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US 8,574,595 B2Granted
Virus purification using ultrafiltration
AssigneeCrucell Holland B.V.
InventorMiranda Weggeman
Life of this patent
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Abstract
Described are methods for purification of recombinant adenovirus from cell culture using a step of ultrafiltration wherein the retentate contains the virus. By applying back pressure on the permeate side during this step, adenovirus can be obtained at high purity without the need for chromatography or ultracentrifugation steps.
Claims17
A method for purification of a recombinant adenovirus to prepare a recombinant adenovirus preparation for clinical use, said method consisting essentially of:
- a) culturing cells that are infected with said recombinant adenovirus,
- b) lysing said cells and removing or fragmenting free nucleic acid, to provide a lysate comprising the recombinant adenovirus,
- c) clarifying the lysate to obtain an adenovirus preparation,
- d) subjecting the adenovirus preparation to ultrafiltration, wherein the adenovirus preparation is in the retentate, to concentrate the adenovirus preparation to obtain a concentrated adenovirus preparation,
- e) subjecting the concentrated adenovirus preparation of step d) to ultrafiltration, wherein the adenovirus preparation is in the retentate, and exchanging it with at least 5 diafiltration volumes (DFVs) of buffer, wherein one DFV is the volume of the retentate after concentration in step d), and
- f) subjecting the adenovirus preparation of step e) to sterile filtration, to obtain the recombinant adenovirus preparation for clinical use;
- wherein in steps d) and e) back pressure of at least 5 kPa is applied on the permeate side, and wherein the method does not utilize a chromatography column and an ion exchange filter.
4 more elements
A method for purification of a recombinant adenovirus to prepare a recombinant adenovirus preparation for clinical use, said method consisting essentially of:
- a) culturing cells that are infected with said recombinant adenovirus,
- b) lysing said cells and removing or fragmenting free nucleic acid, to provide a lysate comprising the recombinant adenovirus,
- c) clarifying the lysate to obtain an adenovirus preparation,
- d) subjecting the adenovirus preparation of step c) to ultrafiltration, wherein the adenovirus preparation is in the retentate, and exchanging it with at least five (5) diafiltration volumes (DFVs) of buffer, wherein one DFV is the volume of the adenovirus preparation after c), and
- e) subjecting the adenovirus preparation of step d) to sterile filtration, to obtain the recombinant adenovirus preparation for clinical use;
- wherein in step d) back pressure of at least 5 kPa is applied on the permeate side, utilize a chromatography column and an ion exchange filter.
3 more elements
A method for purification of a recombinant adenovirus to prepare a recombinant adenovirus preparation for clinical use, the method consisting essentially of:
- a) culturing cells that are infected with the recombinant adenovirus;
- b) adding a nuclease to the cell culture, and thereafter;
- c) lysing the cells to provide a lysate comprising the recombinant adenovirus;
- d) clarifying the lysate to obtain an adenovirus preparation;
- e) subjecting the adenovirus preparation to ultrafiltration, wherein the adenovirus preparation is in the retentate, to concentrate the adenovirus preparation to obtain a concentrated adenovirus preparation;
- and
- f) subjecting the concentrated adenovirus preparation of step e) to ultrafiltration, where the adenovirus preparation is in the retentate, and exchanging it with at least 5 diafiltration volumes (DFVs) of buffer, wherein one DFV is the volume of the retentate after concentration in step e),and
- g) subjecting the adenovirus preparation of step to sterile filtration, to obtain the recombinant adenovirus preparation for clinical use;
- wherein in steps e) and f), backpressure of at least 5 kPa is applied on the permeate side, utilize a chromatography column and an ion exchange filter.
6 more elements
A method for purifying a recombinant adenovirus, the method comprising:
- a) culturing cells infected with the recombinant adenovirus,
- b) lysing the cultured cells and removing or fragmenting free nucleic acid, to provide a lysate comprising the recombinant adenovirus,
- c) clarifying the lysate to obtain an adenovirus preparation,
- d) subjecting the adenovirus preparation to ultrafiltration, wherein the adenovirus preparation is in the retentate, to concentrate the adenovirus preparation to obtain a concentrated adenovirus preparation,
- e) subjecting the concentrated adenovirus preparation of step d) to ultrafiltration, wherein the adenovirus preparation is in the retentate, and exchanging it with at least 5 diafiltration volumes (DFVs) of buffer, wherein one DFV is the volume of the retentate after concentration in step d), and
- f) subjecting the adenovirus preparation of step e) to sterile filtration, to obtain the recombinant adenovirus preparation;
- wherein, in steps d) and e), back pressure of at least 5 kPa is applied on the permeate side, and
- wherein the method does not utilize a chromatography column and an ion exchange filter.
5 more elements
A method for purifying a recombinant adenovirus, the method comprising:
- a) culturing cells infected with the recombinant adenovirus,
- b) lysing the cultured cells and removing or fragmenting free nucleic acid, to provide a lysate comprising the recombinant adenovirus,
- c) clarifying the lysate to obtain an adenovirus preparation,
- d) subjecting the adenovirus preparation of step c) to ultrafiltration, wherein the adenovirus preparation is in the retentate, and exchanging it with at least five (5) diafiltration volumes (DFVs) of buffer, wherein one DFV is the volume of the adenovirus preparation after c), and
- e) subjecting the adenovirus preparation of step d) to sterile filtration, to obtain the recombinant adenovirus preparation;
- wherein, in step d), back pressure of at least 5 kPa is applied on the permeate side, and
- wherein the method does not utilize a chromatography column and an ion exchange filter.
4 more elements
A method for purifying a recombinant adenovirus, the method comprising:
- a) culturing cells infected with the recombinant adenovirus;
- b) adding a nuclease to the cell culture, and thereafter;
- c) lysing the cells to provide a lysate comprising the recombinant adenovirus;
- d) clarifying the lysate to obtain an adenovirus preparation;
- e) subjecting the adenovirus preparation to ultrafiltration, wherein the adenovirus preparation is in the retentate, to concentrate the adenovirus preparation to obtain a concentrated adenovirus preparation;
- and
- f) subjecting the concentrated adenovirus preparation of step e) to ultrafiltration, where the adenovirus preparation is in the retentate, and exchanging it with at least 5 diafiltration volumes (DFVs) of buffer, wherein one DFV is the volume of the retentate after concentration in step e), and
- g) subjecting the adenovirus preparation of step f) to sterile filtration, to obtain the recombinant adenovirus preparation;
- wherein, in steps e) and f), backpressure of at least 5 kPa is applied on the permeate side, and
- wherein the method does not utilize a chromatography column and an ion exchange filter.
7 more elements
Dependencies are inferred from the claim text.