TWI314581B - Method of extracting virus from cell culture - Google Patents

Description

A7 B7 1314581 V. INSTRUCTIONS (1) RELATED APPLICATIONS This application claims the benefit of US Provisional Application Serial No. 60/276,734, the entire disclosure of which is incorporated herein by reference. . FIELD OF THE INVENTION The present invention relates to methods for extracting viruses from cell culture. In particular, this method is suitable for extracting infectious viruses suitable for clinically administered to mammals including humans. references

Berry et al., Biotechnology and Bioengineering, "Production of Reovirus Type-1 and Type-3 from Vero Cells Grown on Solid and Macroporous Microcarriers", Biotechnology and Bioengineering 62: 12-19 (1999).

Bos, J.L., "Res oncogenes in human Cancer: A Review", Cane. Res. 49(17): 4682-4689 (1989).

Chandron and Nibert, "Proteas cleavage of reovirus capsid protein mul and mulC is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle", J. of Virology 72(1): 467-75 (1998).

Coffey, M.C., et al., "Reovirus therapy of tumors with activated Ras pathway", Science 282: 1332-1334 (1998).

Davis, et al., Microbiology, Lippincott, Philadelphia (1990).

Drastini, Y. et al., "Comparison of eight different _______-^4-_ This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm) A7 B7 1314581 V. Invention description (2 ) [procedures For harvesting avian reoviruses grown in Vero cells", J. Virological methods 39: 269-278 (1992) °

Fields, B.N. et al., Fundamental Virology. 3rd Edition, Lippincott-Raven (1996).

Japanese Patent 63044532A, published February 25, 1988.

McRae, M.A. and Joklik, W.K., "The nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3", Virology, 89: 578-593 (1979).

Nibert et al., "Rovirus and their replication", in Fields et al., Fundamental Virology. 3rd Edition, Lippincott-Raven (1996).

Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia PA 19th ed. (1995) e

Smith, R.E., et al., "Polypeptide components of virions, top component and corrs of reovirus type 3", Virology, 39:791-800 (1969).

Strong, J.E. and P.W. Lee, "The v-erbV oncogene confers enhanced cellular susceptibility to reovirus infection", J. Virol. 70: 612-616 (1996).

Strong, J.E., et al., "Evidence that the Epidermal Growth Factor Receptor on Host Cells Confers Reovirus Infection Efficiency", Virology 197(1): 405-411 (1993).

Strong, JE and., "The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus", EMBO J. -5- This paper scale applies to @国标准(CNS) A4 specification (210 X 297 mm) A7 B7 1314581 V. INSTRUCTIONS (3) 17: 3351-3362 (1998).

Taber et al., "The selection of virus-resistant Chinese hamster ovary cells", Cell 8: 529-533 (1976). WO99/08692A1, published February 25, 1999. All publications, patents and patent applications mentioned above The complete disclosure is expressly disclosed in its individual publications, patent applications, or patents, the entire disclosure of which is hereby incorporated by reference in its entirety herein in its entirety in its entirety herein in Therefore, virology has become an area of accumulated research. There is always a constant demand for efficient production of viruses to isolate and purify viral proteins, to produce vaccines, or to provide infectious viruses for experimental research. Recently, new developments in viral therapy have been further improved. It is required to effectively produce infectious virus. Reovirus therapy is an example of viral therapy. Reovirus is a double-stranded RN A virus that binds to most cells. However, most cells are not susceptible to reovirus infection. And the result of reovirus binding to its cellular receptor results in no viral replication or virion production. This may be the reason why the reovirus is not known to be associated with any particular disease. It has recently been found that cells transformed with the ras oncogene become susceptible to reovirus infection, but not to the untransformed control group ( Strong et al, 1988). For example, when the anti-reovirus NIH 3T3 cells are transformed by activated Ras or Sos (a protein that activates Ras), the reovirus infectivity is enhanced. Similarly, Reovirus infection is resistant to small-6- This paper scale applies to Chinese National Standard (CNS) VIII 4 specifications (210 X 297 mm) A7 B7 1314581 V. Description of invention (4) White rat fibroblasts in EGF Receptor genes or v-erbB oncogenes become sensitized after infection, both of which activate the ras pathway (Strong et al., 1993; Strong et al., 1996). Therefore, the reovirus can be activated. Selective infection and replication in cells of the R as pathway. The ras oncogene is the main cause of a significant proportion of mammalian tumors. The activation mutation of the ras gene itself accounts for about 30% of all human tumors (B 〇s, 1989) , mainly occurs in pancreatic cancer (90%), sporadic colorectal cancer (50%) and lung cancer (40%), and bone marrow-like blood cancer (30%). In the ras path, the activation of upstream or downstream factors of ras It is also associated with tumors. For example, excessive expression of HER2/Neu/ErbB2 or epithelial growth factor (EGF) receptors is common in breast tumors (25-30%), platelet-derived growth factor (PDGF) receptors or EGF receptors. Excessive performance is common in gliomas and glioblastomas (40-50%). It is known that both the E G F receptor and the PDGF receptor activate r a s when binding to their individual ligands, and v-erbB encodes a constructive activating receptor lacking an extracellular functional site. Since many human tumor lines are caused by genetic alterations or high Ras activity of the proto-oncogenes, the reovirus therapy is a novel and effective therapy for these conditions (Coffey et al., 1989). Reovirus therapy is highly selective for tumor cells associated with Ras and does not infect normal cells. This therapy has a wide range of uses and can be used in both human and non-human animals. In order to produce a reovirus suitable for clinical administration, a method for rapidly and efficiently producing a reovirus in a culture cell is required. In addition, the traditional method of extracting virus from cultured cells is cumbersome and JL is time consuming, so that the virus is produced into a -7-paper scale applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) A7 B7 1314581 V. Invention Description (5 ) This is too high. Therefore, there is also a need to improve the method of extracting viruses. SUMMARY OF THE INVENTION The present invention relates to methods for extracting viruses from cell culture. Traditionally, the method of extracting virus from cells has been subjected to multiple freeze-thaw cycles and purified by density gradient and ultra-high speed centrifugation. In the present invention, we extracted the virus in the cell culture with a detergent, and as a result, the yield was higher than that obtained by the cold-kang method. In addition, this extraction step can be carried out at a peripheral temperature or a high ambient temperature. Specifically, the extraction method can be carried out at a convenient temperature such as 25 ° C or 37 ° C, and still produces a high viral power price. Accordingly, one aspect of the invention provides a method of producing a virus from a cell culture comprising the steps of: (a) providing a cell culture of the infected virus; (b) adding a detergent to the culture, extracting from the cell. Virus; and (c) collecting the virus. This cleaning agent can be any cleaning agent commonly used to disintegrate cells. Specifically, the detergents in the group consisting of Triton X-100, Tween 20, NP-40 and sodium deoxycholate are selected. The best cleaning agent is Ding riton X-100, with a final concentration of 1%. This virus extraction method can be carried out at any temperature above the freezing point, specifically above 4 °C. A typical extraction method can be carried out at a temperature and does not need to be maintained at a preset temperature. It is best to extract at 25 °C. It is preferably carried out at a culture temperature of the cells, for example, 37 ° C, so that cell culture and virus extraction can be carried out in the same incubator. When the cell culture is incubated with the detergent for a period of time sufficient to disintegrate the cells -8- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) A7 B7 1314581 V. Illustrative (6). The culture time is preferably 60 minutes or less, more preferably 30 minutes or less, and most preferably 10 minutes. In a preferred embodiment, the virus is a non-coated virus. This membrane-free virus is preferably a reovirus. The invention is applicable to any reovirus, in particular a mammalian reovirus. The mammalian reovirus is preferably a human reovirus, preferably a serotype 3 reovirus, and is best for a Deering strain reovirus. The cell can be any cell suitable for the production of a virus of interest, including adsorbent cells or suspension cells. Cell growth is preferred in suspension cultures. When the virus is a reovirus, the cell is preferably HEK 293 cells. Collecting the virus after extraction can exclude cell debris, for example by filtration. In order to increase the filtration flow rate, a fractional filtration method may be practiced in which filtration is carried out before the large pore size and then through at least one filtration step of a smaller pore size. The pore size and type of the filter will vary depending on the virus and cell properties and can be determined by those skilled in the art. For example, when using HEK 293/SF cells to produce reovirus, use a filter of 8 or 5 pores before using a 3 filter and finally a 0.8 filter. This filtrate can be further concentrated to reduce the volume of the virus suspension. If it is desired to change the solution in which the virus is suspended, an ion exchange chromatography method or a dialysis method can be employed. Another aspect of the invention provides a composition comprising a virus collected according to the invention. Specifically, a virus composition suitable for clinical administration is provided, and the preferred virus is a reovirus. The composition may further comprise a pharmaceutically acceptable excipient and/or carrier. Detailed Description of the Invention -9- This paper scale applies to the Chinese National Standard (CNS) Α4 specification (210X 297 mm) A7 B7 1314581 V. Description of the invention (7) The present invention relates to a method for extracting virus from cell culture. We have developed a quick and easy way to replace traditional freeze-thaw techniques. Before the present invention is described in further detail, the terms used in this application are defined as follows unless otherwise indicated. DEFINITIONS As used herein, "viral infection" refers to the entry of a virus into a cell and subsequent replication of the virus in the cell. As used herein, "infection rate" refers to the ratio of the number of viruses to the number of cells when a virus is used in contact with a cell. "Cell lysis" as used herein refers to the collapse of the cell membrane of a cell, followed by the release of all or part of the contents of the cell. As used herein, "culture conditions" refer to conditions for culturing cells, including but not limited to temperature, culture vessel type, humidity, concentration of co2 or any other gas used in the culture vessel, medium format, cultured cells The initial density, and if the cell is infected with a virus, refers to its initial infection rate. The "cell culture" as used herein refers to a cultured cell population found under its culture conditions. In particular, cell cultures include cells and media. Cells that have been aggregated are not considered cell cultures unless they are re-introduced into culture conditions. As used herein, a "cell-associated" virus refers to a virus that attaches to or captures a portion of a cell that has produced a virus. Thus, before the host cell dissolves, the virus binds to the cell. When the cell begins to dissolve, the virus remains attached. Or catch the disintegrated cell parts and remain bound to the cells. However, when the virus is self-cultivated, the paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) A7 B7 1314581 V. Description of the invention ( 8) When the nutrient is released, it is no longer bound to the cells. Therefore, 'cell free virus' refers to a virus that does not bind to cells. The term "extracting" virus as used herein refers to the transformation of a cell-bound virus into a cell-free virus. The process of the virus. As used herein, "cleaner" is a substance having a hydrophilic terminal and a hydrophobic terminal, and the cleaning agent is preferably a synthetic chemical compound, and more preferably a synthetic chemical compound which is biodegradable. Detergents suitable for use in the present invention enhance cell membrane breakdown and promote release of the contents of the disrupted cells. As used herein, "culture" after the addition of a detergent to a cell culture refers to the process of mixing the cell culture with a detergent. As used herein, "collecting" a virus refers to the collection of cells from a culture of a cell that has previously been infected with the virus. The process of virus. The typical collection method of this virus is to separate cell debris and virus and collect the virus-containing part. The virus may be further separated from the dissolved substance as needed, for example, by centrifugation - "week temperature" as used herein refers to about 10°. C to a temperature of about 30 ° C. The ambient temperature is preferably between about 15 ° C and about 30 ° C, more preferably between about 20 ° C and about 25 ° C, and most preferably about 25 ° C. " The cytopathic effect can be explained by double swelling of the cells and the appearance of particles and cell disintegration. Cells showing cytopathic effect are negatively stained in viable cell counts because they absorb dyes. "Cell" refers to cells attached to a culture vessel in cell culture. Examples of the adherent cells include monolayer cells which are cells which form a monolayer of cells on the surface of the culture vessel; "suspended cells" refer to cells which do not adhere to the culture vessel in the cell culture. Suspension cells can be -11 - This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 1314581

The rotary culture method is a culture method in which a continuous perturbation medium is grown in a "rotating culture method" in a culture process. The cell "disintegration" used herein refers to cell membrane disintegration 1 at least - the cell content is released from the cell. The cell disruption method can be, for example, a freezing method, an ultrasonic method, or a detergent treatment method. 7 / - This is also used, the cell viability " or "cell maintenance survival ratio' refers to the percentage of cytopathic effector cells that do not appear in the population. Knife The "no-film virus" used in this article is a virus without a membrane. For example, the beneficial membrane virus can be any adenoviral material (such as adenovirus), picornavirus material (such as encephalomyelitis virus), regut Viral material (such as reovirus), papilloma virus material (such as papilloma virus), small DNA virus material (such as Kilham rat virus) or iridescent virus material (such as mosquito mosquito virus). The "I" reovirus used, refers to any virus classified in the genus Reovirus, including naturally occurring, modified or recombinant. The reovirus is a virus with a double-stranded, segmented RN A genome with a diameter of 60-80 nm and two concentric shells, each of which is icosahedral f. The genome consists of 10-12 isolated double-stranded RN a, and the total genomic size is 16-27 kbp. The individual RN A fragments are diverse in size. Three different but related reovirus types have been collected from many species. These three types share a common complement-fixed antigen. The human reovirus consists of three serotypes: type 1 (Lang [Lang] strain or TIL), type 2 (Jones [Jones] strain, T2J), and type 3 (Dearing strain or Ai [Abney] strain, T3D) These three serotypes are very -12- This paper scale applies to China National Standard (CNS) A4 specification (210X 297 mm) A7 B7 1314581 V. Invention description (1〇) Easy to rely on neutralization Method and hemagglutination inhibition test identification (see, for example, Fields, BN et al, 1996). Reoviruses may occur naturally or be modified. When the reovirus is isolated from the source of the natural world and is not intentionally modified by humans in the laboratory, it is "naturally occurring." For example, a reovirus can be from a "wild source", that is, from a human that has been infected with a reovirus. Reovirus can be modified to still lyse a mammalian cell with active ras pathway The reovirus can be pre-treated by chemical or biochemical treatment (for example, by protease treatment, such as chymotrypsin or trypsin) before administration of proliferating cells. Protease pretreatment can remove the outer shell or outer sheath of the virus. And can increase the viral infectivity. Reovirus can be coated in vesicles or micelles (.Chandron and Nibert, 1998). For example, virions can be in the concentration of alkyl sulfate detergents that can form micelles. In the presence of chymotrypsin, a new infectious subviral particle is produced. The reovirus can be a recombinant (eg, regroup and body) of two or more reoviruses with different pathogenic phenotypes. A solitary virus, thus containing different priming determinants, to reduce or prevent immune responses in mammals that have been exposed to 4 subtypes in the past. This recombinant virion virion can be passed through mammals. The cells are co-infected with reoviruses of different subtypes to cause recombination, and the coat proteins of different subtypes are introduced into the outer sheath of the formed virions. The "HEK 293 cells" indicator used herein is shown as 2 The human embryonic kidney cell line of 9 3 (ACTT CRL-1573) or a derivative thereof, for example, 293/SF cells (ACTT CRL-1573.1) is a HEK 293 cell that has been adapted to grow in serum-free medium. The invention also includes the HEK 293 fine -13- paper scale applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm).

A7 B7 1314581 V. INSTRUCTIONS (11) Cell adaptation to any other species of HEK 293 cells or derivatives thereof that grow in other culture conditions or exogenous DNA transformation, but the constraints are not Destruction of the cells of the invention supports the ability to efficiently produce reovirus. "Clinical administration" of a substance as used herein refers to bringing the substance into contact with any part of the living organism's body in order to improve or maintain the health of the organism. Methods Reovirus is described as a model system to illustrate the present invention. However, the present invention should also be applicable to other viruses, in particular, it is free of enveloped viruses. We have previously developed a method for growing reoviruses on HEK 293 cells. In a short period of time after the cells are infected with the virus, the reovirus will replicate in HEK 293 cells, resulting in a high-valency virus, thus suggesting a simple and efficient method for producing reovirus (Examples 1 and 2). In addition, HEK 293 has been domesticated and can be grown in suspensions in large quantities, and we have developed large-scale production methods. In order to isolate the reovirus from the suspension culture, we initially extracted and purified the virus particles according to conventional methods. Briefly, cells were disrupted via freeze-thaw method and extracted three times with dichloromethane (Freon). The virions were then purified by CsCl gradient and ultra-high speed centrifugation. However, when the virus is produced on a large scale, these steps are too complicated and time consuming. Because we have developed a simplified method of extracting viruses. It has been found that a high amount of infectious reovirus is released into the extract by HEK 293 cells and the detergent for a short period of time. The virus can then be applied to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) by particle size or -14-paper scale. A7 B7 1314581 V. Inventive Note (12) Simple separation method with different density, such as filtration The method, dialysis method or particle size exclusion method will separate the virus from the cell debris, and the resulting virus can be used for reovirus therapy. The reovirus produced according to the present invention is suitable for administration to humans, and this method complies with the procedure recommended by F D A for disintegrating cells in the presence of a detergent. In the present invention, we tested four detergents, including Triton X-100, NP-40, Tween 20 nonionic detergent, and the ionic detergent sodium deoxycholate. Although all four detergents are functional in the present invention, the Triton X-100 can produce more than twice as many viruses as the other two nonionic detergents. Deoxycholic acid steel is as effective as Triton X-100. Other cleaning agents, particularly detergents which are commonly used to disrupt cells, should also be used in the present invention. Examples of such other cleaners include other Triton cleaners, other Tween cleaners (eg Tween-80), sodium lauryl sulfate, lithium lauryl sulfate and lauryl trimethylammonium chloride = this result shows The detergent extraction method is more efficient than the freeze-thaw method (this is a standard step for virus extraction). A method for extracting avian reovirus from Vero cells has been reported in the literature, in which reovirus is combined with high cells, and distilled deionized water is compared with freeze-thaw method, dichlorodioxoethane extraction method, or trypsin treatment. The law is efficient (Drastini et al., 1992). The present invention proposes a rapid, convenient, and effective method because it does not require a process in which the distilled water method must first be agglomerated and then resuspended. High concentration salts such as barium chloride are contemplated for use in the present invention as a replacement cleaner. However, the use of detergents is better than high concentration salts. Therefore, the present invention proposes a rapid extraction of virus from cell culture _ and -15- This paper scale is applicable to China National Standard (CNS) A4 specification (210X 297 mm) A7 B7 1314581 V. Invention Description (13) Simple method . The detergent can be added directly to the culture medium of the suspension culture or adherent cells, in which case it is not necessary to remove the medium first. In addition, there is no need for other ways to disintegrate cells or extract viruses, such as cold-kang-solution; Eastern law or ultrasonic method. An important feature of the invention is that the extraction step can be carried out at temperatures above or above ambient or ambient temperature. Traditionally, in order to preserve the structure and function of proteins, viruses were extracted and purified at low temperatures, typically at 0-4 °C. For the same reason, protease inhibitors are often included in the extraction solution. Thus, the process of the invention surprisingly can be carried out at elevated temperatures without the use of any protease inhibitors. In fact, temperatures up to 37 °C resulted in the same infectious virus yield as at 25 °C (Table 3). Therefore, by directly adding a detergent to the cell culture and continuously stirring the culture to release the virus, the virus can be extracted without changing the temperature. Alternatively, since the virus is extracted in accordance with the present invention, it is not necessary to maintain a constant temperature, this step may occur at ambient temperature, and even the ambient temperature may vary depending on the location, or the ambient temperature at the same location may change over time. The virus can be collected after extraction. Any method established in the related art can be used to purify the virus. For example, cell debris can be removed by sputum. In order to increase the filtration flow rate, a fractional filtration method may be carried out in which the filtration step is carried out with a large pore size, followed by at least one filtration step of a smaller pore size. The size and type of filter pore size will vary depending on the virus and cell properties and can be determined by those skilled in the art. For example, when using HEK 293/SF cells to produce reovirus, use a filter of 8 or 5 pore size before using a 3 μM pore size filter and finally a 0.8 Μ pore size filter. If the amount of filtration volume is too large, the filtrate can be used according to any method known in the art. -16- This paper scale is applicable to China National Standard (CNS) Α4 specification (210X297 mm) A7 B7 1314581 5. Inventive Note (14) Method Concentration. For example, ultrafiltration can be performed using a hollow fiber cartridge (A/G Technology) or a flat disc and a Pall Filtron. The concentrated virus suspension can be subjected to dialysis or ion exchange chromatography in one step. Except for too much salt or adding other ingredients to the virus suspension. The method can be applied to reoviruses produced by other non-HEK 293 cells including, but not limited to, mouse L929, Vero and Chinese hamster ovary cells. This method should also be applicable to other viruses, specifically other membrane-free viruses. Compositions The invention also provides viral compositions prepared in accordance with the present invention. These compositions can be used for the isolation and identification of viral proteins, vaccine production, or compositions containing infectious diseases that can be used as viral stocks or clinically. Compositions for clinical administration purposes are usually mixed with excipients, diluted with an excipient or coated in a carrier which can be formed into capsules, sachets, paper packs or other containers (WO 99/08692 A1 ). When a pharmaceutically acceptable excipient is used as a diluent, it can be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Therefore, the composition may be in the form of a tablet, a pill, a powder, a mouth-shaped key, a sachet, a flat capsule, a granule, a suspension, an emulsion, a solution, a sugar solution, an aerosol (either in a solid or contained in a liquid medium), An ointment containing, for example, up to 10% by weight of an active compound, a soft and hard gelatin capsule, a suppository, a sterile injectable solution, in the form of a sterile packaged powder. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, citrate, tragacanth, gelatin, calcium citrate 'polyvinylpyrrolidone , cellulose, sterilized -17- This paper scale is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1314581 A7 - ~~ -----B7 _. V. Invention description (15) ~ --- Water syrup and methyl cellulose. The formulation may additionally comprise a lubricant (such as talc 'magnesium stearate and mineral oil); a wetting agent; an emulsifier and a suspending agent; a preservative (such as hydroxy hydroxybenzoate and propyl ester); a sweetener; And spices. The compositions of the present invention may be formulated to deliver the active ingredient rapidly, continuously or with a delayed release after administration to a patient by methods known in the art. For the preparation of a solid composition such as a medicinal tablet, the main active ingredient/reovirus is mixed with a pharmaceutically acceptable excipient to form a solid pre-formulation composition containing a homogeneous mixture of the compounds of the invention' when referring to such pre-formulations "Equivalent" means that the active ingredient is uniformly dispersed in the composition, so that the composition can be easily divided into equally effective unit dosage forms such as tablets, pills and sacs. The tablets or pills of this month may be coated or combined to form a dosage form having the advantage of prolonging action. For example, a tablet or pill may contain an internal dose and an external dose component, the latter being the envelope type of the former. These two components can be separated by an enteric layer to resist decomposition in the stomach and delay the release of internal components into the eleven finger. A wide variety of materials can be used for such enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with materials such as shellac, whale alcohol, cellulose acetate, and the like. Liquid aa type wherein the composition of the present invention can be administered orally or by injection, including an aqueous solution, a suitably flavored syrup, an aqueous or oily suspension, novel and using an edible oil such as corn oil, cottonseed oil, sesame oil, coconut oil or peanut oil. Flavoring lotions, and tinctures and similar pharmaceutical agents. Compositions for inhalation or insufflation include pharmaceutically acceptable solutions and suspensions, aqueous or organic solvents, or mixtures thereof, and powders. This liquid or solid ___ -18- This paper scale is suitable for g g home standard (CNS) Μ specifications (21QX 297 public)

Binding A7 B7 1314581 V. INSTRUCTIONS (16) The body composition may comprise a suitable pharmaceutically acceptable excipient composition as described herein. Preferably, the composition is administered orally or nasally for local or systemic effects. Preferably, the composition is contained in a pharmaceutically acceptable solvent for gas atomization using a gas. The aerosolized solution can be inhaled directly by the aerosol device, or the aerosol device can be attached to a mask or intermittent positive pressure breathing apparatus. The solution, suspension or powder composition can be delivered in a suitable manner, preferably by oral or nasal administration. Another preferred formulation for use in the method of the present invention utilizes a transdermal delivery device ("applied"). Such a skin-worn patch can deliver a controlled amount of the reovirus of the present invention in a continuous or discontinuous manner. The construction and use of a transdermal patch for the delivery of a medicinal substance is well known in the art. See, for example, U.S. Patent No. 5,023,252, which is incorporated herein by reference. This patch is constructed to be continuous, pulsed or to deliver medical substances as needed. Other suitable formulations for use in the present invention can be found in Reminton's Pharmaceutical Sciences. The following examples illustrate the invention and are not intended to be limiting in any way. Examples In the following examples, the following abbreviations are defined as follows. Undefined abbreviations are as defined in their general acceptance. CI = confidence interval TCID5〇 = tissue culture infectious dose βΜ = micromolar concentration mM = millimolar concentration 莫 = molar concentration ml = ml-19- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1314581 A7 B7 V. INSTRUCTIONS (17) 仁1 = microliter mg = mgβ% = microgram g/L = gram per liter rpm = revolutions per minute FB S = fetal bovine serum DTT two-one Sulphur tread sugar fresh NP-40 = Nonidet P-40 (octylphenoxypolyethoxyethanol) SDS = sodium dodecyl sulfate PBS = phosphate buffer / 3-ME = no - pickling ethanol MOI or moi = infection rate PFU = plaque formation unit hr = hour °C = Celsius temperature General method Cell and virus Human embryonic kidney cells 2 9 3 (HEK 293 ), Vero (African green monkey kidney) cells and mouse fibroblast L -929 cells were supplied by the manufacturer BioReliance (Rockville, Maryland). HEK 293 cells were grown in medium containing 10% heat-inactivated horse serum and 90% of the following mixture: Eagle's minimum base medium containing 2 mM L-熬醯 and Earle's balanced salt solution (adjusted to 1.5 g) /L sodium bicarbonate), 0.1 mM non-essential amino acid and 1.0 mM C-acid. Mouse L-929 and Vero cells are in accordance with Chinese National Standard (CNS) A4 size (210x 297 mm) A-20 B7 1314581. V. Inventive Note (18) 10% FBS and 90% Mixture: 2 mM L-glycine and Earle's balanced salt solution (adjusted to 1.5 g/L sodium bicarbonate), 0.1 mM non-essential amino acid and 1. mM mM pyruvate medium Hyperplasia. 293/SF cells were supplemented with 4 mM L-branched amine in 293 serum-free medium (Life Technologies, Rockville, Maryland) at 36 °C ± 2 °C '6% ± 2% C〇2, and 80% ± Grown in a rotating flask at 35-40 rpm impeller speed at 5% relative humidity. Residual strains of Reovirus serotype 3 were used in these studies to be grown in suspensions of L-929 cells purified according to Smith (Smith et al., 1969). However, the extraction buffer is omitted without the use of 巯-mercaptoethanol (dot-ME). The particle/PFU ratio of the purified reovirus is typically 100/1. Monolayer cell infection and virus quantification Fusion monolayers of HEK 293, Vero and L-929 cells were grown in 24-well plates and infected with reovirus of known infection rate. After incubating at 7 °C for 1 hour, the monolayer cells were washed with warm medium and then cultured in the medium. At different time points after infection, the mixture of NP-40 and sodium deoxycholate was directly added to the mixture. In the medium of the infected layer cells, the final concentrations were 1% and 0.5%, respectively. The lysate was then collected and the virus yield was determined by plaque titration on L-929 cells, expressed as Log1() TCID5Q/ml. Suspension cell infection method 293/SF cells were grown at 106 cells/ml and infected with reovirus. The culture is grown until the medium is turned from red to orange, or until the cell viability is reduced to the desired level, based on the viable cell count. The number of viable cells without cytopathic efficiency was calculated under the microscope. The cytopathic effect refers to the cells -21 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

A7 B7 1314581 V. INSTRUCTIONS (19) The swelling and appearance of particles and cell mass separation. The number of viable cells can also be calculated using live cell staining methods commonly used in the relevant art. Traditional methods for extracting and purifying viruses When the desired degree of cell viability is reached, the cells are aggregated into pieces after centrifugation and resuspended in 10 mM Tris, pH 7.4, 250 mM NaCl and 0.1% Triton X-100. The cells were then lysed by freeze-thaw method and kept on ice for 20-40 minutes, and the cells were periodically mixed and lysed by inversion. The suspension was mixed with an equal volume of pre-cooled Freon® (1,1,2-trichloro-1,1,2-trifluoro-ethane) for 1 〇 minutes, followed by 4 ° C and 2500 rpm. Centrifuge for 10 minutes. Different layers are separated. The aqueous phase (upper layer) was removed and extracted twice as described above, and the virus was agglomerated by ultracentrifugation at 25,000 rpm for 1 hour at 4 °C. The agglomerates were resuspended in P B S, and the disease was purified by gasification enthalpy gradient. This gradient contained 2 layers of CsCl solution (1.20 g/ml and 1.4 g/ml, respectively) prepared in 10 mM Tris (pH 7.4). The virus suspension was added to the upper layer of this gradient and centrifuged at 26,000 rpm for 2 hours in a SW 28.1 centrifuge at 4 °C. Viral bands (lower bands in the two bands because the upper band contained an empty outer sheath) were collected and dialyzed against sterile P B S. Example 1 Determining the Optimal Cell Line for Producing Reovirus In order to determine whether there is a difference in the production of enterovirus between the susceptible cell lines after infection, analysis of the production of different cell lines exhibiting the receptivity of reovirus The relative yield of reovirus. Among the particularly noteworthy cell lines are those that have been approved by many regulatory authorities for the production of biologics. - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) A7 B7 1314581 V. Description of the invention (2〇) Cell line. Therefore, HEK 293, Vero and L-929 cells were exposed to reovirus and their ability to produce reovirus was compared. The quantitative method of virus production collects infected cells and their growth medium at different time points after infection. The resulting lysate was then subjected to plaque titration and analyzed to determine virus yield. The results are expressed in force price ± 95% CI (Log10TCID50/ml) and are shown in Table 1 below. The results indicate that although all of the test cells are susceptible to reovirus, the virus yield of each cell strain is still quite different. Table 1: Time course of virus production by different cell lines (expressed by force price ±95% CI, Log10TCID50/ml) Time HEK 293 Vero L929 24 hours 8.30±0.51 4.80±0·35 6_68±0.24 3 6 hours 9·05 ±0·43 5.55±0.32 7.93±0.40 48 hours 9.55±0.49 6·68±0·40 8.55±0.49 72 hours 9_30±0.43 8·18±0.36 9.05±0.32 96 hours 9.80±0.35 9·93±0.59 9.30± 0·43 This result clearly shows that HEK 293 cell line is the most efficient cell for producing reovirus. In addition, HEK 293 cells produce more viruses earlier. Therefore, the production time of large-scale manufacturing of reovirus is shortened. Example 2 Effect of initial infection rate on final virus yield In order to determine whether the initial infection rate determines the final virus yield, HEK 293 cells were infected with an initial infection rate (m.o.i.) in the range of 1 to 0.1. Our results are shown in Table 2. Showing the initial m.o.i. and final virus yield -23- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) A7 B7 I314581 V. The invention description (21) is indeed relevant. When the initial m.o.i. is less than 1, it is most suitable for large-scale production of reovirus. Table 2: Effect of MOI on virus yield (expressed by force price ±95% CI, Log丨0TCID50/ml) HEK 293 Vero L929 j4 hours 9.18±0·36 8.55 ± 0.43 7.68 Soil 0.24^hours 8_92±0.52 9 ·30±0.43 9·37±0_48 48 hours 9·55±0.43 10·30±0.37 9·68±0·50 /72 hours 9.55±0·32 9.93±0.40 9.18 Soil 0.40 j hours 9.80±0.00 10.30±0.43 10.18 Soil 0.36 In addition, our results confirm that the most appropriate time to collect cells is also important. We found that the maximum viral production time was 24 hours later. This result is not surprising, as it was insufficient 24 hours before the virus protein was properly synthesized and composed into mature virions. More surprisingly, a reduction in virus was observed at the 72-hour time point, followed by a significant increase in the number of infected particles at the 96-hour time point. It is postulated that a slight decrease in the 72-hour time point is likely to be viral protein degradation, followed by a second round of virus replication at the 96-hour point. Example 3 Evaluation of Virus Extraction Steps At the outset, we extracted and purified virions following the traditional methods described in "Materials and Methods." Briefly, via cold; East-solution; Eastfava cells and dichlorodioxy The methane is extracted three times. The virions are purified by CsCl gradient and ultra-high speed centrifugation. However, when the virus is produced on a large scale, this step-24- This paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) A7 B7 1314581 The invention (22) is too complicated and time consuming. In order to obtain the best collection conditions for large-scale suspension culture, we have tested several other extraction methods as described below. The reovirus is grown in a constant range. Suspension culture of HEK 293 cells for 2, 48, and 72 hours. After that, add detergents such as Dingshao 11乂-100, Tween 20, NP-40 or deoxycholic acid #3 (Na-D_OC) to In the culture, to the final concentration of each experiment as shown in Table 3. Continue to agitate the culture for 10 or 60 minutes at 25 ° C or 37 ° C. Then remove a portion from the culture in L-929 cells. Lysolysis of the solution, determination of virus yield It is also indicated by Log1QTCID5. The cold-thaw method is also tested, in which the culture is frozen and thawed without adding any detergent in order to disintegrate the cells. In the 'untreated' experimental group, one is taken directly from the culture. And determine the virus production. Table 3. Effect of different extraction methods on virus yield

Loading time sample disease yield soil CI (Logi〇TCID5〇) plus virus control group #1 8·30±0·37 plus virus control group #2 7.68±0·40 plus virus control group #3 7.93±0_24 virus control group Confirmation price 7.99±0.24 Negative control group did not detect virus 24 hours 25 ° C, 10 minutes, 0.1% TX-100 10.67 ± 0_24 25 ° C, 10 minutes, 1% Tween-20 10_42 ± 0.40 Freeze / Thaw Method 10.00±0.37 • Line-25- This paper scale is applicable to China National Standard (CNS) A4 specification (210X 297 mm) A7 B7 1314581 V. Invention description (23) Time sample disease yield soil Cl (Logi〇TCID5〇) Untreated 9·88±0·36 48 hours 25°C, 10 minutes, 0.1% TX-100 10.04±0·43 25. . , 10 minutes, 〇 _3% TX-100 10.42 ± 0 · 52 25 ° C, 10 minutes, 1% Tween-20 10 · 17 ± 0 · 36 25 ° C, 10 minutes, 3% Tween-20 9_79 ± 0.32 25°C, 10 minutes, 0.1% Na-DOC 10.42±0.50 25°C, 10 minutes, 1% NP-40 9.77±0·32 25〇C, 60 minutes, 0.1% TX-100 10·17±0· 44 25〇C, 60 minutes, 1% Tween-20 9·92±0·36 37°C, 10 minutes, 0.1% TX-100 10.42±0.40 37°C, 10 minutes, 1% Tween-20 10·29 ±0.32 37. . , 60 minutes, 0.1% TX-100 10·42±0·50 37〇C, 60 minutes, 1% Tween-20 9_42±0·66 Freeze/thaw method 10·38±0·24 Untreated 10·25± 0.32 72 hours 37°C, 10 minutes, 0.1% TX-100 11·29±0_75 37°C, 10 minutes, 1% Tween-20 9·79±0.32 Freeze/thaw method 10.63±0.24 Untreated 10·13± 0·44 -26- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X297 mm) A7 B7 1314581 V. Description of invention (24) These results show Triton X-100, Tween 20, Na-DOC or NP- 40 can be used to extract virus from HEK 293 cell culture, and its virus yield is better or equivalent than the freeze-thaw method. It should be noted that the Triton X-1 method generally produces more than twice as many viruses as the two other nonionic detergents (Tween 20 and NP-40). Na-DOC is a quasi-test ionic detergent under which conditions produce approximately the same amount of infectious virus as the 0.3% Triton X-100 group. The most appropriate concentration of Triton X-100 was explored in further experiments, showing that Triton C-100 at a final concentration of 1% produced the highest viral amount in the extraction mixture. In addition, it is worth noting that by using a detergent to dissolve the cells instead of the freeze-thaw method, the extraction process should be carried out at 25 ° C or even 37 ° C without affecting the production of infectious virions. This is in contrast to the traditional method of performing a complete virus extraction step at low temperatures (usually 4 °C) in the presence of a protein inhibitor in order to preserve the functional protein. Therefore, the virus can be extracted from the cultured cells by a simple and convenient method, and the large-scale virus production can be greatly improved. Although the virus yield of the "untreated" experimental group was quite high, this indicates that many virions may have been released into the medium before extraction, and the detergent treatment further increased the yield by about 10 times. The effect on yield by detergent treatment was particularly pronounced at 24 and 72 hours post infection. However, at the 48-hour time point, the detergent extraction method did not significantly increase virus production. It is therefore shown that most viruses appear to bind to cells at 24 and 72 hours, but not at 48 hours. This observation is consistent with previous discussions that proteolytic degradation may occur at the midpoint of the culture process, after _____-27-_ This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1314581 A7 B7 V. Inventive Note (25) For the second round of virus replication. Following the above extraction step, the cell debris can be removed by a simple method such as filtration or centrifugation, and the resulting virus can be used for clinical administration. If the cell debris is removed by the sputum method, it can be selectively concentrated, for example, more than the sputum method to reduce the volume of the virus preparation. The iron content of the filtrate or concentrated viral preparation can be further adjusted, for example, by ion exchange chromatography or dialysis. -28- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm)

Claims (1)

Hf (§iIl〇4928 Patent Application ^ ^Application for Patent Range Replacement (95 Patent Application ------ A method for producing infectious reovirus, comprising: (a) providing an infected reo disease | (HEK 293) cell culture; (b) borrowing The virus was extracted from the cells by adding MTriton x_1 to the culture and incubated at about 37 for about 10 minutes; and (c) collecting and purifying the reovirus. 2. The method of claim 1, wherein the method comprises the steps of: removing the cell residue by filtration and concentrating the filtrate. 3. The method of claiming the mountain of the mountain, wherein the reovirus a mammalian reovirus. /, the mammalian reintestinal, wherein the human reovirus, wherein the serotype 3 reovirus, wherein the HEK 293 cell is a living 4, as claimed in claim 3 Method The orphan virus is a human reovirus. 5 · The method of claim 4 is serotype 3 virus. 6. The method of claim 5 is the Dearing strain. The method of applying for the scope of patent item 1 is long in suspension. This paper scale is suitable for the price of fresh (CNS) A4 specification (21GX 297 public hair) ' ---------