CN101283085A - Methods and compositions for dried cellular forms - Google Patents

Abstract

Methods and compositions of spray drying cellular material are provided that allow preservation of the cellular material. In one aspect, the cellular material is spray dried with a quantity of excipient. In another aspect, the cellular material is spray dried using a cryoprotectant.

Description

The method and composition that is used for dried cellular forms

The cross reference of related application

The application requires the right of priority of U. S. application serial number of submitting on August 11st, 2,005 60/707,425 and the U. S. application serial number of submitting on March 31st, 2,006 60/788,133, and quotes the full content of incorporating these two applications into as proof.

Background

The dried forms of virion, cell biological body (cellular organisms) and other film binding substance (membranebound materials) can have very big use in pharmaceutical industries and public health industry.Dried cellular forms (DCF) demonstrates effectiveness at aspects such as standing storage, processing convenience, food delivery, agricultural and human hygiene applications.The example of DCF comprises the dry yeast that is used for food applications, the cell of cryopreservation (for example hemocyte), and the full cell (Trsic-Milanovicet al., J Serb.Chem.Soc., 66:435-42,2001 that are used for gene delivery; Diniz-Mendes et al., Biotechnol.Bioeng., 65:572-8,1999; And Seville et al., J.Gene Med., 4:428-37,2002).

DCF is usually by two kinds of method preparations: batch dry (bulk drying) is carried out in i) freeze-drying (lyophilization) or lyophilize (freeze-drying), its aqueous suspensions that relates to the pair cell form; Or ii) cryopreservation (cryopreservation), it relates to high-caliber cryoprotectant (cryoprotectant) is injected the water-containing cell suspension, and with the schedule speed that at utmost reduces necrocytosis the suspension temperature is reduced to below 0 ℃.A shortcoming of freeze-drying (or lyophilize) and cryopreservation is to be difficult to prepare a large amount of DCF (Kirsopand Snell with lower cost in the most of cellular material of protection; eds.; 1984; Maintenance of Microorganisms:A Manual of LaboratoryMethods; London, Academic Press).Two kinds of technology all are subjected to striding the restriction that the double-layer of lipoid membrane mass transfer reaches relevant osmotic stress (osmotic stress).

In the industrialization preparation of bacille Calmette-Guerin vaccine (BCG) vaccine, use freeze-drying.Annual millions of newborn infant is given BCG with defence tuberculosis (TB) by injection, a kind of by the bacterial disease that is called tubercule bacillus (tuberclebacillus) or mycobacterium tuberculosis (Mycobacterium tuberculosis) (Roche et al., Trends Microbiol., 3:307-401,1995).At present, TB is the sixth-largest lethal factor, and this global infectious disease increases with 3% estimation annual growth.It is urgent day by day to the demand of novel vaccine that the appearance of AIDS and it and getting in touch of tuberculosis make, this be since BCG in the vulnerable period of human body, typically in preceding 30 years of people's life to the TB infection, only has medium effectiveness (Fine, Lancet, 346:1339-1345,1995).It is that the BCG vigor is lower in the DCF that produces that BCG lacks one of possible cause of effectiveness.

General introduction

The present invention is based in part on the novel method of spraying drying cellular material and the discovery of composition, and these method and compositions represent significant efficiency of pcr product, higher organism activity (for example vigor) and good powder processing performance.Dried cellular forms, for example, the dried cellular forms by composition disclosed herein and method produce has low water content, applicable to experimenter's inhalation.Dried cellular forms keeps the activity of for some time when the temperature of (above freezing) above freezing stores, provide possibility for storing (for example standing storage) and delivery easily.These character make method and composition as herein described can be used for the vaccine production thing, for example, and for drug administration by injection, the oral or vaccine production thing that sucks.

In one aspect, the present invention includes such dry powder, it has the water (for example free-water) that is less than about 10% (for example being less than about 8%, 5%, 4%, 3%, 2% or 1%), cellular material and with the vehicle of dry weight basis at least 25% (for example at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99% or more).In some embodiments, powder be do not carry out freezing and the preparation.In some embodiments, powder prepares by spraying drying.In some embodiments, cellular material comprises bacterium (for example mycobacterium (Mycobacterium) belong to bacterium, for example mycobacterium tuberculosis (M.tuberculosis), M. smegmatics (M.smegmatis) or bacille Calmette-Guerin vaccine (Bacillus Calmette-Guerin)), virus, eukaryotic microorganisms, mammalian cell (for example red corpuscle, stem cell, granulocyte, inoblast or thrombocyte), film tuberculosis organoid, liposome, based on the bio-reactor of film or based on the drug delivery system of film.In some embodiments, the ratio of the quality of vehicle and the units of cellular material is 0.25pg vehicle per unit cellular material at least (for example at least 0.25,0.5,1,2,5,10,20,50,100,200,500,1000,2000,5000,10,000 or 20,000pg vehicle per unit cellular material).In some embodiments, the ratio of vehicle quality pair cell material mass is at least 0.1 (for example at least 0.25,0.5,1,2,5,10,15,20,25,30,40,50,100,200,500,1000 or 2000).In some embodiments, when described powder comprised viable cell (for example bacterium), the cell of (for example 1%, 2%, 4%, 5%, 6%, 8%, 10%, 12%, 15%, 18%, 20%, 25% or more) was alive more than 0.5%.In some embodiments, after surpassing 0 ℃ (for example above 4 ℃, 10 ℃, 20 ℃, 25 ℃, 30 ℃, 40 ℃ or 50 ℃) and storing time more than 10 days (for example 20,30,40,50,60,70,80,90,100,110 or 120 days), their initial activities of viable cell maintenance in the powder more than 1/1000 (for example more than 1/500,1/200,1/100,1/50,1/20 or 1/10).In some embodiments, vehicle comprises leucine, mannitol, trehalose, dextran (dextran), lactose, sucrose, Sorbitol Powder, white protein, glycerine, ethanol or its mixture.In some embodiments, described powder does not comprise cryoprotectant, for example the cryoprotectant of cryoprotectant of Tian Jiaing or significant quantity (for example not being the cryoprotectant of described vehicle).In some embodiments, described powder does not comprise salt, for example the salt of salt of Tian Jiaing or significant quantity.Described dry powder can be formulated as pharmaceutical composition, for example is used for the pharmaceutical composition of inhalation.

In one aspect of the method, the present invention includes the method for preparing the dry powder that comprises cellular material, described method be by: the aqueous solution is provided, and the wherein said aqueous solution comprises at least 0.01mg/ml (for example, at least 0.1,1,2,5,10,20,50,100 or 200mg/ml) vehicle and at least 10 ⁵Unit/ml (for example, at least 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹Or 10 ¹⁰The cellular material of unit/ml), and comprise under the condition of dry powder described cellular material, that contain the water (for example free-water) that is less than about 10% (for example being less than about 8%, 5%, 4%, 3%, 2% or 1%) weight the described solution of spraying drying in generation.In some embodiments, the ratio of vehicle quality and cellular material units is at least 0.25 pik vehicle per unit cellular material (for example at least 0.25,0.5,1,2,5,10,20,50,100,200,500,1000,2000,5000,10,000 or 20,000pg vehicle per unit cellular material).In some embodiments, the ratio of vehicle quality and cellular material quality is at least 0.1 (for example at least 0.25,0.5,1,2,5,10,15,20,25,30,40,50,100,200,500,1000 or 2000).Some wherein cellular material comprise that in the embodiment of bacterium (for example gram positive bacterium), described solution does not contain the salt or the cryoprotectant of interpolation.Some wherein cellular material comprise in the embodiment of eukaryotic cell (for example mammalian cell) that described solution can contain salt or other is enough to make the minimized solute of osmotic pressure.

In some embodiments, described solution comprises the vehicle with dry weight basis at least 10% (for example at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99% or more).In some embodiments, described solution comprises and is less than 10 ¹⁰Unit/ml (for example is less than 10 ⁹, 10 ⁸, 10 ⁷Or 10 ⁶The cellular material of unit/ml).In some embodiments, described cellular material comprises that bacterium (for example bacterium of Mycobacterium, for example mycobacterium tuberculosis, M. smegmatics or bacille Calmette-Guerin vaccine), virus, eukaryotic microorganisms, mammalian cell (for example red corpuscle, stem cell, granulocyte, inoblast or thrombocyte), film are in conjunction with organoid, liposome, based on the bio-reactor of film or based on the drug delivery system of film.In some embodiments, described vehicle comprises leucine, mannitol, trehalose, dextran, lactose, sucrose, Sorbitol Powder, white protein, glycerine, ethanol or its mixture.In some embodiments, the described aqueous solution does not contain cryoprotectant, for example is not the cryoprotectant of described vehicle.In some embodiments, described method also comprises described dry powder is formulated in the pharmaceutical composition, for example is used for the pharmaceutical composition of inhalation.The present invention also comprises by described novel method dry powder that produce, that comprise cellular material.

In one aspect of the method, the present invention includes with the cellular material spraying, to make the minimized method of the damage of this material by reducing osmotic stress (osmotic stress).Can reduce osmotic stress by following method: the radius initial value (R of cellular material (the claiming cell herein again) unit of spraying drying is treated in acquisition ^c(0)); Select following value: (i) the entrance and exit gas temperature differential of spray-dryer (Δ T); (ii) average droplet size (R ^d); The (iii) gasification latent heat of solvent (λ); (iv) the film of cellular material is for hydraulic diffusibility (hydraulic the permeability) (L of cryoprotectant _p); (v) mole number (the x of extracellular solute ^e _s); (vi) mole number (the x of solute in the cell ⁱ _s); (vii) mole number (the x of extracellular cryoprotectant ^e _Cp); (viii) concentration (C in the initial cell of cryoprotectant ⁱ _Cp(0)); (ix) cell count (n _Cells); Obtain equation 36 with these values

$-\frac{1}{L_p{R}_{\mathrm{gas}}T}\frac{d{R}^c\left(t\right)}{\mathrm{dt}}=\frac{x_s^e}{\frac{4}{3}\mathrm{\π}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3]}-\frac{x_s^i}{\frac{4}{3}\mathrm{\π}{R}^c{\left(t\right)}^3-V_{\mathrm{excluded}}}$

$+\mathrm{\σ}[\frac{x_{\mathrm{cp}}^e}{\frac{4}{3}\mathrm{\π}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3]}-C_{\mathrm{cp}}^i\left(0\right)]2\underset{n=1}{\overset{\∞}{\mathrm{\Σ}}}\frac{{\sin }^2\left({\mathrm{\λ}}_n\right)-{\mathrm{\λ}}_n\sin \left({\mathrm{\λ}}_n\right)\cos \left({\mathrm{\λ}}_n\right)}{{{\mathrm{\λ}}_n}^2-{\mathrm{\λ}}_n\sin \left({\mathrm{\λ}}_n\right)\cos \left({\mathrm{\λ}}_n\right)}{e}^{-{{\mathrm{\λ}}_n}^2\stackrel{\‾}{D_{\mathrm{cp}}^{*}}t/R^c{\left(t\right)}^2}---\left(36\right)$

Value, and, if R ^c(t) in the time of drying of prediction, maintain within minimum limit and the greatest limit, then use the described cellular material of condition spraying drying of selected value so that the damage of described material is minimized.In some embodiments, described method also comprises the time of drying of determining prediction.Can be selected so that the damage of described material is minimized minimum limit and greatest limit.For example, minimum limit can for initial radium at least about 60% (for example at least 70%, 80%, 90%, 95%, 98% or 99%).

For example, greatest limit can be at the most 160% (for example at the most 140%, 125%, 110%, 105%, 102% or 101%) of initial radium.In some embodiments, cellular material comprises that bacterium (for example bacterium of Mycobacterium, for example mycobacterium tuberculosis, M. smegmatics or bacille Calmette-Guerin vaccine), virus, eukaryotic microorganisms, mammalian cell (for example red corpuscle, stem cell, granulocyte, inoblast or thrombocyte), film are in conjunction with organoid, liposome, based on the bio-reactor of film or based on the drug delivery system of film.In some embodiments, the pair cell material adds cryoprotectant (for example add within the cellular material or outside) before being about to carry out spraying drying.In some embodiments, described method also comprises described dry powder is formulated in the pharmaceutical composition, for example supplies in the pharmaceutical composition of inhalation.The present invention also comprises by described novel method dry powder preparation, that comprise cellular material.

In a further aspect, the present invention includes the method that produces dry powder, wherein said dry powder comprises the water (for example free-water) that is less than about 10% (for example being less than about 8%, 5%, 4%, 3%, 2% or 1%), and the bacterium of Mycobacterium, described method be by: the aqueous solution is provided, and the wherein said aqueous solution comprises at least 0.01mg/ml (for example at least 0.1,1,2,5,10,20,50,100 or 200mg/ml) vehicle and at least 10 ⁵Colony-forming unit/ml (for example at least 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹Or 10 ¹⁰The mycobacterium species of colony-forming unit/ml), and contain the water (for example free-water) that is less than about 10% (for example being less than about 8%, 5%, 4%, 3%, 2% or 1%) in generation, and under the condition of the dry powder of described mycobacterium species, the described solution of spraying drying.In some embodiments, described solution relatively the mycobacterium species of each colony-forming unit contain the vehicle of 0.25pg at least (for example each colony-forming unit at least 0.5,1,2,5,10,15,20,25,35 or the vehicle of 50pg).In some embodiments, the described aqueous solution does not contain cryoprotectant, for example is not the cryoprotectant of described vehicle.In some embodiments, described mycobacterium species is mycobacterium tuberculosis, M. smegmatics, Mycobacterium bovis (M.bovis) or bacille Calmette-Guerin vaccine bacterium.In some embodiments, described method further comprises described dry powder is formulated in the pharmaceutical composition, for example is used for inhalation or is used for this powder is restored at the medicinal fluid carrier pharmaceutical composition of (reconstitute) back drug administration by injection.In some embodiments, described method also comprises described dry powder is mixed with vaccine, for example is used for inhalation or is used for this powder is restored at the medicinal fluid carrier vaccine of back drug administration by injection.The present invention also comprises by described novel method dry powder preparation, that comprise mycobacterium species.

In one aspect of the method, the present invention includes the vaccine composition that contains dry powder, described dry powder contains the water (for example free-water) that is less than about 10% (for example being less than about 8%, 5%, 4%, 3%, 2% or 1%), cellular material and with the vehicle of dry weight basis at least 25% (for example at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99% or more).In some embodiments, described dry powder prepares by method as herein described.Described vaccine composition can be formulated as and is used for parenteral or mucous membrane (for example oral or suck) and uses.In some embodiments, cellular material comprises that bacterium (for example mycobacterium species, for example mycobacterium tuberculosis, M. smegmatics or bacille Calmette-Guerin vaccine), virus, eukaryotic microorganisms, mammalian cell (for example red corpuscle, stem cell, granulocyte, inoblast or thrombocyte) or film are in conjunction with organoid.Vaccine composition can comprise one or more adjuvants.In some embodiments, with described one or more adjuvants with described cellular material spraying drying to form described dry powder.In some embodiments, after producing described dry powder, they are mixed with described one or more adjuvants.

The present invention also comprises by experimenter (for example human or animal) being used the immunization method of the vaccine composition that comprises dry powder described herein.In some embodiments, described dry powder prepares by method as herein described.Vaccine composition can be prepared and be used for parenteral or mucous membrane (for example oral or suck) and use.In some embodiments, described experimenter is baby, children or adult.In some embodiments, described cellular material comprises that bacterium (for example bacterium of Mycobacterium, for example mycobacterium tuberculosis, M. smegmatics or bacille Calmette-Guerin vaccine), virus, eukaryotic microorganisms, mammalian cell (for example red corpuscle, stem cell, granulocyte, inoblast or thrombocyte) or film are in conjunction with organoid.The vaccine composition that is used for immunization method can comprise one or more adjuvants.

Further, the present invention includes the method that stores dry powder as herein described, described method is by described powder is remained on temperature above freezing, for example 4 ℃ to 50 ℃ (for example 4 ℃ to 40 ℃, 4 ℃ to 30 ℃, 4 ℃ to 20 ℃, 4 ℃ to 10 ℃, 10 ℃ to 50 ℃, 10 ℃ to 40 ℃, 10 ℃ to 30 ℃) are through at least 1 day time (for example at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or longer).In some embodiments, described dry powder is remained on envrionment temperature.In some embodiments, described dry powder prepares by method as herein described.In some embodiments, described dry powder is formulated as pharmaceutical composition or vaccine composition.

In aspect other, the present invention includes the method for transporting the pharmaceutical composition or the vaccine composition that comprise dry powder, described dry powder contains the water (for example free-water) that is less than about 10% (for example being less than about 8%, 5%, 4%, 3%, 2% or 1%), cellular material and with the vehicle of dry weight basis at least 25% (for example at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99% or more).Described method comprises that preparation comprises the pharmaceutical composition or the vaccine composition of dry powder (for example dry powder for preparing by methods described herein), and in temperature above freezing, for example 4 ℃ to 50 ℃ (for example 4 ℃ to 40 ℃, 4 ℃ to 30 ℃, 4 ℃ to 20 ℃, 4 ℃ to 10 ℃, 10 ℃ to 50 ℃, 10 ℃ to 40 ℃, 10 ℃ to 30 ℃) transport described pharmaceutical composition or vaccine composition.In some embodiments, transport described pharmaceutical composition or vaccine composition in envrionment temperature.

Unless otherwise defined, all technology used herein and identical meaning known to scientific terminology has the general technical staff of the technical field of the invention.Although method and material similar with material to methods described herein or that be equal to can be used for enforcement of the present invention or test, suitable method and material have been described hereinafter.This paper quotes the full content that is incorporated herein all publications, patent application, patent and other reference mentioned as proof.If contradictory, when to comprise that this specification sheets in being defined in is as the criterion.In addition, the material here, method and embodiment only are used for the example explanation, and are not intended to restriction.

Accompanying drawing and following description have provided the details of one or more embodiments of the present invention.According to this specification sheets and accompanying drawing and claims, further feature of the present invention, purpose and advantage are obvious.

Description of drawings

The synoptic diagram of the model of the cellular material that Fig. 1 is surrounded by water.R ^cThe radius of expression cell.C ^e _s, C ^e _Cp, C _sAnd C ' _CpCryoprotection agent concentration in salt concn and the cell in the outer salt concn of difference indicator cells, extracellular cryoprotection agent concentration, the cell.

Fig. 2 A is the X-Y scheme of parallel membrane.

Fig. 2 B is the X-Y scheme of protruding platform border (convex plateau borders).

Fig. 3 is 80: 20 leucines: the electron photomicrograph of the spraying drying product of M. smegmatics.

Fig. 4 is 95: 5 leucines: the electron photomicrograph of the spraying drying product of M. smegmatics.

Fig. 5 is 90: 10 leucines: the fluorescence micrograph of the spraying drying product of M. smegmatics.Used M. smegmatics has been expressed GFP, and shows fluorescence in Photomicrograph.

Fig. 6 is 25 ℃ of 95: 5 leucines after 1 week of storage: the electron photomicrograph of M. smegmatics.

Fig. 7 just describes under the following condition in the exsiccant droplet cell volume (V/V relatively ₀) the figure of numerical solution: (a) amount of intracellular cryoprotectant is greater than the extracellular; (b) no cryoprotectant; (c) amount of the inside and outside cryoprotectant of cell equates.

Fig. 8 is described under the similar osmotic stress effect figure of the influence of the spray-dired M. smegmatics vigor of glycerine and salt pair.

Fig. 9 describes in the spray-dried powders M. smegmatics vigor yield to the figure of vehicle (leucine) solution per-cent.

Figure 10 describes 50: 50 leucine/M. smegmatics (smeg) powder time dependent line chart of M. smegmatics vigor yield under three kinds of conditions of storage.

Figure 11 describes 95: 5 leucine/M. smegmatics (smeg) powder time dependent line chart of M. smegmatics vigor yield under three kinds of stable conditions.Shown in the result be the average of 5 experiments.

Figure 12 A and 12B have been described in and have not had 95: 5 leucine/M. smegmatics (smeg) powder time dependent line chart of M. smegmatics vigor yield under three kinds of stable conditions under the monophogphoryl lipid A condition.

Figure 13 is described in to have tensio-active agent tyloxapol and Pluronic ^TMSpray-dired 95: 5 and 50: 50 leucines under the condition of-F68: the figure of the vigor yield of M. smegmatics.

Figure 14 is described in the time dependent line chart of Mycobacterium bovis BCG vigor yield under two kinds of conditions of storage.

Figure 15 is the fibroblastic Photomicrograph of NIH3T3 fetal mice of work in 1 month after the spraying drying.

Figure 16 is that the rat heart inoblast of gathering in former generation one group of 20X of the 3rd day and the 8th day after spraying drying differs micro-image.

Figure 17 is that NIH 3T3 fetal mice inoblast one group of 20X of the 3rd day and the 8th day after spraying drying differs micro-image.

Describe in detail

The present invention relates to new compositions and method for the preparation of dried cellular forms (DCF). These compositions and method help to prepare large batch of cellular material dried forms with well processed characteristic and cell viability. In preferred embodiments, initial excipient concentration (with dry weight basis) the dried cellular material of typical case of (for example at least 60%, 70%, 80% or 90%) with at least 50%. But initial excipient concentration can hang down and reach 25% in some cases. Can select or process these excipient so that cellular material is dried with cryoprotector, to reduce the osmotic stress in the dry run.

The composition of describing herein and method can be used for dry any cellular material, for example cellular material relevant with medicine, agricultural or food applications. " cellular material " in this article can with " film be combined material " (membrane bound material) Alternate, refer to the material of the film parcel that consisted of by lipid bilayer. Exemplary cellular material comprises bacterium (for example gramnegative bacterium and gram-positive bacterium, and vaccine form), membrane-bound virus (for example HIV), eukaryotic microorganisms (for example yeast), mammalian cell (for example haemocyte (for example cord blood cell), blood platelet, stem cell, granulocyte, fibroblast, endothelial cell (for example vascular endothelial cell), myocyte, Skin Cell, bone marrow cell and other cell), film are in conjunction with organelle (for example mitochondria), liposome, based on bioreactor (the Bosquillon et al. of film, J.Control Release, 99:357-367,2004) with based on drug delivery system (the Smith et al. of film, Vaccine, 21:2805-12,2003).

Other example of cellular material comprises that film is in conjunction with virus (for example influenza virus, rabies viruses, vaccinia virus, West Nile Virus, HIV, HVJ (sendai virus), hepatitis B (HBV), vaccinia subgroup virus (for example variola virus and vaccinia virus), herpes simplex virus (HSV) and other herpesviral). Other exemplary cellular material comprises viral infectious (AIDS for example, AIDS related complication (AIDS Related Complex), fowl pox (varicella), flu, cytomegalovirus infection, colorado tick fever, dengue fever, Ebola hemorrhagic fever, mumps, hand-foot-and-mouth disease, hepatitis, herpe simplex, herpes zoster, HPV (HPV), influenza (influenza), Lassa fever, measles, marburg hemorrhagic fever, infectious mononucleosis, mumps, polio, progressive multifocal leukoencephalopathy, rabies, rubella, SARS, smallpox (acne), viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile disease and yellow fever) pathogenic former, sick (the anthrax for example of bacillary transfection, meningitis, brucellosis, campylobacteriosis, cat scratch disease, cholera, diphtheria, epidemic typhus, gonorrhoea, impetigo, légionaires' disease, leprosy (hansen's disease), leptospirosis, listeriosis, Lyme disease, glander-like disease, methicillin resistance staphylococcus aureus (MRSA) infects, nocardiosis, pertussis (pertussis), the plague, pneumococcal pneumonia, psittacosis, Q heat, Rocky mountain spotted fever (RMSF), salmonellosis, scarlet fever, shigellosis, syphilis, lockjaw, trachoma, tuberculosis, tularemia, typhoid fever, typhus, and urinary tract infection) pathogenic former, parasitic infectious disease (African typanosomiasis nagana for example, amcbiasis, roundworm disease, babesiasis, chagas disease, clonorchiasis, Cryptosporidiosis, cysticercosis, bothrio-cephaliasis, dracunculiasis, hydatidosis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, spontaneous (free-living) amoeba infection, Giardiasis, ganthostomiasis, hymenolepiasis, isosporiasis, kala-azar, leishmaniasis, malaria, metagonimiasis, fly-blown, onchocercosis, pediculosis, retrofection, scabies, snail fever, taeniasis, toxocarasis, toxoplasmosis, trichinosis, trichinosis, trichuriasis, and trypanosomiasis) pathogenic former, and fungal infectious disease (aspergillosis for example, blastomycosis, candidiasis (dandidiasis), coccidioidomycosis (doccidioidomycosis), cryptococcosis (dryptococcosis), histoplasmosis and tinea pedis) pathogenic former. In addition, these related substanceses that cause a disease former attenuation (for example auxotrophy) forms and can promote the anti-former immunity of causing a disease (for example, BCG and bovine vaccine) can be used for method as herein described, for example, for the preparation of the method for vaccine (referring to, Sambandamurthy et al for example, Nat.Med., 9:9,2002; Hondalus et al., Infect.Immun., 68:2888-98,2000; And Sampson et al., Infect.Immun., 72:3031-37,2004).

The excipient that is used for methods described herein and composition includes, but not limited to compatible carbohydrate, natural and synthetic polypeptide, amino acid, surfactant, polymer or its combination. Typical excipient will (be seen for example Adamski and Anderson less than 1.0 (for example less than 0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2 or 0.1) to the reflectance factor (reflection coefficient) of the film of the cellular material in the drying, Biophys J, 44:79-90,1983; And

And Sigler, Physiol.Res., 49:191-195,2000). Suitable carbohydrate comprises monose, such as galactolipin, D-MANNOSE, sorbose, dextrose etc. Also can use disaccharides, such as lactose, trehalose, maltose, sucrose etc. Other excipient comprises cyclodextrin, such as 2-HP-BETA-CD; And polysaccharide, such as gossypose, maltodextrin, dextran etc.; And aldehyde alcohol/sugar alcohol, such as mannitol, xylitol, D-sorbite etc. Suitable polypeptide comprises dipeptides Aspartame (aspartame). Suitable amino acid comprises any natural amino acid that exists that forms powder under the effect of standard medicine process technology, these amino acid comprise nonpolar (hydrophobic) amino acid and polarity (neutral, positively charged and electronegative) amino acid, and these amino acid are regarded as Generally Recognized as safe (GRAS) by FDA. The representative example of nonpolar amino acid comprises alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. The amino acid whose representative example of polarity neutral comprises cysteine, glutamine, serine, threonine and tyrosine. The amino acid whose representative example of polarity zone positive charge comprises arginine, histidine and lysine. The amino acid whose representative example of polarity zone negative electrical charge comprises aspartic acid and glutamic acid. Suitable synthetic organic polymer comprises poly-[1-(2-oxygen-1-pyrrolidinyl) ethene], i.e. PVP (povidone) or PVP.

Dry compositions

Usually, use relatively small amount excipient dried cellular material, often relate to freezing. Do not having in the freezing situation, the gained powder often contains the water of significant quantity, if this is still to keep active because cellular material without freezing, then can not be dried to below certain water content (for example water of about 40% weight). Dry powder with good workability and stability need to be less than 10% weight usually, preferably is less than the water of 5% weight. This is because higher moisture mark causes between the powder particle producing significant capillary force, thereby makes powder reunite (aggregation). Therefore, in order to obtain to have the DCF of good powder processability and stability, just relate to a large amount of excipient and carry out spray-drying. Specifically, be less than 10% or 5% dry powder in order to obtain total moisture content, should be with by weight at least 25% the excipient of (for example at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99% or more) dry with cellular forms, obtain containing the dry powder of the cellular material of less weight fraction, when reservation is enough to keep active moisture, do not make again powder exist excess moisture with the overall processability of induced damage resistive powder like this.

Spray-drying is the standard procedure of using in grocery trade, medicine company and the agricultural. In spray-drying, mix by droplet and the drying medium (for example air or nitrogen) that makes spraying, moisture is evaporated away from the feeding liquid (spraying (spray)) of atomizing. This is crossed range drying and removes volatile substance in the droplet, stays " doing " particle of non-volatile component, and the size of these particles, form, density and volatile content are controlled by dry run. The mixture of being sprayed can be solution, emulsion, suspension or dispersion (dispersion). Many factors of dry run can affect the character of dried particles, the flow velocity and environmental condition (the Sacchetti and Van Oort that comprise bleed type, rotary drum size (drum size), evaporating solution and recyclegas, Spray Drying and Supercritical Fluid Particle Generation Techniques, Glaxo Wellcome Inc., 1996).

Usually, spray-drying process comprises Four processes: mixture is separated into little droplet, spraying mixes with drying medium (for example air), moisture is evaporation from spraying, and desciccate separates (Sacchetti and Van Oort with drying medium, Spray Drying and Supercritical Fluid Particle Generation Techniques, Glaxo Wellcome Inc., 1996).

Mixture is dispersed into the surface area that little droplet greatly increases thing to be dried, so that dry run is rapider. Typically, the higher particulate of gained that causes of energy dispersive is less. Dispersion can realize by any means known in the art, comprise drive nozzle, two-fluid spray nozzle, rotary atomizer and ultrasonic nozzle (Hinds, Aerosol Technology, 2nd Edition, New York, John Wiley and Sons, 1999). In some embodiments, with the press atomization mixture less than 200psi.

Disperse after (spraying) mixture, spraying and the drying medium (for example air) of gained mixed. Typically, be blended in the continuous heated air flow and occur. Hot-air improves the heat transfer for spray droplets, and increases evaporation rate. Air stream can be discharged into after drying in the atmosphere, perhaps circulates and recycles. Circulation of air often provides malleation and/or negative pressure to keep (Sacchetti and Van Oort by the arbitrary end at air-flow, Spray Drying and Supercritical Fluid Particle Generation Techniques, Glaxo Wellcome Inc., 1996).

When droplet and drying medium come in contact, because the droplet specific area is high, size is little, the fast evaporation very. Based on the character of drying system, in the product of drying, can keep the moisture (Hinds, Aerosol Technology, 2nd Edition, New York, John Wiley and Sons, 1999) of residual level.

Then product is separated with drying medium. Typically, the primary separation of product occurs in the bottom of hothouse, then for example using, cyclone separator, electrostatic precipitator, filter or Drechsel system reclaim product (Masters et al., Spray Drying Handbook, Harlow, UK, Longman Scientific and Technical, 1991).

The character of end product comprises granularity, final moisture content and yield, depends on many factors of dry run. Typically, the parameters such as inlet temperature, air velocity, liquid feed stream speed, droplet size and mixture concentration are regulated to produce product (the Masters et al. of expectation, Spray Drying Handbook, Harlow, UK, Longman Scientific and Technical, 1991).

Inlet temperature refers to the drying medium through heating, and normally air is flowing into the temperature that records before the hothouse. Typically, inlet temperature can be regulated according to hope. The drying medium temperature that product reclaims the position is called outlet temperature, and it depends on the character of inlet temperature, drying medium flow velocity and the mixture of being sprayed. Typically, higher inlet temperature makes amount of moisture reduction (Sacchetti and Van Oort in the end product, Spray Drying and Supercritical Fluid Particle Generation Techniques, Glaxo Wellcome Inc., 1996).

Air velocity (air flow rate) refers to that drying medium passes through flow (flow) of system. Air stream can be by providing in the arbitrary end of spray drying system or inner sustain malleation and/or negative pressure. Typically, higher air velocity causes the time of staying (be drying time) of particle in drying equipment shorter, and large (the Masters et al. of the amount that causes residual moisture in the final products, Spray Drying Handbook, Harlow, UK, Longman Scientific and Technical, 1991).

Liquid feed stream speed refers to be transported in each unit interval the amount of the liquid of hothouse. (throughput) is higher for the flux of liquid, and it is more that the droplet evaporation becomes the required energy of particle. Therefore, the higher output temperature (output temperature) that causes of flow velocity is lower. Typically, reduce this flow velocity, keep simultaneously inlet temperature and air velocity constant, can reduce water content (Masters et al., Spray Drying Handbook, the Harlow of end product, UK, Longman Scientific and Technical, 1991).

Droplet size/size is meant by the size of spray nozzle dispersive droplet.Typically, less droplet makes that the water content of final product is lower, granularity less (Hinds, Aerosol Technology, 2 ^NdEdition, New York, John Wiley and Sons, 1999).

The concentration of dry mixture of waiting to spray is also influential to final product.Typically, higher concentration causes the granularity of final product bigger, this is because each is by more (the Sacchetti and Van Oort of the material of spray droplets, Spray Drying and Supercritical Fluid ParticleGeneration Techniques, Glaxo Wellcome Inc., 1996).

Being used for spray-dired system can be from for example Armfield, Inc. (Jackson, NJ), BrinkmannInstruments (Westbury, NY), BUCHI Analytical (New Castle, DE), Niro Inc (Columbia, MD), Sono-Tek Corporation (Milton, NY), Spray Drying Systems, Inc. (Randallstown, MD) and Labplant, Inc. (North Yorkshire England) buys.

The final water content of spray-dried powders can be determined by any means known in the art, for example, pass through thermogravimetry.Thermogravimetry is determined water content (Maa et al., Pharm.Res., 15:5,1998) by the heating powder and the mass loss of measuring in the moisture evaporation process.Typically, for the sample that contains cellular material (for example bacterium), water will divide two phases (phases) evaporation.First is called free-water mutually, mainly is the moisture of dry vehicle.Second is called combination water mutually, mainly is the moisture of cellular material.Can be measured free-water and combination water, be determined whether powder contains desirable moisture content (Snyder et al., Analytica Chimica Acta, 536:283-293,2005) in vehicle or cellular material.

In spray-drying process, reduce osmotic stress

The vehicle introduced in the exsiccant cell solution of waiting to spray can be selected and/or introduces by such mode, makes the overall penetration stress minimum that the film of cellular material bears, thus the activity of keeping.Although owing to above-mentioned reason, it is considerable keeping required vehicle massfraction with respect to the cellular material massfraction, but for cell viability, introduces the mode of these vehicle before the character of these vehicle, the spraying drying, may be important, or even crucial.

For cellular material, the drying of droplet in the spraying drying rotary drum can be regarded as and be similar to the refrigerating process of organism in standard cryopreservation process, (James as shown in Figure 1, " Maintenanceof Parasitic Protozoa by Cryopreservation; " Maintenance of Microorganisms, Academic Press, London, 1984).

When containing the droplet evaporation of organism, the salt concn (C in (and extracellular) in the droplet ^e _s) with respect to the salt concn (C in the organism ⁱ _s) will increase.Reason is that cytolemma is impermeable for the transfer of salt, and is permeable relatively for the transfer of water.The result is that the drying of droplet has increased the salt concn in the droplet that is evaporating, and forms the osmotic stress (due to the salt concn imbalance of film either side) of cell membrane, causes water to be pushed out from cell.This dehydration can be seen that film forming attempts mechanically to reduce osmotic stress (Batycky et al., Phil.Trans.Roy.Soc.Lond., A355:2459-88,1997) by the imbalance of eliminating salt concn.

The process that " dehydration " of cellular material in the droplet evaporative process and cellular material stand to take place when freezing is essentially identical.As mentioned above, excessively dehydration can cause the cracking of cellular material, for fear of excessive dehydration, has developed the technology relevant with the cryopreservation field, just the control of the use of cryoprotectant and freeze-thaw cycle.Cryoprotectant is faster than the pharmacology inert substance of the speed permeation cell film of salt to be slower than water.Because these technology are relevant with the method for spraying drying cellular material, hereinafter they carried out concise and to the point summary (Karlsson and Toner, Biomaterials, 17:243-256,1996).

At first, in view of the semipermeability of film to cryoprotectant, cryoprotectant applies osmotic pressure to film, and the concentration of this osmotic pressure and cryoprotectant is proportional, and for best cryoprotectant, it is in close proximity to the osmotic pressure that salt applied of suitable concentration.This means, be immersed in the remarkably influenced that osmotic stress that cytolemma stood in the aqueous medium that contains the similar big or small cryoprotectant of impermeability salt concn and the condition of oozing such as non-tend to be subjected to the existence of cryoprotectant material.Therefore, the film diffusion of striding of cryoprotectant provides a kind of means of reversed osmos stress, even also be like this under the unequal situation of the salt concn of film either side.Thus, cryoprotectant provides the means of dispersion (diffuse) osmotic stress.The cryoprotectant that is suitable for described novel method includes but not limited to methyl-sulphoxide, ethylene glycol, propylene glycol and glycerine (Chesne and Guillouzo, Cryobiology, 25:323-330,1988).In some embodiments, cryoprotectant is not included in the dried mixture.

In the cryopreservation rules, cryoprotectant is added in the suspension of cellular material to the concentration (C of interpolation ^e _Cp) and the salt concn significant correlation.Should be pointed out that and to control this interpolation, that is to say, can add cryoprotectant, make cryoprotectant to diffuse through cytolemma and do not make cell dehydration with enough slow speed in order to avoid make cell be subjected to too high osmotic stress.Then freezing-because the effect of natural cryoprotectant in the cell; the freezing extracellular that causes forms ice; thereby increase extracellular salt concn-process in, cryoprotectant can diffuse through cytolemma and improve IC, thereby increases the inner concentration (C of cryoprotectant ⁱ _Cp).So just alleviated the osmotic stress on the cytolemma, especially under freezing condition of carrying out with enough slow speed.Like this, cryoprotectant helps to keep cell viability, has explained its purposes (Karlsson and Toner, Biomaterials, 17:243-256,1996) aspect preservation blood, seminal fluid and other useful cell.

Though spraying drying and cryopreservation have similarity, it has special advantages for cellular material, and this advantage is a particularly important for extensive use.The cell cryopreservation of cell suspension in enormous quantities is difficult; this is because when the suspension yardstick is far longer than the cell yardstick, the mass transfer dynamics on two kinds of yardsticks require (add or removal cryoprotectant and frozen cell process in related) be very different.May to be that the blood of employing standard cryopreservation method is freezing be not easy to be applicable to one of complete organ refrigerated reason for this.Spraying drying is divided into cell suspension less amount (being droplet) automatically, and they can regard the cryopreservation subsection roughly as.Scale is amplified the amount that does not need significantly to increase the droplet of spraying: it is to realize by the size that increases spray drying vessel, suspension flow (flow) and other standard scale amplification means that increase by nozzle that scale is amplified.

Therefore, spraying drying can be provided for producing the method for a large amount of DCF, and the specific activity of these DCF is higher by the activity that cryopreservation and freeze drying technology obtain.

Hereinafter described a kind of theory form, it provides the pair cell form to carry out spraying drying, and membrane stress is minimized, thereby makes the maximized rule of vigor.These methods depend on uses cryoprotectant and the spray-dired parameter of control for example type of solvent, gasinlet temperature and spraying drying jet size and speed of rotation (droplet size).

Described method is determined the speed that spray droplets can be dried in heating environment, like this, under the condition that the cryopreservation agent exists, can regulate the film radius of suspended matter.Thus, can prevent that film is retracted to R ^c _MinBelow or be expanded to R ^c _MaxMore than.Illustrate, at R ^c _MinSituation under, be not that all suspended matters all will be retracted to critical radius (R owing to the dehydration that osmotic pressure orders about ^c _Cri) below.If cell walls is an inflexible, can directly this condition be equal to mutually with the critical stress that causes inactivation.At first, idealized geometric condition and concentration in considering a problem are considered two kinetics under the limiting condition then.Then, set up hydromeehanics and mass transfer equation and describe cell radius velocity of variation as the function of system parameter.

For ease of explanation, it is contemplated that a kind of like this cell suspension, wherein cell is to have equilibrium radius R ^c _oSpheroid.Salt and cryoprotectant are arranged in these cells, and they are C in intracellular concentration ⁱ _sAnd C ⁱ _Cp, be C in extracellular concentration ^e _sAnd C ^e _Cp

When spraying drying, form the droplet individuality of suspended matter.Here, we suppose that cell keeps uniform distribution in spray solution and spray process, and therefore the concentration in each droplet of being sprayed equates.Flow velocity (its can be in spraying drying physics control) can clearly be solved by following formula in addition:

$a=\frac{N}{n_{\mathrm{cells}}{t}_o}---\left(1\right)$

Wherein a is the speed that time per unit generates droplet, n _CellsBe the cell count that suspends in each droplet individuality of being sprayed, N is the total cell count in the volume, t _oBe with volume V _oThe required time quantum of spraying.

Waiting to spray the volume fraction of cell in the suspension will be with φ _oExpression, wherein

And N is suspension volume φ _oIn total cell count.

Suppose that these droplets have the radius R of homogeneous ^d _oThereby the mark of cellular material can be expressed as

${\mathrm{\φ}}_o=n_{\mathrm{cells}}{\left(\frac{R_o^c}{R_o^d}\right)}^3---\left(3\right)$

Wherein n is the cell count that suspends in the droplet individuality that each is sprayed.

Under the uniform situation of hypothesis, four concentration C that in original suspension, record ^e _s, C ^e _Cp, C ⁱ _s, C ⁱ _CpEqual each by the starting point concentration of salt and cryoprotectant in the cell of spray droplets.The absolute mole number of salt and cryoprotectant must conservation in known each droplet, and these concentration will change in time based on the variation of diameter of droplets and cell dia.

Make x ⁱ _sAnd x ^e _s, and x ⁱ _CpAnd x ^e _CpRepresent respectively and in the droplet individuality, disperse back salt and cryoprotectant, so just have at the mole number of outside and cell interior:

$C_s^i=\frac{x_s^i}{\frac{4}{3}\mathrm{\π}{R}^{c^3}-{V^c}_{\mathrm{excluded}}}---\left(4\right)$

$C_{\mathrm{cp}}^i=\frac{x_{\mathrm{cp}}^i}{\frac{4}{3}\mathrm{\π}{R}^{c^3}-{V^c}_{\mathrm{excluded}}}---\left(5\right)$

$C_s^e=\frac{x_s^e}{\frac{4}{3}\mathrm{\π}{R}^{d^3}(1-\mathrm{\φ})}=\frac{x_s^e}{\frac{4}{3}\mathrm{\π}[R^{d^3}-n_{\mathrm{cells}}{R}^{c^3}]}---\left(6\right)$

$C_{\mathrm{cp}}^e=\frac{x_{\mathrm{cp}}^e}{\frac{4}{3}\mathrm{\π}{R}^{d^3}(1-\mathrm{\φ})}=\frac{x_{\mathrm{cp}}^e}{\frac{4}{3}\mathrm{\π}[R^{d^3}-n_{\mathrm{cells}}{R}^{c^3}]}---\left(7\right)$

Here, V ^c _ExcludedBe that salt and/or cryoprotectant can not be assigned to wherein volume in each cell individual, it is regarded as time-invariant.Because salt can not penetrate film, parameter x ⁱ _sAnd x ^e _s(representing in the cell and extracellular salt mole number) also is constant to the time.Only time variable is exactly R in the above-mentioned like this expression formula ^cAnd R ^dAnd cell is interior and extracellular cryoprotectant mole number x ⁱ _CpAnd x ^e _Cp

Each droplet individuality will evaporate in the spraying drying rotary drum, and its vaporator rate depends on external conditions, droplet size, droplet volatility or the like.When initial, consider the very rare at first (φ of suspension _o＜＜1) character, cell individual will be on average mutually away from.As time goes on, cell closely contact more and more it is contemplated that two kinds of limit situations like this:

Here, φ (t)＜＜1 in drying process.In the case, suppose that each cell individual is isolated (isolated), be suspended in the infinitely-great bath (bath) as it salt and cryoprotection agent concentration (thereby to osmotic stress) in developing are reacted.Since the symmetry (Consideration of the mass transfer that vide infra) of problem, the equal radial shrinkage of droplet and cell (or expansion).Therefore, consider Fig. 1, around the cell individual neutralization because osmotic stress and can not being expressed as follows because of the velocity distribution due to the liquid motion:

v＝ι _r v _r(t) (8)

ι wherein _rBe in the spherical coordinate system that with the cell centre is initial point along the unit vector of coordinate r, v _r(t) be the size of radial velocity.

In addition, establish cell and the droplet fluid is incompressible.

▽·v＝0 (9)

Or

$\frac{{\∂v}_r}{\∂r}=0---\left(10\right)$

Because the radial velocity at cell centre place is necessary for zero, conclusion is in the everywhere

v＝0 (11)

This conclusion hint, any radial motion of cytolemma must be " incorporeity " (non-material), the meaning is the quality mean motion that the film motion is not equal to continuous fluid.

Therefore, situation 1 is such problem: the differentiation of cell individual in droplet is diffusion-driven.

At φ _oUnder → 1 the limiting condition, just extremely closely contact of the cell individual in the exsiccant droplet.The differentiation that cytolemma takes place because of osmotic stress is limited in such environment: cytolemma or one-tenth plane earth perhaps form near so-called " platform border " (plateau borders) and protrude curved surface by adjacent cell.Such membrane environment is presented among Fig. 2.

In situation 2, several basic assumptions of situation 1 are no longer set up.At first, consider that the droplet that the tight contact of cell and excluded volume owing to cell cause " adjoins " resistance to mass transfer mutually, Yan Buhui is at once with respect to the cross-cell membrane water transport is defeated in the increase of salt and cryoprotection agent concentration in expection outside or the external phase.This just means along with droplet volume continues to reduce; the salt of droplet periphery and cryoprotection agent concentration will significantly increase with respect near the concentration the droplet center; thereby will be subjected to high osmotic stress near the cell of droplet periphery, and the cell that is positioned at the center will be subjected to and even not have osmotic stress few.So, make the radial dilatation of each cell in the drying process or meaning that contraction minimizes this target just indeterminate, because each cell will experience different conditions in time.Target in the situation 2 is: perhaps make the most pregnable cell, the cell expansion that promptly is positioned at those cells of periphery minimizes, and perhaps considers the reasonable time constraint for dry process, the cell of maximum number in the rescue droplet.(note, make the minimum needed final dry restricted condition of necrocytosis of periphery, under limiting condition, may require unlimited slow drying.)

For the purpose of this analysis, remaining consideration will be carried out round situation 1 specially.

Can determine two significant mass transfer problems for situation 1.First problem relates to the just mass transfer of salt and cryoprotectant in the exsiccant droplet, just supposes that the concentration of salt and cryoprotectant increases as the function of time equably in the exsiccant droplet.Because cell suspension is rare, the dry problem of droplet can be considered individually.This back problem is spherical water droplet exsiccant problem in the warm air continuum.

Externally the globoferous cell mass transfer problem in the non-boundary environment of salt and cryoprotection agent concentration homogeneous variation is suddenly solved by people such as Batycky (1997) previously.In their analysis; the cell fluid is described to a kind of continuum; it is (homogenized) of homogenizing that intracellular salt and cryoprotection agent concentration are considered as in whole tenuigenin liquid and inner organoid, or (the specially averaged) of especially flat homogenizing.According to the standard definition of osmotic pressure on the film, promptly Reynold's transport theorem (Reynolds Transport Theorem) and to water the Darcy's law by membrane permeability (Darcylaw description) described, provable film speed is:

$U=\frac{{\mathrm{dR}}_o^c}{\mathrm{dt}}=-L_p{R}_{\mathrm{gas}}T[(C_s^e-C_s^i{|}_{R=R_c\left(t\right)})+\mathrm{\σ}(C_{\mathrm{cp}}^e-C_{\mathrm{cp}}^i{|}_{R=R\left(t\right)})]---\left(12\right)$

L wherein _pIt is the hydraulic diffusibility (m/satm) of film; σ is called reflection coefficient (reflection coefficient) (0＜σ＜1), the mark that the expression film reduces with respect to salt the perviousness of cryoprotectant.

The time rate of change of film place salt and cryoprotection agent concentration can be obtained by the mass transfer conservation equation of decorrelation in the cell.Though salt and cryoprotectant is dense in the cell, still be assumed to be Fick diffusion (Fickian diffusion) for constant salt and cryoprotectant.According to Batycky et al. (1997) and include Edwards in and the result of Davis (Chem.Eng.Sci., 50:1441-54,1995), these diffusivitys are expressed as process-scale coefficient (course-scale coefficients)

The existence of organoid in their reflection cells.

In Batycky et al. (1997), the control differential equation of salt concn can be expressed as:

$\frac{\∂\stackrel{\‾}{C_s^i}}{\∂t}=\frac{1}{r^2}\frac{\∂}{\∂r}\left(\stackrel{\‾}{D_s^{*}}{r}^2\frac{\∂\stackrel{\‾}{C_s^i}}{\∂r}\right)---\left(13\right)$

$\stackrel{\‾}{D_s^{*}}\frac{\∂\stackrel{\‾}{C_s^i}}{\∂r}+\frac{d{R}^c\left(t\right)}{\mathrm{dt}}{C}_s^i=0,\∀r=R\left(t\right),t---\left(15\right)$

If starting condition is

During t=0, $C_s^i=C_s^i\left(0\right),$ Wherein during t=0, R ^c(t)=R _i(16)

In above-mentioned equation, C _s ⁱWith

By the following formula association:

$\stackrel{\‾}{C_s^i}=C_s^i(1-\frac{V_{\mathrm{excluded}}^c}{\frac{4}{3}\mathrm{\π}{R}^c{\left(t\right)}^3})---\left(17\right)$

Separating these equations can get:

$\stackrel{\‾}{C_s^i}=\frac{x_s^i}{\frac{4}{3}\mathrm{\π}{R}^c{\left(t\right)}^3}---\left(18\right)$

$C_s^i=\frac{x_s^i}{\frac{4}{3}\mathrm{\π}{R}^c{\left(t\right)}^3-V_{\mathrm{excluded}}^c}---\left(19\right)$

In Batycky et al. (1997), the control differential equation of cryoprotection agent concentration can be expressed as:

$\frac{\∂\stackrel{\‾}{C_{\mathrm{cp}}^i}}{\∂t}=\frac{1}{r}\frac{\∂}{\∂r}\left(\stackrel{\‾}{D_{\mathrm{cp}}^{*}}{r}^2\frac{\∂\stackrel{\‾}{C_{\mathrm{cp}}^i}}{\∂r}\right),---\left(20\right)$

Its final condition is

$\stackrel{\‾}{D_{\mathrm{cp}}^{*}}\frac{\∂\stackrel{\‾}{C_{\mathrm{cp}}^i}}{\∂r}+\frac{d{R}^c\left(t\right)}{\mathrm{dt}}{C}_{\mathrm{cp}}^i=P_{\mathrm{cp}}(C_{\mathrm{cp}}^e-C_{\mathrm{cp}}^i),\∀r=(R^c\left(t\right),t)---\left(22\right)$

Starting condition is

During t=0, $C_{\mathrm{cp}}^i=C_{\mathrm{cp}}^i\left(0\right)---\left(23\right)$

During t=0, $R^c\left(t\right)=R_o^c---\left(24\right)$

Be associated as:

$\stackrel{\‾}{C_{\mathrm{cp}}^i}=C_{\mathrm{cp}}^i(1-\mathrm{\θ}+\mathrm{\κ\α}+\mathrm{K\θ}),\∀r,t---\left(25\right)$

Wherein θ is the osmotic pressure inert part (organoid) of cell, and κ=Henry's law uptake factor, α are the organoid specific surface areas, and K is the partition ratio that enters organoid.

Separating these methods with equation (14) (Batycky et al.1997) obtains

$-\frac{1}{L_p{R}_{\mathrm{gas}}T}\frac{d{R}^c}{\mathrm{dt}}=C_s^e-\frac{x_s^i}{\frac{4}{3}\mathrm{\π}{R}^c{\left(t\right)}^3-V_{\mathrm{excluded}}}+\mathrm{\σ}[C_{\mathrm{cp}}^e-C_{\mathrm{cp}}^i\left(0\right)]2\underset{n=1}{\overset{\∞}{\mathrm{\Σ}}}\frac{{\sin }^2\left({\mathrm{\λ}}_n\right)-{\mathrm{\λ}}_n\sin \left({\mathrm{\λ}}_n\right)\cos \left({\mathrm{\λ}}_n\right)}{{{\mathrm{\λ}}_n}^2-{\mathrm{\λ}}_n\sin \left({\mathrm{\λ}}_n\right)\cos \left({\mathrm{\λ}}_n\right)}{e}^{-{{\mathrm{\λ}}_n}^2\stackrel{\‾}{D_{\mathrm{cp}}^{*}}t/R^c{\left(t\right)}^2}---\left(26\right)$

Its starting condition is

During t=0, $R^c\left(t\right)=R_o^c---\left(27\right)$

λ wherein _nBe the eigenwert of the non-zero root of following transcendental equation:

βλ _n＝tan(λ _n) (28)

Wherein, P _SpBe the speed that the semipermeability solute enters cell, factor beta is defined as:

$\mathrm{\β}={(1-\frac{P_{\mathrm{sp}}{R}^c\left(t\right)}{\stackrel{\‾}{D_{\mathrm{sp}}^{*}}(1-\mathrm{\θ}+\mathrm{\κ\α}+\mathrm{\κ\θ})})}^{-1}---\left(29\right)$

Note, though λ _nOn the quick time scale of diffusion is substantially invariable, and they are slowly to change in time on the time scale of cytolemma expansion.Equation (28) is with cell radius R ^c(t) be associated with outside salt and cryoprotection agent concentration, outside salt and cryoprotection agent concentration then depend on the speed of droplet evaporation.This incidence relation is described below.

Many investigators have investigated the spherical droplet of exsiccant in gas phase, when especially ignoring the convection effect in the gas.Evaporation in the spray-dryer depends on the control speed (governing rate) of evaporation and the residence time (residence time) of evaporation.The residence time is the function of spraying-air movement in the moisture eliminator.Under the situation of droplet with respect to the ambient air motion, the flow condition around the droplet in the motion influences vaporator rate.At this moment, if the speed of relative movement between air and the droplet is extremely low, then droplet is influenced by airflow fully.According to boundary layer theory, identical with the evaporation in the still air condition with the vaporator rate of the droplet of zero speed of relative movement motion.Therefore, with droplet by spray-dired evaporation as with the still air condition under the evaporation similar mechanism carry out modeling.

Experiment neutralization in theory, the universal relation of deferring between the controlled parameter of droplet radius and spray-drying process by following formula provide (Masters, 1991, Spray Drying Handbook, LongmanScientific and Technical, Harlow, UK):

$\mathrm{dt}=-\frac{\mathrm{\λ}{\mathrm{\ρ}}_{1}D}{K_d\mathrm{LMTD}}\mathrm{dD}---\left(30\right)$

D=2R wherein _c, K _dBe the average thermal conductivity of air film around the droplet in the evaporation, ρ ₁Be the density of gas phase, λ is the gasification latent heat of droplet, and LMTD is the log-mean temperature difference by the following formula definition:

$\mathrm{LMTD}=\frac{\mathrm{\&Delta;}{T}_0-\mathrm{\&Delta;}{T}_1}{2\&CenterDot;303{\log }_{10}({\mathrm{\&Delta;<figure-callout\; id="0"\; label="T"\; filenames="A20068003775700551.png,A20068003775700552.png"\; state="\{\{state\}\}">T</figure-callout>}}_0/{\mathrm{\&Delta;T}}_1)}---\left(31\right)$

Δ T wherein ₀With Δ T ₁It is the temperature difference initial and final between droplet and the gas phase.

(30) integration is obtained:

$R^d\left(t\right)=\sqrt{\mathrm{kt}+R_o^{d^2}}---\left(32\right)$

Wherein

$k=-\frac{K_d\mathrm{LMTD}}{{\mathrm{\&lambda;\&rho;}}_1}---\left(33\right)$

With (32) substitution (6) and (7), the instant concentration and the droplet vaporous parameter of salt and cryoprotectant associated:

$C_s^e=\frac{x_s^e}{\frac{4}{3}\mathrm{\&pi;}{(\mathrm{kt}+R_o^{d^2})}^{3/2}(1-\mathrm{\&phi;})}=\frac{x_s^e}{\frac{4}{3}\mathrm{\&pi;}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3]}---\left(34\right)$

$C_{\mathrm{cp}}^e=\frac{x_{\mathrm{cp}}^e}{\frac{4}{3}\mathrm{\&pi;}{(\mathrm{kt}+R_o^{d^2})}^{3/2}(1-\mathrm{\&phi;})}=\frac{x_{\mathrm{cp}}^e}{\frac{4}{3}\mathrm{\&pi;}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3]}---\left(35\right)$

Spray drying process can be represented with the following differential equation:

$-\frac{1}{L_p{R}_{\mathrm{gas}}T}\frac{d{R}^c\left(t\right)}{\mathrm{dt}}=\frac{x_s^e}{\frac{4}{3}\mathrm{\&pi;}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3]}-\frac{x_s^i}{\frac{4}{3}\mathrm{\&pi;}{R}^c{\left(t\right)}^3-V_{\mathrm{excluded}}}$

$+\mathrm{\&sigma;}[\frac{x_{\mathrm{cp}}^e}{\frac{4}{3}\mathrm{\&pi;}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3}-C_{\mathrm{cp}}^i\left(0\right)]2\underset{n=1}{\overset{\&infin;}{\mathrm{\&Sigma;}}}\frac{{\sin }^2\left({\mathrm{\&lambda;}}_n\right)-{\mathrm{\&lambda;}}_n\sin \left({\mathrm{\&lambda;}}_n\right)\cos \left({\mathrm{\&lambda;}}_n\right)}{{{\mathrm{\&lambda;}}_n}^2-{\mathrm{\&lambda;}}_n\sin \left({\mathrm{\&lambda;}}_n\right)\cos \left({\mathrm{\&lambda;}}_n\right)}{e}^{-{{\mathrm{\&lambda;}}_n}^2\stackrel{\&OverBar;}{D_{\mathrm{cp}}^{*}}t/R^c{\left(t\right)}^2}---\left(36\right)$

By finding the solution above-mentioned equation, can determine in order to make minimise stress, to make $R_{\min }^c<R^c\left(t\right)<R_{\max }^c$ Kept or made the maximizes stress of suspension film binding substance and the speed of rotation (R of essential spray-dryer entrance and exit gas temperature (being Δ T), bleed type and droplet size ^d), type of solvent (λ), cryoprotectant type (L _p) etc. condition.At the cryopreservation cell freezing and melt and to find in the rule of speed and rules that these rules are similar.

Pharmaceutical composition

The dried cellular forms of Miao Shuing for example with the dried cellular forms of described novel composition or novel method preparation, can be prepared as pharmaceutical composition, for example vaccine composition herein.Cellular material can prepare drug powder with multiple pharmacy acceptable diluent, weighting agent, salt, buffer reagent, stablizer, solubilizing agent and other material spraying drying well known in the art.Perhaps, after spraying drying, at least a preparation of product in multiple pharmacy acceptable diluent, weighting agent, salt, buffer reagent, stablizer, solubilizing agent, adjuvant and other material well known in the art can be come pharmaceutical compositions, for example drug powder.Term " pharmacy the is acceptable " meaning is the non-toxic substance of the validity of the biologic activity of interferon activity composition not.The feature of composition can depend on route of administration.In some embodiments, composition can be stored in check temperature before using.

Using of pharmaceutical composition (pharmaceutical composition that for example contains dried cellular forms) can be implemented by multiple usual manner, for example suction, oral cavity absorption or skin, subcutaneous or intravenous injection.Preferably use by suction.In some embodiments, composition is as vaccine administration.

Dried cellular forms can be formulated as and utilizes medical treatment device, and for example sucker (seeing for example United States Patent (USP) 6,102,035 (powder inhalator) and 6,012,454 (dry powder inhalers)) sucks.Sucker can comprise the independent compartment that is used for active compound (it has the pH that is suitable for storing), is used for another compartment of neutralization buffer agent, and is used for before being about to atomizing compound and neutralization buffer agent blended mechanism.In one embodiment, sucker is a metered-dose inhaler.

Being used for topical administration comprises dry powder inhaler (DPI), metered-dose inhaler (MDI) and spraying gun in three kinds of common system of lung airway.MDI uses in the most frequently used inhalation method, can be used for delivering solubilized form or as the medicine of dispersion.Typically, MDI comprises freonll-11 or other high relatively vapour pressure propelling agent, and it forces aerosolized medicine to enter respiratory tract when the device starting.Different with MDI, DPI relies on patient's FI that the medicine of dry powder form is imported in the lung usually fully.Spraying gun applies energy to liquor and forms for the pharmaceutical aerosol that sucks.Also the someone has inquired into use fluorochemicals medium and directly lung has been delivered medicine in liquid ventilation (liquid ventilation) or lung lavage (pulmonarylavage) process.These methods and other method can be used for delivering the stem cell form.United States Patent (USP) 6,732,732 and 6,766,799 have also put down in writing exemplary suction apparatus.

The form of the aerosol spray that described composition can be easily provides with compression packing or spraying gun is delivered, and wherein uses suitable propelling agent, for example Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas.Under the situation of pressurized aerosol, can carry a certain amount of valve to determine dose unit by providing.Can prepare and be used for capsule sucker or insufflator, that contain dried cellular forms and cartridge case.

Though dispensable, can use delivery toughener such as tensio-active agent further to strengthen lung and deliver." tensio-active agent " used herein is meant such compound, and it has hydrophilic segment and lipophilic portion, these parts by with two can not merge mutually between interfacial interaction promote the absorption of medicine.Tensio-active agent is useful for dried particle to be because several reasons for example, reduces particles coalesce, reduces macrophage phagocytic or the like.When with the Curosurf coupling, compound can absorb more efficiently, because tensio-active agent, DPPC for example can go far towards the diffusion of compound.Tensio-active agent is well known in the art, includes but not limited to phosphoglyceride, for example phosphatidylcholine, two palmityl L-α-phosphatidylcholines (DPPC) and diphosphatidylglycerol (DPPG); Hexadecanol; Lipid acid; Polyoxyethylene glycol (PEG); Polyoxyethylene-9; Lauryl (auryl ether); Palmitinic acid; Oleic acid; Sorbitanic trioleate (Span ^TM85); Glycocholate, surfactant protein (surfactin); Poloxamer (poloxomer); Sorbitan aliphatic ester; The sorbitanic trioleate; Tyloxapol (tyloxapol); And phosphatide.

In one aspect of the method, dried cellular forms can be prepared with such pharmaceutical acceptable carrier, and this pharmaceutical acceptable carrier has the granular size that can not suck, and makes that promptly this carrier can be be ingested size in the lung of any significant quantity.Said preparation can be the uniform mixture that is become with the larger particles (for example about 15 to 100 μ m) of carrier than small-particle (for example being less than 10 μ m) of dried cellular forms.When disperseing, smaller particles is inhaled in the lung thereupon, and bigger particle usually remains in the oral cavity.Be suitable for the blended carrier comprise have acceptable taste and on toxicology harmless crystallization or amorphous vehicle, comprise sucking or oral, for example sugar, disaccharides and polysaccharide.Representational example comprises lactose, mannitol, sucrose, Xylitol or the like.

For Orally administered, can use the acceptable vehicle of pharmacy such as tackiness agent (for example pre-gelatinization W-Gum, polyvinylpyrrolidone or Vltra tears); Weighting agent (for example lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (for example Magnesium Stearate, talcum or silica); Disintegrating agent (for example yam starch or Explotab); Or wetting agent (for example Sodium Lauryl Sulphate BP/USP), drug powder for example is mixed with the tablet or the capsule of preparation by conventional methods.Tablet can carry out dressing by means commonly known in the art.Being used for Orally administered liquid prepared product can take, for example, the form of solution, syrup or suspension, perhaps can be used as dryed product provides, and water or other suitable solvent are modulated before use.Such liquid prepared product can use pharmacy acceptable additive such as suspension agent (for example Sorbitol Powder syrup, derivatived cellulose or hydrogenation edible-fat), emulsifying agent (for example Yelkin TTS or gum arabic), non-aqueous solvent (for example Prunus amygdalus oil, oily ester, ethanol or classified vegetables oil) and sanitas (for example methyl p-hydroxybenzoate or propyl ester, or Sorbic Acid), prepared by conventional means.These prepared products also can optionally contain buffer reagent, salt, seasonings, tinting material and sweeting agent.

Described composition can be formulated as and is used for for example injecting or continuous infusion by injection, carries out parenteral administration.Activeconstituents can provide with form of powder, uses suitable solvent before use, and for example aseptic apirogen water is modulated.Injection preparation can provide with unit dosage form, for example is contained in ampoule or the multi-dose container with the sanitas that adds.Composition can be taked the form of suspension, solution or emulsion in oiliness or the aqueous vehicles, and can contain materials such as suspension agent, stablizer and/or dispersion agent.

Adjuvant

Vaccine of the present invention can be prepared with other immunomodulator.Particularly, vaccine composition can comprise one or more adjuvants.The adjuvant that can be used in the vaccine composition described herein includes, but are not limited to:

A. contain mineral composition

The mineral composition that contains that is suitable for use as adjuvant as herein described comprises mineral salt, such as aluminium salt and calcium salt.(for example also comprise mineral salt such as oxyhydroxide (for example oxyhydroxide), phosphoric acid salt (for example hydroxyl phosphate, orthophosphoric acid salt), vitriol or the like, see " Vaccine Design " the 8th and the 9th chapter, (1995) Powell and Newman compile, ISBN:030644867X.Plenum) or the mixture of the different minerals materialization compound (mixture of phosphoric acid salt and oxyhydroxide adjuvant for example, randomly, phosphoric acid salt is excessive), wherein compound is taked any suitable form (example gel, crystallization, amorphous or the like), is preferred to the absorption of salt.The composition that contains mineral substance can also be formulated as the particle of metal-salt (PCT publication No. WO00/23105).

Aluminium salt can be included in the composition as herein described, makes Al ³⁺Dosage be every dose 0.2 to 1.0mg.In one embodiment, the adjuvant based on aluminium that is used for the present composition is alum (potassium aluminium sulfate (AlK (SO ₄) ₂), perhaps alum derivative, for example by antigen is mixed in phosphate buffered saline buffer with alum, then with alkali for example ammonium hydroxide or sodium hydroxide carry out titration and precipitation, thereby generated in-situ alum derivative.

The adjuvant based on aluminium that another kind is used for vaccine composition of the present invention is aluminum hydroxide adjuvant (Al (OH) ₃) or crystalline hydroxy aluminum oxide (AlOOH), the latter is a kind of outstanding sorbent material, has about 500m ²The surface-area of/g.Perhaps, provide aluminum phosphate adjuvant (AlPO ₄) or Adju-Phos, they contain phosphate group, replace the some or all of oh groups of aluminum hydroxide adjuvant.Preferred aluminum phosphate adjuvant provided herein is unbodied, dissolves in acidity, alkalescence and central medium.

In another embodiment, the adjuvant that is used for the present composition comprise aluminum phosphate and aluminium hydroxide the two.In its more particular embodiment, the amount of adjuvant phosphoric acid aluminium is greater than the amount of aluminium hydroxide, and for example aluminum phosphate is 2: 1,3: 1,4: 1,5: 1,6: 1,7: 1,8: 1,9: 1 or greater than 9: 1 to the weight ratio of aluminium hydroxide.More specifically, the amount that aluminium salt exists can be each vaccine dose 0.4 to 1.0mg, or each vaccine dose 0.4 to 0.8mg, or each vaccine dose 0.5 to 0.7mg, or the about 0.6mg of each vaccine dose.

Usually,, make and carry the electric charge opposite, select preferably based on the adjuvant of aluminium with adjuvant at required pH antigen by the electrostatic attraction between the optimization molecule, the ratio of perhaps multiple adjuvant based on aluminium, for example aluminum phosphate is to the ratio of aluminium hydroxide.For example, at pH 7.4, aluminum phosphate adjuvant (iso-electric point=4) adsorbs N,O-Diacetylmuramidase and does not adsorb white protein.If target is a white protein, then select aluminum hydroxide adjuvant (iso-electric point=11.4).Perhaps, can reduce its iso-electric point, it be become be used for more alkaline antigenic preferred adjuvant with phosphoric acid pre-treatment aluminium hydroxide.

B. fat liquor

The oil emulsion compositions that is adapted at being used as in the described composition adjuvant comprises shark alkene-water miscible liquid.Particularly preferred adjuvant is the submicron O/w emulsion.Being preferred for submicron O/w emulsion of the present invention is the optional shark alkene-water miscible liquid that contains different amount MTP-PE, for example contains 4-5%w/v shark alkene, 0.25-1.0%w/v Tween ^TM80 (polyoxyethylene sorbitanic monoleate) and/or 0.25-1.0% Span ^TM85 (sorbitanic trioleates), and, randomly, N-acetyl muramyl-L-alanyl-D-isoglutamine base-L-L-Ala-2-(1 '-2 '-two palmityls-s-n-glycerine-3-hydroxyl phosphinylidyne oxygen)-ethamine [N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1 '-2 '-dipalmitoyl-s-n-glycero-3-huydroxyphosphophoryloxy)-and ethylamine] (MTP-PE) submicron O/w emulsion, for example, submicron O/w emulsion (the international publication WO90/14837 of " MF59 " by name; United States Patent (USP) 6,299,884 and 6,451,325, and Ott et al., " MF59--Design and Evaluationof a Safe and Potent Adjuvant for Human Vaccines " (Powell, M.F. and Newman in " Vaccine Design:TheSubunit and Adjuvant Approach ", M.J. compile) PlenumPress, New York, 1995, pp.277-296).MF59 contains 4-5%w/v shark alkene (for example 4.3%), 0.25-0.5%w/v Tween ^TM80, reach 0.5%w/v Span ^TM85, and the MTP-PE of optional difference amount, (Microfluidics, Newton Mass.) are mixed with submicron particles such as 110Y type Micro Fluid bed to use Micro Fluid bed (microfluidizer).For example, MTP-PE can exist with the amount of about 0-500. μ g/ agent, more preferably 0-250. μ g/ agent, most preferably 0-100 μ g/ agent.For example, " MF59-100 " every dose contains 100 μ g MTP-PE, or the like.MF69, another kind are used for submicron O/w emulsion of the present invention, contain 4.3%w/v shark alkene, 0.25%w/v Tween ^TM80, reach 0.75%w/v Span ^TM85, and optional MTP-PE.Another kind of submicron O/w emulsion is MF75, claims SAF again, contains 10% shark alkene, 0.4 %Tween ^TM80,5%Pluronic ^TMBlock polymer L121 and thr-MDP, same microfluid changes into the submicron emulsion.The MF75-MTP representative comprises MTP, for example the MF75 preparation of every dose of 100-400 μ gMTP-PE.

Be used for the submicron O/w emulsion of described composition, their preparation method and immunostimulant, for example muramylpeptides is documented in international publication WO90/14837 and United States Patent (USP) 6,299,884 and 6,451,325.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) also can be used as adjuvant in theme composition.

C. saponin(e preparation

The saponin(e preparation also can be used as adjuvant in described composition.Saponin(e is heterogeneous steroline of a group and triterpenes glucosides, in being present in bark, leaf, stem, the root of a plurality of plant species even spending.Obtained extensive studies from the isolating saponin(e of bark of Quillaia saponaria (Quillaia saponaria Molina) as adjuvant.Can also from chinaroot greenbrier (Smilax ornata) (sarsaprilla), circular cone Gypsophila acutifolia (Gypsophillapaniculata) (brides veil) and Saponaria officinalis (Saponaria officianalis) (Radix saponariae) industry prepare saponin(e.The saponin adjuvant preparation comprises the preparation of purifying, for example QS21, and lipid formulations, for example immunostimulating complex (ISCOM).

The astragalin composition that utilized efficient thin-layer chromatography (HP-TLC) and RPHPLC (reversed-phase high-performance liquid chromatography) (RP-HPLC) purifying.Use the fraction of these technology specificity purifying to obtain identifying, comprise QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C.Typically, saponin(e is QS21.United States Patent (USP) 5,057,540 disclose the method for a kind of QS21 of preparation.The saponin(e preparation can also comprise sterol, for example cholesterol (seeing that PCT announces WO96/33739).

Can use the combination of saponin(e and cholesterol to form unique particle, be called immunostimulating complex (ISCOM).ISCOM typically also comprises phosphatide, for example phosphatidylethanolamine or phosphatidylcholine.Any known saponin(e all can be used among the ISCOM.Preferably, ISCOM comprises one or more among Quil A, QHA and the QHC.In EP0109942, WO96/11711 and WO96/33739, further described ISCOM.Randomly, ISCOM can not contain other stain remover.See WO00/07621.

At Barr et al., among Advanced Drug Delivery Reviews (1998) 32:247-271 as seen based on the Summary of Development of the adjuvant of saponin(e.Other sees Sjolander et al., Advanced Drug DeliveryReviews (1998) 32:321-338.

D. virosome and virus-like particle (VLP)

Virosome and virus-like particle (VLP) also can be as the adjuvants of the present composition.These structures contain one or more protein from virus usually, optional and phosphatide combination or formulated together.They are non-virulent, nonreplicative normally, and does not contain any natural viral genome usually.Viral protein can be recombinated and be prepared or separate from intact virus.These are suitable for viral protein among virosome or the VLP and comprise and derive from influenza virus (for example HA or NA), hepatitis B virus (for example core or housing albumen), hepatitis E virus, Measles virus, sindbis virus (Sindbis virus), rotavirus, foot and mouth disease virus, retrovirus, norwalk virus (Norwalk virus), human papillomavirus, HIV, the RNA phage, Q phagus beta (for example coat protein), the GA phage, the fr phage, the AP205 phage, and the protein of Ty (for example retrotransposon Ty albumen p1).VLP is at WO03/024480, WO03/024481 and Niikura et al., Virology (2002) 293:273-280; Lenz et al., Journal of Immunology (2001) 5246-5355; Pinto et al., Journal of Infectious Diseases (2003) 188:327-338; And Gerber et al., Journal ofVirology (2001) 75 (10): further discussion is arranged among the 4752-4760.Virosome has further discussion at for example Gluck et al. among Vaccine (2002) 20:B10-B16.Trivalent INFLEXAL in nose ^TMProduct (Mischler ﹠amp; Metcalfe (2002) Vaccine 20 Suppl 5:B17-23) and INFLUVACPLUS ^TMUse immunostimulant reconstruction influenza virus body (IRIV) as the subunit antigen delivery system in the product.

E. bacterium or microorganism derivative

The adjuvant that is suitable in the present composition comprises bacterium or microorganism derivative, such as:

(1) non-toxic derivant of enterobacteria lipopolysaccharides (LPS)

Such derivative comprises monophosphoryl lipid A (MPL) and 3-O-deacylated tRNA base MPL (3dMPL).3dMPL is that the 3-with 4,5 or 6 acylations chains takes off-mixture of O-acyl group monophosphoryl lipid A.Disclosing 3-among the EP 0 689 454 takes off-a kind of preferred " small-particle " form of O-acyl group monophosphoryl lipid A.Such 3dMPL " small-particle " is small enough to the filter membrane (seeing EP 0,689 454) of 0.22 micron of aseptic filtration.Other nontoxic LPS derivative comprises the monophosphoryl lipid A stand-in, for example aminoalkyl glucosaminide phosphate (aminoalkyl glucosaminide phosphate) derivative, for example RC-529.Referring to Johnson et al. (1999) Bioorg.Med.Chem.Lett., 9:2273-2278.

(2) lipid A derivative

The lipid A derivative comprises the lipid A derivative from intestinal bacteria (Escherichia coli), such as OM-174.OM-174 is at for example Meraldi et al., Vaccine (2003) 21:2485-2491; With Pajak et al., on the books among Vaccine (2003) 21:836-842.

(3) immunostimulatory oligonucleotide

The immunostimulatory oligonucleotide that is suitable as adjuvant comprises and contains the CpG motif nucleotide sequence of (a kind of sequence contains non-methylated cytosine(Cyt), and thereafter with guanine, the two links to each other by the phosphoric acid ester bond).Containing the oligonucleotide of the palindrome or poly (dG) sequence or bacterium double-stranded RNA also has been proved to be and has had immunostimulating.

CpG can comprise that nucleotide modification/analogue modifies such as thiophosphatephosphorothioate, and can be double-stranded or strand.Randomly, guanine can be replaced by analogue such as 2 '-deoxidation-7-deazaguanine.Can be about the example that possible analogue replaces referring to Kandimalla et al., Nucleic AcidsResearch (2003) 31 (9): 2393-2400; WO02/26757 and WO99/62923.The adjuvant effect of CpG oligonucleotide is at Krieg, and Nature Medicine (2003) 9 (7): 831-835; McCluskie et al., FEMS Immunology and Medical Microbiology (2002) 32:179-185; WO98/40100; United States Patent (USP) 6,207,646; United States Patent (USP) 6,239,116 and United States Patent (USP) 6,429,199 in further discussion is arranged.

The CpG sequence can be at TLR9, such as motif GTCGTT or TTCGTT.See Kandimalla et al., Biochemical Society Transactions (2003) 31 (part 3): 654-658.The CpG sequence can be to inducing the Th1 immunne response specific, CpG-A ODN for example, and perhaps it can be to inducing the B cell response more specific, for example CpG-B ODN.At Blackwell et al., J.Immunol. (2003) 170 (8): 4061-4068; Krieg, TRENDS inImmunology (2002) 23 (2): CpG-A and CpG-BODN have been discussed among 64-65 and the WO01/95935.Typically, described CpG is CpG-A ODN.

Typically, the mode of CpG oligonucleotide structure makes that 5 ' end is that acceptor identification can reach.Randomly, two CpG oligonucleotide sequences can form " immunizator " (immunomers) in their 3 ' end lap.Referring to, for example, Kandimalla et al., BBRC (2003) 306:948-953; Kandimalla et al., Biochemical Society Transactions (2003) 31 (the 3rd part): 664-658; Bhagat et al., BBRC (2003) 300:853-861 and WO03/035836.

(4) ADP-ribosylation toxin and detoxification derivative thereof

Bacterium ADP-ribosylation toxin and detoxification derivative thereof can be used as adjuvant in described composition.Typically, described protein source is in intestinal bacteria (for example E.coli LT " LT "), cholera (" CT ") or Whooping cough (" PT ").WO95/17211 described the purposes of the ADP-ribosylation toxin of detoxification as mucosal adjuvants, and WO98/42375 has described its purposes as the parenteral adjuvant.Preferably, adjuvant is LT mutant such as LT-K63, LT-R72 and the LTR192G of detoxification.ADP-ribosylation toxin and detoxification derivative, especially LT-K63 and LT-R72 can see in following document as the purposes of adjuvant: Beignon et al., Infection and Immunity (2002) 70 (6): 3012-3019; Pizza et al., Vaccine (2001) 19:2534-2541; Pizza et al., Int.J.Med.Microbiol. (2000) 290 (4-5): 455-461; Scharton-Kersten et al., Infection andImmunity (2000) 68 (9): 5306-5313; Ryan et al., Infection and Immunity (1999) 67 (12): 6270-6280; Partidos et al., Immunol.Lett. (1999) 67 (3): 209-216; Peppoloni et al., Vaccines (2003) 2 (2): 285-293; With Pine et al., J.ControlRelease (2002) 85 (1-3): 263-270.The numbering benchmark of aminoacid replacement is typically according to Domenighini et al., and Mol.Microbiol (1995) 15 (6): the ADP-ribosylation toxin A subunit that provides among the 1165-1167 and the comparison of B subunit.

F. bioadhesive polymer and mucomembranous adhesion agent

Bioadhesive polymer (bioadhesives) and mucomembranous adhesion agent (mucoadhesives) also can be as the adjuvants in the theme composition.Suitable bioadhesive polymer comprises the cross-linked derivant of esterification hyaluronic acid microballoon (Singh et al. (2001) J.Cont.Rele.70:267-276) or mucomembranous adhesion agent such as polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polysaccharide and carboxymethyl cellulose.Chitosan and derivative thereof also can be used as adjuvant in described composition.Referring to for example WO99/27960.

G. particle

Microparticle (microparticles) and nano particle (nanoparticles) (for example polymer/nanoparticle) also can be used as adjuvant in described composition.Microparticle (typically is diameter～100nm to～150 μ m, for example diameter～200nm is to～30 μ m, or diameter～500nm is to the particle of～10 μ m) and nano particle (typically be～10nm is to the particle of～1000nm, for example diameter～10nm arrives～100nm, diameter～20nm arrives～500nm, or diameter～50nm to～300nm) can (for example poly-(alpha hydroxy acid), polyhydroxybutyrate, poe, poly-acid anhydrides, polycaprolactone or the like form with poly-(lactide-co-glycolide) by biodegradable and nontoxic material.Randomly, can handle particle, make them have electronegative surface (for example handling) or positively charged surface (for example handling such as CTAB) with cationic detergent with SDS.Can transform the particulate specificity, making them deliver drug concentrations to desired location increases.Referring to for example Matsumoto et al., Intl.J.Pharmaceutics, 185:93-101,1999; Williams et al., J.Controlled Release, 91:167-172,2003; Leroux et al., J.Controlled Release, 39:339-350,1996; Soppimath et al., J.Controlled Release, 70:1-20,2001; Chawla et al., Intl.J.Pharmaceutics, 249:127-138,2002; Brannon-Peppas, Intl.J.Pharmaceutics, 116,1-9,1995; Bodmeier et al., Intl.J.Pharmaceutics, 43:179-186,1988; Labhasetwar et al., Adv.Drug DeliveryReviews, 24:63-85,1997; Pinto-Alphandary et al., Intl.J.Antimicrobial Agents, 13:155-168,2000; Potineni et al., J.Controlled Release, 86:223-234,2003; Kostet al., Adv.Drug Delivery Reviews, 46:125-148,2001; With Saltzman et al., DrugDiscovery, 1:177-186,2002.

Particle, nano particle preferably can be assembled into and has an aggregate fixed structure, the big small scale of micron, and they have shell or the matrix that the mixture by lipotropy and/or hydrophilic molecule (normally medicinal " vehicle ") constitutes.Nano particle can form in method mentioned above, and can mix cellular material, and wherein cellular material serves as the particle body, is positioned on the particle surface or is wrapped in particle.The shell of aggregated particles or matrix can comprise pharmaceutical excipient such as lipid, amino acid, sugar, polymkeric substance, also can mix nucleic acid and/or peptide and/or protein and/or small molecules antigen.Can also use the combination of antigenicity substance.These aggregated particless can form in following method.

U.S. Patent application serial number 2004/0062718 has been described a kind of preparation method who is used as the porous nano particle aggregate particle (PNAP) of vaccine.Antigen can perhaps can mix in the shell of microparticle by constituting nano particle, being connected in nano grain surface or being wrapped in the nano particle and combining with nano particle, causes humoral immunization and cellular immunization then.Other exemplary PNAP preparation method is at Johnson and Prud ' homme, Austral.J.Chem., and 56:1021-1024, on the books in 2003.

These particles such as Edwards et al., Proc.Natl.Acad.Sci.USA, 19:12001-12005,2002 describedly assemble generation and (are called Troy/wooden horse particle by the larger particles that less subunit's particle is become like that, because they keep the peculiar property of their less subunit, also keep the key feature of larger particles simultaneously).Medicine can be wrapped in subunit's particle, perhaps is wrapped in by in the larger particles that constitutes than microaggregate.

Particle can be taked the dry powder form that is suitable for sucking.In a specific embodiment, particle can have and is less than about 0.4g/cm ³Tap density.This paper will have and be less than about 0.4g/cm ³The particle of tap density be called " aerodynamics light grains " (aerodynamically light particles).More preferably have and be less than about 0.1g/cm ³The particle of tap density.The preferred size of aerodynamics light grains, for example volume intermediate value geometric diameter (VMGD) is about at least 5 microns.In one embodiment, VMGD is about 5 microns to about 30 microns.In another embodiment, particle have about 9 microns to about 30 microns VMGD.In another embodiment, particulate median diameter, mass median diameter (MMD), mass median coating diameter (MMED) or mass median geometric diameter (MMGD) are at least 5 microns, for example about 5 microns to about 30 microns.The aerodynamics light grains preferably has about 1 micron and arrives about 5 microns " mass median aerodynamic diameter " (MMAD), claims again herein " aerodynamic diameter ".In one embodiment, MMAD is about 1 micron to about 3 microns.In another embodiment, MMAD is about 3 microns to about 5 microns.

In another embodiment, particle has less than about 0.4g/cm ³Coating mass density (envelope mass density), claim again herein " mass density ".Isotropy particulate coating mass density is defined as granular mass divided by the minimum spherical coating volume that particle can be wrapped into wherein.

Tap density can be used the instrument that those skilled in the art will know that, and for example two plane microprocessor control tap density testers (Dual Platform Microprocessor Controlled Tap DensityTester) (Vankel, N.C.) or Geopyc ^TM(Micrometrics Instrument Corp., Norcross Ga.30093) is measured instrument.Tap density is the gauge of coating mass density.Tap density can be used USP Bulk Density and Tapped Density, United States Pharmacopiaconvention, and Rockville, Md., 10th Supplement, 4950-4951,1999 method is determined.May facilitate the feature of low tap density to comprise irregular surface structure and vesicular structure.

The particulate diameter, their VMGD for example, can make electricity consumption district sensing instrument (electrical zonesensing instrument) such as Multisizer IIe (Coulter Electronic, Luton, Beds, England) or laser-diffractometer (Helos for example, Sympatec, Princeton, N.J. makes) measure.Other instrument that is used to measure particle diameter is well known in the art.The particulate diameter will depend on factors such as granulometric composition and synthetic method and difference in the sample.Can be selected the distribution of granular size in the sample, to realize the optimum deposition in the respiratory tract target site.

Can be being suitable for selection area to respiratory tract, such as the lung deep or go up air flue or middle air flue carries out local material, surfaceness, diameter and tap density of delivering and prepares particle.For example, last air flue delivered to use higher density or bigger particle, perhaps, can use and have particulate mixture identical or different therapeutical agent, not of uniform size in the sample, with the different zones of target lung in applied once.Aerodynamic diameter is about 3 to be preferred for centering air flue and last air flue to about 5 microns particle and to deliver.Aerodynamic diameter is about 1 to be preferred for the lung deep is delivered to about 3 microns particle.

Aerocolloidal inertial impaction and gravity settling are sedimentation mechanisms (Edwards, J.Aerosol Sci., 26:293-317,1995) main in eupnea condition downtake and the lung acinus.The importance of these two kinds of sedimentation mechanisms and aerosol quality but not increase pro rata with particle (or coating) volume.Because the position of aerosol deposition is by aerosol quality decision (being like this for the average air kinetic diameter greater than about 1 micron particle at least) in lung, reduce tap density by irregularity and the particle porosity that increases particle surface, can under the condition that all other physical parameters equate, allow in lung, to deliver bigger particle coating volume.

Maximum deposition in the lung can the computational aerodynamics diameter be provided, and this point is to utilize diameter less than about 5 microns in the past, and preferred about 2 realize that to about 3 microns nano sized particles these particles are engulfed subsequently.Selection has bigger diameter but the particle of enough light (feature that therefore has " light on the aerodynamics ") can realize equal lung delivery, and larger-size particle is not engulfed.With respect to particle, use particle can obtain better delivery with coarse or uneven surface with smooth surface.

Can prepare or separate suitable particle, for example by filtration or centrifugal, coming provides preselected size distribution for particulate samples.For example, can have at least about the diameter in 5 microns the selected scope more than about particle of 30%, 50%, 70% or 80% in the sample.The particle of certain percentage the selected scope that must fall into can be, for example, about 5 to about 30 microns, perhaps randomly, about 5 to about 15 microns.In a preferred embodiment, the particle of at least a portion has about 9 to about 11 microns diameter.Randomly, can also prepare such particulate samples, wherein at least about 90%, or randomly about 95% or about 99% diameter that has in the selected scope.The existence than large diameter air kinetics light grains of higher proportion in the particulate samples can strengthen the delivery to the lung deep of the therapeutical agent that wherein mixes or diagnostic reagent.The major diameter particle means the intermediate value geometric diameter usually at least about 5 microns particle.

The particle that preferred targeting antigen is delivery cell (" APC ") has the minimum diameter of 400nm, and this is the limit that APC engulfs.Be used to pass tissue transportation and targeted cells have 10nm for the preferred particulates of picked-up minimum diameter.Final preparation can form and be suitable for that lung is delivered and in the dry powder of ambient-temp-stable.

H. liposome

United States Patent (USP) 6,090,406, United States Patent (USP) 5,916,588 and EP 0 626 169 put down in writing the example of the Liposomal formulation that is suitable as adjuvant.

I. Soxylat A 25-7 and polyoxyethylene ester formulation

The adjuvant that is adapted at using in the composition comprises Soxylat A 25-7 and polyoxyethylene ester.Referring to for example WO99/52549.Such preparation can also comprise the combination (WO01/21207) of polyoxyethylene sorbitan esters tensio-active agent and octoxinol (octoxynol), and Voranol EP 2001 or ester surfactant and at least a other the combination (WO01/21152) of nonionic surface active agent such as octoxinol.Preferred Soxylat A 25-7 is selected from down group: polyoxyethylene-9-lauryl ether (laureth9), polyoxyethylene-9-stearyl (steoryl) ether, polyoxyethylene-8-stearyl (steoryl) ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether.

J. poly-phosphorus piperazine (PCPP)

Gather phosphorus piperazine (polyphosphazene) preparation at for example Andrianov et al., Biomaterials (1998) 19 (1-3): 109-115 and Payne et al., Adv.Drug.Delivery Review (1998) 31 (3): on the books among the 185-196.

K. muramylpeptides

The example that is suitable as the muramylpeptides of adjuvant comprises N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-positive muramyl-l-alanyl-d-isoglutamine (nor-MDP) and N-acetyl muramyl-l-alanyl-d-isoglutamine acyl-l-L-Ala-2-(1 '-2 '-two palmityls-s-n-glycerine-3-hydroxyl phosphinylidyne oxygen)-ethamine (MTP-PE).

L. imidazoquinolie compounds

The example that is adapted at being used as in the described compound imidazoquinoline (imidazoquinoline) compound of adjuvant comprises Imiquimod (Imiquimod) and analogue thereof, they are at Stanley, and Clin.Exp.Dermatol. (2002) 27 (7): 571-577; Jones, Curr.Opin.Investig.Drugs (2003) 4 (2): 214-218; With United States Patent (USP) 4,689, further describe in 338,5,389,640,5,268,376,4,929,624,5,266,575,5,352,784,5,494,916,5,482,936,5,346,905,5,395,937,5,238,944 and 5,525,612.

M. thiosemicarbazone compound

The example of thiosemicarbazone (thiosemicarbazone) compound, and preparation, make and screen all and be adapted in the described composition method as the compound of adjuvant, be included in those that put down in writing among the WO04/60308.Thiosemicarbazone is used for the stimulation human peripheral blood mononuclearcell to produce cytokine, and for example TNF-α is especially effective.

N. couroupitine A compound

The example of couroupitine A (tryptanthrin) compound, and preparation, make and screen all and be adapted in the described composition method as the compound of adjuvant, be included in those that put down in writing among the WO04/64759.The couroupitine A compound is used for the stimulation human peripheral blood mononuclearcell to produce cytokine, and for example TNF-α is especially effective.

O. people's immunomodulator

The people's immunomodulator that is adapted at being used as in the described composition adjuvant comprises cytokine, such as interleukin (for example IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), Interferon, rabbit (for example interferon-), macrophage colony stimulating factor, and tumour necrosis factor.

Described composition also can comprise the combined aspects of one or more above-mentioned adjuvants.For example, following adjunvant composition can be used for the present invention:

(1) saponin(e and O/w emulsion (WO99/11241);

(2) saponin(e (for example QS21)+nontoxic LPS derivative (for example 3dMPL) (seeing WO94/00153);

(3) saponin(e (for example QS21)+nontoxic LPS derivative (for example 3dMPL)+cholesterol;

(4) saponin(e (for example QS21)+3dMPL+IL-12 (optional+sterol) (WO98/57659);

(5) combination (seeing european patent application 0835318,0735898 and 0761231) of 3dMPL and for example QS21 and/or O/w emulsion;

(6) SAF contains 10% shark alkene, 0.4 %Tween ^TM80,5%Pluronic ^TMBlock polymer L121 and thr-MDP, perhaps microfluidization becomes the submicron emulsion, and perhaps the vortex vibration forms the bigger emulsion of particle size;

(7) Ribi ^TMAdjuvant system (RAS) (Ribi Immunochem) contains 2% shark alkene, 0.2%Tween ^TM80 and be selected from down one or more bacterial cell wall fractions of group: monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferred MPL+CWS (Detox ^TM);

(8) one or more mineral salts (for example aluminium salt)+nontoxic LPS derivative (for example 3dPML); With

(9) one or more mineral salts (for example aluminium salt)+immunostimulatory oligonucleotide (nucleotide sequence that for example comprises the CpG motif).

Aluminium salt and MF59 are the typical adjuvants that is used for the injectable vaccine.Bacteriotoxin and bioadhesive polymer are to be used for mucous membrane to deliver vaccine, use or suck the typical adjuvant of vaccine such as nose.Other adjuvant that can be used for mucosal vaccine is at for example Stevceva and Ferrari, Curr.Pharm.Des., and 11:801-11,2005 and Cox et al., Vet.Res., 37:511-39 discusses in 2006 to some extent.

Embodiment

Embodiment 1: the spraying drying of M. smegmatics suspension

Illustrate that for example the cells form that does not have vehicle is carried out spraying drying can be produced too moist and can't prepare or processed powders, uses M. smegmatics as the model microorganism.Use B ü chi

Mini spray moisture eliminator B-290 (B ü chi

Mini Spray Dryer B-290) (BrinkmannInstruments, Westbury NY) carry out spraying drying and form dry powder, wherein temperature in, flow velocity and excipient concentration are all controlled.

Under the condition that does not have vehicle with the microorganism spraying drying.With the solution of pure M. smegmatics at PBS-Tween

After the washing, be resuspended in the 90mL water, in 80 to obtain 3x10 ⁸The bacterial concentration of CFU/mL.Under the envrionment conditions of 19.5 ℃ and 48% humidity, with 130 ℃ of temperature ins, 50 ℃ of temperature outs, flow velocity 22mL/min with this M. smegmatics solution spray drying.Bacterial aggregate is assembled in the spray-dryer cylinder body, fails to spray from cyclonic separator with powder type.The material of collecting from spray-dryer is moist, almost can't process.

Embodiment 2: use leucine spraying drying M. smegmatics

The vehicle that relatively small amount is described for example can not produce successful exsiccant powder, use leucine as the model vehicle with the M. smegmatics spraying drying.Exsiccant solution is by the 4mg/mL leucine solution of 80% (by weight) and 20% 3x10 ⁹CFU/mL M. smegmatics suspension is formed 400mL solution.Solution online before being about to arrive nozzle (in line) mixes.Under the envrionment conditions of 20 ℃ and 69% humidity, with 150 ℃ of temperature ins, 60 ℃ of temperature outs, flow velocity 8mL/min with the solution spray drying.Average droplet size is estimated as the 50-60 micron.This process has produced product by the cyclonic separator of spray-dryer, but product is too moist, and yield is low.Obtained containing the micro-yellow powder (Fig. 3) of bacterium alive.But this powder conglomeration, and the flowing property of performance is relatively poor.

Embodiment 3: the leucine spraying drying M. smegmatics that uses higher concentration

The vehicle of higher concentration such as leucine, can produce good spray-dried powders, and higher excipient concentration can increase microbial activity.Once more by with 90% and 95% 4mg/mL leucine solution and 10% and 5% 3x10 ⁹CFU/mL M. smegmatics suspension has been mixed with the solution of 400ml.These solution also are on-line mixing before being about to arrive nozzle.Under the envrionment conditions of 20 ℃ and 69% humidity, with 150 ℃ of temperature ins, 55 ℃ of temperature outs, flow velocity 8mL/min with the solution spray drying.Average droplet size is estimated as the 50-60 micron.

Table 1 provides the result of spraying drying operation gained.In all cases, spraying drying has all obtained trickle, white, great-hearted powder, is suitable for aerosol dispersion, and efficiency of pcr product is higher.Vigor calculates with the colony forming single-digit on the 7H9 agar plate that contains Totomycin.With (leucine: powder M. smegmatics) was compared, and 95: 5 powder observation has arrived remarkable higher organism vigor (about 20-80 doubly) (Fig. 4), illustrate that the vehicle that adds in spray-drying process is to protecting the importance of microorganism in 90: 10.General appearance according to powder is estimated water content.Use thermogravimetry (TGA) in addition quantitative analysis of water content.Fig. 5 is a fluorescence micrograph of describing the M. smegmatics of expressing green fluorescent protein (GFP), and these M. smegmatics use 90: 10 leucines: M. smegmatics has carried out spraying drying.This Photomicrograph shows that only some contains fluorescigenic M. smegmatics (green) in the particles of powder.

Table 1. leucine spraying drying M. smegmatics

Leucine: M. smegmatics ratio	CFU goes into	CFU goes out	Quality is gone into (mg)	Quality goes out (mg)	The % vigor	The % efficiency of pcr product	Water content (1-is low, among the 2-, and the 3-height)
								95∶5	1.50x10 10	7.00x10 8	1016	562	8.4％	55.3％	1
90∶10	3.00x10 10	2.10x10 7	1682	556	0.2％	33.1％	1
								95∶5	1.50x10 10	7.00x10 8	1661	1651	4.7％	99.4％	2
90∶10	3.00x10 10	2.25x10 7	1682	903	0.1％	53.7％	2

Efficiency of pcr product in the table 1 with the quality in the end product than being calculated by the ratio of solute quality in the spray solution.The quality of end product comprises any residuary water in the powder.Usually, a part of quality adheres to drying plant and can't reclaim.

Embodiment 4: with mannitol spraying drying M. smegmatics

Can to come the spraying drying microorganism with other vehicle in order proving, to use the carbohydrate mannitol further to experimentize.Excipient solution is by 95% 10mg/mL mannitol solution and 5% 3x10 ⁹CFU/mL M. smegmatics suspension is formed, and liquor capacity is 200mL, generates by on-line mixing before being about to arrive nozzle.Under the envrionment conditions of 21.9 ℃ and 63% humidity, with 145 ℃ of temperature ins, 55 ℃ of temperature outs, flow velocity 12mL/min with the solution spray drying.Average droplet size is estimated as the 50-60 micron.Spraying drying has obtained trickle, white, great-hearted powder is suitable for aerosol dispersion, and efficiency of pcr product is 50%, contains bacterium alive.

Embodiment 5: the vigor of exsiccant M. smegmatics in storage

In order to measure the vigor of spray-dried M. smegmatics in storage process, as embodiment 3, carried out spraying drying, and the gained powder was stored for 1 to 2 week in 4 ℃, 25 ℃ and 40 ℃ in sealed vessel.Vigor is in the colony forming single-digit on the flat board.95: 5 leucines: the M. smegmatics powder has kept significant vigor after 4 ℃ or 25 ℃ stored for 1 weeks, but does not have remarkable vigor after 40 ℃ of storages.90: 10 leucines: the M. smegmatics powder has kept vigor at 4 ℃ after storing for 1 week, but at higher temperature debility then.Fig. 6 has shown 95: 5 leucines: the electron photomicrograph of M. smegmatics powder after 25 ℃ of 1 weeks of storage.

Embodiment 6: adopt the spray-dired simulation of cryoprotectant

In order to show that the mode that vehicle in the spray-drying process imports may be the important factor that vigor keeps, simulate in no cryoprotectant, cell and there is in isocyatic cryoprotectant and the cell cryoprotection agent concentration in the extracellular greater than the cellular material volume (Fig. 7) in the spray-drying process under these three kinds of different conditions of extracellular cryoprotection agent concentration with equation 36.Purpose is a kind of normal form of proof, according to this normal form, by in cell, the extracellular, or the inside and outside both sides of cell are introduced cryoprotectants (vehicle) membrane stress is minimized.

Mathematica is used in simulation

(Champaign IL) carries out program for Wolfram, Inc..To all three figure, initial cell radius (R ^c(0)) is set at 1 μ m, initial droplet radius (R ^d ₀) be set at 25 μ m, relative cell volume was mapped to the time.L _pBe set at 1.0 μ m/ (atm min); R _GasBe set at 0.08205745867258821 (atm L)/(K mol); T is set at 295.15K.Under all three kinds of situations, k=-(K _dLMTD)/(λ ρ ₁) (equation 33).Obtain LMTD for 65 ℃ by setting 500 ℃ of temperature ins, 200 ℃ of temperature outs, 20 ℃ of initial droplet temperatures and final droplet temperature.With these value input equations 30, obtain LMTD=((500 ℃-20 ℃)-(200 ℃-65 ℃))/(2.303*log ₁₀((500 ℃-20 ℃)/(200 ℃-65 ℃))).K _dBe set at 0.02kcal/ (m hr ℃); λ is set at 530kcal/kg; ρ ₁Be set at 1000kg/m ³Cell count (n _Cells) be set at 100, excluded volume (V _Excluded) be set at 0.46 times of original volume.

Be set at 10 ^-6

For the track among Fig. 7 (a), extracellular cryoprotection agent concentration is lower than intracellular cryoprotection agent concentration, the amount (x of extracellular salt ^e _s) be set at 0.26M and multiply by initial droplet volume (V ^d ₀=4/3 π (R ^d ₀) ³), the amount (x of cell inner salt ⁱ _s) be set at 0.26M and multiply by initial droplet volume, the amount (x of extracellular cryoprotectant ^e _Cp) be set at 0mol, the concentration (C of cryoprotectant in the cell ⁱ _Cp(0)) is set at 1M.Use these conditions, 0 to 0.105 second time is asked the value of equation 36, obtain track (a).

For the track among Fig. 7 (b), there is not cryoprotectant in extracellular or the cell, the amount (x of the amount of extracellular salt and cell inner salt ^e _sAnd x ⁱ _s) be set at 0.26M and multiply by initial droplet volume.Amount (the x of cryoprotectant in the cell ^e _Cp) and concentration (C ⁱ _Cp(0)) is set at 0mol and 0M respectively.Use these conditions, 0 to 0.105 second time is asked the value of equation 36, obtain track (b).

For the track among Fig. 7 (c), intracellular cryoprotection agent concentration equals extracellular cryoprotection agent concentration, the amount (x of the amount of extracellular salt and cell inner salt ^e _sAnd x ⁱ _s) be set at 0.26M and multiply by initial droplet volume.Intracellular cryoprotection agent concentration (C ⁱ _Cp(0)) and extracellular cryoprotection agent concentration be set at 1M, thereby the amount (x of extracellular cryoprotectant ^e _Cp) multiply by initial droplet volume for 1M.Use these conditions, 0 to 0.105 second time is asked the value of equation 36, obtain track (c).

These results show, depend on the method that imports the cryoprotectant vehicle, obtain the great volume skew of difference (volume excursion) (or membrane stress) distribution plan.Can develop the cells form spray drying process of the cell viability minimization of loss of sening as an envoy to from this understanding.

Embodiment 7: by the cell viability of minimum membrane osmotic stress optimization M. smegmatics

Illustrate for example how minimum membrane stress can improve the vigor of dried cellular, as in Example 3,4mg/mL leucine solution with 95% and 5% 3x10 ⁹CFU/mL M. smegmatics suspension mixes, and has prepared the solution of 400ml.But in this case, do not add glycerine to the M. smegmatics suspension.Equally, do not having glycerine, and using under the condition of the distilled water that uses among isotonic saline solution (0.9%NaCl) replacement all embodiment of front, the same solution spraying drying.Solution also is on-line mixing when being about to arrive nozzle.Under 20 ℃, the envrionment conditions of 69% humidity, solution is carried out spraying drying with 150 ℃ of temperature ins, 55 ℃ of temperature outs, flow velocity 8mL/min.Average particle size is estimated as the 50-60 micron.

Table 2. uses and does not use glycerine to 95: 5 (M. smegmatics/leucine) spraying dryings

Glycerine	CFU goes into	CFU goes out	Quality is gone into (mg)	Quality goes out (mg)	The % vigor	The % efficiency of pcr product
							Have	1.50x10 10	7.00x10 8	1016	562	8.4％	55.3％
Do not have	1.50x10 10	1.93x10 9	1520	830	24.1％	53.5％

Table 2 provides usefulness and without glycerine 95: 5 leucine/M. smegmatics mixtures has been carried out the result that spraying drying is operated gained.In all cases, spraying drying has all obtained trickle, white, great-hearted powder, is suitable for aerosol dispersion, and efficiency of pcr product is higher.Vigor calculates with the colony forming single-digit on the 7H9 agar plate that contains Totomycin.With (leucine: M. smegmatics) powder was compared, and did not use the powder observation of glycerine to arrive remarkable higher organism vigor in 95: 5 that use glycerine.Do not use glycerine and use 0.9% isotonic saline solution 95: 5 (leucines: when M. smegmatics) mixture carries out spraying drying; with respect to not using glycerine and not using 95: 5 (leucines: M. smegmatics) of salt; observe very low cell viability (Fig. 8), illustrate from being removed the importance of osmotic pressure active substance the spray-dired solution for protection microorganism in spray-drying process.

These results have proved conclusively the prediction of embodiment 6, and promptly in the drying process of cellular material suspension, the existence of cryoprotectant or salt may cell membrane cause significant stress, causes vigor to descend, and the latter can infer it is in the drying process due to the necrocytosis.

Embodiment 8: cell content increases in the high spray-dired powder of M. smegmatics vigor

Illustrate that for example thereby spray-dired cell keeps high vigor and can cause in the spray-dried powders free water content to reduce and cause cell content to raise, as in Example 7, by mixing 90%, 50%, 40%, 30%, 20% and 10% 4mg/mL leucine solution and 10%, 50%, 60%, 70%, 80% and 90% 3x10 ⁹CFU/mL M. smegmatics suspension one does not use glycerine and does not use salt, has prepared the solution of 400ml.Equally, before being about to arrive nozzle with the solution on-line mixing.Under 20 ℃, the envrionment conditions of 69% humidity, solution is carried out spraying drying with 150 ℃ of temperature ins, 55 ℃ of temperature outs, flow velocity 8mL/min.Average particle size is estimated as the 50-60 micron.

Fig. 9 shows the vigor result of spraying drying operation.The same with the embodiment of front, vigor descends with the reduction of excipient concentration, proves that high-caliber vehicle is essential for the good cell vigor.But different with the embodiment of front is, has obtained the good trickle dry powder of vigor with low 50% the excipient concentration of reaching.As if this shows, when the cell integrity is maintained, and/or when not using as glycerine when room temperature still keeps liquid additive, the vehicle of low concentration (being lower than 90%) may provide good result.Vigor calculates with the colony forming single-digit on the 7H9 agar plate that contains Totomycin, demonstration be the result of four replications of each ratio.

These results show, remove cryoprotectant, cause the cell viability under the excipient concentration condition that reduces to increase.

Embodiment 9: the preservation period stability that contains the spray-dried powders of M. smegmatics

Illustrating is not for example having under the refrigerated condition, cell viability can be kept for some time after drying, with among the embodiment 8 with 50: 50 and 95: 5 leucines: the powder of M. smegmatics preparation places under the batch condition of storage of 4 ℃, 25 ℃ and 40 ℃, and calculates vigor with the colony forming single-digit on the 7H9 agar plate that contains Totomycin.

Figure 10 and 11 has shown the vigor result as two kinds of powder of the function of time.Vigor has been kept the several months, and wherein preceding 3 vigor losses in the middle of the month are the most violent, and then vigor is stable in during longer.Be stored in 4 ℃ of powder under the condition and in 3 months, kept surpassing 1/10th former vigor.The powder that is stored in 25 ℃ of conditions kept be higher than for delivery optimum 10 ⁶The vigor of threshold value, and the powder that is stored in 40 ℃ of conditions is kept vigor and is reached 2 months.Vigor difference in time may be because due to the bacterial concentration difference, bacterial concentration influences the water content in the powder between 50: 50 powder and 95: 5 powder.

Embodiment 10: the stabilizing effect that uses monophogphoryl lipid A

Determined the effect of lipophilic substance monophogphoryl lipid A (MpLA) to the stability of spray-dired M. smegmatics.Experimentize to determine whether to be used as the oiliness shell means of the internal moisture in the retain bacteria, to increase its vigor on the longer time point.4g/ml leucine solution with 95% and 5% M. smegmatics suspension, and 0.25%MpLA, spraying drying M. smegmatics as described above.With the temperature out of 124 ℃ temperature ins and 45 ℃ to this solution spray drying.Envrionment temperature is 31.6 ℃ and 34% relative humidity.These conditions have obtained 66% quality yield.

Shown in Figure 12 A and 12B, to compare with the bacterium that does not have MpLA to handle, the bacterium of handling with MpLA is kept vigor in can be during 16 weeks.After reaching the storage in 1 year, still measure vigor.

Embodiment 11: the effect of kinds of surface promoting agent

Explanation can be used multiple dispersion agent to obtain above-mentioned result and not influence vigor for example, uses 0.05% tyloxapol (dispersion agent that uses in the previous embodiment) and 0.05% and 0.1%Pluronic ^TM-F68 has prepared the mycobacterium Smegmatis preparation of 95: 5 and 50: 50.These result of experiment are shown among Figure 13.Compare with the powder that uses the tyloxapol preparation, use these Pluronic ^TM-F68 has no significant effect the vigor of gained powder.

Embodiment 12: the preservation period stability that contains the spray-dried powders of Mycobacterium bovis BCG

The suitability of our conclusion to the vaccine organism is described for example, and we have carried out similar experiment with Mycobacterium bovis BCG.We use the method identical with embodiment 3; without salt or cryoprotectant; prepared 95: 5 leucines: the powder of Mycobacterium bovis BCG, and dried material is placed under the batch condition of storage of 4 ℃, 25 ℃ and 40 ℃, calculate vigor with the colony forming single-digit on the 7H9 agar plate.Figure 14 is shown to till 3 months the vigor result as two kinds of powder of the function of time.Be stored in 4 ℃ of powder under the condition and in 3 months storage process, kept their former vigor substantially.Be stored in 25 ℃ of powder under the condition and kept similar vigor, some losses were arranged in the time of 3 months.These vigor result is similar with the result of the M. smegmatics bacterium shown in 10 to Fig. 9.

Embodiment 13: the spraying drying of mammalian cell

The preparation of membrane permeation stress minimum can further be applicable to non-bacterial cell in order to prove leucine concentration height, and we test with the rat heart inoblast that NIH 3T3 fetal mice inoblast of cultivating and former generation gather.

We have prepared 3 kinds of preparations: we are with 4 milligrams of every ml distilled waters of leucine every milliliter of 100 ten thousand inoblasts that suspend, and wherein the volume/volume of leucine solution/cell solution ratio is 30/70,50/50 and 70/30.We with among the embodiment 3 to the used simulated condition of M. smegmatics with these preparation spraying dryings.

All experiments show that all the survival ability that rat heart inoblast that former generation gathers and NIH 3T3 fetal mice inoblast are stood spray-drying process about equally.Higher vigor when as if the leucine of higher concentration cause spraying drying; But, in view of fibroblastic cytolemma lower than the membrane rigidity of bacterium, the osmotic stress that osmotic pressure active substance (osmolytically active substances) produces in the pair cell is more responsive, has obtained higher vigor and lower clean osmotic stress by spraying drying cell in PBS (table 3) or " Tyrode " solution (table 4).Be suspended in cell and leucine among PBS or the Tyrode and as above spraying drying, wherein the volume/volume of leucine solution/cell solution ratio is 30/70,50/50 and 70/30.Under latter event, after spraying drying, reclaimed NIH 3T3 fetal mice inoblast alive, and after spraying drying, observed NIH 3T3 fetal mice inoblast alive in 1 month, as shown in Figure 15.

Table 3. phosphate buffered saline (PBS) (PBS) prescription

Component	Concentration (mg/L)
		Potassium primary phosphate	144
Sodium-chlor	9000
		Sodium phosphate dibasic	795

The outer ionogen solution formula of table 4.Tyrode mammalian cell

Component	Concentration (mg/L)
		Calcium chloride	265
D-glucose	901
		HEPES	1192
Magnesium chloride	203
		Repone K	403
Sodium-chlor	7889
		Sodium phosphate	40

After the spraying drying, reclaim the rat fibroblast and the bed board of NIT 3T3 fetal mice inoblast alive and the collection of former generation from 70/30,50/50 and 30/70 preparation.Figure 16 and 17 shows after the spraying dryings bed board cell of the 3rd day and the 8th day.These figure show that higher excipient concentration (leucine concentration) produces higher viable count when drying.

Other embodiment

Several embodiments of the present invention have been described.But, should be appreciated that in without departing from the spirit and scope of the present invention, can make various modifications.Therefore, other embodiment falls into the scope of claims.

Claims (40)

1. dry powder, comprise be less than about 10% water, cellular material and with dry weight basis at least about 25% vehicle.

2. the dry powder of claim 1, wherein said powder does not comprise the salt or the cryoprotectant of significant quantity.

3. the dry powder of claim 1, wherein said cellular material comprise that bacterium, virus, eukaryotic microorganisms, mammalian cell, film are in conjunction with organoid, liposome, based on the bio-reactor of film or based on the drug delivery system of film.

4. the dry powder of claim 3, wherein said cellular material comprises bacterium.

5. the dry powder of claim 4, it is alive wherein surpassing 1% bacterium.

6. the dry powder of claim 4, wherein said bacterium is mycobacterium tuberculosis or M. smegmatics bacterium.

7. the dry powder of claim 4, wherein said bacterium is bacille Calmette-Guerin vaccine (BCG) bacterium.

8. the dry powder of claim 3, wherein said cellular material comprises mammalian cell.

9. the dry powder of claim 8, wherein said mammalian cell comprises red corpuscle, stem cell, granulocyte, inoblast or thrombocyte.

10. the dry powder of claim 1, wherein said cellular material comprises viable cell.

11. each dry powder of claim 1-10, wherein said vehicle comprises leucine, mannitol, trehalose, dextran, lactose, sucrose, Sorbitol Powder, white protein, glycerine, ethanol or their mixture.

12. the method for a pharmaceutical compositions comprises:

Produce claim 1-11 each dry powder and

Described dry powder is formulated in the pharmaceutical composition.

13. the method for claim 11, wherein said pharmaceutical composition are formulated as for sucking and use.

14. a generation comprises the method for the dry powder of cellular material, this method comprises:

The aqueous solution is provided, and this aqueous solution comprises the vehicle and at least 10 of 1mg/ml at least ⁵The cellular material of unit/ml; With

Under the condition that produces the moisture dry powder that comprises described cellular material that is less than about 10% weight, the described solution of spraying drying.

15. the method for claim 14, wherein said cellular material comprise that bacterium, virus, eukaryotic microorganisms, mammalian cell, film are in conjunction with organoid, liposome, based on the bio-reactor of film or based on the drug delivery system of film.

16. the method for claim 15, wherein said cellular material comprises bacterium.

17. the method for claim 16, wherein said bacterium are mycobacterium tuberculosis or M. smegmatics bacterium.

18. the method for claim 16, wherein said bacterium are bacille Calmette-Guerin vaccine (BCG) bacteriums.

19. the method for claim 14, wherein said cellular material comprises mammalian cell.

20. the method for claim 19, wherein said mammalian cell comprises red corpuscle, stem cell, granulocyte, inoblast or thrombocyte.

21. each method of claim 14-20, wherein said vehicle comprises leucine, mannitol, trehalose, dextran, lactose, sucrose, Sorbitol Powder, white protein, glycerine, ethanol or their mixture.

22. each method of claim 14-21 also comprises described dry powder is formulated in the pharmaceutical composition.

23. dry powder that produces by each method of claim 14-22.

24. the method for a spraying drying cellular material, described method comprises:

Acquisition wait the to spray radius initial value (R of cellular material unit of drying ^c(0));

Determine the time of drying of prediction;

Select following value:

(i) the entrance and exit gas temperature differential of spray-dryer (Δ T);

(ii) average droplet size (R ^d);

The (iii) gasification latent heat of solvent (λ);

(iv) the film of cellular material is for the hydraulic diffusibility (L of cryoprotectant _p);

(v) mole number (the x of extracellular solute ^e _s);

(vi) mole number (the x of solute in the cell ⁱ _s);

(vii) mole number (the x of extracellular cryoprotectant ^e _Cp);

(viii) concentration (C in the initial cell of cryoprotectant ⁱ _Cp(0)); With

(ix) cell count (n _Cells);

Obtain equation 36 with these values

$-\frac{1}{L_p{R}_{\mathrm{gas}}T}\frac{d{R}^c\left(t\right)}{\mathrm{dt}}=\frac{x_s^e}{\frac{4}{3}\mathrm{\&pi;}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3]}-\frac{x_s^i}{\frac{4}{3}\mathrm{\&pi;}{R}^c{\left(t\right)}^3-V_{\mathrm{excluded}}}$

$+\mathrm{\&sigma;}[\frac{x_{\mathrm{cp}}^e}{\frac{4}{3}\mathrm{\&pi;}[{(\mathrm{kt}+R_o^{d^2})}^{3/2}-n_{\mathrm{cells}}{\left(R^c\left(t\right)\right)}^3]}-C_{\mathrm{cp}}^i\left(0\right)]2\underset{n=1}{\overset{\&infin;}{\mathrm{\&Sigma;}}}\frac{{\sin }^2\left({\mathrm{\&lambda;}}_n\right)-{\mathrm{\&lambda;}}_n\sin \left({\mathrm{\&lambda;}}_n\right)\cos \left({\mathrm{\&lambda;}}_n\right)}{{{\mathrm{\&lambda;}}_n}^2-{\mathrm{\&lambda;}}_n\sin \left({\mathrm{\&lambda;}}_n\right)\cos \left({\mathrm{\&lambda;}}_n\right)}{e}^{-{{\mathrm{\&lambda;}}_n}^2\stackrel{\&OverBar;}{D_{\mathrm{cp}}^{*}}t/R^c{\left(t\right)}^2}---\left(36\right)$

Value;

And

If R ^c(t) in the time of drying of prediction, maintain within minimum limit and the greatest limit, then use the described cellular material of condition spraying drying of selected value.

25. the method for claim 24 wherein selects described value to make that the damage of pair cell material minimizes in the drying process.

26. the method for claim 24 or 25, wherein said cellular material comprise that bacterium, virus, eukaryotic microorganisms, mammalian cell, film are in conjunction with organoid, liposome, based on the bio-reactor of film or based on the drug delivery system of film.

27. the method for claim 26, wherein said cellular material comprises bacterium.

28. the method for claim 27, wherein said bacterium are mycobacterium tuberculosis or M. smegmatics bacterium.

29. the method for claim 27, wherein said bacterium are bacille Calmette-Guerin vaccine (BCG) bacteriums.

30. the method for claim 26, wherein said cellular material comprises mammalian cell.

31. the method for claim 30, wherein said mammalian cell comprises red corpuscle, stem cell, granulocyte, inoblast or thrombocyte.

32. also being included in soon, each method of claim 24-31, wherein said method add cryoprotectant to cell before the described cellular material of spraying drying.

33. the method for claim 32, wherein said cryoprotectant adds within the cell.

34. the method for claim 32, wherein said cryoprotectant adds to outside the cell.

35. the dry powder that produces by each method of claim 25-34.

36. each method of claim 25-34 also comprises spray-dried cellular material is formulated in the pharmaceutical composition.

37. one kind produces the moisture method that is less than the dry powder that comprises mycobacterium species of about 10% weight, described method comprises:

The aqueous solution is provided, and this aqueous solution comprises the vehicle and at least 10 of 1mg/ml at least ⁵The mycobacterium species of colony-forming unit/ml; With

Under the condition that produces the moisture dry powder that comprises mycobacterium species that is less than about 10% weight, the described solution of spraying drying.

38. the method for claim 36, the wherein said aqueous solution do not contain the salt or the cryoprotectant of interpolation.

39. pass through the dry powder that the method for claim 36 produces.

40. the method for claim 37 or 38 also comprises described dry powder is formulated in the pharmaceutical composition.

Images