Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2001-09-28
2004-08-10
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S306100, C435S308100, C435S320100, C536S025400
Reexamination Certificate
active
06773913
ABSTRACT:
BACKGROUND OF THE INVENTION
(i) Field of the Invention
The present invention relates to a device and a process for purifying vectors, and more particularly, it relates to a device and a process for concentrating a solution containing hosts and/or vectors, replacing the solution with another solution, or removing undesired substances from the solution.
(ii) Description of the Related Art
Along with a rapid development in a genetic engineering industry in recent years, a need for manipulating high-purity vectors on a large scale has been increasing. However, it takes a long time to obtain a large amount of the high-quality vectors, since a complex device must be used to purify them. In addition, the operation of the device is also complicated and requires some technical experience.
In particular, a centrifugal separator has heretofore been used to concentrate a solution in which hosts containing vectors are cultured in a purification process of vectors. Therefore, there exist problems that this culture medium cannot be concentrated in a closed system and that it is difficult to achieve the scale-up of the concentration. For example, “Separate volume of Experimental Medicine, Genetic Engineering Handbook” (issued on Mar. 20, 1991 by Yodo-sha) describes “Mass preparation of vector DNA”, which is carried out by the repetition of centrifugation, on the pages 21 to 23. Furthermore, also in order to remove undesired substances such as small proteins and small DNAs contained in vector DNAs, a centrifugal separator has heretofore been used, and hence the similar problems have been present. In addition, endotoxins cannot be removed by a centrifugal separator or an ion exchange column, and must be removed by a special column such as a hydrophobic column.
Therefore, a device or a process has been desired which can concentrate a solution containing hosts and/or vectors, replace the solution with another solution or remove undesired substances from the solution in a closed system. Further, a device or a process has been desired which can remove endotoxins effectively. Further, a device or a process by which a large amount of high-quality vectors can be easily obtained in a closed system have been desired.
Meanwhile, a filtration film for TFF (Tangential Flow Filtration) (referred to as “TFF film” hereinafter) has been known as a type of filtration films. However, the use of the TFF film for concentrating a solution containing hosts and/or vectors or removing undesired substances from the solution has not been known in the field of genetic engineering.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a device or a process which can concentrate a solution containing hosts and/or vectors, replace the solution with another solution and remove undesired substances from the solution in a closed system.
Further, it is an object of the present invention to provide a device or a process by which endotoxins can be effectively removed.
Further, it is an object of the present invention to provide a device or a process by which a large amount of high-quality vectors can be easily obtained in a closed system.
The present inventors have made intensive studies and completed the present invention by finding that the use of a TFF film or a surfactant facilitates the concentration of the solution containing hosts and/or vectors, replacement of the solution with another solution or removal of undesired substances from the solution. In addition, it has been found that the vectors purified by the present invention are of high purity. In particular, the present invention effectively utilizes a TFF film, which has never been used for gene manipulation before, for gene manipulation for the first time.
According to a first aspect of the present invention, a concentrating device is provided that comprises a TFF film which filtrates a solution containing hosts and/or vectors to concentrate the solution.
According to a second aspect of the present invention, a replacing device is provided that comprises a TFF film which filtrates a mixture of a first solution containing hosts and/or vectors and a second solution while supplying the second solution to replace the first solution with the second solution.
According to a third aspect of the present invention, an undesired substance removing device is provided that comprises a TFF film which removes undesired substances from a solution containing hosts and/or vectors.
According to a fourth aspect of the present invention, an endotoxin removing device is provided that comprises a surfactant supply unit which supplies a surfactant, a mixing vessel in which a crudely purified vector solution containing endotoxins is mixed with a surfactant to separate the solution into a water layer which mainly contains vector DNAs and a surfactant layer which contains the endotoxins, and a discharge unit which discharges the surfactant layer.
According to a fifth aspect of the present invention, a concentrating process is provided that comprises the step of filtering a solution containing hosts and/or vectors by using a TFF film.
According to a sixth aspect of the present invention, a replacing process is provided that comprises the step of filtering a mixture of a first solution containing hosts and/or vectors and a second solution by using a TFF film, while supplying the second solution, to replace the first solution with the second solution.
According to a seventh aspect of the present invention, an undesired substance removing process is provided that comprises the step of removing undesired substances from a solution containing hosts and/or vectors by using a TFF film.
According to an eighth aspect of the present invention, a process for removing endotoxins is provided that comprises the steps of mixing a crudely purified vector solution containing endotoxins with a surfactant, separating the solution into a water layer which mainly contains vector DNAs and a surfactant layer which contains the endotoxins and discharging the surfactant layer.
The TFF film used in the present invention is a type of filtration films and has a number of pores. The TFF filtration using this film is also called “cross-flow filtration”. Its mechanism is shown in FIG.
3
. As shown in
FIG. 3
, as a solution flows parallel to the surface of the film (from left to right in the direction indicated by the arrow A in FIG.
3
), a portion of the solution which may contain specific substances passes through pores (or is filtered) (downward in the direction indicated by the arrow B in FIG.
3
). The sizes of substances which pass through or do not pass through the pores depend on the sizes of the pores. In the present invention, the sizes can be appropriately determined according to application purposes. In general, the direction in which the solution flows is perpendicular to the direction in which the substances pass through the pores. In the case of the present invention, however, the angle between the two directions is not limited to a right angle.
Further, hosts used in the present invention are not particularly limited. Illustrative examples of the hosts include bacteria such as
Escherichia coli, Bacillus subtilis
and yeast which are generally and frequently used in genetic engineering.
Further, vectors used in the present invention are not particularly limited. Illustrative examples of the vectors include plasmids, phages and cosmids which are generally and frequently used in genetic engineering.
The concentrating device and process according to the present invention can be used, for example, for concentrating a culture medium which contains hosts containing vectors and various treatment solutions used in vector purification processes. Further, they can also be used for concentrating a solution which contains hosts containing target endogenous substances.
Further, in the replacing device and process of the present invention, a first solution can be replaced with a desired second solution. That is, by filtering a mixture of the first solution and the second solution by a
Arakaki Sakae
Kikukawa Masanao
Nagamine Masaru
Arakaki Sakae
Ketter James
Parkhurst & Wendel L.L.P.
LandOfFree
Device and process for purifying vectors does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Device and process for purifying vectors, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Device and process for purifying vectors will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3285627