CN1732257A - Immunogenic composition - Google Patents

Abstract

The present invention relates to a method of drying biological and other labile samples so that they can be preserved as a highly viscous liquid. The method involves the steps of preparing a preservation sample by dissolving/suspending an active agent in a solution of a stabilising agent, subjecting the preservation sample to such temperature and pressure conditions that the preservation sample looses solvent by evaporation without freezing or bubbling to form a foam and removing solvent until the preservation sample dries to form a highly viscous liquid.

Description

Drying process

The present invention relates to the preservation of biological sample and other Unstable Sample, the sample of so preserving and the novel process of preserving described sample.Described novel process comprises that the sample that will comprise promoting agent and stablizer adds in the container, makes described sample stand certain temperature and pressure condition so that by evaporation forfeiture solvent, and does not need freezing sample or sample is bubbled to form foam.Next,, keep or adjust described pressure and temperature condition at second drying stage, thus remove desolvate and dry described preservation sample to form the liquid of high viscosity.The present invention further provides the composition of preserving by technology of the present invention, especially vaccine composition through preserving.

Unstable Sample, especially biological sample need prolong its stability and prolong its storage life thus.Adopt freeze-dry process to achieve the above object traditionally, in this technology, prepare the solution of described material, and freezing described sample.At elementary drying stage, under reduced pressure, remove most of moisture content in the deicing by distillation, form porous " cake ".Usually carry out second drying stage after, change pressure and temperature this moment, and moisture content is evaporated from described solid " cake ".Compare with liquid preparation, resulting freeze-drying stability of sample has improved.Yet described freeze-dry process is very consuming time and expensive, forms to be the rate-limiting step in the production process.

Freeze-drying can also cause active or antigenic forfeiture of some promoting agent.For the particular organisms material, live virus for example, its activity may significantly be lost (Pikal (1994) ACS Symposium 567:120-133) in described freeze-dry process.At ambient temperature, manyly remain unsettled (Carpenter etc. (1994) ACS Symposium567:134-147) through freeze dried material.

Adopt stablizer, for example polyol can be avoided the infringement that caused by refrigerating process to a certain extent.In technology, avoid freezing sample and remove moisture content (WO96/40077 by boiling; US6306345), can make further improvement to described freeze-dry process.This method relates to the mixture that the glass matrix of preparation in appropriate solvent forms material and wait to preserve sample, from mixture, evaporate most of solvent to obtain slurries, described slurries are exposed to be enough to make under the described slurries ebullient pressure and temperature, removes remaining solvent.Be similar to above-mentioned method and can be called as the foam mat drying method.In " boiling " stage, the formation by bubble and breaking, the sample that described method will be to be preserved is exposed under the stress.Especially under the situation of preserving unsettled material, it may cause active forfeiture.

US5 has described similar method in 766,520, and wherein said technology relates to partly removes moisture content to form viscous fluid, further described slurries is made its " boiling " through vacuum-treat, carries out further drying being lower than substantially under 100 ℃ the temperature.This method still has some problem of conventional freeze-dry process.When described technology is carried out in big Freeze Drying Equipment, sample according to its on the top of the shelf different positions and with different speed dryings, this causes the activity of the different amounts of different sample losses in described drying process.It causes being lack of consistency in same batch.

Because it has the performance of stabilization, trehalose is a kind of polyol of the people's of being subjected to favor.Trehalose is naturally occurring, inert, irreducibility and disaccharide avirulent, that can form glassy mass, and the drying damage in finding it at first and preventing certain plants and animal is relevant.Trehalose helps to prevent big quantity of material in drying process and memory phase afterwards, comprise the sex change of protein, virus and food, some reasons are that trehalose has higher relatively glass transition temp (being about 120 ℃ under anhydrous state) (US4891319; US5149653; US5026566).Trehalose also makes enzyme remain stable (Argall and Smith (1993) Biochem.Mol.Bio.Int.30; 491).Find that also trehalose and a large amount of stabilization polyols help to improve the preservation of freeze-drying sample, especially are easy under the situation of loss of activity at described sample in freeze-dry process.Other carbohydrate useful in freeze-dry process comprises sucrose and lactose.

The present invention relates to a kind of method of improved preservation promoting agent, if especially described promoting agent is unsettled and is easy to the situation of loss of activity in more conventional drying process.Described technology may further comprise the steps, and promoting agent is dissolved/be suspended in to prepare the preservation sample in the stabiliser solution; Described preservation sample is positioned under certain temperature and the pressure condition, removes the solvent of described preservation sample, and need not make sample freezing or bubble to form foam by evaporation; Remove and desolvate, form the liquid of high viscosity until described preservation sample drying.

Described technology is very gentle, and promoting agent and without freezing or boiling, and therefore freeze-drying and the foam mat drying method than routine is more favourable, and in described freeze-drying and foam mat drying method, sample need be exposed in described one or two stress.When promoting agent to be preserved is unstable, adopt method of the present invention to increase active and/or antigenic shelf time.It can followingly be measured: the exsiccant promoting agent is restored again, described solvent is preferably the water or the aqueous solution, measure its activity or antigenicity by standard detecting method (for example passing through ELISA), and with described result and undried sample or by freeze-drying or foam mat drying method drying then the resulting result of restorative sample compare.

It is particularly advantageous adopting the dry IPV of technology of the present invention (poliovirus of deactivation, it is the immunogen in the injectable Poliomyelitis Vaccine).IPV is present in the known vaccine as liquid preparation (WO99/48525).Because standard lyophilization technology causes the forfeiture of IPV antigenicity, therefore attempt in vaccine, to adopt solid IPV preparation to have certain problem.Part is because the needed time of technology of the present invention has shortened, and therefore technology of the present invention has increased the antigenic confining force of poliovirus greatly.

Technology of the present invention is more favourable than common freeze-dry process, and this is because its cycle of operation is shorter, refrigerated is required lower, and makes it that efficiency more be arranged.Because described drying process is generally the rate-limiting step in the technology, so adopt method of the present invention when reducing expense, to improve production level.

Accompanying drawing is described

Fig. 1 is for being arranged in the photo of the high viscosity liquid of being inverted bottle (inverted vial).

Describe in detail

Method of the present invention is used for preserving activating agent, and it may further comprise the steps:

-activating agent suspended or be dissolved in the stabiliser solution preserve sample with preparation;

-make described preservation sample stand certain temperature and pressure condition, described temperature and pressure makes this preservation sample by evaporation forfeiture solvent, and does not carry out freezing or bubble to form foam, thereby forms viscous liquid;

And randomly further may further comprise the steps:

-remove and desolvate, until the dry high viscosity liquid that forms of described viscous liquid.

A kind of method of preserving promoting agent is produced the promoting agent form that can prolong storage time, and, keep activity and/or the antigenicity and/or the immunogenicity of described promoting agent at memory period.Preferably, described promoting agent can keep at least 40%, 50%, 60%, 70%, preferred 80%, 90%, 95% of its original activity 4 ℃ of storages at least 3,6,9,12,24 months the time.Can measure antigenicity or immunogenicity by standard detecting method described below.

This method is particularly suitable for prolonging the storage life of unstable product, and described unstable product is in being stored in solution or take place freezing or when bubble forming foam, soon loss of activity.

Unstable product is stored in solution and/or freezing and/or stand stress, for example in the foam forming process after the relevant blistered stress, is easy to loss of activity and/or forfeiture antigenicity and/or forfeiture immunogenicity.

It is specially adapted to following situation, and the low concentration (for example 3%-15%w/v) that promptly forms the polyol of glass is favourable, and short drying process (being less than 4,6,8,10 or 12 hours) of preferred time.

Viscous liquid is defined as the product that removes the starting stage of desolvating, and when the described starting stage finishes, removes most of solvent from sample.Can identify this point,, make that the temperature of sample is back to envrionment temperature because the endothermic effect of a large amount of evaporations disappears because the speed of evaporation is slack-off.

After starting stage, another section obtained the liquid of high viscosity after the period under the viscous liquid that obtains when initial stage of drying is finished was exposed to and reduces pressure at exsiccant.The solvent of high viscosity liquid is less than or equal to 15,12,10,8,5,4,3,2 or 1% (w/w), and preferably (Eur.J.Pharm.Biopharm. (2000) 50 by Karl Fischer coulomb moisture analyser; 277-284) measure.The preferable range of solvent is 1-3%, 3-5%, 5-10% or 10-15% (w/w).Described high viscosity liquid has enough low solvent, thereby described promoting agent is in 4 ℃ of preservations at least 3,6,9,12 or still be in steady state 24 months the time, allow that described promoting agent keeps at least 40%, 50%, 60% in this time, preferred 70%, 80%, 90%, 95% activity and/or antigenicity and/or immunogenicity.Preferably, described high viscosity liquid has solid appearance, but be rubber-like or glassy, is preferably glassyly, and they can be at 2,4 or 6 days, preferred 1,2,3 or 4 weeks, more preferably 2,4,6,8,10 or 12 months in the utmost point mobile lentamente.The container that contains described high viscosity liquid can be inverted and be positioned under the room temperature until observing flowing of described high viscosity liquid, thereby measure described extremely slow flowing.In preferred embodiments, at 2,4 or 6 days, preferred 1,2,3 or 4 weeks, more preferably 2,4,6,8,10 or 12 months after, the liquid that is positioned at the described high viscosity of upside down position does not show mobile.Preferably, the liquid of described high viscosity has limpid, transparent outward appearance.

Preserve the preparation of sample

Any stablizer all is suitable for first step of the present invention.Suitable material includes but not limited to all many alkyl compounds, comprises hydro carbons and non-hydrocarbons polyol.Preferably, the many alkyl compounds of described stabilization can make promoting agent not be stored by sex change, gathering or alternate manner loss of activity basically.Particularly suitable material comprises carbohydrate, sugar alcohol and hydrocarbon derivative.Preferably, many alkyl compounds of described formation glassy mass are the hydro carbons or derivatives thereof, comprise glucose, maltulose (maltulose), Palatinose, lactulose, sucrose, maltose, lactose, isomaltose, maltose alcohol, Saccharum lactis, palatinit, trehalose, raffinose, stachyose, melizitose or dextran, more preferably trehalose, sucrose, sorbyl alcohol, raffinose, N.F,USP MANNITOL, lactose, Saccharum lactis or palatinit most preferably are sucrose, sorbyl alcohol, lactose or trehalose.

Bacterial polysaccharides is particularly advantageously as the stablizer in the immune composition, because it can be simultaneously as stablizer and immunogen.

Hydro carbons includes but not limited to polyol, hydroxyethylamyle and the carbohydrate multipolymer of the hydro carbons of monose, disaccharide, trisaccharide, oligosaccharides and corresponding sugar alcohol, for example hydrocarbon derivative and chemical modification.Natural and synthetic hydro carbons all is fit to adopt.Synthin includes but not limited to that wherein glycosidic link is by the hydro carbons of mercaptan key (thiol bond) or carbon bond replacement.Can adopt the hydro carbons of D and L shaped formula.Described hydro carbons can be irreducibility or reductibility.When adopting the reductibility hydro carbons, preferably add the inhibitor of Maillard reaction.

Being suitable for reductibility hydro carbons of the present invention is hydro carbons known in the art, includes but not limited to glucose, maltose, lactose, fructose, semi-lactosi, seminose, maltulose and lactulose.The irreducibility hydro carbons includes but not limited to, is selected from the irreducibility glucosides of the polyol of sugar alcohol and other straight chain polyvalent alcohol.Other useful hydro carbons comprises gossypose, stachyose, melizitose, dextran, sucrose, cellobiose, mannobiose and sugar alcohol.Described sugar alcohol glucosides is preferably monoglycosides, especially by the reduction disaccharides, and for example lactose, maltose, lactulose and maltulose and the compound that obtains.

Particularly preferred hydro carbons is trehalose, sucrose, sorbyl alcohol, maltose alcohol, Saccharum lactis, palatinit and glucopyranosyl-1 → 6-N.F,USP MANNITOL.

Amino acid can be used as stablizer and can self use, and also preferred and polyol is used in combination.Preferred amino acids comprises glycine, L-Ala, arginine, Methionin and glutamine, although any amino acid or amino acid, peptide, protolysate or for example sero-abluminous proteinic combination all can be used as stablizer.

The concentration of the stablizer that adopts in the technology of the present invention can be 1% to 50% weight/volume, and preferred 1-5%, 5-10%, 15-20%, 20-25% or 25-50% most preferably are less than or equal to 15% or 10% (w/v).The amount of needed stablizer and the amount of existing salt are proportional.Therefore,, may need 10% to 25% greater concn, come drying to have the high content of salt sample of (being higher than 100mM, 200mM, 300mM, 400mM or 500mM) although stabiliser content is preferably 2% to 10%.

Preferably, described preservation sample contains and can suppress high viscosity liquid formation crystalline component of the present invention.Salt and comprise amino acid and phenol red other molecules in inhibiting crystalline forms.

Container

Can handle different mixtures and various container shapes and size simultaneously.Ideally, employed container size is large enough to hold original mixture and formed its solid volume.Usually, it is by the surface-area and the decision of glassy formation condition of the glassy quality that forms material, container.The quality of described glassy formation material must enough produce the actual minimum mass on the per unit area vessel surface of being converted into.Mixture that this ratio value is different and used container and change, but those skilled in the art can rule of thumb determine described ratio easily according to step as herein described.Can use any above-mentioned bottle, comprise that Wheaton is molded and cut the bottle of pipe.

Optimal process of the present invention adopts has anti-solvent, the container of preferred waterproof internal surface.By applying hydrophobic composition, for example achieve the above object by siliconization at inner surface of container.By well known to a person skilled in the art that technology realizes siliconization.In a method, the inside with silicone emulsion rinsing container at high temperature is generally 350 ℃ then, handles by baking oven, thereby container is carried out siliconization.Alternatively, make container, to obtain the waterproof internal surface with water-proofing composition.

The drying products that the waterproof internal surface of container obtains this technology is easier to restore, because assemble moisture content still less on the container side wall.

Although can adopt singulative herein, can exist more than a kind of glassy matrices formation material, more than a kind of additive and more than a kind of material.Those skilled in the art are easy to determine the significant quantity of these components.

Solution

The solvent that stablizer and promoting agent are dissolved in wherein can be aqueous, organic or its mixture.The water-containing solvent that can adopt q.s with the organic solvent that dissolves described glassy matrices and form material and adopt q.s with the solubilizing hydrophobic material, thereby form the hydrophobic substance that is mixed with glassy mass.

Choice of Solvent depends on the character of the vitrophyric material of selected formation, and any additive and/or the Substance Properties that will mix.The character of described solvent and q.s volume should make glassy matrices form material and additive and/or material solubilising fully arbitrarily.If described material is a water wetted material, then described liquid is preferably aqueous, to avoid owing to disadvantageous solvent phase mutual effect causes any possible loss of activity.Preferably, described water-containing solvent comprises any suitable water-containing solvent well known in the art, and it includes but not limited to water and biological buffered soln.Preferably, the volume percent of described water-containing solvent is 5% to 98%, and more preferably 80-98% most preferably is 85-98%.

The volume of solvent can change, and it depends on that glassy matrices forms material and will the blended material and additive arbitrarily.Needed minimum volume is the required amount of the various components of dissolving.Yet, can also adopt the suspension of homodisperse described material.The appropriate amount of each component is easy to be determined according to embodiment provided herein by those skilled in the art in the particular.

Different additive can be added in the described preservation sample.Preferred additives is the Maillard reaction suppressor.Preferably, contain carbonyl and amino, imino-or guanidine radicals if described material and/or glassy matrices form material, then described composition further contain at least a physiologically acceptable, prevent the Maillard reaction suppressor of the significant quantity of amino group and reactive carbonyl group condensation in the composition substantially.The Maillard reaction suppressor is well known in the art.The amount of described inhibitor should be enough to prevent or prevent substantially the condensation of amino group and reactive carbonyl group.General described amino group is present on the described material, and carbonyl group is present in described glassy matrices and forms on the material, or opposite.Yet described amino acid and carbonyl group can be at the intramolecularly of described material or hydro carbons.

Known different types of compound has the effect that suppresses the Maillard reaction, therefore can be used for composition as herein described.These compounds are generally the competitiveness or the noncompetitive inhibitor of Maillard reaction.Competitive inhibitor includes but not limited to amino-acid residue (D and L), the composition of amino-acid residue and peptide.Particularly preferably be Methionin, arginine, Histidine and tryptophane.Methionin and arginine are for the most effective.There are many known noncompetitive inhibitors.Include but not limited to aminoguanidine and derivative thereof and amphotericin B.Also described suitable Maillard inhibitor among the EP-A-0433679, it comprises 4-hydroxyl-5,8-dioxo quinoline.

It is favourable mixing illuminating colour in the preservation sample, its objective is to make through the inventive method institute exsiccant product to be easy to more observe.This is even more important in recuperation, thereby guarantees before using, and the liquid of high viscosity is thoroughly reduced.Preferably, described illuminating colour keeps its color when neutral pH, and is fit to be injected in the patient body.Most preferred illuminating colour is phenol red.

By evaporation forfeiture solvent (evaporation drying-step b)

Technology of the present invention relates to the preservation sample is stood certain pressure and temperature condition, thereby makes described preservation sample lose solvent by evaporation, and does not carry out freezing to sample or bubble to form foam.

Because the temperature in the endothermic nature of described evaporation technology, described preservation sample often is different from the outside temperature of described sample.Mentioned temperature is the condition in the described preservation sample external world, for example, under the situation that adopts big industrial Freeze Drying Equipment, is meant the temperature of shelf.It is usually corresponding to the temperature setting of Freeze Drying Equipment.

Randomly, have in the method for the invention preserving the initial step that sample outgases.Before pressure is further reduced, pressure is reduced to or is lower than 200mBar, preferred 200 to 35mBar, and continues at least 5 minutes.

The preferred embodiments of the invention realize evaporation drying by reducing pressure and controlled temperature condition.Described pressure is adjusted to or is lower than 30,25,20, and is preferred 15,12, and most preferably 10,8,7,6,5,4,3,2 or 1mbar, keep temperature is arranged on more than 0 ℃ preferred 5 ℃ to 37 ℃, 4 ℃ to 10 ℃, 10 ℃ to 15 ℃ simultaneously; 15 ℃ to 20 ℃; 20 ℃ to 25 ℃; 25 ℃ to 30 ℃; 30 ℃ to 37 ℃ or 37 ℃ to 45 ℃.These conditions were kept 1,2,3,4,5,8,10,12,16 or 24 hour preferred 2-4 hour, 4-6 hour, 6-8 hour, 8-12 hour or 12-18 hour at least.In a particularly preferred embodiment, described pressure maintains greater than 2mbar, and wherein temperature is set to 15 ℃, thereby prevents that sample is freezing.In preferred embodiments, described temperature maintenance is at 15 ℃, and pressure is set to 5-10mBar, more preferably 6-9mBar, most preferably about 8mBar.When adopting higher temperature to be provided with, can adopt lower slightly pressure and not freezing sample, when adopting lower temperature to be provided with, it is freezing to prevent that described pressure should remain on higher level.Preferably, keep one competent period of described condition,, and make the temperature of sample be similar to the temperature in the sample external world so that described vaporator rate slows down.

Preferably, described preservation sample is not freezing or non-foaming/and boiling is to form foam, and its forfeiture solvent is to form the liquid of viscous liquid or high viscosity.

Remove and desolvate to form the liquid of high viscosity

The ensuing stage of the inventive method relates to removing desolvates, until the sample drying of described preservation to form the liquid of high viscosity.At described second drying stage, described sample is neither freezing also non-foaming to form foam.

High viscosity liquid is defined as solvent is less than or equal to 15,12,10, more preferably the material of 8,5,4,3,2 or 1% (w/w) is preferably measured by Karl Fischer coulomb moisture analyser.The liquid of described high viscosity has enough low solvent, thereby described promoting agent is 4 ℃ of storages at least 3,6,9,12 or keep steady state 24 months the time, make described promoting agent in this time, keep at least 40%, 50%, 60%, preferred 70%, 80%, 90%, 95% activity and/or antigenicity and/or immunogenicity.Preferably, the liquid of described high viscosity has solid and/or limpid outward appearance, but be glassy, and they can be at 2,4 or 6 days, preferred 2,3 or 4 weeks, more preferably 2,4,6,8,10 or 12 months in mobile lentamente.The container that contains described high viscosity liquid can be inverted and be positioned under the room temperature until observing flowing of described liquid, thereby measure described utmost point slow flow.In preferred embodiments, at 2,4 or 6 days, preferred 2,3 or 4 weeks, more preferably 2,4,6,8,10 or 12 months after, the described high viscosity liquid that is positioned at upside down position does not show mobile.

In one embodiment of the invention, the condition that adopted can reach this point the first evaporation drying stage that the pressure and temperature condition was remained on.For example, described pressure maintains or is lower than 30,25,20, and is preferred 15,12, and most preferably 10,8,7,6,5,4,3,2 or 1mbar, keep temperature to be arranged on simultaneously and be higher than 0 ℃, preferred 5 ℃ to 37 ℃, 5 ℃ to 10 ℃, 10 ℃ to 15 ℃; 15 ℃ to 20 ℃; 20 ℃ to 25 ℃; 25 ℃ to 30 ℃; Or 30 ℃ to 37 ℃.Be set to 15 ℃ for temperature, pressure maintains 5-10mBar, preferred 6-9mBar, about 8mBar most preferably, and keeping 4-24 hour, preferred 1-4,4-8,8-12 or 12-16 hour.These temperature and pressure conditions were kept 1,2,3,4,5,6,8,10,12,18 hour or the longer time, to obtain the liquid of high viscosity, its solvent is less than or equal to 15,12, preferred 10,8,5,4,3,2 or 1% (w/w), it is preferably measured by Karl Fischer coulomb moisture analyser.

Another embodiment of the present invention increases to the temperature setting in the solvent removal process and is higher than the temperature setting that this technology is kept early the time.It makes solvent leave sample with faster rate, thereby can finish method of the present invention in the shorter time.For example, described temperature setting increases to and is higher than 0 ℃, more preferably is higher than 20 ℃, preferred 5 ℃ to 37 ℃, 5 ℃ to 10 ℃, 10 ℃ to 20 ℃; 20 ℃ to 30 ℃; More preferably 30 ℃ to 40 ℃; More preferably 40 ℃ to 50 ℃; Most preferably 50 ℃ to 60 ℃, simultaneously pressure maintains or is lower than 30,25,20, and is preferred 15,12, and most preferably 10,8,7,6,5,4,3,2 or 1mbar.These temperature and pressure conditions were kept 1,2,3,4,5,6,8,10,12 or 18 hour or the longer time, to obtain solid, solvent in the described solid is less than or equal to 15,12,10,8,5,4,3,2 or 1% (w/w), and it is preferably measured by Karl Fischer coulomb moisture analyser.This embodiment require described promoting agent under the temperature that described method adopts for thermostability, thereby the described method of successful implementation.

(the pressure setting in the step c) is reduced to and is lower than the pressure setting (step b) that is adopted before this technology the preferred embodiments of the invention with the removal of solvents process.It makes solvent leave sample with faster rate, thereby can finish method of the present invention in the shorter time.It can also make more a high proportion of solvent forfeiture.For example, described pressure is set to or is lower than 7,6, and is preferred 5,4,3, and more preferably 2,1.5,1, most preferably 0.8,0.5,0.2,0.1,0.05,0.02,0.01 or 0.005mBar, simultaneous temperature maintains or is higher than 0 ℃, preferred 10 ℃ to 20 ℃; 20 ℃ to 30 ℃; 30 ℃ to 35 ℃ or be higher than 40 ℃.These temperature and pressure conditions kept 1,2,3,4,5,6,8,10,12 or 18 hour or the longer time, to obtain solid, solvent in the described solid is less than or equal to 15,12, preferred 10,8,5,4,3,2 or 1% (w/w), it is preferably measured by Karl Fischer coulomb moisture analyser, and (Eur.J.Pharm.Biopharm. (2000) 50; 277-284).

Preferably, step b) and c) (or only b)) should be equal to or less than 18 hours, preferred 16,12,10 hours, most preferably finish in 8,6,5 or 4 hours time.

Promoting agent

Method of the present invention can be used for preserving any promoting agent, but it preserves in technology in loss of activity and/or antigenicity and/or the immunogenic unstable promoting agent particularly useful being kept at other.

The promoting agent that adopts the inventive method to preserve can comprise and is selected from following biosystem: cell, ubcellular composition, bacterium, outer membrane vesicles preparation and virus, virus component or virus-like particle.It can also comprise molecule, for example protein, peptide, amino acid, polynucleotide, oligonucleotide, polysaccharide, oligosaccharides, polysaccharide-protein binding substances (conjugate), oligosaccharides-protein conjugate.

Can adopt the example of the promoting agent that the inventive method preserves to comprise the material of any biologically active, the material that for example has drug effect, include but not limited to anti-inflammatory drug, anodyne, tranquilizer, anxiolytic medicament, Anticonvulsants, antidepressive, antipsychotic drug, tranquillizer, anxiolytic medicament, narcotic antagonist, antiparkinsonism drug, cholinergic antagonist, chemotherapeutics, immunosuppressor, antiviral, biocide, appetite-inhibiting agent, anticholinergic agents, antimetrics, antihistaminic, antimigraine, coronary artery, the vasodilator of brain or periphery, hormone medicine, contraceptive bian, antithrombotic, diuretic(s), antihypertensive drug, cardiovascular agent, class opium etc.

Suitable reagent also comprises treatment and preventative medicine.Include but not limited to any effectively bio-modification agent in treatment.Described material includes but not limited to ubcellular composition, cell, bacterium, outer membrane vesicles preparation, virus and molecule, described molecule includes but not limited to lipid, organism, protein and peptide (synthetic and natural), peptide mimics, hormone (peptide, steroid and reflunomide), D and L type aminoacid polymers, oligosaccharides, polysaccharide, Nucleotide, oligonucleotide and nucleic acid, and described nucleic acid comprises DNA and RNA, protein nucleic acid heterocomplex, small-molecule substance and physiologically active analogue thereof.Further, described modifier can comprise analogue, antagonist and homologue derived from natural origin or by reorganization or synthesis mode preparation.

" protein " also refers to peptide and polypeptide herein.Described protein includes but not limited to enzyme, biological agent, tethelin, somatomedin, Regular Insulin, comprises mono-clonal and polyclonal antibody and fragment, Interferon, rabbit, interleukin and cytokine.

The therapeutic preparation based on nucleic acid by the methods described herein preparation is also included within the present invention.In this article, " nucleic acid " comprise well known in the art any in treatment effective nucleic acid, include but not limited to DNA, RNA and physiologically active analogue thereof.Described Nucleotide can encoding gene maybe can be any vehicle of recombinant DNA well known in the art, includes but not limited to plasmid, retrovirus and adeno associated virus.

The present invention further comprises the material with prophylactic activity and the preservation of carrier thereof.Preferred composition comprises immunogen, for example vaccine.After restoring, vaccine can be oral or can be injected.Suitable vaccine includes but not limited to antigen and/or the hapten that adds adjuvant and be coupled in carrier with the Nucleotide vehicle of virus deactivation, coding for antigens, bacterium, protein, polysaccharide, oligosaccharides and/or boivin antigen, antigen with deactivation alive that live.Special preferred pin is to following effective vaccine: diphtheria, tetanus, Whooping cough, Clostridium botulinum, cholera, singapore hemorrhagic fever, the first type, B-mode, third type and hepatitis E, hemophilus influenzae b, streptococcus pneumoniae, Neisseria meningitidis, Diplococcus gonorrhoeae, streptococcus aureus, staphylococcus epidermidis, B family suis, A family suis, simplexvirus, helicobacter pylori, influenza, Japanese encephalitis, meningococcus A, B, C, Y, W, measles, parotitis, papillomavirus, pneumococcus, poliovirus, (IPV-preferably includes 1 to the poliovirus of deactivation, 2,3 types, as the standard vaccine in vaccine field, most preferably be the Salk Poliomyelitis Vaccine), rubella, rotavirus, respiratory syncytial virus, Shigellae, tuberculosis, varicella zoster virus, yellow jack and combination thereof.The antigen component of vaccine can also be made by molecular biology method, to obtain containing recombinant peptide or the fusion rotein that is derived from the former proteinic one or more parts of causing a disease.For example, show that the fusion rotein that contains antigen and b subunit of cholera toxin can be induced at described antigenic immune response.Sanches etc. (1989) Proc.Natl.Acad.Sci.USA 86:481-485.Vaccine is particularly suitable for mixing in the composition of single dose.They are that indefinitely is stable in the peripheral ring border, can be dissolved in the sterile diluent again at once before immunization.

In preferred embodiments, described immunogenic composition can comprise capsular polysaccharide, and it is derived from one or more serologic group A, C, W-135 and the Y of Neisseria meningitidis.Can comprise capsular polysaccharide in another preferred embodiment derived from streptococcus pneumoniae.Described pneumococcal capsular polysaccharide antigen is preferably selected from serotype 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F (most preferably being selected from serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F).Another preferred embodiment can contain the PRP capsular polysaccharide of haemophilus influenzae type b.Another preferred embodiment can contain 5 types, 8 types, 336 or the PNAG capsular polysaccharide of streptococcus aureus.The I type, II type, III type or the PLA capsular polysaccharide that contain staphylococcus epidermidis in another preferred embodiment.Another preferred embodiment contains the streptococcic Ia type of B family, Ic type, II type or III type capsular polysaccharide.Another preferred embodiment contains the streptococcic capsular polysaccharide of A family, preferably further comprises at least a M protein, more preferably contains polytype M protein.

In one embodiment of the invention, described bacterial polysaccharides is natural polysaccharide total length, purified.In alternative embodiment of the present invention, described polysaccharide is played little (size) 2 to 20 times of repetitions, preferred 2-5 doubly, 5-10 doubly, 10-15 doubly or 15-20 doubly, make it be easy to more handle thereby described polysaccharide is less.Adopt oligosaccharides in preferred embodiments.Oligosaccharides contains 2 to 20 repeating units usually.

Polysaccharide and oligosaccharides can as mentioned belowly be unconjugated or bonded.

Adopt store method of the present invention can preserve the composition of two or more above-mentioned promoting agents.Adopt store method of the present invention can preserve part or all of vaccine.

The preferred promoting agent that adopts technology of the present invention to preserve comprises IPV (mixture of the poliovirus strain of deactivation).IPV, especially 3 type components are to the freeze-drying and the foam mat drying method sensitivity of routine, and it shows as, and lose antigenicity after freeze-drying or foam mat drying and recovery subsequently.

IPV is defined as the poliovirus (preferably include 1,2,3 types, as the standard vaccine in vaccine field, most preferably be the Salk Poliomyelitis Vaccine) of deactivation.The vaccine dose of IPV contains 1 type (Mahoney), the 4-16 of 20-80, preferred 40 or 80 D-antigen units, 2 types (MEF-1) of preferred 8 or 16 D-antigen units, and 3 types (Saukett) of 20-64, preferred 32 or 64 D-antigen units.

When adopting method of the present invention dry, preferably keep a kind, 2 kinds or whole 3 kinds of antigenicities of 1,2 and 3 type polioviruses; More preferably, keep 1 type; 2 types; 3 types; 1 type and 2 types; 1 type and 3 types; 2 types and 3 types; Or the antigenicity level of 1 type, 2 types and 3 types is antigenic at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% with reference to sample, the described processing of not passing through described drying process with reference to sample.After the liquid with described high viscosity restored in the aqueous solution, this can measure by any appropriate means, and described method comprises that employing is at the polyclone of 1 type, 2 types and/or 3 type polioviruses and/or the ELISA of monoclonal antibody.

When adopting method of the present invention to carry out drying, preferably keep a kind, 2 kinds or whole 3 kinds immunogenicities of 1,2 or 3 type polioviruses; More preferably, keep 1 type; 2 types; 3 types; 1 type and 2 types; 1 type and 3 types; 2 types and 3 types; Or the immunogenicity level of 1 type, 2 types and 3 types is immunogenic at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% with reference to sample, the described processing of not passing through described drying process with reference to sample.After the liquid with described high viscosity restores in the aqueous solution, can measure by any appropriate means.In a preferred method, the liquid of described high viscosity restores in the aqueous solution, and immunization is in animal body, preferred mouse.After one section reasonable time, from the inoculation animal, gather antiserum(antisera), detect seroconversion.Preferably, with comparing of undried, can obtain at least 0.4,0.5,0.6,0.7,0.8 or 0.9 relative potency with reference to sample.

Preferably, IPV can with one or more combinations in Hib (haemophilus influenzae type b) PRP polysaccharide or oligosaccharides and/or meningococcus A, C, W and/or Y polysaccharide or oligosaccharides and/or pneumococcal polysaccharide or the oligosaccharides.Most preferably, described promoting agent comprises IPV and Hib; IPV and MenC; IPV, Hib and MenC; Hib and MenC; IPV and MenA and C; Hib and Men A and C; IPV, Hib, MenA and C; Hib, Men C and Y; Or IPV, Hib, Men C and Y.

The promoting agent of above-mentioned particularize can also comprise the pneumococcal capsular polysaccharide that one or more are as described below.

In the composition of above-mentioned employing polysaccharide, can also adopt oligosaccharides (such as hereinafter definition).

Although it is not described composition can add adjuvant (as mentioned below), that they preferably add adjuvant or preferably do not comprise aluminium salt.

Preferably, described polysaccharide or oligosaccharides can combine with the peptide or the carrier proteins that comprise T-helper epitopes (as described below).

Annexing ingredient

Through technology exsiccant preferred composition of the present invention can with other antigen combination in the combination-vaccine, described combination-vaccine be drying or be preferably the liquid preparation that can be used to restore described dried ingredients.Comprise in diphtheria toxoid, Toxoid,tetanus, whole cell pertussis (Pw), acellular pertussis (Pa) (as described below), hepatitis B virus surface antigen, hepatitis A virus (HAV), hemophilus influenzae b polysaccharide, gonococcus polysaccharide, Neisseria meningitidis serotypes B protein, pneumococcal polysaccharide, pneumoprotein matter or the following listed arbitrary antigen one or more with the preferred antigens of above-mentioned promoting agent combination.Bacterial polysaccharides can combine with carrier protein, Toxoid,tetanus, Toxoid,tetanus fragment C, diphtheria toxoid, CRM197, pneumolysin and 3-protein d (US6342224) that described carrier protein is for example as described below.

The promoting agent that adopts technology of the present invention to preserve can be prepared with capsular polysaccharide, and described polysaccharide is derived from following one or more: Neisseria meningitidis, hemophilus influenzae b, streptococcus pneumoniae, A family suis, B family suis, streptococcus aureus or staphylococcus epidermidis.In preferred embodiments, described immunogenic composition can comprise one or more the capsular polysaccharide among serotype A, C, W-135 and the Y that belongs to derived from Neisseria meningitidis.Another preferred embodiment comprises the capsular polysaccharide derived from streptococcus pneumoniae.Described pneumococcal capsular polysaccharide antigen is preferably selected from serotype 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F (most preferably being selected from serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F).Another preferred embodiment comprises the PRP capsular polysaccharide of haemophilus influenzae type b.Another preferred embodiment comprises 5 types, 8 types, 336 or the PNAG capsular polysaccharide of streptococcus aureus.Another preferred embodiment contains I type, II type, III type or the PLA capsular polysaccharide of staphylococcus epidermidis.Another preferred embodiment contains the streptococcic Ia type of B family, Ic type, II type or III type capsular polysaccharide.Another preferred embodiment contains the streptococcic capsular polysaccharide of A family, preferably further comprises at least a M protein, more preferably contains polytype M protein.

In one embodiment of the invention, described bacterial polysaccharides is natural polysaccharide total length, purified.In another embodiment of the present invention, described polysaccharide is beaten little 2 to 20 times, preferred 2-5 doubly, 5-10 doubly, 10-15 doubly or 15-20 doubly, make it be easy to more handle thereby described polysaccharide is less.Adopt oligosaccharides in preferred embodiments.Oligosaccharides contains 2 to 20 repeating units usually.

Described capsular polysaccharide can be unconjugated or be bonded to carrier protein, for example Toxoid,tetanus, Toxoid,tetanus fragment C, diphtheria toxoid, CRM197, pneumolysin, 3-protein d (US6342224).By transgenation and/or preferably tetanus toxin, diphtheria toxin and pneumolysin are carried out detoxification by chemical treatment.

Described polysaccharide conjugate can prepare by the coupling method of any known.For example, described polysaccharide can pass through the thioether bond coupling.This combining method depends on and with 1-cyano group-4-Dimethylamino pyridine a tetrafluoro borate (CDAP) polysaccharide is activated and form cyanate.Like this, described activated polysaccharide can be directly or is coupled to amino on the carrier protein by spacer groups.Preferably, described cyanate and hexanediamine coupling adopt different band chemical process (heteroligation chemistry) that described amination deutero-polysaccharide is incorporated into carrier proteins, and described chemical process relates to the formation of thioether bond.The PCT of Uniformed Services university openly applies for having described among the WO93/15760 this binding substances.

Described binding substances is all right, prepares by direct reductive amination method, as described in US4365170 (Jennings) and US4673574 (Anderson).Other method has been described among EP0161188, EP208375 and the EP0477508.

Other method relate to hexanodioic acid hydrazides (ADH) deutero-through the polysaccharide of cyanogen bromide-activated by the carbodiimide condensation reaction be coupled on the protein carrier (Infect.Immunity such as Chu C, 1983,245-256).

Preferred pneumoprotein antigen is the pneumoprotein matter that is exposed to the streptococcus pneumoniae outside surface (pneumococcally can be discerned by host immune system to the small part life cycle described), or by streptococcus pneumoniae secretion or the protein that discharges.Most preferably, described protein is lipoprotein or its fragment of toxin, conglutnin, 2-constituent signals conversion, streptococcus pneumoniae.Particularly preferred protein includes but not limited to: pneumolysin (preferably removing toxicity by chemical treatment or sudden change) [Mitchell etc., Nucleic Acids Res.1990 July 11; 18 (13): 4010 " Comparison of pneumolysin genes and proteinsfrom Streptococcus pneumoniae types 1 and 2. ", BiochimBiophy Acta such as Mitchell on January 23rd, 1989; 1007 (1): 67-72 " Expression of thepneumolysin gene in Escherichia coli:rapid purification and biologicalproperties ", WO96/05859 (A.Cyanamid), WO90/06951 (Paton etc.), WO99/03884 (NAVA)]; PspA and stride film disappearance variant (US5804193-Briles etc.); PspC and stride film disappearance variant (WO97/09994-Briles etc.); PsaA and stride film disappearance variant (Berry﹠amp; Paton, Infect Immun in December, 1996; 64 (12): 5255-62 " Sequence heterogeneity of PsaA, a 37-kilodalton putativeadhesin essential for virulence of Streptococcus pneumoniae "); Streptococcus pneumoniae choline binding protein and stride film disappearance variant; CbpA and stride film disappearance variant (WO97/41151; WO99/51266); Glyceraldehyde-3-phosphate dehydrogenase (Infect.Immun.199664:3544); HSP70 (WO96/40928); PcpA (Sanchez-Beato etc., FEMSMicrobiol Lett 1998,164:207-14); M sample protein, (EP0837130) and conglutnin 18627, (EP0834568).Other preferred pneumoprotein matter antigen is disclosed among the WO98/18931, particularly selected antigen among WO98/18930 and the PCT/US99/30390.

The preferred Neisseria protein of preparing with immunogenic composition of the present invention comprises TbpA (WO93/0681; EP586266; WO92/03467; US5912336), TbpB (WO93/06861; EP586266), Hsf (WO99/31132), NspA (WO96/29412), Hap (PCT/EP99/02766), PorA, PorB, OMP85 (being also referred to as D15) (WO00/23595), PilQ (PCT/EP99/03603), PldA (PCT/EP99/06718), FrpB (WO96/31618 sees SEQ ID NO:38), FrpA or FrpC or both total at least 30,50,100,500,750 amino acid whose preservation parts (WO92/01460), LbpA and/or LbpB (PCT/EP98/05117; Med.Microbiol.199932:1117 such as Schryvers), FhaB (WO98/02547), HasR (PCT/EP99/05989), lipo02 (PCT/EP99/08315), MltA (WO99/57280) and ctrA (PCT/EP00/00135).The preferred protein purification that adds Neisseria protein as an adventitial formulation part.

Vaccine is preferably prepared with antigen, thereby one or more the protection during diphtheria, tetanus and Bordetella pertussis infected is provided.Whole cell pertussis Bordetella (Pw) that described Bordetella component can be deactivation or acellular pertussis (Pa), it contains from least a antigen of PT, FHA and 69kDa pertacin (preferred two or all three kinds).Some other acellular vaccine also contains agglutinogen, for example Fim2 and Fim3, and these vaccines also can be used for the present invention.It is diphtheria toxoid and Toxoid,tetanus that antigen at diphtheria and tetanic protection generally is provided.Described toxoid is the toxin (after for example, with formaldehyde treated) through chemical ablation or passes through to introduce one or more point mutation and the toxin of deactivation.

High viscosity liquid alternatively of the present invention can be used as test kit to be provided, and contains the glassy liquid of described high viscosity in one of them container, contains liquid D TPa or DTPw in another container.Described test kit for example can comprise the dual cavity syringe, and but the component of drying and liquid ingredient are arranged in same syringe are arranged in different chamber.Before injecting, restore the component of described drying immediately with described aqueous vaccine as single vaccine.Therefore for example, the liquid composition of high viscosity of the present invention restores with liquid D TPa or DTPw vaccine (preferred interim preparation) and carries out administration as single vaccine.DTPa or DTPw vaccine generally add aluminium hydroxide at least in part as adjuvant (for example the Infanrix  of GlaxoSmithKline Biologicals s.a. and Tritanrix  vaccine).

Described vaccine also can randomly comprise one or morely can make the host resist the hemophilus influenzae of non-somatotype, the antigen of RSV, and/or one or more antigen that can make the host resist influenza virus.

The hemophilus influenzae proteantigen of preferred non-somatotype comprises Fimbrin protein (US5766608) and comprises the syzygy of resultant peptide (for example LB1 syzygy) (US5843464-Ohio State Research Foundation), OMP26, P6,3-protein d, TbpA, TbpB, Hia, Hmwl, Hmw2, Hap and D15.

Preferred influenza antigen comprises that the totivirus that grows in egg or mdck cell or Vero cell, virus that live or deactivation, splitted influenza virus or full influenza virus particles are (as R.Gluck, Vaccine, 1992,10,915-920 is described) or its purifying or recombinant protein, for example HA, NP, NA or M protein, and combination.

Preferred RSV (respiratory syncytial virus) antigen comprises F glycoprotein, G glycoprotein, HN albumen, M albumen or derivatives thereof.

It should be noted that antigen composition of the present invention can comprise one or more capsular polysaccharides from single bacterium kind.Antigenic composition can also comprise the capsular polysaccharide from one or more bacterium kinds.

Immunogenic composition and vaccine

Another aspect of the present invention comprises immunogenic composition or the vaccine that contains acceptable vehicle on high viscosity liquid of the present invention and the pharmacology.

Preferably, described immunogenic composition or vaccine contain a certain amount of adjuvant, and it is enough to increase at immunogenic immune response.Suitable adjuvant includes but not limited to aluminium salt, shark alkene mixture (SAF-1), muramyl peptide, Saponin/TSM derivative, mycobacterium cell walls preparation, monophosphoryl lipid A, mycolic acid derivatives, non-ionic type block copolymer surfactant, Quil A, b subunit of cholera toxin, polyphosphonitrile and derivative thereof and immunostimulating complex (ISCOM), those described in (1990) Nature 344:873-875 such as Takahashi.For the application of animal doctor aspect and in animal, produce antibody, can adopt the mitogenetic component of Freund adjuvant.

The same with all immunity compositions or vaccine, immunogenic immune significant quantity must be determined by experience.The factor of need considering comprise immunogenicity, immunogen whether with adjuvant or carrier proteins or other carrier complexing or covalent attachment, the number of route of administration, the immunizing dose that need give.Above-mentioned factor is that the vaccine field is known, and the immunologist does not need to carry out the over-drastic test just can determine that this is the technical ability that the immunologist possesses.

The promoting agent that can contain different concns in the liquid of high viscosity of the present invention or the vaccine.The Cmin of general described material is to reach its expection to use required amount, and peak concentration is for staying in the solution or evenly being suspended in maximum in the original mixture.For example, the minimum of therapeutical agent is preferably provides the single dosage with result of treatment.For biologically active substance, the necessary amount of biologically active when Cmin is recovery, peak concentration is the concentration in the time of can not keeping unit for uniform suspension.Under the situation of single dosage unit, described amount is the amount that single treatment is used.Usually, each dosage estimates to comprise 1-100 μ g proteantigen, and preferred 5-50 μ g most preferably is 5-25 μ g.The preferred dose of bacterial polysaccharides is 10-20 μ g, 10-5 μ g, 5-2.5 μ g or 2.5-1 μ g.The preferred amounts difference of various materials, but those skilled in the art are easy to determine.

The high viscosity liquid that comprises promoting agent

Another aspect of the invention is and comprise high viscosity liquid promoting agent, that preferably can obtain or obtain by the inventive method.Adopt after the dry also recovery of the inventive method, described promoting agent preferably keeps its activity and/or antigenicity and/or immunogenicity.Activity, antigenicity or the immunogenicity of the promoting agent of preferred maintenance at least 40,50,60,70,80,90 or 95%.It can for example as indicated abovely be determined by any appropriate means.

The liquid of high viscosity of the present invention preferably includes and forms glass polyol, and it is selected from glucose, maltulose, Palatinose, lactulose, sucrose, maltose, lactose, sorbyl alcohol, isomaltose, maltose alcohol, Saccharum lactis, palatinit, trehalose, gossypose, stachyose, melizitose and dextran.

High viscosity liquid of the present invention can contain arbitrary promoting agent mentioned above.Promoting agent by the high viscosity fluid preservation can comprise biosystem, for example cell, ubcellular composition, bacterium, outer membrane vesicles goods and virus.It can optionally contain or further comprise molecule, for example protein, peptide, amino acid, polynucleotide, oligonucleotide, polysaccharide, oligosaccharides, polysaccharide-protein binding substances, oligosaccharides-protein conjugate.It can also comprise the composition that contains two or more above-mentioned promoting agents.

Preferred embodiment comprises preferably the liquid by the inventive method preparation or the high viscosity that can be made by the inventive method, and wherein said promoting agent is or comprises vaccine or vaccine component.The preferred ingredient of vaccine as mentioned above and comprise IPV, more preferably IPV and bacterial polysaccharides, preferably belong to polysaccharide or the oligosaccharides of A, C, W and Y from hemophilus influenzae b and Neisseria meningitidis.

The preferred vaccine component comprises IPV (the deactivation mixture of poliovirus strain).Preferably, IPV and one or more following combinations: Hib PRP polysaccharide and/or meningococcus A, C, W and/or Y polysaccharide and/or pneumococcal polysaccharide (as mentioned above), more preferably IPV and Hib; IPV and MenC; IPV, Hib and MenC; Hib and MenC; IPV and MenA and C; Hib, MenA and C; IPV, Hib, MenA and C; Hib, MenC and Y; Or IPV, Hib, MenC and Y.

In the composition of above-mentioned employing polysaccharide, can also adopt oligosaccharides (as mentioned above).

Although these compositions can add adjuvant (as mentioned above), they preferably do not add adjuvant or preferably do not comprise aluminium salt.

Preferably, described polysaccharide or oligosaccharides combine (as mentioned above) with peptide that comprises the t helper cell epi-position or carrier protein.

High viscosity liquid of the present invention preferably with combination-vaccine in optionally drying the combination of other antigen or preferably make up with liquid preparation, described liquid preparation can be used to restore each component of described exsiccant.Comprise following one or more with the preferred antigens of the content of container of the present invention combination: diphtheria toxoid, Toxoid,tetanus, whole cell pertussis (Pw), acellular pertussis (Pa) (as mentioned above), hepatitis B virus surface antigen, pneumococcal polysaccharide, pneumoprotein, Neisseria polysaccharide, Neisseria protein.Bacterial polysaccharides can combine with carrier protein, for example aforesaid Toxoid,tetanus, Toxoid,tetanus fragment C, diphtheria toxoid, CRM197, pneumolysin, 3-protein d (US6342224).

Another aspect of the invention is the method for preparing vaccine, it is included in the step of restoring described high viscosity liquid in the aqueous solution.In preferred embodiments, the described aqueous solution comprises diphtheria toxoid, Toxoid,tetanus, Whooping cough (acellular or full cell) antigen, and randomly further comprises hepatitis B virus surface antigen.The DTP vaccine randomly adds aluminium salt adjuvant to small part, preferred aluminium hydroxide or aluminum phosphate.

Another embodiment of the present invention is a test kit, and high viscosity liquid wherein of the present invention is positioned in first container, comprises that the vaccine of liquid D TP (acellular or full cell) is positioned in second container.Can adopt aforesaid dual cavity syringe.

All reference or the patent application of quoting in the patent specification all are hereby incorporated by.

Embodiment

Adopt standard method to implement following examples, except special detailed description, described standard method all is known in those skilled in the art and is conventional.These embodiment are exemplary, do not limit the present invention.

The foundation of embodiment 1 freezing conditions

With sucrose 1%, 5%, 10% and 20% the solution of obtaining soluble in water with the preparation sample.Sample is put into Heto Drywinner 8-85 Freeze Drying Equipment, and wherein shelf temperature is controlled in 1 ℃, and the outlet temperature of condenser is-85 ℃, by discharging pressure regulating valve, has 6 thermopairs to be used to measure product temperature.In whole process, described shelf temperature setting maintains 15 ℃.Described pressure initially is reduced to 200mBar, maintains this level 10 minutes, then pressure further is reduced to 50mBar, 5mBar, 2.5mBar, 0.75mBar, 0.4mBar and 0.2mBar.Make each stress level keep 20 minutes, thereby make temperature reach balance, the temperature of sample is carried out reading with thermopair.Thermopair is connected to the sample with different sucrose, and the temperature of record is the mean value of temperature in the table 1.

The result

No matter existing concentration of sucrose, all samples is freezing between 1.66 to 1.11mBar.The temperature of measuring under the different pressures situation is in close proximity to the temperature by described three-poing curve prediction.Therefore, the existence of sucrose does not produce remarkably influenced to the temperature that is positioned at the sample under the different pressures.

Be frozen for fear of sample, when shelf temperature was 15 ℃, pressure should maintain more than the 2mBar.Under lower temperature, described pressure should maintain higher level, and adopts higher temperature that pressure is further reduced, and sample can be not freezing.

Table 1

Pressure	The temperature of measuring	Theoretical temperatures	Liquid/freezing
				1000mBar	15℃		Liquid
50mBar	15℃		Liquid
				5mBar	1℃	1℃	Liquid
2.5mBar	-5℃	-7℃	Liquid
				0.75mBar	-21℃	-21℃	Freezing
0.4mBar	-22℃	-27℃	Freezing
				0.2mBar	-27℃	-32℃	Freezing

Embodiment 2 is not freezing or do not form foam and carry out the exsiccant method

Preparation contains the preservation sample of 5%, 10%, 15% and 25% sucrose, and adds in the bottle.Sample is positioned over temperature in the whole process to be arranged in 15 ℃ the Freeze Drying Equipment.Described pressure initially is reduced to 200mBar, maintains this level 10 minutes, further reduce pressure then with the degassing.Further pressure is reduced to 8mbar and kept 2-3 hour, the thermopair in the sample shows during this period, because transpiration cooling, the temperature of described sample is reduced to 4 ℃.After 2-3 hour, the temperature of described sample is back to 15 ℃, and it shows that the evaporation of carrying out will finish under these temperature and pressure conditions.In this stage of described technology, sample does not seethe with excitement with formation foam or freezing, thereby makes the promoting agent in the sample be exposed to the least possible stress.Described sample has the outward appearance of viscous liquid.

Pressure further is reduced to 0.1mBar, simultaneously described shelf temperature setting is maintained 15 ℃, thereby make sample further dry.Kept these conditions again 10-16 hour.In this stage, sample temperature is remained on 15 ℃, because vaporator rate slows down.Further carry out drying, resulting sample has solid appearance.If sample is sidelong along one puts, in a couple of days, described sample contents flows very slow, and it shows that described sample is the liquid glass with high viscosity.Accompanying drawing 1 has shown the outward appearance of described high viscosity liquid.

Embodiment 3 is not freezing or do not form foam and after the drying, IPV keeps immunogenicity

Described sample does not stand and foaming or the freezing relevant stress of following foam to form.Experimentize, to determine whether this method produces high-caliber antigen retention when being used for dry IPV.

Carry out three independent experiments, wherein IPV is suspended in the aqueous solution again, contain 10% sucrose or 10% trehalose in the described aqueous solution as stablizer.Sample is put into bottle through siliconization, bottle is positioned in the Heto Drywinner8-85 Freeze Drying Equipment, temperature is set to 15 ℃.Pressure initially is reduced to 35mBar to remove the gas in the sample.After 10 minutes, pressure further is reduced to 8mBar, and kept this level 2 hours.In this stage, keep temperature to be arranged on 15 ℃, and the temperature in the monitoring sample.When water was evaporated from sample, temperature was reduced to 4 ℃, but when finishing in described two hours, described temperature is back to 15 ℃, and this is because the speed of evaporation has slowed down.Do not bubble under these conditions or the formation foam.Further described pressure is reduced to 0.1mBar then, in preceding two experiments, continued to keep these conditions 16 hours, in the 3rd experiment, continued to keep these conditions 10 hours.

Restore described sample in water, the antigenicity that adopts ELISA to estimate three poliovirus strains keeps situation.The monoclonal antibody of anti-3 type IPV is used for ELISA, keeps situation with the antigen of estimating described restorative, freeze-drying sample, compares with the antigen reservation situation that does not have the refrigerated control sample.Shown result is the percentage ratio of reading of the sample that do not stand drying process.

The result

Described exsiccant sample has solid appearance, but they show as the liquid/glass form of high viscosity, and this is because if in a couple of days described container is inverted, the sample of described drying can flow.

Table 2 adopts monoclonal antibody to determine the antigenic reservation situation of 3 type IPV (no by ELISA Form foam or freezing and carry out drying)

Prescription	First experiment (18 hours period)	Second experiment (18 hours period)	The 3rd experiment (12 hours period)
				Do not contain sugar	0％
2.5% sucrose	0％
				10% sucrose	75％	78％	91％
10% trehalose	82％	79％	93％

Compare with following shown freeze-drying result, 3 type IPV antigen reservation levels are very favorable, in following result, have shown when employing resists 3 type monoclonal antibodies, find low-down value usually in identical ELISA table.

Table 3 adopts mono-clonal and polyclonal antibody to determine 1 type, 2 types and 3 type IPV by ELISA Antigenic reservation situation (freeze-drying)

Drying means	Polyol content	ELISA-1/2/3 type %
				Polyclone	Mono-clonal
		Freeze-drying	3.15% sucrose			46/49/58 *	19/25/0
Freeze-drying	10% trehalose			47/43/58	25/0/0

^*Repeat to contain for 5 times the freeze-drying experiment of 3.15% sucrose, shown result is from a representational experiment.

Embodiment 4 is as the prolonged storage stability of the dry IPV of high viscosity liquid/glass storage

Adopting embodiment 3 described methods to carry out exsiccant IPV stored 9 months down at 4 ℃.Sample is restored in the water with 150mM NaCl, adopt the antigenicity of three poliovirus strains of ELISA assessment to keep situation.Adopt three monoclonal antibodies to estimate the antigen reserving degree in restorative storage sample in each ELISA, described three monoclonal antibodies are respectively at different virus strain.Before storage, the recovery sample from same batch is carried out similar ELISA detection.All results all do not compare with there being the exsiccant control sample.Shown result does not stand the percentage ratio of the sample reading of drying process.

The result

Table 4 is as 9 months reservation situations afterwards of liquid IPV antigen storage of high viscosity

Handle	1 type ELISA	2 type ELISA	3 type ELISA
				Exsiccant/through restorative, not storage	72％	75％	88％
Exsiccant/, stored 9 months down at 4 ℃ through restorative	70％	94％	90％

Therefore, do not lose antigenicity through embodiment 3 described methods IPV dry and that under 4 ℃, stored at least 9 months.

Embodiment 5 and undried IPV compare the interior immunogenicity of body that drying forms high viscosity liquid and restores IPV afterwards

With the IPV immunization of 10 one group Wistar rat with different extension rates, described IPV carries out dry liquid with the formation high viscosity through embodiment 2 described methods in the presence of 10% sucrose, and restores.Other 10 one group Wistar rat is used IPV control sample immunization, the described IPV of preparation that uses the same method, but do not carry out drying.

After 21 days, gather serum from all rats, and adopt 1 type, 2 types and 3 type polioviruses in different immune precipitation determinations, described serum to be detected.

Result shown in the table 5 comprises: a) the rat quantity that each IPV extension rate is responded, b) ED50, it is to guarantee the required dosage of 50% rat generation seroconversion, it is assessed by the immune precipitation determination method, and c) with the comparing of undried with reference to IPV, described drying and through the relative potency of restorative IPV.

Table 5 is compared with reference to IPV (JLE097) with undried, forms high viscosity in drying Liquid (JLE017/05) and restore after the immunogenicity of IPV

Sample		Quantity	Reaction		ED50	RP is relative
								Undiluted	1/1.25	1/3.125	1/7.81		Tire
JLEO17/05
							1 type	10	9	6	5	6.37	0.956
2 types	6	4	3	3	7.14	0.825
							3 types	6	8	2	1	18.18	1.051
JLE097
							1 type	10	10	10	7	3.33	1.120
2 types	8	6	5	2	3.12	0.951
							3 types	7	6	4	1	16.91	1.172
Reference
							1 type		10	8	4	6.37
2 types		7	5	2	2.93
							3 types		5	3	0	22.57

JLEO17/05 is that drying forms the liquid of high viscosity and next carries out restorative IPV batch.JLE097 is the object of reference of undried.

Table 5 shown, with the IPV of each extension rate carry out rat quantity that the generation of immunization replys drying and restorative IPV and undried be similar between with reference to sample.Substantially, 1 type IPV causes the strongest immunne response, and 2 types cause immune response in less slightly rat, and 3 types cause the most weak immunne response.

The dry ability of not damaging immunoprecipitating antibody in the IPV primosome with the technology that forms high viscosity liquid.Relative potency (RP) reading is 1.0 o'clock, and it represents that described sample has caused and replying that the reference sample is equal to.Two dry samples of all three PLDs viruses are all produced approximate 1.0 RP reading, and it shows that described drying process does not exert an influence to the ability that sample causes immunne response.

It is dry with the influence to the immunne response ability of immunoprecipitation in the IPV primosome of the liquid that forms high viscosity that embodiment 6 adopts sucrose or trehalose to carry out as stablizer

10 one group Wistar rat is used under the situation that 10% sucrose or 10% trehalose exist carries out drying, carry out restorative IPV then and carry out immunization, as described in embodiment 2.To other 10 one group Wistar rat with undried, carry out immunization as the equivalent IPV of reference sample.

After 21 days, gather serum from all rats, and adopt as in the embodiment 5 described immunity and the measuring method evaluation respectively at the amount of the immune neutralizing antibody of 1 type, 2 types and the generation of 3 type polioviruses.

Compare with the immunne response with reference to sample caused of described undried, calculate the relative potency of each sample.

The result is as shown in table 6.

Table 6 carries out exsiccant relatively in sucrose and trehalose

Lot number	Existing sugar	The relative potency of 1 type in the body/2 types/3 types	Humidity %	Time length (hour)
					Jle017	10% trehalose	0.95/0.82/1.05	nd	7
31CO3/01	10% sucrose	0.69/1.20/0.97	4.6％	18
					31CO3/02	10% trehalose	0.60/0.94/0.9	11.5％	18
03D02/01	10% sucrose	0.74/1.05/0.96	5.9％	12
					03D02/02	10% trehalose	0.58/0.98/1.06	10.6％	12

When adopting sucrose as stablizer, remaining moisture content is about 5% in the sample, and it is lower than when adopting trehalose as stablizer remaining about 10% moisture content in the sample, and described moisture content is measured by Karl Fischer coulomb moisture analyser.

In drying process, sucrose and trehalose all are effectively stablizing aspect the IPV, thereby for most of dissimilar polioviruses, describedly obtain being approximately 1.0 relative potency reading through restorative IPV.Described relative potency is for the immunogenic 3 type polioviruses of forfeiture are especially favourable comparatively easily.

Embodiment 7 measures humidity by Karl Fischer

Adopt Karl Fischer titrimetry instrument (Aqua30.00-Elektrochemie Halle) to analyze.The described sample of weighing, and put into the baking box that temperature is set to 80 ℃.Use the nitrogen purging sample, add hydranal reagent (Riedel de Hahn) then thus analyze by coulometry.

Claims (39)

1. method of preserving promoting agent may further comprise the steps:

A) promoting agent is dissolved/be suspended in the stabiliser solution, sample is preserved in preparation;

B) described preservation sample is placed under certain temperature and the pressure condition, thus make this preservation sample by the vaporization losses solvent forming viscous liquid, and do not carry out freezing or relate to the foaming that foam forms.

2. the method for claim 1 further may further comprise the steps:

C) further described preservation sample is placed under certain temperature and the pressure condition, thereby make described viscous liquid drying to form high viscosity liquid.

3. claim 1 or 2 method wherein in step b), are reduced to 20mbar or lower with described pressure.

4. the method for claim 1-3, wherein in step b), the outside temperature of described preservation sample is 5 ℃ to 37 ℃.

5. the method for claim 2-4, wherein in step c), the outside temperature of described preservation sample is 5 ℃ to 37 ℃.

6. the method for claim 2-5, the outside temperature of wherein preserving sample described in the step c) is higher than in step b).

7. the method for claim 6, wherein in step c), the outside temperature of described preservation sample increases to more than 20 ℃.

8. the method for claim 2-7, wherein with respect to the pressure in the step b), the pressure in the step c) has reduced.

9. the method for claim 8, wherein in step c), described pressure is reduced to 1mbar or lower.

10. the method for claim 1-9, wherein step b) is finished in less than 4 hours time.

11. the method for claim 2-10, wherein step b) and c) in less than 12 hours time, finish.

12. the method for claim 1-11, wherein said stablizer comprises the polyol that forms glassy mass, and it is selected from glucose, maltulose, Palatinose, lactulose, sucrose, maltose, lactose, sorbyl alcohol, isomaltose, maltose alcohol, Saccharum lactis, palatinit, trehalose, raffinose, stachyose, melizitose and dextran.

13. the method for claim 12, wherein said stablizer are sucrose.

14. the method for claim 12-13, the concentration of wherein said stablizer is lower than 15%.

15. the method for claim 1-14 contains phenol red in the wherein said preservation sample.

16. the method for claim 1-15 is wherein at the described preservation sample of the container inner drying with anti-solvent internal surface.

17. comprising, the method for claim 1-16, wherein said promoting agent be selected from following molecule: protein, peptide, amino acid, polynucleotide, oligonucleotide, polysaccharide, oligosaccharides, polysaccharide-protein binding substances and oligosaccharides-protein conjugate.

18. comprising, the method for claim 1-16, wherein said promoting agent be selected from following biosystem: cell, subcellular component, bacterium, virus, virus component and virus-like particle.

19. the method for claim 18, wherein said promoting agent comprise IPV (poliovirus of deactivation).

20. the method for claim 18-19, wherein said promoting agent comprise Hib (b type hemophilus influenzae) polysaccharide or oligosaccharides.

21. the method for claim 18-20, wherein said promoting agent comprise polysaccharide or the oligosaccharides of Neisseria meningitidis C.

22. the method for claim 1-21, wherein said promoting agent comprises vaccine.

23. a high viscosity liquid that comprises promoting agent, the antigenicity or the activity of wherein having preserved described promoting agent.

24. the high viscosity liquid of claim 23 can obtain by the method for claim 1-22.

25. the high viscosity liquid of claim 23 or 24 comprises the polyol that is selected from following formation glassy mass: glucose, maltulose, Palatinose, lactulose, sucrose, maltose, lactose, sorbyl alcohol, isomaltose, maltose alcohol, Saccharum lactis, palatinit, trehalose, raffinose, stachyose, melizitose and dextran.

26. the high viscosity liquid of claim 25, the polyol of wherein said formation glassy mass are sucrose.

27. comprising, the high viscosity liquid of claim 23-26, wherein said promoting agent is selected from following molecule: protein, peptide, amino acid, polynucleotide, oligonucleotide, polysaccharide, oligosaccharides, polysaccharide-protein binding substances and oligosaccharides-protein conjugate.

28. comprising, the high viscosity liquid of claim 23-27, wherein said promoting agent is selected from following biosystem: cell, subcellular component, bacterium, virus, virus component and virus-like particle.

29. the high viscosity liquid of claim 23-28, wherein said promoting agent comprises vaccine.

30. the high viscosity liquid of claim 23-29, wherein said promoting agent comprises IPV.

31. the high viscosity liquid of claim 23-30, wherein said promoting agent comprises bacterial polysaccharides or oligosaccharides.

32. the high viscosity liquid of claim 31, wherein said promoting agent comprise Hib (b type hemophilus influenzae) polysaccharide or oligosaccharides, preferably it combines with carrier protein.

33. the high viscosity liquid of claim 23-32, wherein said promoting agent comprise the polysaccharide or the oligosaccharides of Neisseria meningitidis serogroup C, preferably it combines with carrier protein.

34. the high viscosity liquid of claim 23-33, it places the container with anti-solvent internal surface.

35. immune composition or vaccine, it comprises as acceptable vehicle on described high viscosity liquid of claim 23-24 and the pharmacology.

36. a method for preparing vaccine, it is included in the step of restoring in the aqueous solution as the described high viscosity liquid of claim 23-35.

37. the method for claim 36, the wherein said aqueous solution comprise diphtheria antigen, tetanus antigen and pertussis antigen (acellular or full cell).

38. the method for claim 37, wherein the DTP vaccine adds adjuvant aluminium hydroxide at least in part.

39. a test kit, it comprise be contained in first container as the described high viscosity liquid of claim 23-34 and be arranged in the aqueous vaccine component of second container.

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