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Method for the production and purification of adenoviral vectorsMethod for the production and purification of adenoviral vectors description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080050770, Method for the production and purification of adenoviral vectors. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001]The present application is a continuation-in-part of co-pending U.S. patent application Ser. No. 08/975,519 filed Nov. 29, 1997 which is based on U.S. Provisional Patent Application Ser. No. 60/031,329 filed Nov. 20, 1996. The entire text of the above-referenced disclosures are specifically incorporated by reference herein without disclaimer. 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]Adenoviral vectors, which carry transgenes that can be transcribed and translated to express therapeutic proteins, are currently being evaluated in the clinic for the treatment of a variety of cancer indications, including lung and head and neck cancers. As the clinical trials progress, the demand for clinical grade adenoviral vectors is increasing dramatically. The projected annual demand for a 300 patient clinical trial could reach approximately 1.08.times.10.sup.16 viral particles. [0006]Traditionally, adenoviruses are produced in commercially available tissue culture flasks, "cellfactories," or RB. Virus infected cells are harvested and subjected to multiple freeze-thaws to release the viruses from the cells in the form of crude cell lysate. The produced crude cell lysate (CCL) is then purified by multiple CsCl gradient ultracentrifugation steps. The typically reported virus yield from 100 single tray cellfactories is about 1.times.10.sup.14 viral particles. Clearly, it becomes unfeasible to produce the required amount of virus using this traditional process. New scaleable and validatable production and purification processes have to be developed to meet the increasing demand. [0007]The purification throughput of CsCl gradient ultracentrifugation is so limited that it cannot meet the demand for adenoviral vectors for gene therapy applications. Therefore, in order to achieve large scale adenoviral vector production, purification methods other than CsCl gradient ultracentrifugation have to be developed. Reports on the chromatographic purification of viruses are very limited, despite the wide application of chromatography for the purification of recombinant proteins. Size exclusion, ion exchange and affinity chromatography have been evaluated for the purification of retroviruses, tick-borne encephalitis virus, and plant viruses with varying degrees of success (Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al., 1978, Smith and Lee, 1978; O'Neil and Balkovic, 1993). Even less research has been done on the chromatographic purification of adenovirus. This lack of research activity may be partially attributable to the existence of the effective, albeit non-scalable, CsCl gradient ultracentrifugation purification method for adenoviruses. [0008]Recently, Huyghe et al. (1996) reported adenoviral vector purification using ion exchange chromatography in conjunction with metal chelate affinity chromatography. Virus purity similar to that from CsCl gradient ultracentrifugation was reported. Unfortunately, only 23% of virus was recovered after the double column purification process. Process factors that contribute to this low virus recovery are the freeze/thaw step utilized by the authors to lyse cells in order to release the virus from the cells and the two column purification procedure. [0009]Clearly, there is a demand for an effective and scaleable method of adenoviral vector production that will result in a high yield of product to meet the ever increasing demand for such products. Recently Blanche et al in WO 98/00524, based on U.S. Ser. No. 60/026,667, describe adenoviral production methods that are useful as descriptive art. PCT publication No. WO 98/00524 and U.S. Ser. No. 60/026,667 are specifically herein incorporated by reference for their description of techniques for production and purification of recombinant adenovirus. SUMMARY OF THE INVENTION [0010]The present invention describes a new large scale process for the production and purification of adenovirus. This new production process offers not only scalability and validatability but also virus purity comparable to that achieved using CsCl gradient ultracentrifugation. [0011]The present invention relates to a process for preparing large scale quantities of adenovirus. Indeed, it is believed that very large quantities of adenovirus particles can be produced using the processes of the present invention, quantities of up to about 1.times.10.sup.18 particles, and preferably at least about 5.times.10.sup.14 particles. This is highly desirable, as there are currently no techniques available to produce the very large, commercial quantities of adenovirus particles required for clinical applications at the high level of purity needed. [0012]In one embodiment, the process generally involves preparing a culture of producer cells by seeding producer cells into a culture medium, infecting cells in the culture after they have reached a mid-log phase growth with a selected adenovirus (e.g., a recombinant adenovirus), and harvesting the adenovirus particles from the cell culture. This is because it has surprisingly been discovered by the inventors that maximal virus production is achieved in the producer cells when they are infected in the later part of log phase growth and prior to stationary growth. Preferably, the adenovirus particles so obtained are then subjected to purification techniques either known in the art or set forth herein. [0013]In certain preferred embodiments of the present invention, therefore, the producer cells are infected with adenovirus at between about mid-log phase and stationary phase of growth. The log phase of the growth curve is where the cells reach their maximum rate of cell division (i.e. growth). The term mid-log phase of growth refers to the transition mid-point of a logarithmic growth curve. Stationary phase growth refers to the time on a growth curve (i.e. a plateau) in which cell growth and cell death have come to equilibrium. [0014]In even more preferred embodiments, the producer cells are infected with the adenovirus during or after late-log phase of growth and before stationary phase. Late-log phase is defined as cell growth approaching the end of logarithmic growth, and before reaching the stationary phase of growth. Late-log phase can typically be identified on a growth curve as a secondary or tertiary point of inflection that occurs as the log-growth phase slows, approaching stationary growth. [0015]In a preferred embodiment of the present invention, the producer cells are seeded into the cell culture medium using an essentially homogeneous pool of cells. The inventors have surprisingly discovered that the use of a homogeneous pool of cells for seeding can provide much improved confluency and cell density as well as better maturation of the virus, which in turn provides for larger production quantities and ultimate purity of the virus recovered. Indeed, seeding through the use of separate rather than homogeneous cell populations, for example from individual cell culture devices used in the cell expansion phase, can result in uneven cell density, and therefore uneven confluency levels at the time of infection. It is believed that the use of a homogeneous cell pool for seeding overcomes these problems. [0016]In another preferred embodiment of the present invention, the culture medium is at least partially perfused during a portion of time during cell growth of the producer cells or following infection. Perfusion is used in order to maintain desired levels of certain metabolites and to remove and thereby reduce impurities in the culture medium. Perfusion rates can be measured in various manners, such as in terms of replacement volumes/unit time or in terms of levels of certain metabolites that are desired to be maintained during times of perfusion. Of course, it is typically the case that perfusion is not carried out at all times during culturing, etc., and is generally carried out only from time to time during culturing as desired. For example, perfusion is not typically initiated until after certain media components such as glucose begin to become exhausted and need to be replaced. [0017]The inventors have discovered that low perfusion rates are particularly preferred, in that low perfusion rates tend to improve one's ability to obtain highly purified virus particles. The inventors prefer to define perfusion rate in terms of the glucose level that is achieved or maintained by means of the perfusion. For example, in the present invention the glucose concentration in the medium is preferably maintained at a concentration of between about 0.5 g/L and about 3.0 g/L. In a more preferred embodiment, the glucose concentration is maintained at between about 0.70 g/L and 2.0 g/L. In a still more preferred embodiment, the glucose concentration is maintained at between about 1.0 g/L and 1.5 g/L. [0018]Also in certain preferred embodiments, the inventors prefer to recirculate the cell culture media while carrying out processes in accordance with the present invention, and even more preferably, the recirculation is carried out continuously. Recirculation is desirable in that it affords a more even distribution of nutrients throughout the cell growth chamber. [0019]In certain other embodiments, the cells are seeded into the culture medium and allowed to attach to a culture surface for between about 3 hours and about 24 hours prior to initiation of medium recirculation. Attachment of cells to a cell surface generally allows for a more consistent and uniform cell growth and higher virus production rate, which in turn allows for the production of higher quality virus. It has been found by the inventors that recirculation can sometimes impede consistent and uniform cell attachment, and that ceasing recirculation during cell attachment phases can provide significant advantages. [0020]With respect to seeding, in a preferred embodiment of the present invention, the cell culture medium is seeded with between about 0.5.times.10.sup.4 and about 3.times.10.sup.4 cells/cm.sup.2, and more preferably with from about 1-2.times.10.sup.4 cells/cm.sup.2. The reason for this is that it has been found that in order to achieve maximal cell expansion and growth, it is most preferable to inoculate the selected growth chamber with a lower number of cells that one might typically use in other cell growth situations. The inventors have found that higher numbers of cells used in the cell inoculation step results in a cell density that is too high and can result in an over-confluence of cells at the time of viral infection, thus lowering yields. It is well within one of skill in the art to determine that in other types of cell culturing systems, similar optimization of the seeding density for a particular system could easily be determined. Nevertheless, in a particularly preferred embodiment, the cell culture medium is seeded with between about 7.5.times.10.sup.3 and about 2.0.times.10.sup.4 cells/cm.sup.2. In an even more preferred embodiment, the cell culture medium is seeded with between about 9.times.10.sup.3 and 1.5.times.10.sup.4 cells/cm.sup.2. [0021]In another preferred embodiment of the present invention, the harvested adenovirus is purified and placed in a pharmaceutically acceptable composition. A pharmaceutically acceptable composition is defined as one that meets the minimal safety required set forth by the FDA or other similar pharmaceutical governing body, and can thus be administered safely to a patient. The present invention provides processes for the purification of the adenovirus. For example, the adenovirus is purified by steps that include chromatographic separation. While more than one chromatography step can be used in accordance with the present invention to purify the adenovirus, this will often result in significant losses in terms of yield. Thus, the inventors have discovered that surprising levels of purity can be achieved where only a single chromatography step is carried out, particularly where that chromatography step is carried out using ion-exchange chromatography. Ion-exchange chromatography is an excellent choice for purification of adenovirus particles due to the presence of a net negative charge on the surface of adenoviruses at physiological pH, permitting high purity isolation of adenovirus particles. [0022]In particular embodiments of the present invention, the recombinant adenovirus is a replication-deficient adenovirus encoding a therapeutic gene operably linked to a promoter. A replication deficient adenovirus carrying a therapeutic gene linked to a promoter allows the controlled expression of the therapeutic gene by activating the promoter. The precise choice of a promoter further allows tissue specific regulation and expression of the therapeutic gene. In particular embodiments, the promoter is an SV40 IE, RSV LTR, .beta.-actin, CMV-IE, adenovirus major late, polyoma F9-1, or tyrosinase promoter. Continue reading about Method for the production and purification of adenoviral vectors... Full patent description for Method for the production and purification of adenoviral vectors Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for the production and purification of adenoviral vectors patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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