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Novel method for the protection and purification of adenoviral vectors

Inactive Publication Date: 2006-12-07
JANSSEN VACCINES & PREVENTION BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0015] The present invention is related to methods for producing purified viral compositions including adenovirus compositions of sufficient purity for therapeutic administration without the necessity for elaborate purification steps. More specifically, the invention relates to the discovery that size partitioning purification techniques may be used to provide adenoviral preparations of sufficient purity that they may be therapeutically administered without additional purification steps such as chromatographic and other methods previously considered necessary. Without intending to be bound by any particular theory of the invention it is believed that the steps of processing viral host cells in a cell suspension culture in a serum free media results in a viral particle product with a reduced load of contaminants. Moreover, the contaminants are of a size and nature that they may be readily separated from viral particles by a simple size partitioning purification step.
[0016] The ability to produce purified adenoviral preparations without traditional chromatographic purification steps provides significant improvements in viral production yields while reducing expense.

Problems solved by technology

Similarly, if the pore or inclusion size is too small, some undesirable contaminants may be retained.

Method used

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  • Novel method for the protection and purification of adenoviral vectors
  • Novel method for the protection and purification of adenoviral vectors
  • Novel method for the protection and purification of adenoviral vectors

Examples

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example 1

[0308] According to this example, cells were cultured and adenoviral vectors produced with medium perfusion using a 10 L (5 L working volume) Wave Bioreactor® (20 / 50EH (Wave Biotech, LLC) equipped with a YSI-2700 SELECT™ biochemistry analyzer according to the production and purification process depicted in FIG. 1. FIG. 3 depicts a perfusion Wave bioreactor (10) comprising an inflated plastic bag (12) containing cell culture media (14) and an internal flat perfusion filter (16) to provide separation between the cells and spent medium. Media is fed to the bioreactor from a feed bag (18) by feed pump (22) Spent culture medium is withdrawn through the floating filter (16) to a harvest bag (20) by harvest pump (24). Controller (26) controls the functions of the pumps and bioreactor (10). No medium recirculation is required, and consequently this mode of medium perfusion is very gentle to the cells in culture. The wave action minimizes filter clogging during perfusion. The culture volume ...

example 2

[0312] According to this example, the product of Example 1 was subjected to diafiltration using a tangential flow filtration (TFF) membrane using a Pellicon 2 mini system fitted with a 500 KD Biomax membrane cassette The clarified harvest was concentrated 20-fold using the Pellicon 2 mini system prior to diafiltration using a 500 mM Tris buffer at pH 8.0. Diafiltration was performed by the consistent volume method. Fresh diafiltration buffer was continuously added to the system as filtrate was permeated out of the membrane. Studies carried out using the 100 L production scale are set out in Table 5 below. The lack of fetal bovine serum in the culture medium makes is feasible to use TFF membrane partitioning diafiltration as a method of virus purification with high recovery.

TABLE 5TiterHPLC PurityRecoveryTotal Yield(vp / mL)(%)(%)(vp)Clarified Harvest1.2 × 10115.3NA1.20 × 101610-fold DF2.3 × 101278.6901.08 × 101620-fold DF2.2 × 101289.5891.07 × 101630-Fold DF2.3 × 101293.5891.06 × 10...

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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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of U.S. Provisional Application Ser. No. 60 / 624,627 filed Nov. 3, 2004 the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION [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] A variety of cancer and genetic diseases currently are being addressed by gene therapy. Viruses are highly efficient at nucleic acid delivery to specific cell types, while often avoiding detection by the infected host's immune system. These features make certain viruses attractive candidates as gene-delivery vehicles for use in gene therapies (Robbins and Ghivizzani, 1998;...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCA61K48/00C12N7/00C12N15/1017C12Q1/70C12N2710/10343C12N2710/10351C12N15/86
Inventor PHAM, HAIZHANG, SHUYUANCLARKE, PETER
Owner JANSSEN VACCINES & PREVENTION BV
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