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. 2019 Sep 19;14(9):e0222199.
doi: 10.1371/journal.pone.0222199. eCollection 2019.

Sequential two-step chromatographic purification of infectious poliovirus using ceramic fluoroapatite and ceramic hydroxyapatite columns

Yae Kurosawa  1   2 , Shigehiro Sato  2 , Tsuneo Okuyama  1   3   4 , Masato Taoka  4
Affiliations

Sequential two-step chromatographic purification of infectious poliovirus using ceramic fluoroapatite and ceramic hydroxyapatite columns

Yae Kurosawa et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

I (YK) receive only salaries from HOYA Technosurgical Corporation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Standardized two-step purification procedure.
CFAp, ceramic fluoroapatite; CHAp, ceramic hydroxyapatite; NaPB, sodium phosphate buffer.
Fig 2
Fig 2. Purification of Sabin type 2 virus by pH gradient elution on a ceramic fluoroapatite (CFAp) column.
(A) Chromatogram obtained under the following conditions: column, CFAp; sample (volume), cell culture supernatant containing Sabin type 2 virus (5 mL); column wash, 10 mM sodium phosphate buffer (NaPB; pH 6.4, 10 mL) and 300 mM NaPB (pH 6.4, 20 mL); equilibration, 300 mM NaPB (pH 5, 15 mL); elution, linear gradient at 300 mM NaPB from pH 5 to pH 8.2 for 10 mL; wash after separation, 300 mM NaPB (pH 8.2, 5 mL) and 600 mM NaPB (pH 8.2, 10 mL). Blue line, ultraviolet (UV) absorbance at 280 nm; black broken line, conductivity; red line, infectivity in median tissue culture infectious dose (TCID50); and purple line, double-stranded DNA (dsDNA) contents. Fr. A was pooled for the evaluation. (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The cell culture supernatant and pooled Fr. A were concentrated 10-fold and 30-fold, respectively, by ultrafiltration using a molecular weight cutoff of 10,000. The molecular weights of the marker proteins are given in kDa.
Fig 3
Fig 3. Chromatograms of Sabin type 2 virus-containing cell culture supernatant in the presence of NaCl.
Separation was carried out in the presence of (A) 1 M NaCl and (B) 1.5 M NaCl under the following conditions: column, ceramic hydroxyapatite (CHAp); sample (volume), cell culture supernatant containing Sabin type 2 virus (5 mL); buffer pH, 7.2; column wash, 10 mM sodium phosphate buffer (NaPB; 9 mL); equilibration, 10 mM NaPB with NaCl (14 mL); elution, linear gradient from 10 mM to 600 mM NaPB with NaCl for 30 mL; wash after separation, 600 mM NaPB (10 mL). Lines are the same as in Fig 2. TCID50, median tissue culture infectious dose.
Fig 4
Fig 4. Sequential two-step purification of Sabin type 2 virus.
(A) Purification of Sabin type 2 virus by pH gradient elution on a ceramic fluoroapatite (CFAp) column (Step 1). Column, CFAp; sample (volume), cell culture supernatant containing Sabin type 2 virus (10 mL); wash, 10 mM sodium phosphate buffer (NaPB) (pH 6.4, 9 mL) and 300 mM NaPB (pH 6.4, 20 mL); equilibration, 15 mL of 300 mM NaPB (pH 5); elution, linear gradient from pH 5 to pH 8.2 at 300 mM NaPB for 10 mL; wash after separation, 300 mM NaPB (pH 8.2, 5 mL) and 600 mM NaPB (pH 8.2, 10 mL). Lines are the same as in Fig 2. Fr. B was pooled and further purified in Step 2. (B) Removal of double-stranded DNA (dsDNA) from the Fr. B fraction by NaPB elution with 1 M NaCl on a ceramic hydroxyapatite (CHAp) column (Step 2). Column, CHAp; sample (volume), Fr. B obtained in (A) diluted 6.7-fold with 0.9% NaCl (17 mL); buffer, pH 7.2; column wash, 10 mM NaPB (13 mL); equilibration, 10 mM NaPB (14 mL) with 1 M NaCl; elution, linear gradient from 10 mM to 187 mM NaPB with 1 M NaCl for 10 mL; wash, 600 mM NaPB (10 mL). Fr. C was pooled for further evaluation. (C) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the pooled fraction obtained through the two-step purification process. The pooled Fr. C fraction obtained in (B) and the cell culture supernatant were concentrated 100- and 10-fold, respectively, by ultrafiltration using a molecular weight cutoff of 10,000. The sizes are given in kDa on the left. Arrows indicate the major proteins in Fr. C. TCID50, median tissue culture infectious dose.
Fig 5
Fig 5. Tandem column system.
CFAp, ceramic fluoroapatite; CHAp, ceramic hydroxyapatite; NaPB, sodium phosphate buffer; PV, poliovirus.
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I (YK) receive only salaries from HOYA Technosurgical Corporation. The funder provided support in the form of salaries for author (YK), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the “author contributions” section.

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