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).