Protein Expression and Purication xxx (2015) xxxxxx

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Protein Expression and Purication

journal homepage: www.elsevier.com/locate/yprep

A hydrophobic interaction chromatography strategy for purication

of inactivated foot-and-mouth disease virus
Hao Li a,b,1, Yanli Yang a,b,1, Yan Zhang a, Songping Zhang a, Qizu Zhao c, Yuanyuan Zhu c, Xingqi Zou c,
Mengran Yu a,b, Guanghui Ma a, Zhiguo Su a,
a
b
c

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
University of Chinese Academy of Sciences, Beijing 100049, PR China
China Institute of Veterinary Drug Control, Beijing 100081, PR China

a r t i c l e

i n f o

Article history:
Received 8 April 2015
and in revised form 28 April 2015
Available online xxxx
Keywords:
Foot-and-mouth disease virus
Hydrophobic interaction chromatography
Purication
Downstream processing

a b s t r a c t
A purication scheme based on hydrophobic interaction chromatography was developed to separate
inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and
8.8-fold purication were achieved on Butyl Sepharose 4FF. Further purication on Superdex 200
resulted in another 29-fold purication, with 92% recovery. The columns were coupled through an intermediate ultraltration unit to concentrate the virus. The entire process was completed in about 3.5 h,
with 75% nal FMDV recovery, and 247-fold purication. The nal product had purity above 98%, with
over 99.5% of host cell DNA removed. High-performance size exclusion chromatography (HPSEC),
Western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM) indicated that
the puried virus contained the required antigen, and was structurally intact with a spherical shape and a
particle size of 28 nm.
2015 Elsevier Inc. All rights reserved.

Introduction
Foot-and-mouth disease is an acute and contagious disease of
cloven-hoofed livestock such as pig, sheep, and cattle [1].
Recently, widespread breakouts were reported in the United
Kingdom, Argentina, and Uruguay, with serious consequences on
agriculture and international trade [24]. New vaccines, for example, the self-assembling capsid protein of the foot-and-mouth disease virus (FMDV)2, have been developed [57]. Nevertheless,
vaccines prepared from inactivated FMDV remains the most
effective [2].
FMDV is a spherical particle consisting of one molecule of
single-stranded positive-sense RNA and a protein capsid [8]. The
capsid contains four structural proteins: VP1, VP2, and VP3 with
molecular weights between 25 and 34 kDa, and VP4 of 810 kDa
[8]. Typically, FMDV is produced in baby hamster kidney cells, separated from cell debris, and then inactivated [9,10]. It remains
Corresponding author. Tel.: +86 10 62561817.
E-mail address: zgsu@ipe.ac.cn (Z. Su).
Both authors contributed equally to this work.
2
Abbreviations used: FMDV, foot-and-mouth disease virus; IEC, ion-exchange
chromatography; HIC, hydrophobic interaction chromatography; SEC, size exclusion
chromatography; HPSEC, high-performance size exclusion chromatography; TEM,
Transmission electron microscopy; DLS, dynamic light scattering; HBsAg, hepatitis B
surface antigen; DRT, dimensionless residence time.
1

quite common to formulate the vaccine from this relatively crude

inactivated antigen [9]. However, as the public has become
increasingly concerned with the safety and quality of livestock
products, regulatory agencies are considering raising the quality
standard for FMDV vaccines. Therefore, more stringent purication
of inactivated FMDV may become necessary to remove host cell
proteins and DNA, which may cause unwanted side effects [11].
FMDV has been puried by PEG precipitation [10,12], sodium
sulfate precipitation [13], ultraltration [13,14], and aqueous
two-phase partition [13]. These methods are useful, but not adequate to achieve high purity [15]. High purity has been achieved
in the laboratory by ultracentrifugation in a sucrose density
gradient [2,12]. However, ultracentrifugation is unsuitable for
industrial-scale production, because it suffers from low capacity
as well as high capital and operational cost.
Chromatographic techniques may provide an alternative strategy, on the basis of high selectivity and scalability [16]. Indeed,
many vaccines, including inuenza virus and recombinant hepatitis B surface antigen (HBsAg), have been successfully produced
through chromatography [17,18]. In addition, size exclusion
chromatography (SEC) was recently used as an analytical tool to
quantify inactivated FMDV [2]. Nevertheless, we are not aware of
any report that describes a complete, effective program to purify
inactivated FMDV by chromatography.

http://dx.doi.org/10.1016/j.pep.2015.04.011
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Please cite this article in press as: H. Li et al., A hydrophobic interaction chromatography strategy for purication of inactivated foot-and-mouth disease
virus, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.04.011

H. Li et al. / Protein Expression and Purication xxx (2015) xxxxxx

A classic chromatographic technique is ion-exchange chromatography (IEC), which is often used to purify antigens such
viruses [19,20] and virus-like particles [21,22]. However, interaction with IEC media may sometimes alter the structure of purication targets, with disastrous results. For example, HBsAg
disassembles during IEC, resulting in substantial loss [23,24]. For
some viruses like FMDV, structural integrity is critical. The virus,
with sedimentation coefcient 146S when live, is highly unstable
and tends to dissociate into smaller particles with sedimentation
coefcient 12S [25]. The immunogenicity of 12S particles is extremely low and unprotective [26]. What is worse, the virus was
found more unstable after being inactivated, even though the inactivating agents like binary ethyleneimine were thought to only
alter the viral RNA without effects on the capsid proteins [27].
Therefore, any chromatographic separation should preserve the
intact FMDV structure.
In this paper, we describe our efforts to purify inactivated FMDV
through chromatography. We initially attempted to use IEC to purify FMDV from cell culture supernatant. However, it was difcult to
achieve both satisfactory recovery and purication fold even after
screening of IEC media and separation conditions. In addition,
desalting by dialysis or dilution was typically required as an extra
preliminary step to reduce the salt concentration in the initial
crude.
Thus, we evaluated hydrophobic interaction chromatography
(HIC) as an alternative, which separates molecules based on
hydrophobicity instead of charge. The technique has never been
used to purify FMDV; indeed, HIC is far less frequently used than
IEC in vaccine preparation. Nevertheless, recovery of over 98%
was achieved when HBsAg was puried by HIC, more than twice
the recovery from IEC [28]. In addition, HIC may be more appropriate than IEC for inactivated viruses, the structure of which may be
sensitive to ionic interactions with IEC media.
The nal purication scheme combines HIC with ultraltration
and SEC. The scheme was optimized by screening various chromatographic media and separation conditions. High-performance
size exclusion chromatography (HPSEC), Western blotting,
dynamic light scattering (DLS), and transmission electron microscopy (TEM) were used to evaluate the puried product.

Materials and methods

Materials
All reagents were analytical grade, and solutions were prepared
using Milli-Q water (Millipore, USA). Separations were executed on
an KTA Purier 100 from GE Healthcare (USA). HIC, IEC, and SEC
media were obtained from GE Healthcare (Uppsala, Sweden). For
HIC, we evaluated Butyl-S Sepharose 6FF, Butyl Sepharose 4FF,
Octyl Sepharose 4FF and Phenyl Sepharose 6FF (high sub).
DEAE-Sepharose FF and ANX Sepharose 4FF (high sub) were evaluated for ion-exchange. SEC was performed on chromatography
media with different separation range, including Sepharose 4FF,
Sepharose 6FF and Superdex 200. Chromatography columns
(200  16 mm I.D., 1000  26 mm I.D.) were from GE Healthcare
(USA).

Virus
FMDV strain O China 1999 was propagated at industrial scale in
BHK-21 cell suspension cultures and inactivated by binary ethyleneimine [2]. Cell debris was removed, and the supernatant containing virus particles was collected. The FMDV supernatant was
originally obtained from Lanzhou Veterinary Research Institute

(Chinese Academy of Agricultural Sciences, China) after cultivation

and pretreatment as described above.
Evaluation of hydrophobic interaction chromatography media
To evaluate the suitability of hydrophobic interaction to separate FMDV, we rst analyzed the media by static adsorption with
increasing hydrophobicity, including Butyl-S Sepharose 6FF, Butyl
Sepharose 4FF, Octyl Sepharose 4FF and Phenyl Sepharose 6FF
(high sub). The effect of ionic strength was studied using 01 M
(NH4)2SO4. Generally, about 0.2 g of drained media,
pre-equilibrated in an appropriate buffer at pH 8.0 with a dened
concentration of (NH4)2SO4, was mixed with 0.8 mL FMDV suspension that had been pre-diluted with 5.6 mL buffer. After static
adsorption for 24 h at 25 C on a shaking incubator at 170 rpm,
the supernatant was analyzed for remaining FMDV and proteins.
Purication by hydrophobic interaction chromatography and ionexchange chromatography
For chromatographic purication by HIC, (NH4)2SO4 was added
to 125 mL FMDV supernatant in amounts required to achieve a target concentration, and the resulting mixture was gently stirred
until the salt dissolved. Then the supernatant was loaded onto a
HIC column (50  16 mm I.D.) individually packed with the HIC
media pre-equilibrated with phosphate buffer (pH 8.0) containing
a desired concentration of (NH4)2SO4. FMDV was eluted stepwise
with decreasing (NH4)2SO4 concentration. Fractions were collected
and analyzed.
To compare purication efciency with HIC, FMDV was also
puried on IEC media, including DEAE-Sepharose FF and ANX
Sepharose 4FF (high sub). Thus, 125 mL FMDV was diluted with
20 mM phosphate buffer pH 8.0 until the ionic strength dropped
below 4 mS/cm. Suspensions were then passed through IEC media
packed in 50  16 mm I.D. columns and pre-equilibrated with
20 mM phosphate buffer pH 8.0. Samples were eluted stepwise
with increasing NaCl concentration. As before, fractions were collected and analyzed.
All experiments were executed on KTA Purier 100 at
2.0 mL/min. Residence time is presented as dimensionless residence time (DRT), which is dened as DRT = RT/(CV/Flow rate),
where RT is residence time and CV is column volume.
Concentration of HIC-puried FMDV
FMDV fractions from HIC were concentrated by pump ltration
(Masterex L/S, USA) through a PES membrane with molecular
weight cut-off 100 kDa (VivaFlow50, Sartorius, Germany). Briey,
about 60 mL FMDV fractions were concentrated to about 20 mL
at room temperature and 130 mL/min. The membrane was ushed
between samples using 0.2 M NaOH at pressure less than 1 bar
until ux returned to the initial value.
Size exclusion chromatography
Size exclusion chromatography was used to rene purication
products. SEC columns with different separation ranges, namely
Sepharose 4FF, Sepharose 6FF, and Superdex 200, were evaluated.
Samples of 510 mL concentrated, HIC-puried FMDV was loaded
on a column packed with one of the three media (900  26 mm
I.D.), followed by elution with pH 8.0 phosphate buffer containing
0.15 M NaCl. The ow rate was 3.0 mL/min controlled by KTA
purier 100 and the detection wavelength was set to 259 nm.
Residence time was converted to dimensionless residence time
as described.

Please cite this article in press as: H. Li et al., A hydrophobic interaction chromatography strategy for purication of inactivated foot-and-mouth disease
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H. Li et al. / Protein Expression and Purication xxx (2015) xxxxxx

Fig. 1. The effect of (NH4)2SO4 concentration on binding of FMDV to Butyl-S Sepharose 6FF (A), Butyl Sepharose 4FF (B), Octyl Sepharose 4FF (C) and Phenyl Sepharose 6FF
(high sub) (D).

HPSEC quantication of intact FMDV

Intact FMDV was quantied by high-performance size exclusion
chromatography (HPSEC) as described previously [29]. The analysis was performed on a TSK G4000 SWXL (300  7.8 mm I.D.) analytical column (Tosohaas, Stuttgart, Germany) using an Agilent
1100 HPLC series system (Agilent, USA) with an inline degasser
and variable-wavelength detector operating at 259 nm. Samples
of 100 lL were injected and eluted at 0.6 mL/min with 50 mM
phosphate buffer pH 7.2 containing 100 mM Na2SO4.
A standard curve for intact FMDV was constructed using preparations puried by sucrose gradient ultracentrifugation according
to published methods [2,12]. The concentration of the standard
was calculated from UV absorbance, using extinction coefcient

Fig. 2. Elution prole of FMDV from a 50  16 mm I.D. column of Butyl Sepharose

4FF. A sample of 125 mL crude FMDV was loaded at 0.8 M (NH4)2SO4 and 20 mM
phosphate, and then eluted in one step to 0.6 M at a ow rate of 2 mL/min.

E1%
259;1cm = 76 [30]. A dilution series of the standard was then analyzed by HPSEC. The standard eluted as a single peak at 13.4 min,
as previously reported [29]. The peak area at 259 nm was linearly
proportional to FMDV concentration, with R2 = 0.998 over the
tested range between 0 and 60.2 lg/mL. Thus, the FMDV concentration in samples was obtained from the peak area at 259 nm.
Quantication of residual host cell protein and DNA

Table 1
Purication efciency on HIC and IEC.

Media

Protein recoverya
(%)

FMDV recoveryb
(%)

Purication
foldc

Butyl Sepharose
4FF
Phenyl Sepharose
6FF
DEAE Sepharose FF
ANX Sepharose
4FF

12

89

7.2

8.2

26

3.2

13
12

69
68

5.2
5.4

Protein recovery is the ratio between proteins eluted after chromatography and
the total protein loaded, as determined by Bradford assay.
b
FMDV recovery is the ratio between total FMDV eluted from chromatographic
columns and total FMDV loaded, as measured by HPSEC.
c
Purication fold is the ratio between FMDV and proteins recovered.

Protein concentration was determined according to a modied

Bradford method, as described elsewhere [31]. Bovine serum albumin was used as reference standard.
Host cell DNA was determined by selectively staining double
stranded DNA using Quant-iT PicoGreen reagent and kits
(Invitrogen), following the manufacturers instructions. A microplate reader (Varioskan ash, Thermo scientic, USA) was used to
analyze samples at excitation and emission wavelengths 480 nm
and 520 nm, respectively.
Western blot, dynamic light scattering and transmission electron
microscopy
Puried FMDV was analyzed by Western blot as described elsewhere [29], except that we used a secondary antibodies labeled

Please cite this article in press as: H. Li et al., A hydrophobic interaction chromatography strategy for purication of inactivated foot-and-mouth disease
virus, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.04.011

H. Li et al. / Protein Expression and Purication xxx (2015) xxxxxx

Table 2
Separation efciency on different SEC columns.

Column

Separation range (kDa)

Resolution

Peak Widtha (DRT)

Sepharose 4FF
Sepharose 6FF
Superdex 200

6020,000
104000
10600

0.71
0.89
2.96

0.29
0.26
0.05

Peak width is dimensionless residence time, as dened in the text.

with Alexa Fluor 790 (Jackson ImmunoResearch, USA). Following

the manufacturers protocol, uorescence was measured at 800 nm
using the Odyssey imager (Li-Cor, USA).
The size distribution of puried FMDV was determined by
dynamic light scattering (DLS) in a Zetasizer Nano ZS (Malvern
Instruments, Malvern, UK). Samples were ltered through a
pre-rinsed 0.22-lm lter, followed by equilibration to 25 C. A
minimum of three measurements were collected from each sample, and results are expressed in number-weighted percentage
distribution.
Finally, the structure of the puried virus was analyzed by
transmission electron microscopy using a Philips FEI Tecnai 20
TEM (Royal Philips Electronics, Amsterdam). Samples obtained by
chromatography was spotted on a 400-mesh copper grid, dried,
stained with 1% uranyl acetate and visualized under TEM.
Results and discussion
Characterization of the FMDV crude
The crude supernatant obtained from industrial-scale cell culture at Lanzhou Veterinary Research Institute was characterized
after virus inactivation and separation from cell debris. The pH of
the FMDV supernatant was about 8.6, with ionic strength about
17.7 mS/cm. The suspension contained 0.4 mg/mL total protein,
but only 1.9 lg/mL FMDV, indicating that the initial virus pool is
contaminated with many unwanted proteins.
Purication of FMDV by hydrophobic interaction chromatography
Recombinant hepatitis B surface antigen was successfully puried with high recovery through HIC [17,28]. To test if HIC is also
suitable to purify FMDV, as well as to optimize separation parameters, several HIC media were rst evaluated by static adsorption

Fig. 3. Elution of HIC-puried FMDV from a 900  26 mm I.D. Superdex 200

column. HIC-puried FMDV was concentrated by ultraltration to 7 mL and passed
through SEC at 3 mL/min using phosphate buffer containing 0.15 M NaCl.

Fig. 4. SDSPAGE of samples from each step of purication. Lane 1, crude FMDV; 2,
HIC-puried virus; 3, retentate in ultraltration; 4, SEC-puried FMDV. The identity
of the product from SEC was veried by Western blot against VP1, shown as lane 5.

over a range of ionic strength (Fig. 1). In most cases, FMDV binding
increased with (NH4)2SO4 concentration, until complete binding is
achieved near 1.0 M (NH4)2SO4. However, less than 60% of the virus
bound to Butyl-S Sepharose 4FF even at this concentration, suggesting that this media is not hydrophobic enough to adsorb
FMDV efciently. Indeed, FMDV binding increased with the
hydrophobicity of the media. Thus, more than 90% of FMDV bound
to Phenyl Sepharose 6FF in 0.6 M (NH4)2SO4, while only about 25%
bound to Butyl Sepharose 4FF. Nevertheless, the virus bound only
weakly to Octyl Sepharose 4FF, even though it is more hydrophobic
than Butyl Sepharose 4FF. One possible explanation is that impurities displace FMDV from Octyl Sepharose 4FF. Conversely, interaction between impurities and Butyl Sepharose 4FF may also be too
weak to interfere with FMDV binding. On the other hand, interaction between FMDV and Phenyl Sepharose 6FF may be sufciently
strong to overcome interference by contaminants.
Taken together, these results suggest that HIC purication may
be achieved by loading FMDV in high salt, and then eluting in low
salt. To maximize recovery, we selected the concentration step
over which FMDV binding changed most dramatically. This step
is 0.80.6 M for Butyl Sepharose 4FF, and 0.60.4 M for Phenyl
Sepharose 6FF.
The chromatographic prole by Butyl Sepharose 4FF is shown in
Fig. 2. Fraction A contains impurities owing through the column,
but not FMDV. The UV absorbance of fraction A was high, but the
protein recovery was only 16%. DNA determination showed more
than 80% of the DNA in supernatant ew through the column in
fraction A, which is because of the weak hydrophobicity of nucleic
acids. As would have been expected from the initial binding experiments, most FMDV was eluted in fraction B, which was collected
at 0.6 M (NH4)2SO4, leading to 89% FMDV recovery with a purication fold of 7.2. Residual bound FMDV as well as contaminants
were eluted in fraction C. Notably, when FMDV was eluted from
Phenyl Sepharose 6FF over several step gradients, resulting in
low fold-purication of 3.2 and low FMDV recovery of only 26%.
Thus, FMDV is simply too strongly bound to this media to enable
enrichment.
As seen from Fig. 1(D), the binding of FMDV to Phenyl
Sepharose 6FF was extremely weak at low salt concentration.
Therefore, another strategy by passing the supernatant through
Phenyl column at low concentration of (NH4)2SO4 might be possible to obtain a high FMDV recovery in breakthrough fraction while

Please cite this article in press as: H. Li et al., A hydrophobic interaction chromatography strategy for purication of inactivated foot-and-mouth disease
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H. Li et al. / Protein Expression and Purication xxx (2015) xxxxxx

Fig. 5. HPSEC analyses of crude (A), HIC-puried (B), concentrated (C) and SEC-puried (D) FMDV. Samples of 100 lL were analyzed at 0.6 mL/min on TSK G4000 SWxL
(300  7.8 mm, I.D.) in 50 mM phosphate buffer pH 7.2. Elution was monitored by UV absorbance at 259 nm.

Table 3
Purication efciency at each step.
Sample

FMDV
100
HIC
UF
SEC
Total

Total
proteina
(mg)
crude
21.8 1.8
20.0 1.2
0.64 0.07

FMDV

Residual host
cell DNAc (%)

Amountb
(lg)

Recovery
(%)

Purication
fold

208

940

100

864 48
770 62
709 38

92 5
89 2
92 2
75 4

8.8 0.2
1.0 0.1
28.8 1.8
247 11

3.3 0.3
2.3 0.8
0.5 0.1

Errors are from duplicate experiments.

a
Total protein was determined by Bradford assay.
b
Total FMDV was measured by HPSEC.
c
Residual host cell DNA is the ratio between DNA remaining after each step and
total DNA in the initial crude. DNA concentration was determined by uorimetry.

removing most of the non-virus protein by adsorption. However,

the host cell DNA would also probably breakthrough the column
together with FMDV, and thus another step is needed to remove
it. Moreover, since FMDV cannot be concentrated by breakthrough
strategy, additional concentration step and more time may be
required for subsequent processing. Therefore, Butyl Sepharose
4FF was deemed more suitable than Phenyl Sepharose 6FF for
FMDV purication.

Comparison of FMDV purication by IEC and HIC

Purication efciency on Butyl Sepharose 4FF and Phenyl
Sepharose 6FF is summarized in Table 1, along with purication

efciency on DEAE Sepharose FF and ANX Sepharose 4FF. The last

two media were screened from several of frequently-used IEC
media in previous work, with relative good purication efciency.
As seen from the table, the highest FMDV recovery and
fold-purication are highest on Butyl Sepharose 4FF. Recovery
from DEAE Sepharose FF and ANX Sepharose 4FF was acceptable,
but IEC requires preliminary dilution or desalting of the crude,
and therefore requires more time. Therefore, Butyl Sepharose 4FF
was retained as the method of choice.
Renement by size exclusion chromatography
FMDV eluted from Butyl Sepharose 4FF HIC was polished by SEC
to remove residual contaminants. We evaluated SEC columns with
separation range beyond, close to, and below 5500 kDa, the
approximate molecular weight of FMDV [29]. Separation efciency
is listed in Table 2.
SEC with Superdex 200 produced the sharpest FMDV peak, with
excellent resolution of 2.96. The virus is excluded from Superdex
200, and was eluted in the exclusion volume while impurities were
eluted much later (Fig. 3), indicating that impurities were of low
molecular weight. In contrast, both FMDV and impurities are fractionated over Sepharose 4FF and Sepharose 6FF. Unfortunately,
separation was poor on both, with resolution below 1. Therefore,
Superdex 200 column was selected to polish FMDV preparations,
even though preparative SEC often suffers from limited efciency
at industrial scale [32]. However, in this particular case, separation
on SEC in negative mode is advantageous, and produces
high-purity and high-concentration products, even in industrial
scale.

Please cite this article in press as: H. Li et al., A hydrophobic interaction chromatography strategy for purication of inactivated foot-and-mouth disease
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H. Li et al. / Protein Expression and Purication xxx (2015) xxxxxx

Fig. 6. DLS analysis (A) and TEM imaging (B) of the puried FMDV.

Purication efciency of the entire scheme

FMDV may thus be puried in three steps, which include HIC on
Butyl Sepharose 4FF, concentration by ultraltration, and SEC on
Superdex 200.
To verify efciency and test reproducibility, FMDV purication
was repeated three times. The initial crude was also scaled up from
125 mL to 500 mL. The size of the HIC column was increased
accordingly, although ultraltration and SEC parameters were not
changed. After purication, the identity of the nal FMDV product
was veried by Western blot (Fig. 4), with purity above 98%, as
determined by SDSPAGE (Fig. 4) and HPSEC (Fig. 5D). The major
protein impurity in the initial crude was serum albumin, which
showed a molecular weight near 70 kDa as shown in Fig. 4. The
serum albumin was the main culture medium which is essential
for cell growth. After HIC, the serum albumin was reduced, and
then was completely removed by SEC. Host cell DNA was efciently reduced to 0.5% after purication, with most being removed
at the rst HIC step.
The purication efciency of each step is shown in Table 3, with
recovery over 90% after HIC and SEC. The average total recovery
was 75%, with 247-fold increase in purity. The standard deviation
from duplicate experiments was less than 6%, suggesting that the
process is reproducible. On balance, the performance of this
scheme is sufcient for industrial-scale production.

Structure of puried FMDV

The structure of puried FMDV was analyzed by DLS and TEM.
Samples exhibit a single DLS peak with diameter 28.0 nm and polydispersity index 0.21 (Fig. 6A), indicating monodispersity. This
result is in good agreement with data from Ravindra Acharya
[33]. Based on TEM imaging (Fig. 6B), the majority of puried virus
are spherical, with calculated mean diameter 28 nm. These results
conrm that puried FMDV is structurally intact, and thus appropriate as vaccine.

Conclusions
Hydrophobic interaction chromatography (HIC) is suitable to
purify inactivated FMDV. Of the HIC media evaluated, Butyl
Sepharose 4FF was found to perform best to achieve recovery
about 92% with 8.8-fold purication. HIC is more efcient than
IEC by eliminating the need for preliminary desalting and reducing
processing time.

Superdex 200 was used to polish FMDV preparations, because it

was the most efcient among the SEC media evaluated. On this column, the virus was excluded from the matrix, and thus separated
from contaminants, resulting in resolution near 3, 92% recovery,
and 28-fold purication. However, optimum and efcient SEC
purication requires an intermediate concentration step by
ultraltration.
On balance, 75% of FMDV could be reliably recovered from
crude, and puried 247-fold to purities above 98%. More than
99% of host cell DNA was removed. Western blot, HPSEC, DLS
and TEM indicate that the nal product contained the required
antigen, and was structurally intact with spherical shape and particle size 28 nm. Thus, our work demonstrates that HIC could be
useful in purifying inactivated viruses whose structural integrity
may be sensitive to industrial processing.
Acknowledgments
Financial supports from the Natural Sciences Foundation of
China (Nos. 21336010, 21306207), National High Technology
Research and Development Program of China (863 Program, Nos.
2012AA02A406 and 2014AA021006), National Key Scientic
Instrument
and
Equipment
Development
Project
(No.
2013YQ14040508), and Special Fund for Agro-scientic Research
in the Public Interest (No. 201303046) are acknowledged.
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Please cite this article in press as: H. Li et al., A hydrophobic interaction chromatography strategy for purication of inactivated foot-and-mouth disease
virus, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.04.011