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Formulation of adenovirus for gene therapy

a technology of adenovirus and gene therapy, which is applied in the field of molecular biology, virus production and gene therapy, can solve the problems of affecting the viability of a viral vector, preventing its use as a gene therapy vector, and not only being expensive to store at ultra-low temperature, and achieves high infectivity

Inactive Publication Date: 2005-08-11
JANSSEN VACCINES & PREVENTION BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides an improved method for producing stable viral formulations for gene therapy. The invention involves the use of pharmaceutical excipients, such as bulking agents and protectants, to create a stable adenovirus composition that can be stored for extended periods without losing its effectiveness. The composition should have an infectivity of at least 60% and should not contain more than 5% moisture. The invention also provides a method for freeze drying the adenovirus composition to further improve its stability. The invention further includes the use of specific cryoprotectants, such as sucrose or trehalose, to protect the adenovirus particles during cryopreservation. Overall, the invention provides a way to produce a stable viral formulation for gene therapy that can be stored and transported without losing its effectiveness.

Problems solved by technology

The structural integrity of a viral vector often is compromised during the formulation process, thus precluding its use as a gene therapy vector.
This ultra-low temperature storage not only is very expensive, but creates significant inconvenience for storage, transportation and clinic use.
However, there have not been reported studies on the lyophilization of live adenovirus for gene therapy.

Method used

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  • Formulation of adenovirus for gene therapy
  • Formulation of adenovirus for gene therapy
  • Formulation of adenovirus for gene therapy

Examples

Experimental program
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Effect test

example 1

Materials and Methods

Lyophilizer

[0198] A Dura-stop μp lyophilizer (FTSsystems) with in process sample retrieving device was used. The lyophilizer is equipped with both thermocouple vacuum gauge and capacitance manometer for vacuum measurement. Condenser temperature is programmed to reach to −80° C. Vials were stoppered at the end of each run with a build-in mechanical stoppering device.

Residual Moisture Measurement

[0199] Residual moisture in freeze dried product was analyzed by a Karl-Fisher type coulometer (Mettler DL37, KF coulometer).

HPLC Analysis

[0200] HPLC analysis of samples was done on a Beckman Gold HPLC system.

Vials and Stoppers

[0201] Borosilicate 3 ml with 13 mm opening lyo vials and their corresponding butyl rubber stoppers (both from Wheaton) were used for both lyophilization and liquid formulation development. The stoppered vials were capped with Flip-off aluminum caps using a capping device (LW312 Westcapper, The West Company).

example 2

Lyophilization: Initial Cycle and Formulation Development

[0202] There are three main process variables that can be programmed to achieve optimal freeze-drying. Those are shelf temperature, chamber pressure, and lyophilization step duration time. To avoid cake collapse, shelf temperature need to be set at temperatures 2-3° C. below the glass transition or eutectic temperature of the frozen formulation. Both the glass transition and eutectic temperatures of a formulation can be determined by differential scanning calorimetry (DSC) analysis. Chamber pressure is generally set at below the ice vapor pressure of the frozen formulation. The ice vapor pressure is dependent on the shelf temperature and chamber pressure. Too high a chamber pressure will reduce the drying rate by reducing the pressure differential between the ice and the surrounding, while too low a pressure will also slow down drying rate by reducing the heat transfer rate from the shelf to the vials. The development of a ly...

example 3

Cycle and Formulation Development with Virus in Formulation

[0213] Effect of Sucrose Concentration in Formulation. Cycle and formulation were further optimized according to virus recovery after lyophilization analyzed by both HPLC and plaque forming unit (PFU) assays. Table 5 shows the virus recoveries immediate after drying in different formulations using the above drying cycle. Variation of the percentage of sucrose in the formulation had significant effect on virus recoveries.

TABLE 5Recoveries of Virus After LyophilizationFormulationResidualRecoveryM % / S % / HSA %Appearancemoisture(%)6 / 0 / 0.5good cake0.44% 06 / 3.5 / 0.5good cake2.2%566 / 5 / 0.5good cake2.5%816 / 6 / 0.5good cake2.7%1206 / 7 / 0.5good cake2.8%1206 / 8 / 0.5good cake3.3%936 / 9 / 0.5good cake3.7%120

[0214] Residual moisture in the freeze-dried product increased as the sucrose percentage increased. A minimum sucrose concentration of 5% is required in the formulation to maintain a good virus recovery after lyophilization. Similar sucrose ef...

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Abstract

The present invention addresses the need to improve the long-term storage stability (i.e. infectivity) of vector formulations. In particular, it has been demonstrated that for adenovirus, the use of bulking agents, cryoprotectants and lyoprotectants imparts desired properties that allow both lyophilized and liquid adenovirus formulations to be stored at 4° C. for up to 6 months and retain an infectivity between 60-100% of the starting infectivity.

Description

[0001] The present application claims priority to the contents of U.S. Provisional Patent Application Ser. No. 60 / 108,606, filed Nov. 16, 1998 and U.S. Provisional Patent Application Ser. No. 60 / 133,116, filed May 7, 1999. The entire text of the above-referenced disclosure is specifically incorporated by reference herein without disclaimer.BACKGROUND OF THE INVENTION [0002] A. Field of the Invention [0003] The present invention relates generally to the fields of molecular biology, virus production and gene therapy. More particularly, it concerns methods for the formulation of highly purified lyophilized and liquid adenovirus particles stable for long-term storage. An important embodiment of the present invention is the use of such long-term storage virus preparations for gene therapy treatments of viral disease, genetic disease and malignancies. [0004] B. Description of Related Art [0005] Viruses are highly efficient at nucleic acid delivery to specific cell types, while often avoid...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/19A61K35/76A61K35/761A61K47/02A61K47/18A61K47/20A61K47/26A61K47/32A61K47/38A61K47/42A61K47/46A61K48/00C12N15/861
CPCA01N1/02A61K35/761A61K9/19A61K47/02A61K47/183A61K47/20A61K47/26A61K47/32A61K47/38A61K47/42A61K47/46A61K48/00C12N7/00C12N15/86C12N2710/10051C12N2710/10332C12N2710/10343C12N2710/10351A61K9/0019
Inventor WU, ZHENGZHANG, SHUYUAN
Owner JANSSEN VACCINES & PREVENTION BV
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