Method for the production and purification of adenoviral vectors
AssigneeCrucell Holland B.V.
InventorsHai Pham, Shuyuan Zhang, Peter Clarke
Life of this patent
Estimating the term…
Abstract
The present invention relates to improved methods for producing adenovirus compositions wherein host cells are grown in a bioreactor and purified by size partitioning purification to provide purified adenovirus compositions.
Claims28
A method for removing contaminants from a adenovirus-containing composition comprising obtaining an aqueous composition comprising a selected adenovirus and undesirable contaminants and subjecting the aqueous composition to size partitioning purification using a size partitioning membrane having partitioning pores that retain virus and permit the passage of contaminants there through to remove contaminants and thereby provide a purified virus composition, wherein the virus composition is subjected to at least 10-fold diafiltration, and
- wherein the aqueous composition is obtained by a method comprising:
- growing host cells in a serum-free medium;
- providing nutrients to said host cells;
- infecting said host cells with the adenovirus;
- and
- lysing said host cells to provide an aqueous composition comprising adenovirus.
A method for isolating adenovirus comprising obtaining an aqueous composition comprising subjecting an aqueous composition comprising a selected adenovirus and undesirable contaminants to filtration through a membrane, wherein the membrane has a cutoff threshold below 1000 kDa, wherein the virus composition is subjected to at least 10-fold diafiltration, and wherein contaminants are eliminated from an adenovirus-containing composition and the adenovirus is retained, and wherein the aqueous composition comprising adenovirus and undesirable contaminants is obtained by a method comprising:
- growing host cells in a serum-free medium;
- providing nutrients to said host cells;
- infecting said host cells with the adenovirus;
- and
- lysing said host cells using detergent to provide the aqueous composition.
A method for removing contaminants from a adenovirus-containing composition comprising obtaining an aqueous composition comprising a selected adenovirus and undesirable contaminants and subjecting the aqueous composition to size partitioning purification using a size partitioning membrane having partitioning pores that retain virus and permit the passage of contaminants therethrough to remove contaminants and thereby provide a purified virus composition, wherein the virus composition is subjected to at least 10-fold diafiltration, and wherein the aqueous composition is obtained by a method comprising:
- growing host cells in a medium;
- providing nutrients to said host cells;
- infecting said host cells with the adenovirus;
- and
- lysing said host cells to provide an aqueous composition comprising adenovirus, wherein the lysis step is carried out by a process that includes hypotonic solution, hypertonic solution, impinging jet, microfluidization, solid shear, detergent, liquid shear, high pressure extrusion, autolysis or sonication.
A method for isolating adenovirus comprising obtaining an aqueous composition comprising subjecting an aqueous composition comprising a selected adenovirus and undesirable contaminants to filtration through a membrane, wherein the membrane has a cutoff threshold below 1000 kDa, wherein the virus composition is subjected to at least 10-fold diafiltration, and wherein contaminants are eliminated from an adenovirus-containing composition and the adenovirus is retained, and wherein the aqueous composition comprising adenovirus and undesirable contaminants is obtained by a method comprising:
- growing host cells in a medium;
- providing nutrients to said host cells;
- infecting said host cells with the adenovirus;
- and
- lysing said host cells to provide the aqueous composition, wherein the lysis step is carried out by a process that includes hypotonic solution, hypertonic solution, impinging jet, microfluidization, solid shear, detergent, liquid shear, high pressure extrusion, autolysis or sonication.
Dependencies are inferred from the claim text.