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Method for the production and purification of adenoviral vectors

a technology of adenoviral vector and purification method, which is applied in the field of cell culture and virus production, can solve the problems of inability to meet the demand for adenoviral vector for gene therapy applications, inability to produce the required amount of virus using this traditional process, and inability to meet the demand for adenoviral vectors

Inactive Publication Date: 2007-07-05
JANSSEN VACCINES & PREVENTION BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] The present invention describes a new process for the production and purification of adenovirus. This new production process offers not only scalability and validatability but also virus purity comparable to that achieved using CsCl gradient ultracentrifugation.
[0011] Thus the present invention provides a method for producing an adenovirus comprising growing host cells in media at a low perfusion rate, infecting the host cells with an adenovirus, harvesting and lysing the host cells to produce a crude cell lysate, concentrating the crude cell lysate, exchanging buffer of crude cell lysate, and reducing the concentration of contaminating nucleic acids in the crude cell lysate.

Problems solved by technology

Clearly, it becomes unfeasible to produce the required amount of virus using this traditional process.
The purification throughput of CsCl gradient ultracentrifugation is so limited that it cannot meet the demand for adenoviral vectors for gene therapy applications.
Reports on the chromatographic purification of viruses are very limited, despite the wide application of chromatography for the purification of recombinant proteins.
This lack of research activity may be partially attributable to the existence of the effective, albeit non-scalable, CsCl gradient ultracentrifugation purification method for adenoviruses.
Unfortunately, only 23% of virus was recovered after the double column purification process.

Method used

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  • Method for the production and purification of adenoviral vectors
  • Method for the production and purification of adenoviral vectors
  • Method for the production and purification of adenoviral vectors

Examples

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example 1

Materials and Methods

[0260] A) Cells

[0261] 293 cells (human epithelial embryonic kidney cells) from the Master Cell Bank were used for the studies.

[0262] B) Media

[0263] Dulbecco's modified Eagle's medium (DMEM, 4.5 g / L glucose)+10% fetal bovine serum (FBS) was used for the cell growth phase. For the virus production phase, the FBS concentration in DMEM was lowered to 2%.

[0264] C) Virus

[0265] AdCMVp53 is a genetically engineered, replication-incompetent human type 5 adenovirus expressing the human wild type p53 protein under control of the cytomegalovirus (CMV) immediate early promoter.

[0266] D) Celligen Bioreactor

[0267] A Celligen bioreactor (New Brunswick Scientific, Co. Inc.) with 5 L total volume (3.5 L working volume) was used to produce virus supernatant using microcarrier culture. 13 g / L glass coated microcarrier (SoloHill) was used for culturing cells in the bioreactor.

[0268] E) Production of Virus Supernatant in the Celligen Bioreactor

[0269] 293 cells from master c...

example 2

Effect of Medium Perfusion Rate in Cellcube™ on Virus Production and Purification

[0291] For a perfusion cell culture system, such as the Cellcube™, medium perfusion rate plays an important role on the yield and quality of product. Two different medium perfusion strategies were examined. One strategy was to keep the glucose concentration in the Cellcube™≧2 g / L (high perfusion rate). The other one was to keep the glucose concentration ≧1 g / L (low medium perfusion rate).

[0292] No significant changes in the culture parameters, such as pH, DO, was observed between the two different perfusion rates. Approximately equivalent amount of crude viruses (before purification) were produced after harvesting using 1% Tween-20 lysis solution as shown in Table 5. However, dramatic difference was seen on the HPLC profiles of the viral solutions from the high and low medium perfusion rate production runs.

TABLE 5Effect of medium glucose concentration on virus yieldGlucoseconcentration (g / L)≧2.0≧1.0...

example 3

Methods of Cell Harvest and Lysis

[0294] Based on previous experience, the inventors first evaluated the freeze-thaw method. Cells were harvested from the Cellcube™ 45-48 hr post-infection. First, the Cellcube™ was isolated from the culture system and the spent medium was drained. Then, 50 mM EDTA solution was pumped into the Cube to detach the cells from the culture surface. The cell suspension thus obtained was centrifuged at 1,500 rpm (Beckman GS-6KR) for 10 min. The resultant cell pellet was resuspended in Dulbecco's phosphate buffered saline (DPBS). The cell suspension was subjected to 5 cycles of freeze / thaw between 37° C. water bath and dry-ice ethanol bath to release virus from the cells. The crude cell lysate (CCL) thus generated was analyzed on HPLC.

[0295]FIG. 2 shows the HPLC profile. No virus peak is observed at retention time of 9.32 min. Instead, two peaks at retention times of 9.11 and 9.78 min are produced. This profile suggests that the other contaminants having si...

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Abstract

The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production with cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge.

Description

BACKGROUND OF THE INVENTION [0001] The present application is a continuation-in-part of co-pending U.S. Provisional Patent Application Ser. No. 60 / 031,329 filed Nov. 20, 1997. The entire text of the above-referenced disclosure is specifically incorporated by reference herein without disclaimer. [0002] 1. Field of the Invention [0003] The present invention relates generally to the fields of cell culture and virus production. More particularly, it concerns improved methods for the culturing of mammalian cells, infection of those cells with adenovirus and the production of infectious adenovirus particles therefrom. [0004] 2. Description of Related Art [0005] Adenoviral vectors, which express therapeutic proteins, are currently being evaluated in the clinic for the treatment of a variety of cancer indications, including lung and head and neck cancers. As the clinical trials progress, the demand for clinical grade adenoviral vectors is increasing dramatically. The projected annual demand...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/06C12N15/00A61K35/76C12N15/09A61K35/761A61K38/00A61K48/00C07K14/47C12N5/02C12N7/01C12N7/02C12N15/86C12N15/861
CPCA61K48/00C07K14/4746C12N7/00C12N15/86C12N2710/10045C12N2710/10051A61K35/761C12N2710/10332C12N2710/10343C12N2710/10351C12N2810/6018A61K38/00C12N2710/10322
Inventor ZHANG, SHUYUANTHWIN, CAPUCINEWU, ZHENGCHO, TOOHYONWILSON, DEBORAHCASTON, LUCETTA
Owner JANSSEN VACCINES & PREVENTION BV
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