Process for purifying hepatitis a virus (HAV), and vaccine compositions containing hepatitis a virus
AssigneeBiocine S.p.A.
InventorsVittoria Pellegrini, Nicoletta Fineschi, Arie J. Zuckerman
Life of this patent
Estimating the term…
Abstract
A process is described for the purification of the hepatitis A virus, which allows one to obtain with good yields a pure product, in which organic material collected by centrifugation after lysis of the culture cells is submitted to gel filtration and successively to ion exchange chromatography.
Claims12
A process for purifying hepatitis A virus (HAV) from cell culture consisting of the following steps:
- a) lysing cells infected with HAV in the presence of a first concentration of detergent;
- b) removing cell fragments to produce a supernatant;
- c) performing gel filtration of the supernatant in the presence of a second concentration of detergent;
- d) submitting the eluate obtained in step c) to ion exchange chromatography;
- and
- e) eluting the virus adsorbed in step d) using a detergent-free elution buffer.
A process for purifying hepatitis A virus (HAV) from cell culture consisting of the steps of:
- a) lysing cells infected with HAV in the presence of 2% Triton-X-100;
- b) centrifuging the lysed cells;
- c) submitting the supernatant obtained in step b) to gel filtration on SEPHAROSE CL-4B resin (Pharmacia) equilibrated with 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% Triton-X-100, pH 7.4;
- d) submitting the eluate obtained in step c) to anion exchange chromatography on DEAE SEPHAROSE CL-6B resin (Pharmacia) equilibrated with 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% Triton-X-100, pH 7.4;
- and
- e) eluting the virus adsorbed in step d) in a continuous gradient employing phosphate buffer having a continuous pH gradient of from 7.4 to 4 and an ionic strength gradient from to 0.3M NaCl.
A process for preparing a composition to be used as a vaccine for Hepatitis A consisting of the following steps:
- a) lysing cells infected with HAV in the presence of a first concentration of detergent;
- b) removing cell fragments to produce a supernatant containing virus;
- c) performing gel filtration of the supernatant in the presence of a second concentration of detergent;
- d) submitting the virus-containing eluate obtained in step c) to ion exchange chromatography;
- e) eluting the virus adsorbed in step d) using a detergent-free elution buffer;
- f) filtering the virus obtained in step e) through a 0.22 micron filter;
- and
- g) inactivating the virus obtained in step f) by treatment with 1:2000 formalin.
Dependencies are inferred from the claim text.