Ta, Duy Tien & Chu, Kai & Soonaan, Izzati & Chin, Christine & Ng, Say Kong & Zhang, Wei. (2021). A New and Simplified Anion Exchange Chromatographic Process for The Purification of Cell-Grown Influenza A H1N1 Virus. Separation and Purification Technology. 263. 118412. 10.1016/j.seppur.2021.118412.
Abstract:
A new and simplified anion exchange chromatographic process for the purification of both live and inactivated cell-grown influenza A H1N1 viruses was established with a recently developed anion exchanger. The resin has been designed to have optimized surface extender in addition to optimized pore size to provide good accessibility as well as good binding capacity for large biomolecules, aiming to overcome common drawbacks of current existing ion exchangers for virus purification. Live H1N1 virus produced from Vero cells was first used as model virus for HP-Q process development and optimization. A traditional hemagglutination (HA) assay and a recently developed, highly sensitive droplet digital PCR (ddPCR) assay were compensatorily used as main quantitative assays throughout the whole study. It was shown that HP-Q allowed direct capture of live H1N1 virus in cell culture supernatant clarified by microfiltration only, and demonstrated an excellent dynamic binding capacity (DBC) of 2.77 × 1012 H1N1 viral particles/ml resin (1.71 × 106 HAU/ml resin) at 1.4 min residence time in the presence of abundant amount of host cell impurities and free viral RNA and at high conductivity. Under optimized conditions, 59.5-80.0% HA recovery was obtained in HP-Q eluate with highly mono-dispersed H1N1 viral particles (99.8% monomer determined by multi-angle dynamic light scattering or MADLS) with an average size at 128.0 nm. Above 95% of host cell protein (HCP) and above 99% of host cell DNA (hcDNA) were removed in a single HP-Q run with purity of purified product meeting regulatory requirement for culture-derived human vaccines. Tangential flow filtration (TFF) and nuclease treatment were not necessary for HP-Q based process due to its excellent capability of removing free nucleic acids (both hcDNA and free viral RNA), HCP, aggregated virus, thus allowing direct loading of virus culture supernatant. Without further process optimization, HP-Q process optimized for live H1N1 virus was directly adapted to the purification of both formaldehyde- and heat-inactivated viruses. Moderate HA recovery (50.4–62.5%) and good impurity removal (87.5–94.9% for HCP and 100% for hcDNA) were achieved for both processes, which can be potentially further improved by individual process optimization. The established HP-Q process for H1N1 virus purification was simple, efficient, robust, cost-effective and scalable, thus being an excellent alternative to existing chromatographic tools for the production of cell-based influenza vaccines.
License type:
Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
Funding Info:
This research is supported by Bio-Rad Laboratories (USA) and the Agency for Science, Technology and Research under its Industrial Alignment Fund (Pre-Positioning) (H17/01/a0/012).
Description:
Please find the link to the article at the publisher's URL: https://doi.org/10.1016/j.seppur.2021.118412