CN101163794A - Formulations for therapeutic viruses having enhanced storage stability - Google Patents

Abstract

Therapeutic viral formulations having enhanced storage stability are described. The formulations comprise a viral vector in addition to one or more of an aqueous cosolvent, a reversible viral-encoded protease inhibitor and a mild reducing agent or other agent that prevents specific degradation of viral components.

Description

Formulations for therapeutic viruses with package stability of raising

The cross reference of related application

The interests of the Application No. 60/656,883 that the application's request on March 1st, 2005 submits to are incorporated herein by reference the full content of the document.

Background technology

Invention field

The present invention relates generally to formulations for therapeutic viruses, and more particularly, the present invention relates to comprise one or more stablizers, comprise aqueous cosolvent, the formulations for therapeutic viruses of reversible capsid endoproteinase inhibitor and/or gentle reductive agent.

Technical background

Plan is applied to the virus particle of human body therapy application must keep its structural integrity so that keep biological activity.Yet, may cause biological activity to descend in the storage of the time of elongated segment inner virus carrier formulation.The generally present viral therapy agent of preparation in the damping fluid of the time that allows storage one elongated segment.Yet, must keep and transport this class preparation so that keep its biological activity under the low temperature relatively.Loss of activity usually occurs in the storage process.

Under relative low temperature, store and transport the reduction that can be used for titre and be reduced to bottom line, but, its for the viral therapy agent lack store under proper condition the cost that uses in the clinical setting of this viral equipment and the ability very important.

Many known viruses comprise that those viruses as the therapeutic virus carrier comprise outer capsid (being adenovirus, parvovirus, papovavirus).Allow the outer capsid shelling in the process in order to enter at cell, most of capsid protein combines with adjacent capsid protein is non-covalent.The intensity of these noncovalent interactions is enough to maintain one effective period of capsid confined state in the outer medium of born of the same parents, but stable inadequately under some biotic condition (promptly low pH, factor receptor are in conjunction with the specificity conformational change that causes, enzymatic degradation etc.).This makes them separate assembling in course of infection.Make capsid protein-protein binding keep stable reagent to can be applicable to the therapeutic virus product formulation.

Known adenovirus and the assembling of capsid inner virus encoded protein enzyme.The adenovirus shelling is released into nuclear and the dissociated step-by-step procedure of viral capsid for causing viral DNA.The verified degraded that is positioned at the L-Cysteine HCL Anhydrous L3/p23 inhibitor blocks protein VI of capsid inside, showing needs L3/p23 proteolytic enzyme to assemble virus before entering, and the virus that enters is dissociated.Other virus also can depend on the part of capsid endoproteinase as its life cycle.

Cause that protease activated incident can cause preparing the degraded of virus in the preparation virus, thereby cause unstable and inactivation.

Therefore, there is demand to being used for the treatment of the degraded that under commercial rational condition, shows minimum level of application and the virus vector preparation of package stability.

Summary of the invention

One aspect of the present invention relates to the virus of producing and storing from culturing cell, includes, but are not limited to the method for the improvement of adenovirus.This production method provides and has caused the new preparation that titre descends and reduces in virus stability improvement and the storage process.

Second aspect of the present invention relates to the preparation of storing the virus after processing.Said preparation can be under freezing and non-freezing conditions and is particularly at room temperature kept virus activity.In one aspect of the invention, the degraded by arrestin VI realizes this purpose.

In one embodiment, described preparation comprises one or more aqueous cosolvent, reversible encoding viral proteinase inhibitor and prevents gentle reductive agent or other reagent that the virus composition specificity is degraded.

In another embodiment, described preparation comprises adenovirus carrier, ARCA damping fluid and is selected from the aqueous cosolvent of propylene glycol, DMSO, PEG, sucrose, glycerine and Tetrahydrofurfuryl polyethylene glycol ether, and wherein said preparation shows under 2 ℃-30 ℃ and is higher than the stability of formulation that lacks described aqueous cosolvent.

Described aqueous cosolvent can be about the propylene glycol of 3-20% for concentration; Concentration is about the Tetrahydrofurfuryl polyethylene glycol ether of 5-20%; Total concn, promptly 10%, 20%, 30%, 40%, 50% or 60% sucrose.

In another embodiment, described preparation comprises the adenovirus carrier and the reversible protease inhibitors of the capsid endoproteinase that depends on the encoding viral that is suitable for entering cell, and wherein said preparation shows under 2 ℃-30 ℃ and is higher than the stability of formulation that lacks described reversible protease inhibitors.

Described reversible protease inhibitors can be about the thioglycerin of 0.5-2.0% or 50-200mM for concentration; Concentration is about the dimethyl thioether of 10-100mM; Concentration is about 20-100mM, the dithiothreitol (DTT) (DTT) of preferred 50mM; Concentration is at least 1% or the halfcystine of 150mM; Gsh; And methionine(Met).

The present invention further provides the preparation that is used to store adenovirus carrier, it comprises aqueous cosolvent and reversible protease inhibitors (as mentioned above).

Preferred said preparation shows when storing down for 5 ℃ or 30 ℃ and is higher than the stability of formulation of not adding aqueous cosolvent and/or reversible protease inhibitors.

The accompanying drawing summary

Accompanying drawing 1A and 1B are for representing for the Ad/GM-CSF (1 * 10 that is stored under 5 ℃¹²Vp/ml), 10wt.%PEG preparation (accompanying drawing 1A) and be stored in Ad/GM-CSF (1 * 10 under 25 ℃¹²Vp/ml), ARCA preparation (accompanying drawing 1B) is as the synoptic diagram of anionresin (AE) the HPLC retention time variation of the function of period of storage.

Accompanying drawing 2A is for representing for the Ad/GM-CSF in the ARCA damping fluid (1 * 10 that is stored under 25 ℃¹²Vp/ml) preparation is as the synoptic diagram of the retention time change (Δ RT) at the AE-HPLC color atlas peak of the function of period of storage, and complete capsid virosome (ICV) and empty penton virosome (PVV) are represented in described color atlas peak.

Accompanying drawing 2B is the synoptic diagram of expression as the per-cent of the initial complete capsid virosome (ICV) of the function of the period of storage of the adenovirus preparation described in the accompanying drawing 2A and empty penton virosome (PVV).

Accompanying drawing 2C for expression as the initial complete capsid virosome (ICV) of the function of the period of storage of the adenovirus preparation described in the accompanying drawing 2A, virosome particle (VP) (ICV+PVV) and the synoptic diagram of GM-CSF excretory per-cent.

Accompanying drawing 3 is that the conduct of the preparation dependent change of expression retention time is stored in the various Ad/GM-CSF (2 * 10 under 5 ℃¹²Vp/ml) RT of the function of the period of storage of adenovirus preparation (minute) synoptic diagram that changes.

Accompanying drawing 4A and 4B are anti-phase (RP) HPLC figure of illustration adenovirus protein degraded, and wherein accompanying drawing 4A is 0: 1 * 10¹²The figure of the ARCA solution of the Ad/GM-CSF of vp/ml and accompanying drawing 4B are the figure of identical adenovirus solution after storing 12 months under 15 ℃.

Accompanying drawing 5A is for representing as Ad/GM-CSF (1.2 * 10¹²Vp/ml) the ARMWG of the effect that demonstrates various cosolvent (0.75M) (10mM Tris, 25mM NaCl, 2.5% glycerine, pH8) preparation in is together with the complete capsid virosome (ICV of ARCA control formulation at the function of 30 ℃ of following periods of storage₀) synoptic diagram of per-cent.

Accompanying drawing 5B is for representing as Ad/GM-CSF (1.2 * 10¹²Vp/ml) the ARMWG of the effect that demonstrates various additives (12% weight) (10mM Tris, 25mM NaCl, 2.5% glycerine, pH8) preparation in and ARCA reference substance are at the complete capsid virosome (ICV of the function of 30 ℃ of following periods of storage₀) synoptic diagram of per-cent.

Accompanying drawing 6 is for representing as CG7060 adenovirus (2 * 10¹²Vp/ml) preparation in the ARCA of the effect of the sucrose additive that demonstrates various concentration is at the complete capsid virosome (ICV of the function of 5 ℃ of following periods of storage₀) synoptic diagram of per-cent.

Accompanying drawing 7 illustrates for L3/p23 proteolytic enzyme structure.

Accompanying drawing 8A is using and is not using under the pretreated situation of NEM the solution in the ARCA damping fluid at the complete capsid virosome (ICV of the function of 30 ℃ of following periods of storage for expression as Ad/GM-CSF₀) synoptic diagram of per-cent and GM-CSF secretion rate (GSR).

Accompanyingdrawing 8B is using and is not using under the pretreated situation of NEM the preparation in the ARCA damping fluid at the albumen VI of the function of 30 ℃ of following periods of storage (VI for expression as Ad/GM-CSF₀) synoptic diagram of per-cent.

Accompanying drawing 9A is for representing as Ad/GM-CSF (1 * 10¹²Vp/ml) demonstrate the independent pre-treatment of DTT or successively the preparation to the ARCA damping fluid of the pretreated effect of NEM at the complete capsid virosome (ICV of the function of 30 ℃ of following periods of storage₀) synoptic diagram of per-cent.

Accompanying drawing 9B is for representing as Ad/GM-CSF (1 * 10¹²Vp/ml) comprise 15 or the ARCA damping fluid of 50mMDTT in preparation at the complete capsid virosome (ICV of the function of 30 ℃ of following periods of storage₀) synoptic diagram of per-cent.

Accompanyingdrawing 9C is for representing as Ad/GM-CSF (1 * 10¹²Vp/ml) using and do not using under the pretreated situation of 5mM diamide (N, N, N ', N '-tetramethyl-azodicarboxy acid amides) preparation in the ARCA damping fluid at the complete capsid virosome (ICV of the function of 25 ℃ of following periods of storage₀) synoptic diagram of per-cent.

Accompanying drawing 10 is for representing as Ad/GM-CSF (1 * 10¹²Vp/ml) preparation in comprising the ARCA damping fluid of various L3/p23 proteinase inhibitor or gentle reductive agent is at the complete capsid virosome (ICV of the function of 30 ℃ of following periods of storage₀) synoptic diagram of per-cent.

Accompanying drawing 11 is for representing as Ad/GM-CSF (1 * 10¹²Vp/ml) preparation in the ARCA of L3/p23 proteinase inhibitor that comprises selection or gentle reductive agent is at the albumen VI of the function of 30 ℃ of following periods of storage (VI₀) synoptic diagram of per-cent, confirm in advance that wherein the L3/p23 proteinase inhibitor selected or gentle reductive agent cause at 30 ℃ of complete capsid virosome (ICV down₀) per-cent is maintained certain hour.

Describe in detail

Definition

Unless otherwise stated, otherwise implement of the present invention aspect in use the chemistry, molecular biology, microbiology, recombinant DNA, science of heredity, immunology, cell biology and the cell that belong in those skilled in the art's skill to cultivate routine techniques.

Unless otherwise stated, otherwise all terms used herein all have and identical implication known to the person of ordinary skill in the field, and implement the routine techniques that the present invention will use chemical, microbiology and recombinant DNA technology.

In describing process of the present invention, use following term and specify definition as described below they.

Term " carrier ", " polynucleotide carrier ", " polynucleotide carrier construct ", " nucleic acid carrier construct " and " vector construction body " are used interchangeably in this article, mean any nucleic acid construct that is used for transgenosis as skilled in the art to understand.

Term used herein " viral vectors " uses according to its implication of generally acknowledging. It refers to the element that comprises at least a viral source and the nucleic acid carrier construct that can be packaged into the viral vectors particle. The viral vectors particle can be used in external or body DNA, RNA or other nucleic acid are changed over to the purpose of cell.

Term used herein " virus ", " virion ", " carrier particle ", " viral vectors particle " and " virion " can Alternate and broadly be understood to imply the infectious virus particle that forms when for example viral vectors of the present invention being transduceed into suitable cell or clone in order to producing infected particle. Virion of the present invention can be used in external or body nucleic acid being changed over to the purpose of cell. The carrier that uses among the present invention can be chosen the codes selection mark wantonly.

Term used herein " adenovirus " and " adenovirion " (being used interchangeably) mean to be classified as any of adenovirus and all viruses, comprise any adenovirus that infects the human or animal, comprise all monoids, hypotype and serotype.

Therefore, unless otherwise stated, otherwise " adenovirus " used herein and " adenovirion " means viral or derivatives thereof own and comprises all serotype and hypotype and natural existence and recombinant forms. This class adenovirus can or can or be modified according to variety of way as known in the art as disclosed herein for wild type. This class is modified and is comprised that modification is packaged in the adenoviral gene group in the particle in order to form infectious virus. This class is modified and is comprised disappearance as known in the art, such as E1a, and E1b, E2a, E2b, the one or more disappearance in E3 or the E4 code area.

" duplicate " with " propagation " and can exchange the ability of using and mean virus vector regeneration or propagation.These terms are well-known in the art.With regard to purpose of the present invention, duplicate generation that comprises adenovirus protein and the regeneration that relates generally to adenovirus.Can use in this area and standard determination method as herein described, measure such as virus yield mensuration, cracking mensuration or plaque assay and duplicate." duplicate " and " propagation " comprise any activity that directly or indirectly relates in the virus production process, include, but are not limited to viral gene expression; The generation of viral protein, nucleic acid or other composition; Virus composition is packaged into intact virus; And lysis.

Used term " reorganization " referred to and uses recombinant DNA technology to connect into the combination of the nucleic acid molecule of filial generation nucleic acid molecule jointly when this paper mentioned nucleic acid molecule.When mentioning virus, cell and organism, term used herein " reorganization ", " conversion " and " genetically modified " refer to host's virus, cell or the organism that imports the heterologous nucleic acids molecule or lacked or modified the natural acid sequence.With regard to importing the heterologous nucleic acids molecule, can stably be integrated into nucleic acid molecule in the host genome or nucleic acid molecule also can be used as extrachromosomal molecule and exists.This extrachromosomal molecule can self-replacation.Should understand the end product that recombinant virus, cell and organism not only comprise conversion process, and comprise the filial generation of its reorganization." non-conversion ", " not genetically modified " or " nonrecombinant " host refer to wild-type virus, cell or the organism that does not contain the heterologous nucleic acids molecule.

Term used herein " rf " means preferentially and duplicates in the cell or tissue of some type, but duplicates degree is lower or do not duplicate carrier and virus particle in other type.In one embodiment of the invention, carrier is at tumour cell or abnormality proliferation tissue, such as the replication type adenovirus carrier and/or the particle of copy choice in solid tumor and other vegetation.They are included in U.S. Pat 5,677, and 178, US 5,698,443, US 5,871, and 726, US 5,801,029, US 5,998, and 205 and US 6,432,700 and PCT open source literature WO95/19434, WO 98/39465, WO 98/39467, WO 98/39466, WO 99/06576, WO 98/39464 and WO 00/15820 in the virus that discloses.This viroid can be known as " oncolytic virus " or " oncolytic vectors " and can be regarded as " cytolytic " or " cytopathogenic " and target cell carried out " selecting cell dissolving ".

Term " copy condition virus ", " preferential replication-competent virus ", " specificity replication-competent virus " and " copy choice virus " be for being used interchangeably and for preferentially to duplicate in the cell or tissue of some type, but the degree of duplicating at other cell type or in organizing is lower or the duplicating virus carrier that do not duplicate and the term of particle.

" host cell " comprise can for or for being used to implement the individual cells or the cell culture of virus vector acceptor of the present invention.Host cell comprises the filial generation of single host cell, and this filial generation is because of natural, accidental or deliberately suddenly change and/or changes that not necessarily (on the form or on total DNA complement) and original parental cell are identical.The related host cell of this paper comprises and uses virus vector in vivo or the cell of in-vitro transfection or infection.

Term " virus allows " means virus or virus vector can be finished life cycle in the whole born of the same parents in the cellular environment of host cell system.

Term " pharmaceutically or acceptable on the pharmacology " is giving molecule body and the composition that Shi Buhui produces bad, irritated or other unwanted reaction if refer to suitablely to the animal or human.

" pharmaceutically acceptable carrier " used herein comprises any He all solvents, dispersion medium, coating material, biocide and anti-mycotic agent, isotonic agent and absorption delay agent etc.This class medium and reagent being applied as in pharmaceutically active substance is well-known in the art.Except that any typical media or reagent were incompatible with active ingredient, its application in therapeutic composition received publicity.The active ingredient of replenishing can also be mixed in the composition.

" stablizer " used herein refers to and is used for improving the stability of preparation virus or virus particle or keeping its bioactive composition.Can be based on the per-cent (%ICV of initial complete capsid virosome₀), the per-cent (%VI of initial albumen VI₀), the stability measured according to the infection titer of virus particle (VP/mL) among every ml; Six adjacent body FACS, GM-CSF secretion level wait to determine the virus stability of raising.

Term used herein " aqueous cosolvent " refers to the little organic molecule easily miscible with water, is higher than sucrose, glycerine and the Tetrahydrofurfuryl polyethylene glycol ether of 5wt.% such as propylene glycol, Sericosol N, DMSO, low molecular poly (PEG), concentration.

Term used herein " reversible capsid endoproteinase inhibitor " refers to the reversible inhibitor of the L3/p23 L-Cysteine HCL Anhydrous of capsidation encoding viral.The examples for compounds that can play " reversible capsid endoproteinase inhibitor " effect comprises thioglycerin, halfcystine, gsh, dithiothreitol (DTT) (DTT), methionine(Met) and dimethyl thioether.

Term used herein " gentle reductive agent " generally refers to sulfo-or sulfhydryl compound, such as thio-alcohol and thioether class.

Term used herein " ARCA " refers to and is used to comprise about 5% sucrose, 1% glycine, 1mM MgCl₂Damping fluid with the virus formulation of 10mM Tris+0.05% polysorbate80 (being also referred to as "Tween 80 ").

So-called term " individuality ", " experimenter ", " " or its phraseological equivalent means individual Mammals to mammalian subject.

Preparation of the present invention and method

This paper has described the various stabilization strategies of storing the adenovirus carrier preparation under the temperature of-20 ℃-room temperature.Verified these stabilization strategies can provide secular physical and chemical stability and keep or strengthen in that for some time inner virus is infective.

Preparation comprises the Injectable composition as liquor or suspension; Can also prepare and be suitable for before injection, being dissolved in or be suspended in solid form in the liquid.Can also make these preparation emulsification.The exemplary composition that is used for this purpose comprises pharmaceutically acceptable carrier.For example, said composition can comprise the human serum albumin of about 100mg in every milliliter of phosphate buffered saline (PBS).Other pharmaceutically acceptable carrier comprises the aqueous solution, and the nontoxicity vehicle comprises salt, sanitas, damping fluid etc.

In preparation, sanitas be can comprise, biocide, antioxidant, sequestrant and rare gas element comprised.Be adjusted at the pH and the exact concentration of various compositions in the pharmaceutical composition according to virus concentration, storage temperature etc., and this class is adjusted into well known by persons skilled in the art.Can further optimize preparation according to storage requirement required for the present invention.In one embodiment of the invention, particularly, store sample, preferably it stored at low temperatures, be usually less than about 10 ℃ with liquid form with regard to regard to the virus of clinical application preparation, more generally be about 5 ℃ or be lower than 5 ℃ temperature.

With regard to refrigerated storage, for example at the sample of-20 ℃ or-80 ℃ following refrigerated storages, suitable damping fluid as mentioned above.Generally with about 10¹¹-Yue 2 * 10¹³The virus concentration of individual particle/ml is stored adenovirus preparation, and wherein when storing with the lower form of concentrating degree, obviously stability is higher.

Pharmaceutical composition of the present invention is mixed with compatible with its expection route of administration.The example of route of administration comprises: non-enteron aisle, for example in intravenously, coordination, intradermal, subcutaneous, the tumour, transdermal (part), intramuscular, intraperitoneal, through mucous membrane, intravenous injection, oral (for example sucking); And rectal administration.

General with this based composition as the pharmaceutically acceptable composition administration that comprises acceptable carrier on the physiology, damping fluid or other vehicle.With regard to tumour is used, consider tumor bed, zone (being lymph) or the whole body administration of injection in the direct tumour, injection excision.Also need be in a few hours or a couple of days by conduit to disease location, for example tumour or tumor locus carry out continuous perfusion.Significant quantity realizes useful or required effect for being enough to, and comprises the amount of clinical effectiveness.Can be in single or divided doses to give significant quantity.With regard to purpose of the present invention, the significant quantity of adenovirus carrier is the amount that is enough to alleviate, improve, stablize, reverse, slow down or postpone the morbid state progress.Determine the amount that gives according to the number of times of the degree of the healthy state of individuality, disease, route of administration, dosage and desired destination.

In one embodiment of the invention, particularly, store sample, preferably it stored at low temperatures, be usually less than about 10 ℃ with liquid form with regard to regard to the virus of clinical application preparation, more generally be to be lower than about 5 ℃.With regard to this class situation, this paper has described the various stabilization strategies of the liquid preparation of adenovirus carrier.Verified these stabilization strategies can provide secular physical and chemical stability and keep or strengthen in that the regular hour inner virus is infective.These strategies comprise and use one or more the preparation comprise in the following composition: (1) aqueous cosolvent; (for example propylene glycol or glycerine) is as the reagent that promotes capsid drift angle albumen (capsid vertex protein) preferential hydration; (2) reversible inhibitor of the L3/p23 proteolytic enzyme L-Cysteine HCL Anhydrous of capsidation encoding viral (for example thio-alcohol or thioether class); (3) Wen He reductive agent or other prevent the reagent of virus composition specificity degraded.

Although do not wish to be subjected to theory constraint, the mechanism of action of the reversible L2/p23 proteinase inhibitor that proposes is to prevent to bind as drift angle the albumen VI digestion (Greber etc. (1996) of albumen (vertex cement protein); EMBO is (8) J.15), prevent that thus the penton mixture from dissociating from the capsid drift angle.The penton mixture dissociates from viral capsid and makes virosome at inactivation biologically.Therefore, prevent that by proteinase inhibitor or the gentle reductive agent that in adenovirus preparation, comprises aqueous cosolvent, reversible encoding viral this incident of dissociating from causing viral infection to keep stable in the regular hour.

The mode of action of inhibitor can be a direct arrestin enzyme active sites or by inducing conformational change to suppress avtive spot (Jones etc. (1996) indirectly through the distal region of proteolytic enzyme and/or pVIc proteolytic enzyme cofactor and the interaction partners reactive site of inhibitor; J.Gen.Vir.77,1821-1824).The evidence of relevant proteolytic enzyme restraining effect mechanism is provided in embodiment and attached data.One class L3/p23 inhibitor, promptly Wen He reductive agent also is used for suppressing capsid protein, the especially oxidation of six adjacent bodies by removing the oxidation kind.Antioxidant general (for example BHT and BHA) can prevent the capsid protein oxidation and comprise them as composition of the present invention.

Cosolvent can be used to promote the drift angle composite surface the preferential hydration (Gekko and Timasheff (1981),Biochemistry 20,4667-4676; Arakawa and Timasheff (1982),Biochemistry 21,6536-6544), thereby improve penton adjacent body Saturday outside penton and adjacent body Saturday dissociated potential energy from facet six adjacent bodies outside the penton.Cosolvent show to the rrna mixture (Douzou (1986), Cryobiology 25,38-47) and to the assembling tubulin (Pittz and Timasheff (1978); Biochemistry 17, and structural stability 615-623) is provided.The cosolvent of enough high densitys also can suppress L3/p23 protease activities site and thus serve as numerous inhibitor class stablizers of having described such as sucrose, glycerine, Sericosol N, propylene glycol or Tetrahydrofurfuryl polyethylene glycol ether.Preferential hydration's effect of cosolvent can be used for reducing the conformational change speed of drift angle, thereby causes bonded " release " between proteolytic enzyme contactin VI or other the drift angle albumen.The trigger point that confirms the penton-incident of dissociating is that the additional method of (as shown in data provided herein) prompting cosolvent stabilization of virosome concentration dependent may be to have reduced due to the virosome collision frequency because of the viscosity by increase solution.

Preparation of the present invention is applied to stable comprise the procedure of processing that the thick of virus or half pure processing intermediate and virus carry out and the original position in the product process that is caused final preparation by series of steps is stable under liquid state.For example; can when collecting step, add the L3/p23 proteinase inhibitor so that prevent the digestion of albumen VI in the course of processing of downstream, and can be at chromatography eluant and in causing using in the tangential flow filtration process of high regional virus concentration by the time the viral consistency cosolvent of high density as the means of protection virus titer.

Carried out many researchs and tested one or more various preparations in the proteinase inhibitor that comprises aqueous cosolvent, reversible encoding viral and the gentle reductive agent, as what further describe in the embodiment part hereinafter.

Adenovirus carrier

The present invention pay close attention to any He all adenoviral serotypes in making up preparation of the present invention adenovirus carrier and the application in the virus particle.The operable adenovirus deposit of the present invention comprises any adenoviral serotype.Adenoviral serotype 1-51 can derive from American type culture collection (American Type Culture Collection) at present, and (ATCC, Manassas VA), and the present invention includes any other serotype of the adenovirus that can derive from any source.The operable adenovirus of the present invention can derive from people or inhuman, such as ox, pig, dog, ape and monkey, bird.For example, adenovirus can belong to hypotype A (for example serotype 12,18,31), hypotype B (for example serotype 3,7,11,14,16,21,34,35,50), subtype C (for example serotype 1,2,5,6), hypotype D (for example serotype 8,9,10,13,15,17,19,20,22-30,32,33,36,39,42,47,49,51), hypotype E (serotype 4), hypotype F (serotype 40,41) or any other adenoviral serotype.A large amount of examples of humans and animals adenovirus can derive from American type culture collection, for example canWww.atcc.org/Search Catalogs/Cell Biology.cfm.On find.

Adenovirus carrier of the present invention comprises no replication type (defective type) and replicating vector.No replication type carrier can't duplicate in target cell or duplicate with extremely low level, is the typical case with the Ad/GM-CSF carrier in this article.In one aspect, no replication type carrier makes E1a, E1b, E2a, at least one the coding region inactivation among E2b or the E4 by the part or all of coding region of disappearance or sudden change usually.It is well-known in the art being used to make the method for these carrier propagation.

In one aspect of the method, adenovirus carrier is a rf.Replicating vector can duplicate in target cell, is the typical case with the CG7060 carrier in this article.Duplicating virus comprises wild-type virus and through transforming so that the virus of duplicating in target cell.They comprise duplicates specificity virus.To duplicate specificity virus is designed in a kind of cell type and compares specificity or preferentially duplicate in another kind of cell type.This term also comprises and duplicates the specificity adenovirus; That is, preferentially in the cell or tissue of some type, duplicate, but in other type, duplicate the virus that degree is lower or do not duplicate.This viroid be called sometimes " molten cell " or " cytopathogenic effect " virus (or carrier), and if they neoplastic cell is had this class effect, be called so " oncolytic cell " virus (or carrier).In certain embodiments, adenovirus carrier of the present invention comprises the therapeutic gene sequence, for example the cytokine gene sequence.

In one embodiment of the invention, virus vector and/or particle are at tumour cell and/or abnormality proliferation tissue, such as copy choice in solid tumor and other vegetation.

With regard to regard to the adenovirus carrier of copy choice in the target cell, researched and developed the duplicating virus carrier of specificity attenuation, for it, copy choice preferentially destroys those cells in cancer cells.Preferentially duplicate in some cell type described in the various cell-specific replication type adenovirus constructs such as following document of (and destroying them thus): for example WO 95/19434, WO 96/17053, WO 98/39464, WO 98/39465, WO 98/39467, WO 98/39466, WO 99/06576, WO 99/25860, WO 00/15820, WO 00/46355, WO 02/067861, WO 02/06862, U.S. Patent Application Publication US 20010053352 and U.S. Pat 5,698,443, US 5,871,726, US 5,998,205 and US 6,432,700.Designed the replication type adenovirus carrier of copy choice in tumour cell.

The typical adenovirus carrier of the present invention comprises, but be not limited to DNA, be encapsulated in the DNA in the adenoviral coat, be packaged in the adenovirus DNA in another kind of virus or the viral sample form (such as herpes simplex and AAV), be encapsulated in the adenovirus DNA in the liposome, with polylysine compound adenovirus DNA, compound with synthetic polycation molecule, put together with Transferrins,iron complexes or with such as this compounds compound adenovirus DNA of PEG, they serve many purposes, including, but are not limited to immunity " shelters " antigenicity and/or increases the virus transformation period or be used for puting together with non-viral protein.

The adenovirus carrier particle can also comprise fibrinous further modification.In one embodiment, adenovirus carrier of the present invention further comprises the target part in the capsid protein that is included in particle.With regard to the example of target adenovirus, for example, referring to WO 00/67576, WO 99/39734, US 6,683,170, US 6,555,368, US 5,922,315, US 5,543,328, US 5,770,442 and US 5,846,782.

In addition, adenovirus carrier of the present invention can also comprise the modification to other viral capsid proteins.The example of these sudden changes includes, but are not limited to those and is described in U.S. Pat 5,731, and 190, the sudden change among US 6,127,525 and the US 5,922,315.The adenovirus of other modification is described in U.S. Pat 6,057, and 155, among US 5,543,328 and the US 5,756,086.

The modular system that is used to produce the adenovirus carrier of expressing insertion sequence is as known in the art and can derives from merchandise resources, for example available from the Adeno X expression system (PaloAlto of Clontech, CA) (Clontechniques (in January, 2000) p.1012), all available from Qbiogene (Carlsbad, CA) Adenovator adenovirus system and AdEasy.

Virus produces and purifying

The host cell that is used to produce virus formulation of the present invention can be supported duplicating of candidate's virus." host cell " as defined herein comprise can by or by the individual cells of virus infection or cell culture.Host cell comprises the filial generation of single host cell, and this filial generation is because of natural, accidental or deliberately suddenly change and/or changes not necessarily and original parental cell identical (on the form or on total DNA complement).Host cell comprises in vivo or the cell of external use virus vector transfection of the present invention or infection.

Host cell of the present invention derives from mammalian cell and preferably derives from primate cell.Although preferred various primate cell and human body cell most preferably, the cell that can support any kind of virus replication all is acceptable in the embodiment of this invention.Being used to implement cell type of the present invention, to include, but are not limited to the African green monkey kidney cell strain be cell, Chinese hamster ovary celI or for any eukaryotic cell of the Virus Type permission that produced.Can pass through methods known in the art, for example by cell that one deck do not infect or the cell that infects with one or more helper viruses contacts with virus particle, incubated cell and definite virus replication and the ability of candidate cell system being tested its support virus replication subsequently.

Multiple host cell can be supported duplicating of adenovirus.Although preferred various primate cell and human body cell most preferably, the cell that can support any kind of virus replication all is acceptable in the embodiment of this invention.The preferred cell that is used for commercial scale production adenovirus is as for example in the HeLa-S3 clone described in the U. S. application sequence number (SN) 10/824796.

The in addition preferred cell that is used for commercial scale production adenovirus is A549 cell, PERC6 cell and people's 293 embryonic kidney cells, and they express adenovirus EIA and EIB gene product etc.The clone that can suitably produce target adenovirus comprises people LNCaP (prostate cancer), HBL-100 (breast epithelium), OVCAR-3 (ovarian cancer) etc.

Cell culture

Host cell is grown in the perfusion system, and this can keep the good culture environment of pH, CO2 and O2 in the cell growth.Perfusion can be removed active metabolite, and nutrition is provided simultaneously.The suitable medium and the condition that are suitable for cultivating the clone that is used to produce virus vector of the present invention are well-known in the art, and can use any suitable medium, for example RPMI, DMEM etc.Substratum can comprise serum, for example foetal calf serum or can not contain serum.The serum that enters the anchorage-dependent cell of the suspension culture that does not contain serum is broken off adapting to and has been used to produce recombinant protein and virus vaccines, and can be applied to produce virus vector.

In certain embodiments, it can be used to use the selective system of removing the unwanted cells growth.This purpose can be to realize by means of the lasting transformation cell lines of carrier that uses the coding selective marker or virus vector transduction or cells infected by using the coding selective marker.Under any situation, the cell that uses suitable medicine or alternative cpd to cultivate conversion/transduction all can cause those to carry the copy choice of the cell of described mark.The copy choice that carries the cell of described mark mean cell that the cell of cultivating conversions/transduction in the presence of medicine that suitable type and concentration are arranged or alternative cpd causes carrying mark with respect to the cell that does not carry mark preferentially or exclusiveness duplicate.The example of mark includes, but are not limited to the HSV thymidine kinase (TK) in TK, HGPRT-or APRT-cell, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyltransferase (APRT) gene respectively.In addition, the metabolic antagonist resistance can be given the Tetrahydrofolate dehydrogenase (DHFR) of methotrexate resistance with electing; Give the gpt of mycophenolic acid resistance; Give aminoglycoside G418 the neo of resistance; Basis with the hph gene of giving hygromycin resistance.

The serum that enters the anchorage-dependent cell of the suspension culture that does not contain serum is broken off adapting to and has been used to use 293 cells (Williams burg BioProcessing Conference, Nov.18-21,1996; WO 98/22588) and A549 cell (Morris etc., WilliamsburgBioProcessing Conference, Nov.18-21,1996) produce recombinant protein (Berg etc., BioTechniques, 14:972-978,1993) and virus vaccines (Perrin etc., Vaccine, 13:1244-1250,1995; Gilbert).

Can make some anchorage-dependent cell adapt to suspension and cultivate, this can help the production of commercial scale.The present invention can depend on the application of bioreactor technology in production virus.Cell is grown in bio-reactor allow to be used to the cell of the viral vector infection in the inventive method and the preparation with commercial scale production.Be applied to purifying by this system of running under the perfusion condition and with improved plan, the invention provides the strategy of the commercial useful virus vector that is easy to the large-scale production q.s.

Bio-reactor has been widely used in by suspension and adherent dependence animal cell culture and has produced biologic.Can be by using multiple device, entrapped cell such as fiber disk, pore revolving filter, hollow fiber or flat sheet membrane filter, sedimentation pipe and the fresh culture perfusion is crossed culture for example.Simple perfusing course exists the inflow of substratum and the outflow of cell and product.Make substratum enter reactor with predetermined and constant rate of speed, described predetermined and constant rate of speed is kept the thinning ratio of culture with the value of the maximum specific growth rate that is lower than cell.

In producing viral process, by making host cell contact under the physiological condition that allows picked-up virus viral with this cell of virus infection.Can be with high infection multiplicity (MoI) cells infected so that optimize productive rate.The host cell replication-competent virus can be after infection be collected them 2-5 days the time.

Cell lysate

Committed step in the virus production process is that virus discharges from host cell when the lysing cell film.The traditional method of lysing cell can break virus and usually cause the productive rate of biological activity viral product lower such as the circulation of mechanical stirring (for example high-pressure extrusion, solid shearing, liquid shear or sonication) and freeze thaw.Another kind of cleavage method is the washing composition cracking, and it generally depends on adds the nonionic detergent that final concentration is about the 0.5%-2.5 weight/volume in the cell that infects.Nonionic detergent commonly used comprises: the washing composition of TritonTM family (TritonTM X-15 for example; TritonTM X-35; TritonTM X-45; TritonTM X-100; TritonTM X-102; TritonTM X-114; TritonTM X-165, all these multi-phase detergents all have the 8-carbon side chain that is connected with aromatic ring); The TweenTM washing composition that belongs to the polyoxyethylene sorbitan ester class family of non-sex change, nonionic lipid acid; With the zwitterionic detergent CHAPS that is the sultaine derivative of cholic acid.Yet this class nonionic detergent also may have destructiveness to virus.

Can collect and use and comprise one or more and knownly combine and help cell that the lytic reagent lytic virus of the nonionic surface active agent of its solubilising infects with epicyte protein (for example, referring to Taylor etc., Biochim Biophys Acta.1612:65-75,2003; Santoni etc., Proteomics, 3:249-253,2003; With Hazard etc., ArchBiochem Biophys.407:117-124,2002).This class nonionic surface active agent can cause in purge process the destructiveness to virus lower and improve stability in the storage process.Nonionic surface active agent in lytic reagent preferably has wetting ability and lipotropy two portions.The example of nonionic surface active agent include, but are not limited to the list that alkyl replaces-, two-and polyose; The list of cycloalkyl substituted-, two-and polyose alkyl alcohol; Polyethenoxy ether class; Dialkyl group-glycerols; And isomer.

Have any list of lipophilic substituent-, two-and polysaccharide all can be as the nonionic surface active agent in the lytic reagent of the present invention.Typical two sugar compounds comprise sucrose, lactose, maltose, isomaltose, trehalose and cellobiose.Lipophilic substituent preferably comprises alkyl or alkenyl.According to the preferred embodiments of the invention, lipophilic substituent is the paraffinic acid residue.

Lipophilic substituent can be (for example straight chain just-alkane or alkene) or nonlinear (for example ring-type or branched paraffin or alkene) of linearity.Lipophilic substituent can also be the paraffinic acid residue.The length of lipophilic substituent can change so that reach the ideal hydrophile-lyophile balance.

Preferred nonionic surfactants comprises the monose that alkyl replaces, the disaccharides that alkyl replaces, the polyose that alkyl replaces, the monose of cycloalkyl substituted, the disaccharides of cycloalkyl substituted, the polyose alkyl alcohol of cycloalkyl substituted, polyethenoxy ether class, dialkyl group-glycerols and isomer thereof, such as just-dodecyl-b-D-maltoside, just-dodecyl-b-D-maltoside, just-dodecyl-b-L-maltoside, just-dodecyl-b-L-maltoside, 6-cyclohexyl hexyl-b-D-maltoside, 6-cyclohexyl hexyl-b-D-maltoside, 6-cyclohexyl hexyl-b-L-maltoside, 6-cyclohexyl hexyl-b-L-maltoside, 6-cyclohexyl hexyl-b-D-maltoside, sucrose monolaurate, just-tridecyl-b-D-maltoside, just-tridecyl-b-D-maltoside, just-tridecyl-b-L-maltoside, just-tridecyl-b-L-maltoside, just-tetradecyl-b-D-maltoside, just-tetradecyl-b-D-maltoside, just-tetradecyl-b-L-maltoside, just-and tetradecyl-b-L-maltoside and laureth 9, as further described in U.S. Patent application sequence number (SN) 10/743813 and 60/631434.

Make cracking agent and cells contacting be enough to lysing cell and from system, remove for some time of extra attached cell.In this type systematic, before purified virus, generally clarify thick virolysis thing, promptly remove the striping fragment.By using degree of depth filter to clarify, described filter is made up of the packed column of the non-absorbing material with certain porosity, makes to hold back bigger film fragment under the situation of not losing virus particle.Thickness and intensity selected depth filter based on the machinery reservation of particle, absorption characteristic, pH value, surface quality, filter.Commercially available cylinder has merged the filter of several types, for example polypropylene, glass filter, Nitrocellulose etc.

Viral purification

It is well-known in the art being used for from the technology of cell lysate separation infectious virus.For example, can by density gradient centrifugation or chromatography isolated viral (for example, referring to U.S. Pat 6,194,191 and US 6,689,600).Kind and information as known in the art according to virus vector to be separated are used the centrifugal proper density condition that is suitable for.

But in order to make viral purifying become the process of mass-producing, preferably adopt such as methods such as chromatographys, under the centrifugal situation, this method can be removed cell debris from cell lysate.In standard procedure, on Filter column, pass through ion exchange chromatography, isolated viral particle from the clarification cell lysate, a large amount of examples of this process are as known in the art.In viral purification, can use the various chromatographic materials that can obtain on the market.The available supported matrix includes but not limited to polymeric material, such as Mierocrystalline cellulose or silica gel type resin or film, and perhaps cross-linked polysaccharides (for example agarose) or other resins.Chromatographic material can also comprise various functional group or the active groups that are attached on the matrix that can be used for separation of biomolecules.

Like this, can use the affinity groups purified virus that is incorporated on the supported matrix, virus interacts by various non-covalent mechanism with these groups, subsequently they is removed.Preferred separation method comprises ion-exchange (especially anionresin).Other specificity affinity groups comprise heparin and with supported matrix bonded special viral antibody.What select that affinity groups in the viral purification will consider is this virus and the interactional avidity of selected affinity groups, and the easy degree of therefrom removing, and the while is the biological function/infectivity of break virus particle not again.Typical polymeric material comprises product Heparin Sepharose HighPerformance (Pharmacia); The macropore hydroxylapatite, such as Macro-Prep CeramicHydroxya patite (Bio-Rad, Richmond, CA); And cellufinesulfate (Amicon).The affinity ligand that can be used for purified virus comprises the antiviral antibody that is attached to suitable resin, and these are well known by persons skilled in the art.

Can adopt various funtion part as known in the art to carry out anion-exchange chromatography, described funtion part includes but not limited to DEAE (diethylaminoethyl-), QAE (QAE(quaternary aminoehyl)) and Q (quaternary ammonium).Can make these funtion parts be attached to any suitable resin, comprise Mierocrystalline cellulose and silicone resin.For example, can make DEAE be attached to various resins in the post, comprise celluosic resin, such as DEAE-MemSep^TM(Millipore, Bedford, MA), Sartobind^TMThe film absorption agent (Sartorius, Edgewood, N.J.) and silicone resin, such as ACTI-MOD^TM(American International Chemical, Natick, MA).Typical resin comprises also and the crosslinked polystyrene of Vinylstyrene pearl that it is found in PharmaciaSource Q, and the dextran that is attached to highly cross-linked spherical sepharose 4B, and it is found in Pharmacia Q-Sepharose XL.For example referring to WO 00/40702.

In viral purification, also can use cation-exchange chromatography, include but not limited to use following chromatographic column, such as SP MemSep^TM(Millipore, Bedford, MA), CMMemSep^TM(Millipore, Bedford, MA), Fractogel^TM(EM SeparationTechnology, Gibbstown is NJ) with Macroprep S for SO3^TM(BioRad, Melville, NY), and based on the resin of heparin.Heparin ACTI-MOD^TMCylinder (AmericanInternational Chemical Inc., Natick, MA) and POROS^TMThe example that perfusion chromatographic media (Boehringer Mannheim) representative is other.

Nuclease is handled

Available nuclease is handled the virus of wash-out.With adopt after handle at once and compare, handle with nuclease that after chromatography the amount of required nuclease is minimized.After handling, nuclease can comprise second chromatographic step, to remove fragmentation DNA and nuclease.

Knownly in this area have many nucleases, preferred nuclease comprises the combination of a kind of or extensive specificity endonuclease, for example enzyme classification 3.1.27.5 (pancreatic ribonuclease) and 3.1.31.1 (micrococcal nuclease) or the like.BenzonaseTM is a kind of genetically engineered enzyme, and it had both had the DNA enzymic activity, also has the RNA enzymic activity, and it can be used in particular for this step of viral purification process.The ability of the quick hydrolysed nucleic acid of BenzonaseTM makes this enzyme can be used for reducing the viscosity of cell lysate, and can be used for being reduced in nucleic acid load during the purifying, therefore can eliminate to disturb and improve productive rate.In case complete digestion, the free length nucleic acid that then will exist in solution shortens to the oligonucleotide of 2 to 4 bases.After nuclease digestion, can on the ion-exchange filter, make the virus operation for the second time, this filter can be identical or different with first kind of filter.

Filtration/sterilization

Can randomly pass through ordinary method, for example use the concentrated and diafiltration of tubular fibre thickener the virus of wash-out.In for the whole preparation that uses, can carry out sterile filtration to the purified virus sample, for example can be for clinical use.The multiple filter that is suitable for reaching this purpose is as known in the art, for example Nitrocellulose membrane filter, rhodia membrane filter, PVDF (modified polyvinilidene difluoroethylene) membrane filter etc.Preferred pvdf membrane filter (for example Millipore Millipak filter).Can improve productive rate by with damping fluid filter being carried out pre-wash, this damping fluid for example is pharmaceutically acceptable vehicle.Have been found that due to illness poison is adhered to filter productive rate is descended, and make this adhesion saturated after reaching certain level.Therefore, load more substantial particle and can improve productive rate, lose percentage and minimize so that make.According to concentration of degree of purification, used virus vector etc., the specified conditions of filtration sterilization are suitably set.

Virus characterizes

Determine to concentrate or isolated viral tire and the method for purity is known in the art.For example, allow the proteic expression of encoding viral in the cell, can determine the quantitatively characterizing of viral infection by measuring.Typically, virus infection with serial dilution allows cell, it is hatched for some time (for example 1-2 days), detect the encoding viral expression of gene then, this gene can virogene (for example the DNA of adenovirus is conjugated protein) or the transgenosis (for example beta-galactosidase enzymes) of being carried by virus.Also can determine some virus, such as the infectivity of adenovirus and vaccinia virus by plaque assay.

Can pass through reversed-phased high performace liquid chromatographic (RP-HPLC) and determine the purity of virus.In the chromatography process, intact virus is dissociated into its constituent, and promptly six adjacent bodies, penton base (Pb) and fiber produce a kind of characteristic fingerprint, and this fingerprint can be based on the integrity of various compositions and changed.By to the concentration that quantitatively also can measure virus of structural protein (for example referring to Lehmberg etc., J Chromatogr B Biomed Sci Appl.732:411-423,1999 and Roitsch etc., J Chromatogr B Biomed Sci Appl., 752:263-280,2001).See Table 1.

Also can use anion-exchange chromatography (AEX) to determine purity, the concentration of virus and tire.This method has been used to the adenovirion (Shabram etc., Hum Gen Ther., 8:453-465,1997) in quantitatively thick lysate or the highly purified sample.It can be used to estimate the particle in interior dilution of extensive dynamicrange and the concentrating sample.

Embodiment

The stability of different adenovirus preparations

With HEK 293 cells of selected serotype adenovirus infection cultivation and by centrifugal results.The granular precipitation resuspending of cell in lysis buffer, and is prepared it with following different damping fluids.The ARCA damping fluid comprises 5% sucrose, 1% glycine, 1mM MgCl₂With 10mM Tris, add 0.05% Polysorbate 80 (being also referred to as "Tween 80 ").Every kind of preparation is carried out sterile filtration by 0.2 micron filter, in the vial with its 1ml that packs into, with the sillicon rubber blocking jam-pack of coating tetrafluoroethylene.Described bottle is stored under 5 ℃, 25 ℃ or 30 ℃.Under 25 ℃ or 30 ℃, store and be considered as at room temperature storing.At the times selected point, the sample of every kind of preparation is studied by methods such as anion-exchange chromatography, reverse-phase chromatographies.AE-HPLC as a result the sample peak type of alleged occurrence in independent ARCA damping fluid variation greater than be present in ARCA damping fluid composition add aqueous cosolvent or with the combination of the reductive agent of gentleness or reversible protease inhibitors in the variation of sample peak type.For example, referring to accompanying drawing 2A-C and accompanying drawing 4A and B.

Use AE-HPLC to estimate complete composition, six adjacent bodies, penton base (Pb) and the time dependent percentage of fiber, period of storage is long more, the percentage of complete penton base and fiber is obviously low more, and complete six adjacent body percentage keep relative stability (for example table 1).

For example, 37 ℃ of following 0 time points are contained in the Ad/GM-CSF (1 * 10 in the GTS/ARMWG damping fluid¹²Vp/ml) the AE-HPLC color atlas of solution shows the peak that only occurred ICV at 6.113 minutes.Storing the interlude point, two peaks (is IVC, and is PVV) occurring at 6.106 and 6.650 minutes, at period of storage point subsequently, preponderate in the peak of PVV in the time of 6.701 minutes.

Table 1 shows, with Ad/GM-CSF (1 * 10¹²Vp/ml) in the time of in the GTS/ARMWG damping fluid of preparation stored under 37 ℃, along with the time changes live virus (showing as the peak about 6.1 minutes the time) and changes non-infectious virus (peak as at about 6.7 minutes the time shows) into.Table 1 shows that also the composition at the HPLC peak that the retention time that had 0,0.4 and 0.7 minute changes shows the transformation (as by as indicated in six adjacent bodies, penton base (Pb) and the complete fibre content) between the live virus.The percentage of the high percent of the peak that changes of time and live virus and complete capsid virosome (ICV) is relevant without reserve, and has the peak relevant with non-infectious virus (as by as indicated in empty penton virosome or the PVV) of 0.7 minute retention time change.

Table 1Six adjacent bodies (RP), penton base (Pb) and complete fiber(WB) percentile AE-HPLC estimates

	Complete six adjacent body (RP) %	Complete Pb (RP) %	Complete fiber (WB) %
				The RT variation (minute)
0	94	100	87
				0.4	95	100	82
0.7	85	32	26

Consistent with table 1, accompanying drawing 2A shows, is stored in 1 * 10 in the ARCA damping fluid under 25 ℃¹²The Ad/GM-CSF preparation of vp/ml changes in time and degrades, and makes the percentage of complete capsid virosome (ICV) descend like this, and this increases consistent with empty penton virosome (PVV) and GM-CSF excretory.

Accompanying drawing 2C shows that it is relevant with the percentile decline of complete capsid virosome (ICV) that the GM-CSF excretory reduces gradually, and this shows that the ICV percentage represents that the Ad/GM-CSF virus formulation influences GM-CSF excretory ability.

Carried out a large amount of research, estimated the influence of aqueous cosolvent adenovirus preparation's stability.As shown in Figure 3, the preparation that comprises 20%PEG, 20% glycerine only causes the less variation of retention time, shows that virus stability is improved.

For complete capsid virosome percentage (%ICV₀) as ARMWG preparation (10mM Tris, 25mM NaCl, 2.5% glycerine, pH8) middle Ad/GM-CSF (1.2 * 10¹²Vp/ml) function of 30 ℃ of following periods of storage of solution has carried out other researchs so that estimate the effect of different stabilization additives under the concentration of 0.75M (accompanying drawing 5A) or 12% weight (accompanying drawing 5B), and compares with the ARCA contrast.By accompanying drawing 5A and accompanying drawing 5B as seen, 30 ℃ store at least 50 days down after, the preparation that comprises propylene glycol or Tetrahydrofurfuryl polyethylene glycol ether demonstrates maximum stability, and the ARCA control formulation demonstrates minimum package stability.

Based on as Ad/GM-CSF (1.2 * 10¹²Vp/ml) the complete capsid virosome percentage (%ICV of the function of 30 ℃ of following periods of storage of solution₀) also estimated of the influence of the sucrose of different concns to virus stability.The sucrose preparation of 5% weight is the ARCA preparation (because ARCA contains this fact of sucrose of 5% weight) of standard.By accompanying drawing 6 as seen, sucrose concentration is high more, and adenovirus stability is big more.

Accompanying drawing 5A, 5B and 6 show that percentile the decline gradually of viewed ICV can reduce by comprising different aqueous cosolvent such as propylene glycol and Tetrahydrofurfuryl polyethylene glycol ether when being stored in virus among the independent ARCA.

Carry out a large amount of research, sounded out the mechanism that reversible protease inhibitors and/or gentle reductive agent help adenovirus preparation's stability.In order to illustrate this potential mechanism, before adding specific formulation, virus particle has been carried out pre-treatment.Accompanying drawing 7 provides the structural representation of L3/p23 proteolytic enzyme, and it shows and has disulfide linkage and free sulfhydryl groups.Describe Study on pretreatment at this, the latent effect mechanism of particular type reagent only is described for example, and be not to plan it is included in the preparation of the present invention.For example, NEM or N-ethyl maleimide are a kind of irreversible proteinase inhibitor, and diamide is a kind of oxygenant.The two all is used to prove conceptual approach, rather than will make it become a kind of composition in the composition that is used for the treatment of.DTT is a kind of reductive agent, and its reversibility ground suppresses disulfide linkage with the maintenance free sulfhydryl groups, and its constitutes one aspect of the present invention.

Accompanying drawing 8A, 8B and 9A-9C show, with NEM, DTT and diamide pre-treatment, can make viewed ICV percentage reduced minimum in time when being stored in virus in 30 ℃ the ARCA damping fluid, this prompting is when being stored in virus formulation in the ARCA damping fluid, and suppressing virus protease may be a kind of means that improve virus stability.For the Ad/GM-CSF among the ARCA (1 * 10¹²Vp/ml) preparation, will 15 and 50mM DTT under the DTT pre-treatment with do not have pretreated effect to compare.Complete capsid virosome (ICV₀) percentage understands as the function table of 30 ℃ of following periods of storage and contain 50mM DTT stability of formulation greater than the preparation that does not contain DTT with contain 15mM DTT stability of formulation (accompanying drawing 9B).

Accompanying drawing 10 and 11 shows initial complete capsid virosome percentage (%ICV₀) and initial albumen VI percentage (%VI₀) respectively as Ad/GM-CSF (1 * 10 in the ARCA preparation that contains different proteinase inhibitor¹²Vp/ml) function of 30 ℃ of following periods of storage of preparation.The result shows that the stability of formulation that contains 50mM DTT, 150mM halfcystine and 15mM dimethyl thioether is based on to initial complete capsid virosome percentage (%ICV₀) and initial albumen VI percentage (%VI₀) both analytical results.

Table 2 shows, the stability of formulation that comprises 50mM DTT, 150mM thioglycerol and 15mM dimethyl thioether respectively is also based on the GM-CSF secretion, and it is as the function of 30 ℃ of following periods of storage, and compares with the ARCA preparation that does not contain proteinase inhibitor.

Table 2Preparation stability

Preparation	％ICV0	％GSR0
			ARCA	41％	31％
+50mM DTT	99％	88％
			+ 150mM thioglycerol	92％	83％
+15mM DMS	96％	89％

Shown a large amount of different preparations (cited in the table 3), they can improve the stability of Ad/GM-CSF virus reserve, and this is by the initial complete capsid virosome percentage (%ICV as the function of 30 ℃ of following periods of storage₀); Initial albumen VI percentage (%VI as the function of 30 ℃ of following periods of storage₀); And the percentage of initial GM-CSF secretion rate (GSR) is determined.Every kind of stability of formulation enumerating in the table 3 is provided in the table 4.

Table 3The preparation abbreviation

#	Preparation
			1	ARCA (5% sucrose, 1% glycine, 1mM MgCl2, 10mM Tris adds 0.05% Polysorbate 80)
2	The above sucrose of ARCA+30%+1% thioglycerol
		3	The above sucrose of ARCA+15%+1% thioglycerol
4	ARCA-sucrose/polysorbate+1% thioglycerol that contains 10% glycerine
		5	ARCA-sucrose/polysorbate+15mM the DMS that contains 10% glycerine
6	ARCA-sucrose/polysorbate+1% thioglycerol that contains 5% glycerine
		7	ARCA-sucrose/polysorbate+1% thioglycerol that contains 10% propylene glycol
8	ARCA-sucrose/polysorbate+1% thioglycerol that contains 5% propylene glycol
		9	ARCA-sucrose/polysorbate+15mM the DMS that contains 5% propylene glycol
10	ARCA-sucrose/polysorbate+15mM DMS+1% the thioglycerol that contains 5% propylene glycol

Table 4Preparation stability

#	％ICV0			％VI0			％GSR0
								16days	30 days	43 days	16days	30 days	43 days	43days
	1	26％	1％	1％	44％	0％	0％								0％
2								91％	96％	89％	70％	74％	67％	51％
	3	92％	95％	79％	83％	75％	51％								24％
4								94％	97％	92％	81％	76％	67％	39％
	5	90％	98％	88％	83％	70％	55％								29％
6								89％	96％	70％	75％	72％	38％	20％
	7	96％	101％	98％	79％	75％	56％								56％
8								94％	99％	95％	70％	67％	49％	59％
	9	93％	97％	95％	69％	60％	55％								40％
10								94％	97％	95％	68％	67％	52％	50％

Period of storage (about 36.5 months) back Ad/GM-CSF (CG6444) preparation (50wt.% glycerine, 10mM Tris, pH7.4) permanent stability under-20 ℃ that table 5 illustrates even prolonging.

Table 550% glycerine, 10mM Tris are done in preparation, among the pH7.4 and be stored under-20 ℃The permanent stability of CG6444

Time	AEX-HPLC		D RT	0.2mm filter	GM-CSF	Hexon-FACS
									Titre	Percentage		Reclaim	Secretion	Infection titer	Infect ratio
(moon)	(VP/mL)	Initially	(min)	(％)	(SU100)	(IU/mL)	(VP/IU)
								0	1.1E+12	100％	0.035	96％	2.7	5.3E+09	189
0.5	1.1E+12	100％	0.021	95％	21.2	4.3E+10	23
								1	1.1E+12	97％	0.050	98％	17.3	4.3E+10	23
3	1.2E+12	103％	0.062	104％	9.7	2.5E+10	40
								6	9.5E+11	84％	0.091	101％	7.0	1.9E+10	53
9	9.3E+11	82％	0.054	95％	6.9	2.8E+10	36
								15	1.1E+12	93％	0.035	93％	11.5	4.3E+10	23
27.3	9.3E+11	82％	-0.005	ND	11.6	2.18E+10	46
								36.5	1.0E+12	92％	0.029	ND	ND	ND	ND

Carried out many other researchs, estimated the influence of aqueous cosolvent adenovirus preparation's stability.

Use many different cosolvent preparation (cited in the table 6) tests to contain the CG0070 virus reserve of 1E12 VP/mL, every kind all shows the stability that has improved CG0070 virus reserve, this stability is the function of-20 ℃ of following periods of storage, and period of storage reaches 16 months.The titre analysis of every kind of preparation enumerating in the his-and-hers watches 6 and the result of stability study are provided in the table 7.Every kind of preparation being tested all demonstrates and reaches 16 months satisfactory stability.

Table 6The preparation of being tested

Sequence number	Titre (VP/mL)	pH	The Tris damping fluid	MgCl2	Cosolvent	Glycine
							1	1.0E+12	7.8	6mM	0.6mM	50% (v/v) glycerine	0.6％(w/v)
2	1.0E+12	7.8	6mM	0.6mM	50% (v/v) propylene glycol	0.6％(w/v)
							3	1.0E+12	7.8	6mM	0.6mM	50% (v/v) glycerine	-
4	1.0E+12	7.8	6mM	0.6mM	50% (v/v) propylene glycol	-
							5	1.0E+12	7.8	6mM	0.6mM	25% glycerine+25% propylene glycol	0.6％(w/v)
6	1.0E+12	7.8	6mM	0.6mM	25% glycerine+25% propylene glycol	-

Table 7Titre is analyzed and stability study

Time (moon):			0	6			9		12	16
											Use the titre analysis [VP/ml] of AEX-HPLC
1 2 3 4 5 6		1.16E+12 1.09E+12 1.12E+12 1.11E+12 1.18E+12 1.12E+12		1.09E+12 1.05E+12 1.04E+12 1.04E+12 1.03E+12 1.02E+12		1.23E+12 1.06E+12 1.16E+12 1.05E+12 1.00E+12 1.02E+12			1.25E+12 1.15E+12 1.13E+12 1.13E+12 1.07E+12 1.04E+12	1.14E+12 1.03E+12 1.16E+12 9.74E+11 1.02E+12 1.06E+12
											Use the titre analysis (initial percentage) of AEX-HPLC
1 2 3 4 5 6	100％ 100％ 100％ 100％ 100％ 100％			94％ 96％ 93％ 94％ 87％ 91％		106％ 97％ 104％ 95％ 85％ 91％			108％ 106％ 101％ 102％ 91％ 93％	98％ 94％ 104％ 88％ 87％ 95％
											The retention time mutation analysis of application AEX-HPLC (minute)
1 2 3 4 5 6	-0.014 -0.035 -0.017 -0.034 -0.013 -0.015			0.021 -0.044 0.039 -o.053 -0.033 -0.033			0.046 -0.036 0.078 -0.042 -0.010 -0.013		0.067 -0.034 0.092 -0.049 -0.020 -0.013	0.075 -0.056 0.108 -0.071 -0.037 -0.038
											The pVI that uses RP-HPLC analyzes (initial percentage)
1 2 3 4 5 6 Ref.		100％ 100％ 100％ 100％ 100％ 100％ 100％		84％ 99％ 80％ 92％ 90％ 96％ 105％		87％ 97％ 86％ 93％ 81％ 84％ 87％			93％ 91％ 75％ 89％ 80％ 85％ 89％	107％ 108％ 104％ 99％ 94％ 104％ 94％
											GM-CSF secretion level (with respect to-70 ℃ of CTL)
1 2 3 4 5 6 R-15		1.1 0.9 1.0 0.7 0.9 0.9 1.0			1.0 0.9 1.0 0.9 0.9 0.7 1.0	1.1 0.9 1.1 1.0 1.1 1.1 1.0		0.9 1.0 1.0 1.0 0.9 0.9 1.0		1.0 1.0 1.1 1.1 1.1 1.1 1.0
											Plaque (Pfu/ml)
1 2 3 4 5 6		ND ND ND ND ND ND		ND ND ND ND ND ND		8.8E+10 9.0E+10 8.0E+10 1.6E+11 9.3E+10 9.8E+10			1.3E+11 1.2E+11 1.3E+11 1.3E+11 1.2E+11 1.4E+11	5.5E+10 7.5E+10 3.6E+10 6.5E+10 5.8E+10 7.5E+10

Use many different cosolvent preparations (cited in the table 8) to test to storing the viral reserve that contains 1E12 and 2E12VP/mL that reaches 20 months down at 5 ℃.Compare with the ARCA preparation, the stability that every kind of cosolvent preparation all demonstrates viral reserve is improved.The titre analysis of every kind of preparation enumerating in the his-and-hers watches 8 and the result of stability study are provided in the table 9.Every kind of preparation being tested all demonstrates and reaches 20 months satisfactory stability, and this determines by following analysis: the complete capsid virosome of %ICV=; [VP] of [ICV]=0 time point; The variation of RT, plaque (VP/PFU) and GSR=are at the standardized GM-CSF secretion rate of reference standard; Wherein＜and #〉expression %[VP]₀, it is the empty penton virosome that does not have ICV to exist.

Table 8The preparation of being tested

#	Preparation	Primary particles titre (VP/mL)
			H1	ARCA	1.8E+12
H2	ARCA+1% thioglycerol+15DNS	1.9E+12
			H5	ARCA-P80+15% sucrose	1.8E+12
H8	ARCA-P80+30% sucrose	1.8E+12
			H11	ARCA-P80-sucrose+20% glycerine	1.8E+12
H18	ARCA-P80-sucrose+15%P.Glycol	1.9E+12
			H22	ARCA-P80-sucrose+5%P.Glycol+15mM DMS	1.8E+12
M1	ARCA	9.2E+11
			M2	The ARCA+1% thioglycerol	9.0E+11
M4	ARCA+15mM DMS	8.9E+11
			M6	ARCA-P80+15% sucrose	9.4E+11
M8	ARCA-P80+30% sucrose	9.1E+11
			M10	ARCA-P80-sucrose+20% glycerine	9.6E+11
M17	ARCA-P80-sucrose+5% glycerine+15mM DMS	9.3E+11
			M20	ARCA-P80-sucrose+10%P.Glycol	9.1E+11
M22	ARCA-P80-sucrose+5%P.Glycol	9.3E+11

ARCA=5% sucrose, 1% glycine, 10mM Tris, 1mM magnesium chloride, RT-pH7.8; The DMS=dimethyl thioether; The P80=Tween-80; The P.Glycol=propylene glycol

Table 9ABe formulated in in the different preparations of 1E12 and 2E12 VP/mL and be stored in 5CG0070 under ℃ (2-8 ℃)

0 month					January			April			June
															％[ICV] o	The variation of RT	Plaque	GS R	％[ICV] o	The variation of RT	GS R	％[ICV]o	The variation of RT	GS R	％[ICV] o	The variation of RT	GS R
	％	(min)	VP/pf u		％	(min)		％	(min)		％	(min)
														H1	100％	-0.025	8	1.0	100％	-0.002	0.8	<50％>	0.441	0.0	＜1％	0.629	0.0
H2	100％	-0.019	8	1.0	97％	-0.028	0.8	100％	0.001	0.7	83％	0.026	0.9
														H5	100％	-0.015	9	1.0	100％	0.002	0.9	105％	0.065	0.8	96％	0.113	1.1
H8	100％	-0.036	10	1.1	104％	-0.014	0.6	108％	0.041	0.8	100％	0.073	1.0
														H11	100％	-0.035	21	1.0	102％	-0.001	0.7	107％	0.054	1.0	98％	0.087	1.1
H18	100％	-0.033	15	0.9	102％	-0.013	0.9	107％	0.041	1.0	99％	0.091	1.0
														H22	100％	-0.033	13	1.1	102％	-0.032	0.9	106％	0.048	0.9	99％	0.078	0.9
M1	100％	-0.041	9	1.1	ND	ND	ND	94％	0.073	0.8	79％	0.227	0.2
														M2	100％	-0.038	9	1.1	ND	ND	ND	104％	0.045	0.9	95％	0.042	0.9
M4	100％	-0.038	10	1.2	ND	ND	ND	97％	0.068	0.8	94％	0.115	0.9
														M6	100％	-0.034	8	1.0	ND	ND	ND	97％	0.058	0.7	94％	0.117	0.8
M8	100％	-0.034	16	1.1	ND	ND	ND	100％	0.033	0.5	ND	ND	ND
														M1 0	100％	-0.031	15	1.2	ND	ND	ND	101％	0.037	0.7	ND	ND	ND
M1 7	100％	-0.030	17	1.1	ND	ND	ND	101％	0.020	0.8	96％	0.081	0.9
														M2 0	100％	-0.036	12	1.0	ND	ND	ND	105％	0.024	0.9	98％	0.093	1.0
M2 2	100％	-0.033	11	1.1	ND	ND	ND	104％	0.015	0.7	98％	0.089	0.9

Table 9BBe formulated in in the different preparations of 1E12 and 2E12 VP/mL and be stored in 5CG0070 under ℃ (2-8 ℃)

September					December				16months				20 months
																		％[ICV ]o	□RT	Plaque	GSR	％[ICV] 0	□RT	Plaque	GSR	％[ICV] ]o	□RT	Plaque	GSR	％[ICV ] 0	□RT	Plaque	GSR
	％	(min)	VP/pfu		％	(min)	VP/pfu		％	(min)	VP/pfu		％	(min)	VP/ pfu
																	H1	＜1％	0.677	＞1000	0.0	＜1％	1.102	ND	ND	＜1％	0.758	ND	ND	＜1％	0.758	ND	ND
H2	38％	0.371	＞1000	0.0	＜1％	0.827	ND	＜0.01	＜1％	0.585	ND	ND	＜1％	0.584	ND	ND
																	H5	98％	0.151	21	1.0	94％	0.295	22	0.4	89％	0.227	22	0.9	93％	0.256	11	0.9
H8	100％	0.125	16	1.0	99％	0.233	18	0.9	98％	0.199	22	1.0	98％	0.230	13	1.2
																	H11	99％	0 136	12	1.2	96％	0.272	15	1.0	96％	0.220	18	1.0	95％	0.251	11	0.9
H18	ND	ND	ND	ND	100％	0.289	20	0.9	97％	0.216	22	0.9	98％	0.236	14	1.0
																	H22	100％	0.118	14	1.1	100％	0.258	20	0.8	95％	0.199	11	1.0	97％	0.214	8	1.0
M1	＜1％	0.508	＞100	0.0	＜1％	0.948	ND	＜0.01	＜1％	0.694	＞100	＜0.01	＜1％	0.718	ND	ND
																	M2	75％	0.103	48	0.7	19％	0.350	154	0.1	＜1％	0.544	＞100	0.1	＜1％	0.529	ND	0.01
M4	92％	0.165	14	0.9	82％	0.325	24	0.7	56％	0.290	22	0.5	46％	0.333	ND	0.02
																	M6	94％	0.150	17	0.8	92％	0.293	16	1.0	88％	0.236	16	1.2	92％	0.250	10	0.6
M8	97％	0.124	19	1.0	96％	0.249	19	0.8	94％	0.196	20	1.0	101％	0.235	12	0.8
																	M10	96％	0.135	20	1.0	95％	0.275	16	1.0	92％	0.205	14	1.0	95％	0.245	6	0.9
M17	95％	0.124	20	0.9	92％	0.248	16	1.0	88％	0.200	22	0.8	96％	0.241	10	0.9
																	M20	99％	0.136	19	0.9	98％	0.273	15	0.8	96％	0.220	19	0.9	94％	0.225	11	0.7
M22	98％	0.134	22	1.0	97％	0.271	19	1.0	91％	0.209	16	1.0	96％	0.238	17	0.8

Use many different dimethyl thioethers and propylene glycol cosolvent preparation (cited in the table 10) to test to storing the viral reserve that contains 4E12 VP/mL that reaches 4 months down at 5 ℃.Compare with the ARCA preparation, the stability that every kind of cosolvent preparation all demonstrates viral reserve is improved.The titre analysis of every kind of preparation enumerating in the his-and-hers watches 10 and the result of stability study are provided in the table 11.Every kind of preparation being tested all demonstrates and reaches 4 months satisfactory stability, and this determines by following analysis: the complete capsid virosome of %ICV=; [VP] of [ICV]=0 time point; The variation of RT, plaque (VP/PFU) and GSR=are at the standardized GM-CSF secretion rate of reference standard; Wherein＜and #〉expression %[VP]₀, it is the empty penton virosome that does not have ICV to exist.

Table 10The preparation of being tested

#	Preparation	Titre (VP/mL)
			T75-1	ARCA-70℃	4.3E+12
T75-2	ARCA	4.1E+12
			T75-3	ARCA+15mM DMS	4.2E+12
T75-9	ARCA-P80-sucrose+10%PropGol	4.0E+12
			T75-10	ARCA-P80-sucrose+10%PropGol+15mM DMS	4.0E+12
T75-11	ARCA-P80-sucrose+10%PropGol+20mM Met	4.0E+12
			T75-12	ARCA-P80-sucrose+5%PropGol	4.0E+12
T75-13	ARCA-P80-sucrose+5%PropGol+15mM DMS	4.0E+12
			T75-14	ARCA-P80-sucrose+5%PropGol+20mM Met	4.2E+12

ARCA=5% sucrose, 1% glycine, 10mM Tris, 1mM magnesium chloride, RT-pH7.8; The DMS=dimethyl thioether; The P80=Polysorbate 80; The PropGol=propylene glycol

Table 11Be formulated in in the different preparations of 4E12 VP/mL and be stored in 5 ℃ of (2-8℃) under CG0070

#	Time [moon]
																		0				1			1.5			2			4
	％[ICV]	ΔRT	Plaque	GSR	％[ICV] o	ΔRT	GSR	％[ICV] o	ΔRT	GSR	％[ICV] o	DRT	GSR	％[ICV]	ΔRT	Plaque	GSR
																		％	(min)	VP/pfu		％	(min)		％	(min)		％	(min)		％	(min)	VP/pfu
	T75-1	100％	0.026	19	1.0	98％	0.023	0.6	ND	ND	ND	97％	0.026	1.0	96％	0.020	10																		1.1
T75-2																		100％	0.020	11	1.0	<54％>	0.539	＜0.01	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
	T75-3	100％	0.025	19	1.0	95％	0.032	0.7	76％	0.049	0.9	<44％>	0.534	0.3	ND	ND	ND																		ND
T75-9																		100％	0.018	22	1.0	101％	0.034	1.3	ND	ND	ND	100％	0.052	1.1	97％	0.073	20	1.1
	T75-10	100％	0.021	21	1.0	99％	0.039	1.2	ND	ND	ND	99％	0.056	1.1	96％	0.072	21																		1.1
T75-11																		100％	0.014	17	1.0	99％	0.029	1.2	ND	ND	ND	101％	0.048	1.0	94％	0.066	21	1.2
	T75-12	100％	0.013	24	1.0	101％	0.028	1.1	89％	0.016	1.1	99％	0.042	1.1	99％	0.068	16																		1.1
T75-13																		100％	0.005	18	1.0	99％	0.031	1.1	95％	0.013	1.1	100％	0.041	0.9	99％	0.066	13	1.0
	T75-14	100％	0.008	18	1.0	99％	0.027	1.0	91％	0.009	1.1	93％	0.048	1.0	93％	0.064	16																		1.2

The complete capsid virosome of ICV=; [VP] of [ICV]=0 time point;＜#〉expression %[VP]₀, it is the empty penton virosome that does not have ICV to exist;

GSR=is at the standardized GM-CSF secretion rate of reference standard;

ND=does not detect

Contain the raising of six adjacent body stability in the preparation of ARCA and DMS

Carried out the mechanism of research with further understanding thio-compounds reversibility inhibition adenovirus protease.In these researchs, virus formulation is formulated in the ARCA damping fluid that is added with DMS.It is oxidized that the forced oxidation experiment shows that six adjacent bodies have precedence over albumen VI, and determine that by the analysis to the GM-CSF secretion rate oxidation of six adjacent bodies and albumen VI does not influence biological function.But, virus is stored in 30 ℃ following time of enzymatic activity temperature, demonstrate the degraded that has precedence over six adjacent bodies as the albumen VI of the function of time.In this research, adding DMS is suppressed in the determined degraded warp-wise preparation relevant with biological function as analyzing by GM-CSF.

In correlative study, show, increase DMS concentration (being respectively 15mM and 150mM) with because of oxygenizement six adjacent body modification are less relevant, and in the ARCA preparation, comprise 15mM DMS has significantly suppressed six adjacent bodies in 20 months by a definite date search time oxidation.

Although in order to be expressly understood the present invention, the front is described in detail the present invention by explanation and embodiment, those skilled in the art can clearly realize that and can carry out some change and modification to the present invention.All respects of the present invention realize by a series of experiments,

Wherein some is that non-limiting example by subsequently is described.Therefore, specification sheets and embodiment should not taken to limit the scope of the invention as scope of the present invention is recorded and narrated in detail by accompanying Claim.

Claims (41)

1. preparation comprises adenovirus carrier, glycine, MgCl₂, Tris damping fluid and be selected from the aqueous cosolvent of propylene glycol, DMSO, PEG, sucrose, glycerine and Tetrahydrofurfuryl polyethylene glycol ether, wherein said preparation shows under 2 ℃-30 ℃ and is higher than the stability of formulation that lacks described aqueous cosolvent.

2. the described preparation of claim 1, wherein said aqueous cosolvent is a propylene glycol.

3. the described preparation of claim 2, the concentration that exists of wherein said propylene glycol is about 3-20%.

4. the described preparation of claim 1, wherein said aqueous cosolvent is a Tetrahydrofurfuryl polyethylene glycol ether.

5. the described preparation of claim 4, the concentration that exists of wherein said Tetrahydrofurfuryl polyethylene glycol ether is about 5-20%.

6. the described preparation of claim 1, wherein said aqueous cosolvent is a sucrose.

7. the described preparation of claim 6, the total concn that exists of wherein said sucrose is at least 20%.

8. the described preparation of claim 1, wherein said aqueous cosolvent is a glycerine.

9. the described preparation of claim 8, the concentration that exists of wherein said glycerine is about 10-50%.

10. the described preparation of claim 1, wherein with described preparation stored under about 5 ℃.

11. the described preparation of claim 1, wherein with described preparation stored under about room temperature (15-30 ℃).

12. preparation comprises the virus vector and the reversible protease inhibitors of the capsid endoproteinase that depends on the encoding viral that is suitable for entering cell, wherein said preparation shows under 2 ℃-30 ℃ and is higher than the stability of formulation that lacks described reversible protease inhibitors.

13. the described preparation of claim 12, the capsid endoproteinase of wherein said encoding viral is an adenovirus protease.

14. the described preparation of claim 13, wherein said reversible protease inhibitors are the L3/p23 cystatin.

15. the described preparation of claim 14, wherein said reversible protease inhibitors is selected from thio-compounds, comprises thioglycerin, dimethyl thioether, dithiothreitol (DTT) (DTT), halfcystine, gsh and methionine(Met).

16. preparation comprises adenovirus carrier, glycine, MgCl₂, Tris damping fluid and thio-compounds, wherein said preparation shows under 2 ℃-30 ℃ and is higher than the stability of formulation that lacks described thio-compounds.

17. the described preparation of claim 16, wherein said thio-compounds are thioglycerin.

18. the described preparation of claim 17, the existing concentration to be about 0.5-2.0% or be about 50-200mM of wherein said thioglycerin.

19. the described preparation of claim 16, wherein said thio-compounds are dimethyl thioether.

20. the described preparation of claim 19, the concentration that exists of wherein said dimethyl thioether is about 10-100mM.

21. the described preparation of claim 16, wherein said thio-compounds are DTT.

22. the described preparation of claim 21, the concentration that exists of wherein said DTT is about 20-100mM.

23. the described preparation of claim 22, the concentration that exists of wherein said DTT is 50mM.

24. the described preparation of claim 16, wherein said thio-compounds are halfcystine.

25. the described preparation of claim 24, the concentration that exists of wherein said halfcystine is at least 1% or 150mM.

26. the described preparation of claim 12, wherein with described preparation stored under 5 ℃.

27. the described preparation of claim 16, wherein with described preparation stored under 5 ℃.

28. the described preparation of claim 12, wherein with described preparation stored under room temperature (15-30 ℃).

29. the described preparation of claim 16, wherein at room temperature with described preparation stored.

30. the preparation of claim 12, wherein said preparation further comprises the aqueous cosolvent that is selected from propylene glycol, DMSO, PEG, sucrose, glycerine and Tetrahydrofurfuryl polyethylene glycol ether.

31. the preparation of claim 16, wherein said preparation further comprises the aqueous cosolvent that is selected from propylene glycol, DMSO, PEG, sucrose, glycerine and Tetrahydrofurfuryl polyethylene glycol ether.

32. the described preparation of claim 31, wherein said aqueous cosolvent are propylene glycol.

33. the described preparation of claim 32, the concentration that exists of wherein said propylene glycol is about 3-20%.

34. the described preparation of claim 31, wherein said aqueous cosolvent are Tetrahydrofurfuryl polyethylene glycol ether.

35. the described preparation of claim 34, the concentration that exists of wherein said Tetrahydrofurfuryl polyethylene glycol ether is about 5-20%.

36. the described preparation of claim 31, wherein said aqueous cosolvent are sucrose.

37. the described preparation of claim 36, the total concn that exists of wherein said sucrose is at least 35%.

38. the described preparation of claim 31, wherein said aqueous cosolvent are glycerine.

39. the described preparation of claim 38, the concentration that exists of wherein said glycerine is about 10-50%.

40. the described preparation of claim 31, wherein with described preparation stored under 5 ℃.

41. the described preparation of claim 31, wherein with described preparation stored under room temperature (15-30 ℃).

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