind help.
Fig. 11. The SDS-PAGE analysis of eluted fractions of skim milk on adsorbents
A-G. Lane 1) skim milk, lane 2) fraction eluted from adsorbent A, lane 3)
fraction eluted from adsorbent B, lane 4) fraction eluted from adsorbent C, lane
5) fraction eluted from adsorbent D, lane 6) fraction eluted from adsorbent E,
lane 7) fraction eluted from adsorbent F, lane 8) fraction eluted from adsorbent
G, lane 9) standard protein mark.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ab.2020.113693.
confirmed that the novel synthesized adsorbents are able to purify the
IgG antibody in one step with high purity, and in the least time.
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