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

Figures

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  Fig 1. Standardized two-step purification procedure. CFAp, ceramic fluoroapatite; CHAp, ceramic hydroxyapatite; NaPB, sodium phosphate buffer.

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  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.

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  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.

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  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)…

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  Fig 5. Tandem column system. CFAp, ceramic fluoroapatite; CHAp, ceramic hydroxyapatite; NaPB, sodium phosphate buffer; PV, poliovirus.