Sequential two-step chromatographic purification of infectious poliovirus using ceramic fluoroapatite and ceramic hydroxyapatite columns
Autoři:
Yae Kurosawa aff001; Shigehiro Sato aff002; Tsuneo Okuyama aff001; Masato Taoka aff004
Působiště autorů:
R&D Department, HOYA Technosurgical Corporation, Akishima-shi, Tokyo, Japan
aff001; Laboratory of Infectious Disease and Immunology, Department of Microbiology, Iwate Medical University, Shiwa, Iwate, Japan
aff002; Protein Technos Institute, Atsugi-shi, Kanagawa, Japan
aff003; Laboratory of Biophysics and Biochemistry, Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji-shi, Tokyo, Japan
aff004
Vyšlo v časopise:
PLoS ONE 14(9)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0222199
Souhrn
Infectious virus purification techniques are important for vaccine development and gene therapy applications. However, the standardized one-step purification technique using ceramic hydroxyapatite (CHAp) has proven unsuitable for poliovirus. Therefore, we designed a sequential two-step chromatographic technique for purification of the infectious Sabin type 2 vaccine strain of poliovirus from the cell culture supernatant. In the first step, we removed protein contaminants from the Sabin type 2 virus fraction by pH gradient elution on a ceramic fluoroapatite column. In the second step, we removed double-stranded DNA derived from host cells by diluting the virus fraction, directly loading it on a CHAp column, and purifying it using a phosphate gradient with 1 M sodium chloride. This process achieved removal rates of more than 99.95% and 99.99% for proteins and double-stranded DNA, respectively, and was highly reproducible and scalable. Furthermore, it is likely that it will be applicable to other virus species.
Klíčová slova:
Research and analysis methods – Biological cultures – Cell cultures – Separation processes – Elution – Physical sciences – Materials science – Materials – Ceramics – Chemistry – Chemical compounds – Phosphates – Sodium phosphate – Biology and life sciences – Organisms – Viruses – RNA viruses – Enteroviruses – Poliovirus – Flaviviruses – Dengue virus – Orthomyxoviruses – Influenza viruses – Microbiology – Medical microbiology – Microbial pathogens – Viral pathogens – Medicine and health sciences – Pathology and laboratory medicine – Pathogens – Infectious diseases – Infectious disease control – Vaccines
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