1. Introduction
From biopharmaceutical point of view, the downstream procedures becoming
a critical bottleneck, which usually requires up to 80% of the overall
construction cost of a protein product (Kol et al., 2020). Foot and
mouth disease (FMD) is a severe and acute systemic viral vesicular
trans-boundary disease of cloven-hoofed animals such as cattle, pigs,
and sheep with a remarkable economic impact (Kleid et al., 1981;
MacDonald, 2018). The etiological agent of FMD is foot-and-mouth disease
virus (FMDV), which belongs to the genus Aphthovirus in the
family of Picornaviridae (Azeem et al., 2020). FMDV particles
consist of a non-enveloped icosahedral capsid with a single-stranded
positive-sense RNA genome of approximately 8,500 nucleotide which is
surrounded by four structural proteins (VP1-4). The FMDV
particle is roughly spherical in shape and about 25–30 nm in diameter
(Domingo et al., 2002; Grubman & Baxt, 2004; MacDonald, 2018). The
current FMD vaccines are produced in a multi-step procedure by
replicating the virulent FMDV in baby hamster kidney cell lines (BHK)
and is formulated with different adjuvants after inactivating with
binary ethylenimine (BEI) chemically (Hassan, 2016; S.-Y. Lee et al.,
2017). However, there are a number of concerns with this type of
vaccines due to different amounts of critical impurities such as viral
non-structural proteins (NSPs) and residual DNA (rDNA) of host cell. The
existence of NSPs in vaccinated animals develop antibody responses in
addition to the active ingredient of vaccine against these contaminate
proteins, false positive differentiation infected from vaccinated
animals, and rDNA leading to some unintended adverse effects in
susceptible animals or human consumers (Lee, et al. 2006). The
traditional approaches used for purifying and concentrating FMD vaccine
are based on ultracentrifugation on sucrose or CsCl gradient (Lee et
al., 2020; Yamamoto et al., 1988), and salting out with anti-chaotropic
agents such as ammonium sulphate or polyethylene glycol precipitation
(Ferreira, et al. 2000; Gavier-Widen et al., 2012; Kim et al., 2019;
Kleid et al., 1981; Lee et al., 2006). These processes cannot meet the
aforementioned requirements since they are usually time-consuming,
difficult to scale up, and affect biological activities considerably.
Membrane ultrafiltration (UF) could not produce a high quality for the
final vaccine product although it has been used for concentration,
buffer exchange, and partial purification of FMD vaccine (Kim et al.,
2019). Various chromatographic techniques have shown a growing trend in
downstream process of vaccine and proteins purification (Heath et al.,
2018; Santry et al., 2020; Tseng et al., 2018; Valkama et al., 2020;
Wang et al., 2019). In general, FMDV particles have been purified using
standard and scalable chromatographic techniques which separate them
based on different properties such as biorecognition or ligand
specificity, like affinity chromatography (Kramberger, et al. 2015;
Rodrigues, et al. 1991; Zhao et al., 2019), isoelectric point (ion
exchange chromatography, IEX) (Namatovu et al., 2013), surface
hydrophobicity (hydrophobic interaction chromatography) (Giebel et al.,
2010; Li et al., 2015; Namatovu et al., 2013), and size or hydrodynamic
diameter (size exclusion chromatography) (Klein & Helfferich, 1970;
Rhee & Amundson, 1982). The main drawback of SEC is related to low
loading capacity, which is usually 0.5-4% total column volume in a
group separation mode, and limits its application in the downstream of
biopharmaceutical production (Chisti & Moo-Young, 1990). IEX is
generally performed in the bind-elute mode instead of flow-through mode
(target passes contaminant bind). Thus, most of the host cell protein
(HCP) impurities are collected in the flow-through, while virus
particles and some other impurities are absorbed on the ion-exchanger
groups immobilized on surface media (Li et al., 2015). Anion or cation
exchange resins have been used according to the p I of the active
ingredient of proteins and vaccines (Benedini et al., 2020).
The performance of IEX can be accomplished mathematically with different
models such as Plate Model and Rate Model to simulate elution curve,
predict retention time, develop scale-up or process in various
operational conditions, and reduce cost and time significantly during
the process development (Benedini et al., 2020; Klein & Helfferich,
1970; Rhee & Amundson, 1982). For instance, Schmidt et al. (Schmidt et
al., 2014), employed a stoichiometric displacement model in order to
detect the retention behavior of a kind of protein at various pH
gradient elution in a cation-exchange chromatography column. They also
applied the model for studying the separation of an acid variant of the
antibody. Benedini et al. (Benedini et al., 2020), developed a
mathematical model to describe an anion exchange chromatography (AEC)
column, which was expedient for purifying the real and unknown mixtures
of proteins.
In our previous work, we applied two-dimensional SEC method assisted
with mathematical modeling for purifying FMDV and investigation dynamic
behavior of the SEC media towards FMDV active ingredient and some
critical impurities such as NSPs and rDNA (Bagheri, et al., 2018;
Helfferich & James, 1970) . In the present study, a combination of AEC
as the first column and SEC as the second column (2D -AEC×SEC) has been
introduced to improve purifying the FMDV in larger sample size as well
as in less period of time. In addition, 2D -AEC×SEC was accompanied with
mathematical modeling in both dimensions for evaluation separation
procedure to produce NSP free FMD vaccine. This new method significantly
improves loading capacity and reduces time for FMDV purification.
Finally, the purified virus particles were partially characterized by
sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light
scattering (DLS), high performance size exclusion chromatography
(HP-SEC), Matrix-Assisted Laser Desorption/Ionization Time-of-Flight
Mass Spectrometry (MALDI-TOF MS), and transmission electron microscopy
(TEM).