Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1999-04-22
2002-05-07
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S005000
Reexamination Certificate
active
06383795
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the efficient purification of adenovirus.
BACKGROUND OF THE INVENTION
Traditionally, adenoviral particles have been isolated through the use of density gradient purification protocols, such as through the use of cesium chloride (CsCl) gradients. While suitable for small-scale preparations, density gradient purification is tedious and time consuming and can not be easily scaled-up. Accordingly, the process is frequently considered commercially undesirable.
An alternative method for purifying adenovirus is to use column or batch chromatography. Early attempts to isolate viral particles by chromatographic techniques using diethylaminoethyl (DEAE) chromatography resins were first reported from 1959 to 1961. Haruna et al. (
Virology
13: 264-267 (1961)) reported using DEAE ion exchange chromatography for purification of types 1, 3, and 8 adenoviruses, while Klemperer and Pereira (
Virology
9: 536-545 (1959)) and Philipson (
Virology
10: 459-465 (1960)) reported difficulties using the same method with other types of adenoviruses. These techniques were not widely used after about 1965, most likely as a result of the tendency of the chromatographic matrix to collapse during use. In addition, the selectivity of the chromatography resins available at the time made chromatographic purification of viruses inferior to the density gradient purification techniques.
Recently, there has been renewed interest in purifying viruses by chromatography. For example, Shabram et al. (WO 96/27677) and Huyghe et al. (
Human Gene Therapy
6: 1403-1416 (1995)) disclose methods of using chromatography resins to purify viruses. Newer packing materials for chromatography also have been developed in the last decade and a half. These packing materials can be classified in four groups: (i) homogeneous cross-linked polysaccharides, which include soft gels (e.g., agarose) having good capacity, but poor resolution and a tendency to compress; (ii) macroporous polymers based on synthetic polymers, which include perfusion chromatography resins with large “throughpores,” allowing for better diffusivity and leading to improved column efficiency, speed, and resolution; (iii) “tentacular” sorbents, which have tentacles that were designed for faster interactions with proteins (e.g. fractogel); and (iv) materials based on a soft gel in a rigid shell, which exploit the high capacity of soft gels and the rigidity of composite materials (e.g., Ceramic HyperD™ F) (see Boschetti,
J. Chromatogr.
658: 207 (1994); Rodriguez,
J. Chromatogr.
699: 47-61 (1997)).
It is desirable to increase the speed, ease of use, and efficiency of purification, particularly large-scale commercial purification, of these prior art techniques. The present invention provides such a process for purification of adenovirus. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of enriching a solution of an adenovirus. The method comprises: (i) obtaining a mixed solution comprising adenovirus and at least one undesired type of biomolecule; (ii) applying the mixed solution to an anion exchange chromatography resin containing a binding moiety selected from the group consisting of dimethylaminopropyl, dimethylaminobutyl, dimethylaminoisobutyl, and dimethylaminopentyl; and (iii) eluting the adenovirus from the purification chromatography resin with an eluant. The method can further comprise applying the mixed solution comprising adenovirus and at least one undesired type of biomolecule to an anion exchange pre-resin prior to applying the adenovirus to the anion exchange chromatography resin.
The present invention also provides a method of purifying an adenovirus from cells infected with adenovirus. The method comprises lysing cells infected with adenovirus, applying the lysate to a single chromatography resin, eluting the adenovirus from the chromatography resin, and collecting a fraction containing adenovirus, wherein the adenovirus is substantially as pure as triple CsCl density gradient-purified adenovirus.
The present invention further provides a method of accurately quantifying the number of adenoviral particles in a solution of adenovirus, such as a solution obtained from a crude lysate from cells infected with adenovirus, comprising (i) applying to and eluting from an anion exchange chromatography resin containing a binding moiety selected from the group consisting of dimethylaminopropyl, dimethylaminobutyl, dimethylaminoisobutyl, and dimethylaminopentyl a sample solution of adenovirus, (ii) determining the absorbance of the sample solution of adenovirus eluted from the chromatography resin and the absorbance of a standard solution of adenovirus, (iii) comparing the absorbance of the sample solution of adenovirus eluted from the chromatography resin with the absorbance of the standard solution of adenovirus, and quantifying the number of adenoviral particles in the sample solution.
The invention may best be understood with reference to the accompanying drawings and in the following detailed description of the preferred embodiments.
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patent: WO 96/27677 (1996-09-01), None
patent: 97/08298 (1997-03-01), None
patent: WO 98/00524 (1998-01-01), None
patent: 98/00524 (1998-01-01), None
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Afeyan et al.,J. Chromatogr., 519, 1-29 (1990).
Haruna et al.,Virology, 13, 264-267 (1961).
Huyghe et al.,Human Gene Therapy, 6, 1403-1416 (1995).
Klemperer et al.,Virology, 9, 536-545 (1959).
Philipson,Virology, 10, 459-465 (1960).
Shabram et al.,Human Gene Therapy, 8, 454-456 and 458-461 (1997).
Carrión Miguel E.
Kovesdi Imre
Menger Marilyn
GenVec, Inc.
Leydig , Voit & Mayer, Ltd.
Mosher Mary E.
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