Concentration and purification of enterovirus 71 using a weak anion-exchange monolithic column

Abstract

BackgroundEnterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five. It is a major concern for public health issues across Asia-Pacific region. The most effective way to control the disease caused by EV-71 is by vaccination thus a novel vaccine is urgently needed. Inactivated EV-71 induces a strong, virus-neutralizing antibody response in animal models, protecting them against a lethal EV-71 challenge and it has been shown to elicit cross-neutralizing antibodies in human trials. Hence, the large-scale production of purified EV-71 is required for vaccine development, diagnosis and clinical trials.MethodsCIM® Monolith columns are single-piece columns made up of poly(glycidyl methacrylate co-ethylene dimethacrylate) as support matrix. They are designed as porous channels rather than beads with different chemistries for different requirements. As monolithic columns have a high binding capacity, flow rate and resolution, a CIM® DEAE-8f tube monolithic column was selected for purification in this study. The EV-71 infected Rhabdomyosarcoma (RD) cell supernatant was concentrated using 8% PEG 8000 in the presence of 400 mM sodium chloride. The concentrated virus was purified by weak anion exchange column using 50 mM HEPES + 1 M sodium chloride as elution buffer.ResultsHighly pure viral particles were obtained at a concentration of 350 mM sodium chloride as confirmed by SDS-PAGE and electron microscopy. Presence of viral proteins VP1, VP2 and VP3 was validated by western blotting. The overall process achieved a recovery of 55%.ConclusionsEV-71 viral particles of up to 95% purity can be recovered by a single step ion-exchange chromatography using CIM-DEAE monolithic columns and 1 M sodium chloride as elution buffer. Moreover, this method is scalable to purify several litres of virus-containing supernatant, using industrial monolithic columns with a capacity of up to 8 L such as CIM® cGMP tube monolithic columns.

Improved virus purification processes for vaccines and gene therapy

“…Their use for virus purification has been extensively evaluated in the last years, both for enveloped and non‐enveloped viruses. Recent examples include lentivirus (Bandeira et al, ), baculovirus (Gerster et al, ), rubella (Forcic et al, ), enterovirus 71 (Venkatachalam et al, ), and canine adenovirus purification (Fernandes et al, ). However, monoliths have shown to be prone to clogging, especially when high host cell DNA is present in the bioreactor bulk, although increasing the monolith pore size up to 6 m is expected to solve this issue (Bencina et al, ).…”

Optimal Use of Vaccines for Control of Influenza A Virus in Swine

“…A more recent experimental challenge study gave similar evidence that polyvalent commercial vaccines containing cluster IV H3N2 can protect against a drifted contemporary cluster IV strain better than a commercial vaccine containing only cluster I H3N2 [ 155 ]. Although antigenic drift is likely diminishing the quality of protection from the newer polyvalent vaccines [ 153 , 156 , 157 ], it is reasonable to assume these will protect herds more reliably than bivalent vaccines that contain even older IAV-S strains.…”

Next generation vaccines and vectors: Designing downstream processes for recombinant protein‐based virus‐like particles

“…Typical precipitants for large biomolecules are ammonium sulfate and polyethylene glycol (PEG) [19]. PEGs with molecular weights ranging from 4000 to 8000 Da have been used in the past for concentrating numerous viruses [61–63] and VLPs such as cowpea mosaic VLPs, noro‐VLPs, and chikungunya‐VLPs [64–66]. Tsoka et al purified VLPs derived from Saccharomyces cerevisiae by PEG‐induced precipitation and scaled up the process to pilot scale in a 100 L stirred tank [47].…”

“…Precipitation with PEG 6000 and perfusion chromatography media with a strong Q ligand yielded in a final purity of 95% [64]. In contrast, Venkatachalam et al recovered 55% of Entero‐VLPs derived from insect cells by using a weak DEAE ligand on a monolithic column after VLP precipitation with PEG 8000 [63]. Thus, a weaker ion exchange ligand and larger pore sizes promote higher VLP recoveries.…”