Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2002-03-01
2004-08-10
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S239000, C435S325000, C424S233100
Reexamination Certificate
active
06773909
ABSTRACT:
FIELD OF THE INVENTION
This invention relates, e.g., to a method to prepare viruses (e.g., Adenoviruses) or other intracellular organisms.
DESCRIPTION OF THE INVENTION
This invention relates, e.g., to a method to release (prepare) intracellular biological entities (e.g., organisms, such as, e.g., viruses or virus particles, particularly Adenoviruses; organelles; or biological molecules), comprising subjecting cells which contain said biological entities to a continuous centrifugation procedure such that, following the concentration of the cells into a cell pellet and subsequent ejection from the centrifuge, the cells are sufficiently lysed to allow preparation of a high yield (per cell of the input material) of the biological entities. No additional steps effective to achieve cell lysis (e.g., a freeze-thaw step or microfluidization) are performed following ejection of the cell pellet from the centrifuge. In a preferred embodiment, organisms thus prepared retain a high degree of viability and/or infectivity; and entities such as organelles or biological molecules are substantially intact and/or biologically active.
One advantage of the method is that it allows one to concentrate and lyse cells in a single process step, thereby simplifying and reducing the cost of isolating intracellular (e.g., subcellular) biological entities from cells. Another advantage of the method is that a higher yield of biologically active intracellular material can be obtained than with other methods. Another advantage of the method is that it allows gentle lysis of the cells, such that intracellular biological entities, e.g., viruses, remain substantially intact and/or viable during the lysis procedure.
One embodiment of the invention is a method to prepare intracellular organisms from host cells containing said organisms, comprising subjecting the cells to continuous centrifugation under conditions effective to concentrate the cells into a cell pellet; and ejecting the pelleted cells from the centrifuge into a collection receptacle, under conditions effective to lyse cells; wherein no additional step effective to achieve cell lysis is performed. Another embodiment is a method to prepare intracellular organisms from host cells containing said organisms, comprising subjecting the cells to continuous centrifugation under conditions effective to concentrate the cells into a cell pellet; and ejecting the pelleted cells from the centrifuge into a collection receptacle, under conditions effective to lyse cells.
Another embodiment is a method as above, wherein the cells in the “ejectate” (the ejected cells) are substantially lysed and/or predominantly lysed; at least 50%, preferably at least 90%, of the cells are lysed; the cells are lysed as they are ejected; the intracellular organisms are viruses; the viruses are Adenoviruses; the Adenoviruses are recombinant Adenoviruses suitable for gene therapy; the yield/cell of Adenovirus particles or infectious Adenovirus is greater than that obtainable when cells containing said Adenovirus are lysed by a freeze-thaw procedure, e.g., the yield/cell of Adenovirus particles is about 1.2 to about 1.6 fold greater, or the yield/cell of infectious Adenovirus is about 1.5 to about 1.9 fold higher; the cells are animal cells, preferably mammalian or insect cells; the cells being ejected are under a relative centrifugal force of about 6,500 to 10,000 g, preferably about 7,000 to 9,000 g; said centrifugal force is about 7000 or 8,000 g; the pelleted cells are ejected through one or more ejection outlets having a rectangular shape and a cross-sectional area of about 50 to 500 mm
2
; the cells are centrifuged in a Westfalia Centrifuge, Model CSA-1 or CSC-6; or the forces exerted on the cells at the time of ejection are effective to substantially lyse the cells.
Another embodiment is in a method of preparing intracellular viruses from cells containing said viruses by continuous centrifugation, the improvement comprising harvesting viruses from the ejected cell pellet directly, without performing an additional step effective to achieve cell lysis.
Another embodiment is a method to prepare a cell lysate, consisting of subjecting cells to continuous centrifugation to form a cell pellet; and ejecting said pelleted cells into a collection receptacle, wherein the collected cells are in the form of a cell lysate.
Another embodiment is a method to prepare a cell lysate, comprising subjecting cells to continuous centrifugation under conditions effective to concentrate the cells into a cell pellet; and ejecting the pelleted cells from the centrifuge into a collection receptacle, wherein the cells are in the form of a cell lysate; wherein no additional step effective to achieve cell lysis is performed.
Another embodiment is organisms (e.g., viruses, particularly Adenoviruses) prepared by any of the above methods.
Another embodiment is a method to prepare an organelle or biological molecule, comprising subjecting cells comprising said organelle or biological molecule to continuous centrifugation under conditions effective to concentrate the cells into a cell pellet; ejecting the pelleted cells from the centrifuge into a collection receptacle, wherein the cells in the “ejectate” (the ejected cells) are substantially lysed; wherein no additional step effective to achieve cell lysis is performed; and harvesting the organelle or biological molecule from the ejectate.
In the method of the invention, a culture of cells comprising a biological entity (e.g., an organism, organelle or biological material) of interest is fed through a continuous centrifuge. Typically, a concentrated “cell pellet” (which, of course, can comprise portions of cells or cell debris, in addition to intact cells) collects in a centrifuge container (e.g., a bowl), while spent medium is continuously released through a first outlet into a first collection receptacle. At predetermined time intervals, a feed of wash buffer (any suitable wash buffer, e.g., PBS freezing buffer) is optionally started in order to exchange any supernatant remaining in the centrifuge container with the wash buffer. Following this exchange (if employed), the wash buffer feed is discontinued and the concentrated cells are expelled from the centrifuge container through a second outlet into a second collection receptacle. Depending on the volume in which the cells are ejected, the cell concentration can be significantly greater than the initial concentration in the feed (e.g. about a 10-fold to 50-fold concentration typically about 30-fold).
Without wishing to be bound to any theory, it is proposed that the cells remain substantially intact as they are centrifuged into the cell pellet. That is, a small enough fraction of cells is lysed during centrifugation so that a significant quantity of intracellular organisms, organelles, or biological molecules is not prematurely released into the supernatant and shunted to the collection receptacle with the spent medium. Conditions of centrifugation can be routinely optimized for any cell of interest in order to minimize the amount of cell lysis during centrifugation. See, e.g., Examples 2 and 4 for typical centrifugation conditions.
It is also proposed that as the concentrated (pelleted) cells are then ejected from the centrifuge through an ejection outlet, they are subjected to forces (e.g., shear forces), such that the cells are substantially lysed. A variety of ejection means by which cells are ejected through an ejection outlet are encompassed by the invention. In general, cells are forced out of (expelled from) the centrifuge by centrifugal force through an ejection outlet when a barrier (e.g., a gate) is removed (e.g., lifted or displaced), at an opportune time, to expose the ejection outlet. Such barriers can be operated by any of a variety of mechanims, e.g., electronic (e.g., solenoid), pneumatic, magnetic or by a mechanical linkage. In a preferred embodiment, a gate is opened by a hydraulically operated drive, using water which does not contact the cells. Typically, the supernatant is continuously and
Monica Thomas J.
Whiteley Erik M.
Chen Stacy B
Housel James
Millen White Zelano & Branigan P.C.
Schering Aktiengesellschaft
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