CN1735430B - Immunogenic composition - Google Patents

Immunogenic composition Download PDF

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CN1735430B
CN1735430B CN2003801081843A CN200380108184A CN1735430B CN 1735430 B CN1735430 B CN 1735430B CN 2003801081843 A CN2003801081843 A CN 2003801081843A CN 200380108184 A CN200380108184 A CN 200380108184A CN 1735430 B CN1735430 B CN 1735430B
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sample
ipv
immunogenic composition
oligosaccharide
polysaccharide
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CN1735430A (en
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Y·马耶尔斯
J·斯蒂芬尼
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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Priority claimed from GB0225532A external-priority patent/GB0225532D0/en
Priority claimed from GB0225543A external-priority patent/GB0225543D0/en
Priority claimed from GB0317371A external-priority patent/GB0317371D0/en
Priority claimed from GB0317381A external-priority patent/GB0317381D0/en
Priority claimed from GB0317380A external-priority patent/GB0317380D0/en
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Priority claimed from PCT/EP2003/012160 external-priority patent/WO2004039399A1/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to immunogenic compositions comprising a dried solid or highly viscous liquid formulation of inactivated polio virus (IPV) and a stabilising agent wherein the IPV retains its antigenicity and/or immunogenicity. Methods of producing a dried formulation of IPV which retains its antigenicity/immunogenicity are described.

Description

Immunogenic composition
The present invention relates to comprise the drying solid of the immunogenic inactivated poliovirus of reservation (IPV) or the immunogenic composition of high viscosity liquid preparation.The present invention also comprises the drying solid that comprises IPV or the vaccine of high viscosity liquid preparation.Another aspect of the present invention is to preserve the method for inactivated poliovirus (IPV) for drying solid or high viscosity liquid.This method comprises by suspending in stabiliser solution or dissolving IPV and bacterial polysaccharides prepare sample, and makes described sample stand to cause the temperature and pressure condition of solvent from described sample forfeiture.Keep or regulate the pressure and temperature condition and desolvate and dry described sample formation solid or high viscosity liquid so that remove.But above-mentioned preparation is reprovision or directly use before use.
As everyone knows, IPV is as a kind of composition of vaccine, and still, it is formulated as liquid, for example is Infanrix penta
Figure 10003_0
Confirmed already that IPV lyophilization processing was relevant with antigenic loss, so be difficult to prepare the effective vaccine that comprises the IPV dried forms.Known exsiccant bacterin preparation is particularly in the presence of bacterial polysaccharides.B type Haemophilus influenzae (Haemophilusinfluefzzae b) PRP polysaccharide (Hib) often is formulated as drying solid, for example is Infanrixhexa (WO99/48525).
The favourable part of IPV drying agent has several respects reason.Drying agent has good storing property, and can increase the pot-life of the vaccine that contains IPV.Exsiccant IPV also may make IPV become the vaccine composition that has more motility, and the new combined vaccine that institute can not existence before making it to be formulated as.Some contains the vaccine of liquid and solid constituent, only mixes (Infanrix hexa for example before administration
Figure 10003_2
).Infanrix Hexa contains exsiccant Hib composition, only before use with the DTPa-HepB-IPV reprovision.IPV just may add more multicomponent, unless described composition may be incompatible with IPV by being formulated as drying solid with Hib in the liquid component of vaccine.
Several technology that is used for dry vaccine composition known in this field.From traditionally, use freeze-drying method to reach this purpose already, prepare substance solution and freezing sample in the method.In preliminary drying stage, in reduced pressure, remove most of moisture by the ice distillation, formed porous ' cake '.Usually following the redrying stage, when pressure and temperature changed, moisture just was evaporated from solid " cake ".Compare with liquid preparation, the lyophilizing sample that is produced has improved stability.But freezing dry process is tediously long, can become the rate-limiting step in the production process.
When a large amount of samples in big exsiccator equipment in batches during lyophilizing, the product diversity also is an a difficult problem.Condition on the freeze dryer shelf is different in different positions, caused sample under different condition with different speed lyophilizing.For some such as for the biomaterial of live virus, loss of activity quite big (Pikal (1994) ACS Symposium567:120-133) in freezing dry process.Still unstable at ambient temperature (Carpenter etc. (1994) the ACS Symposium 567 of many freeze dried materials; 134-147).
The damage that is caused by using cryoprotective agent such as polyol to prevent refrigerating process to a certain extent.In this process,, also obtained further improvement (WO96/40077 to freeze drying process by avoiding freezing sample and removing moisture by boiling; US63063450).This method is included in preparation glass matrix moulding material and the mixture that will preserve sample in the suitable solvent, the a large amount of solvents of evaporation are to obtain slurry from this mixture, slurry is exposed to enough causes under the ebullient pressure and temperature of this slurry, and remove residual solvent.
Similar approach is described in US 5,766, and in 520, wherein said method comprises that part removes moisture to form viscous liquid, further to the slurry evacuation to cause ' boiling ', then be lower than drying under 100 ℃ the temperature basically.This method still runs into some traditional lyophilizing difficult problem.When in big freeze dryer, carrying out this method, depend on that their position samples on the shelf can be with different speed dryings, it has caused in dry run, the activity of different sample loss varying numbers.It has caused conforming shortage in a collection of product.
Up to now, the report that does not prepare the examples of many successful that keeps high-level antigenicity and/or immunogenic IPV drying solid bacterin preparation.
Therefore, the invention discloses the immunogenic composition of a kind of IPV of comprising and stabilizing agent, be formulated as dry compositions or high viscosity liquid, behind reprovision, can produce the immunne response of poliomyelitis virus.The existence of stabilizing agent is crucial for antigenic preservation, and polyol is proved to be effective stabilizer.IPV is more suitable for drying in the presence of bacterial polysaccharides, can make original antigen keep more a high proportion of antigenicity and/or immunogenicity.The present invention includes and preserve the method for compositions that comprises IPV, preferably have polyol and bacterial polysaccharides, wherein the antigenicity of IPV and/or immunogenicity obtain keeping.Compare with the independent lyophilizing of IPV, in the presence of polysaccharide, the lyophilizing of IPV causes the IPV antigen conservation rate improved.In addition, by be formulated as drying solid or high viscosity liquid with IPV, the immunogenicity of Hib also is enhanced.Particularly, when with the instant reprovision of DTP vaccine (as described below), the inventor has found that the Hib titre not because of the aluminium hydroxide of DTP vaccine becomes branch to reduce, and is the same with the situation that does not have IPV.
Employed drying means also can influence IPV antigenicity and/or immunogenic conservation rate.For dry IPV, the conventional Freeze Drying Technique of foaming drying means is more effective aspect the antigenicity that keeps IPV.Surprisingly, in the foaming drying means, comprise freezing step and do not cause the antigenicity loss, can promote the development of quick and effective store method all the better.The another kind of preferable methods of the present invention has kept high-caliber IPV antigenicity and/or immunogenicity, contains the sample of IPV by drying, need not freezing or foaming, forms drying agent, preferred high viscosity liquid preparation.
The invention provides the drying agent of a kind of IPV, it has the advantage of storage-stable.Described drying agent can be just before administration reprovision fast and easily.Using under the preferred foaming drying means situation, because the huge surface area of described cake, the cake of foaming is reprovision especially easily.
The added advantage of IPV and Hib drying solid or high viscosity liquid preparation comprises the enhanced immunogenicity of Hib composition.As everyone knows in multicomponent vaccine, other composition of bacterin preparation can cause to the immunogenic interference of Hib (WO96140242, WO97/00697).In drying agent, comprise IPV and Hib and can alleviate this problem, particularly before administration, exsiccant IPV-Hib compositions is mixed with diphtheria, tetanus and pertussis composition.
Though in the presence of bacterial polysaccharides, might use conventional freeze-drying method lyophilizing IPV, the preferred drying means that uses the foaming dry technology or do not comprise the gentleness of freezing or foaming.These methods have caused in IPV even more antigenicity and/or immunogenicity conservation rate, the also easier and reprovision more quickly of formed cake.Than the Freeze Drying Technique of standard, described method also has quicker and the advantage of high energy efficiency more.Because step of freeze drying rate-limiting step normally in production of vaccine, use preferable methods can produce the more vaccine product of high-magnitude, and need not equipment is carried out additional investment.In preferred foaming drying means, introduce the repeatability in batches that freezing step has also produced improvement.
Description of drawings
Fig. 1-contain is at the photo of the phial of the preservation sample of foaming drying means different phase.
A-has shown the outward appearance of preserving sample when carrying out lyophilization with liquid preparation.
B-has shown the outward appearance of preserving sample when pressure is reduced to 1.5 millibars.Based on the different condition of each phial, sample begins with discrepant slightly speed freezing.
C-has shown the outward appearance of preserving sample under 0.1 millibar, and wherein all samples has become freezing fully.
D-has shown the outward appearance of preserving sample when pressure is increased to the 0.8-3.5 millibar.When preserving sample foaming and solvent evaporation, formed foam glass.
Fig. 2-the photo of high viscosity liquid in inverted phial.
Detailed Description Of The Invention
Immunogenic composition of the present invention
The present invention includes immunogenic composition, be formulated as drying solid or high viscosity liquid, comprise IPV and stabilizing agent, wherein the antigenicity of IPV and/or immunogenicity obtain keeping behind reprovision. The drying solid of IPV or high viscosity liquid preparation can produce immune response, protective immune response preferably, and poliomyelitis virus is preferably after reprovision and inoculation.
IPV is defined as the poliovirus (preferably being contained in 1,2 and 3 types of thinking standard in the vaccine field, most preferably the Salk polio vaccine) of deactivation. The vaccine dose of IPV comprises 20-80, preferred 40 or 1 type (Mahoney) of 80D-antigen unit, 4-16, preferred 8 or 2 types (MEF-1) of 16D-antigen unit and 20-64, preferred 32 or 3 types (Saukett) of 64D-antigen unit.
When dry by method of the present invention, preferred 1,2 or all 3 kind 1, the antigenicity of 2 and 3 type polioviruses obtain keeping; More preferably 1 type; 2 types; 3 types; 1 type and 2 types; 1 type and 3 types; The antigenicity of 2 types and 3 types obtains keeping; Or 1 type, 2 types and 3 types keep the antigenicity level of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% reference sample, and described reference sample is not accepted dry the processing. Behind the reprovision, comprise by ELISA by any suitable method in the aqueous solution that at drying solid or high viscosity liquid it has used polyclone and/or the monoclonal antibody of anti-1,2 and/or 3 type polioviruses, can measure antigenicity.
When by method of the present invention when dry, preferred 1,2 or all 3 kind 1, the immunogenicity of 2 and 3 type polioviruses obtain keeping; More preferably 1 type; 2 types; 3 types; 1 type and 2 types; 1 type and 3 types; The immunogenicity of 2 types and 3 types obtains keeping; Or 1 type, 2 types and 3 types keep the immunogenicity level of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% reference sample, and described reference sample is not accepted dried.In aqueous solution, behind the reprovision, can measure antigenicity at drying solid or high viscosity liquid by any suitable method.In a kind of preferable methods, described drying agent obtains reprovision in aqueous solution, and gives animal inoculation, preferred rat.After suitable a period of time, from the animal of inoculation, collect antiserum and test sera conversion.Preferably, compare, reach at least 0.4,0.5,0.6,0.7,0.8 or 0.9 relative potency with undried reference sample.
The drying solid compositions is a kind of by lyophilizing, distillation, evaporation or the dry preparation that desolvates that removes, described lyophilizing, distillation, evaporation or drying make dissolvent residual be less than or equal to 15%, 12%, 10%, 7%, 5%, 4%, preferred 3%, 2% or more preferably 1%.Term " drying solid " comprises glass, rubber or has the crystalline solid of solid appearance.Arbitrary said method can be used for preparing described drying solid.By distillation, boiling or evaporation, preferably remove and desolvate by evaporation.
High viscosity liquid is defined as to have solvent and is less than or equal to 15,12,10, preferred 8,5,4,3,2 or 1% material.High viscosity liquid has enough low solvent, make activating agent under 4 ℃, preserve at least 3,6,9,12 or 24 months with stable state, after during this period of time, allow that activating agent keeps at least 40,50,60, preferred 70,80,90,95% its antigenicity and/or immunogenicity.High viscosity liquid is not exposed to and relates to the foaming formation thing that foam forms.Preferably, high viscosity liquid has the solid appearance except glass, after several days, in preferred several weeks, more preferably behind the some months, can flow very lentamente.
Immunogenic composition of the present invention is formulated as drying solid or high viscosity liquid, comprises IPV and stabilizing agent and preferred bacterium polysaccharide.Described stabilizing agent is arbitrary following compositions.Described bacterial polysaccharides comprises the capsular polysaccharide that is derived from any antibacterial, preferred one or more Neisseria meningitidiss (Neisseria meningitidis) of described antibacterial, b type Haemophilus influenzae (Haemophilusinfluenzae b), streptococcus pneumoniae (Streptococcus pneumoniae), A group B streptococcus, B group B streptococcus, staphylococcus aureus (Staphylococcus aureus) or staphylococcus epidermidis (Staphylococcus epidermidis).
The PRP capsular polysaccharide of preferred b type Haemophilus influenzae (Haemophilus influenzae b) is present in drying solid or the high viscosity liquid.In another preferred embodiment, described immunogenic composition comprises the drying solid or the high viscosity liquid preparation of capsular polysaccharide, and described capsular polysaccharide is derived from serum group A, C, W-135 and the Y (meningococcal polysacharide) of one or more Neisseria meningitidiss (Neisseria meningitidis).Another embodiment preferred comprises drying solid or the high viscosity liquid preparation that is derived from streptococcus pneumoniae (Streptococcus pneumoniae) capsular polysaccharide.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 from serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F).Another embodiment preferred comprises 5 types, the 8 or 336 type capsular polysaccharides of staphylococcus aureus (Staphylococcus aureus).Another embodiment preferred comprises I type, II type or the III type capsular polysaccharide of staphylococcus epidermidis (Staphylococcusepidermidis).Another embodiment preferred comprises Ia type, Ic type, II type or the III type capsular polysaccharide of B group B streptococcus.Another embodiment preferred comprises the capsular polysaccharide of A group B streptococcus, preferably further comprises at least a M albumen and more preferably comprises polytype M albumen.
In one embodiment of the invention, described bacterial polysaccharides is a total length, the natural polysaccharide of purification.In an optional embodiment of the present invention, described polysaccharide be arranged in by size 2-20 doubly between, preferred 2-5 doubly, 5-10 doubly, 10-15 doubly or 15-20 doubly between, so more little just being easy to more of the big minispread of polysaccharide handled.Used oligosaccharide in preferred embodiments.Oligosaccharide has 2-20 recurring unit usually.
The present invention further comprises the immunogenic composition that comprises more than one bacterial polysaccharideses and IPV as drying solid or high viscosity liquid.Preferably, IPV and one or more Hib (b type Haemophilus influenzae) PRP polysaccharide and/or meningococcus A, C, W and/or Y polysaccharide and/or pneumococal polysaccharide combination.Most preferably, described activating agent comprises IPV and Hib; IPV and MenC; IPV and Hib and MenC; IPV and MenA and C; IPV and Hib and Men A and C; IPV and Hib and Men A and C and Y; Or IPV and Hib and Men C and Y.
The above-mentioned activating agent of enumerating also comprises one or more pneumococcal capsular polysaccharides as described below.
In above-mentioned composition, use polysaccharide, also can use oligosaccharide (as defined above).
Though these compositionss can be used adjuvant (as described below), they preferably do not use adjuvant or preferably do not comprise aluminum salt.
Preferably, described polysaccharide or oligosaccharide are incorporated into peptide or the carrier protein (as described below) that comprises the auxiliary epi-position of T-.
To be present in capsular polysaccharide in the immunogenic composition of the present invention be unconjugated or be incorporated into carrier protein such as tetanus toxoid, tetanus toxoid fragment C, diphtheria toxoid, CRM197, pneumolysin, protein D (US6342224).Tetanus toxoid, diphtheria toxoid and pneumolysin are arbitrary by genetic mutation and/or preferably detoxify by chemical treatment.The preferred embodiment of the invention contains the Hib that is incorporated into tetanus toxoid.
Have more than one bonded polysaccharide in immunogenic composition of the present invention, described polysaccharide is incorporated into identical carrier protein or different carrier proteins.The preferred embodiment of the invention comprises the meningococcal polysacharide that is incorporated into carrier protein.In the presence of bonded Hib and meningitis polysaccharide, they are incorporated into identical carrier protein or different carrier proteins.
Can prepare described polysaccharide conjugate by any known coupling technology.In a kind of preferred coupling technology, described polysaccharide is through the thioether bond coupling.This associated methods depends on the activation of polysaccharide and Tetrafluoroboric acid 1-cyano group-4-dimethylaminopyridine (CDAP) to form cyanate.Therefore, activatory polysaccharide can be directly or by the amino coupled on base at interval and the carrier protein.Preferably, use to comprise xenogenesis connection (heteroligation) chemical reaction that forms thioether bond, be incorporated into carrier protein with cyanate and hexamethylene diamine coupling and with the deutero-polysaccharide of amino.The PCT that above-mentioned conjugate is described in Uniformed Services University openly applies among the WO93/15760.
Described conjugate also can be by direct reductive amination method preparation, and described method is described among US 4365170 (Jennings) and the US 4673574 (Anderson).Other method is described among EP-0-161-188, EP-208375 and the EP-0-477508.
Another kind method comprises the polysaccharide of the deutero-cyanogen bromide-activated of adipic acid hydrazides (ADH) by carbodiimide condensation and protein carrier coupling Infect.Immunity such as (, 1,983,245 256) Chu C..
Mix and not to be adsorbed on the adjuvant as its a part of polysaccharide in the immunogenic composition of the present invention or to be adsorbed on the adjuvant the preferred aluminum salt of described adjuvant (aluminum phosphate or aluminium hydroxide), most preferably aluminum phosphate.
Immunogenic composition of the present invention has comprised can help to prevent the stabilizing agent that damages in dry run.Arbitrary stabilizing agent is described below, and comprises the glass shape polyol that can mix immunogenic composition, no matter is to use the prepared drying solid of method of the present invention, foamed glass or high viscosity liquid compositions.Preferred stabilizing agent comprises sucrose, Sorbitol, lactose and trehalose.
Described preferred chemical compound by method drying of the present invention, can make up with other antigen in combined vaccine, and described vaccine is exsiccant or is used for the liquid preparation of reprovision dry ingredient.
Extra composition
Having mixed the drying solid of the present invention or the high viscosity liquid preparation of IPV and stabilizing agent can prepare with another kind of vaccine composition in addition.The preferred vaccine that comprises IPV drying solid or high viscosity liquid preparation and bacterial polysaccharides can mix with the liquid preparation that comprises extra vaccine composition.Behind solid constituent and liquid component reprovision, complete vaccine is used by injection.
Extra composition comprises the capsular polysaccharide that is derived from one or more Neisseria meningitidiss (Neisseriameningitidis), streptococcus pneumoniae (Streptococcus pneumoniae), A group B streptococcus, B group B streptococcus, staphylococcus aureus (Staphylococcus aureus) or staphylococcus epidermidis (Staphylococcus epidermidis).In a preferred embodiment, described immunogenic composition comprises the capsular polysaccharide of serum group A, the C, W-135 and the Y that are derived from one or more Neisseria meningitidiss (Neisseria meningitidis).Another embodiment preferred comprises and is derived from streptococcus pneumoniae (Streptococcuspneumoniae) capsular polysaccharide.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 from serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F).Another embodiment preferred comprises 5 types, the 8 or 336 type capsular polysaccharides of staphylococcus aureus (Staphylococcus aureus).Another embodiment preferred comprises I type, II type or the III type capsular polysaccharide of staphylococcus epidermidis (Staphylococcusepidermidis).Another embodiment preferred comprises Ia type, Ic type, II type or the III type capsular polysaccharide of B group B streptococcus.Another embodiment preferred can comprise the capsular polysaccharide of A group B streptococcus, preferably further comprises at least a M albumen and more preferably comprises polytype M albumen.
Immunogenic composition of the present invention can be prepared with proteantigen.Preferred pneumoprotein antigen is those pneumoproteins that are exposed to the streptococcus pneumoniae outer surface (can be discerned by host immune system at least a portion of streptococcus pneumoniae biocycle), or is the albumen that streptococcus pneumoniae is secreted or discharge.Most preferably, described albumen is the lipoprotein of toxin, adhesin, binary signal transducin or streptococcus pneumoniae (Streptococcus pneumoniae), or their fragment.Concrete preferred albumen includes but not limited to: pneumolysin (preferably by the chemical treatment or the antidotal that suddenlys change) [.Nucleic Acids Res.1990 Jul11 such as Mitchell; 18 (13): 4010 " Comparison of pneumolysin genes and proteins fromStreptococcus pneumoniae types 1 and 2. " .Biochim BiophysActa such as Mitchell 1989 Jan 23; 1007 (1): 67-72 " Expression of the pneumolysin genein Escherichia coli:rapid purification and biological properties. ", WO96/05859 (A.Cyanamid), WO 90/06951 (Paton etc.), WO 99/03884 (NAVA)]; PspA and stride film disappearance variant (US 5804193-Briles etc.); PspC and stride film disappearance variant (WO 97/09994-Briles etc.); PsaA and stride film disappearance variant (Berry and Paton, Infect Immun 1996 Dec; 64 (12): 5255-62 " Sequenceheterogeneity of PsaA, a 37-kilodalton putative adhesin essential forvirulence of Streptococcus pneumoniae "); Streptococcus pneumoniae choline binding protein and stride film disappearance variant; CbpA and stride film disappearance variant (WO 97/41151; WO99/51266); Glyceraldehyde-3-phosphate-dehydrogenase (Infect.Immun.1996 64:3544); HSP70 (WO 96/40928); PcpA (.FEMS Microbiol Lett1998 such as Sanchez-Beato, 164:207-14); M sample albumen, (EP 0837130) and adhesin 18627, (EP 0834568).Another kind of preferred pneumoprotein antigen is to be disclosed among the WO98/18931 those, particularly in WO 98/18930 and PCT/US99/30390 selected those.
Comprise TbpA (WO93/06861 with the preferred eisseria albumen of immunogenic composition preparation of the present invention; EP586266; WO92/03467; US5912336), TbpB (WO93/06861; EP586266), Hsf (WO99/31132), NspA (WO96/29412), Hap (PCT/EP99/02766), PorA, PorB, OMP85 (also being known as D15) (WO00/23595), PilQ (PCT/EP99/03603), PIdA (PCT/EP99/06718), FrpB (WO96/31618 is referring to SEQ ID NO:38), FrpA or FrpC or with both total at least 30,50,100,500,750 amino acid whose conservative 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), MItA (WO99/57280) and ctrA (PCT/EP00/00135).Albumen or partial outer membrane vesicle prepared product that eisseria albumen can be used as purification add.
Immunogenic composition is preferably prepared with the antigen that provides anti-one or more diphtheria, tetanus and Bordetella pertussis (Bordetella pertussis) to infect protection.But described pertussis composition deactivation, it is (Pw) or preferably acellular pertussis (Pa) of whole cell pertussis Bordetella (B.pertussis), described acellular pertussis contains from least a antigen of PT, FHA and 69kDa Bordetella pertussis adhesin (preferred two kinds or all three kinds), some other acellular vaccine also comprises agglutinogen, for example Fim2 and Fim3 also consider to use these vaccines in the present invention.In general, providing the antigen of diphtheria and tetanus protection is diphtheria toxin, diphtherotoxin and tetanus toxin.Described toxin is the toxin of chemical ablation, for example uses formaldehyde treated subsequently, or by importing the toxin of one or more point mutation deactivations.
Alternatively, immunogenic composition of the present invention can provide by medicine box, contains described drying solid, foamed glass or high viscosity liquid and contain liquid D TPa or DTPw in a kind of container in another kind of container.Above-mentioned medicine box can for example comprise double-chamber syringe, contains described drying solid and liquid component in the different chamber of said syringe.Then, before injection, use the described dry ingredient of aqueous vaccine reprovision immediately, as single vaccine.Therefore for example, with liquid D TPa or DTPw vaccine reprovision drying solid of the present invention, foamed glass or high viscosity liquid (preferably immediately) and with single vaccine administration.Described DTPa or DTPw vaccine use aluminum salt as adjuvant usually at least in part, as aluminum phosphate and/or the aluminium hydroxide (Infanrix of GlaxoSmithKlineBiologicals s.a. for example And Tritanrix
Figure 10003_4
Vaccine).
Described immunogenic composition randomly with can protect the host anti-can't type (non-typeable) moral Haemophilus influenzae (Haemophilus influenzae), one or more antigens of RSV, and/or can protect one or more antigens preparations of host's resisiting influenza virus.Preferably can't type moral Haemophilus influenzae (H.influenzae) proteantigen comprise Fimbrin albumen (US 5766608) and comprise fusion rotein (as the LB1 fusion rotein) (US 5843464-Ohio State Research Foundation) from the peptide among OMP26, P6, protein D, TbpA, TbpB, Hia, Hmw1, Hmw2, Hap and the D15.
Preferred influenza antigen comprises entirely, lives or inactivation of viruses, the division influenza virus, in egg or mdck cell, grow, or vero cells (Vero cells) or full influenza virus corpusculum are (as R.Gluck, Vaccine, 1992,10,915-920 describes) or albumen their purification or reorganization, for example HA, NP, NA or M albumen, or their combination.
Preferred RSV (respiratory syncytial virus) antigen comprises F glycoprotein, G glycoprotein, HN albumen, M albumen or derivatives thereof.
The combined vaccine that comprises DTP-Hib is known in this field.But exist a difficult problem relevant with some preparation, described preparation relates to Hib and other antigenic simple mixing.Unless preparation modestly, otherwise because the interference of other composition of vaccine, the antibody titer of the anti-Hib composition of increase may be lower than by independent inoculation same dose Hib caused those.Even this difficult problem is that this area is well-known, and has solved by the whole bag of tricks already, for this difficult problem, wherein Hib and the IPV immunogenic composition of the present invention that is formulated as drying solid or high viscosity liquid together provides a kind of optional solution.
Immunogenic composition of the present invention can consist of the part of vaccine medicine box, and wherein there are another kind of composition in IPV and Hib with a kind of composition form of medicine box, as mentioned above, exist with the second composition form, for instance, double-chamber syringe as described in this article.Only before using described vaccine, two kinds of compositions are mixed together.In above-mentioned preparation, comprise the preferred drying solid of composition, foamed glass or the high viscosity liquid of IPV and Hib, even it randomly is formulated as liquid.The antibody titer of the anti-Hib composition that said preparation causes is acceptable clinically, so that the protection of anti-b type Haemophilus influenzae pathogen to be provided.In general, the antibody titer in the combined vaccine be in the unit price Hib vaccine the caused titre of same dose Hib at least 85%, 90%, preferred about 100% or more.
Vaccine of the present invention
The immunogenic composition of the invention described above preferably is formulated as vaccine.Preferably, described vaccine comprises an amount of adjuvant of enough enhancings to immunogenic immunne response.Suitable adjuvant includes but not limited to, such as aluminum salt, Squalene mixture (SAF-1), muramyl peptide, saponin derivative, mycobacteria cell wall preparation, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactant, Quil A, b subunit of cholera toxin, polphosphazene and derivant and the immunostimulating complex (ISCOMs) of aluminium hydroxide and aluminum phosphate, be Takahashi etc. as those. (1990) Nature 344:873-875 is described.Use or be used for producing for the antibody for the veterinary, can use the mitogenesis composition of Freund adjuvant animal.
Bacterin preparation of the present invention is reprovision before use preferably.The liquid component that reprovision relates to vaccine mixes with drying solid of the present invention, foamed glass or high viscosity liquid preparation.The present invention also relates to a kind of container with waterproof inner surface, described container contains immunogenic composition of the present invention or vaccine.It is favourable using this container, because it makes dry compositions place the test tube bottom, exists with the form that is easier to reprovision.
The favourable part of mixing coloured dyestuff in preserving sample is to manifest so that allow that dry compositions of the present invention is easier.It is a particular importance in the reprovision process, can guarantee drying solid or the abundant reprovision of high viscosity liquid before use.Preferably, described coloured dyestuff keeps its color under neutral pH, and compatible with the injection that injects the patient.Most preferred coloured dyestuff is phenol red.
When using all immunogenic compositions or vaccine, immunogenic immune effective dose should be determined by experience.The factor that should consider comprises immunogenicity, and no matter immunogen and adjuvant or carrier protein or other carrier are compound or covalently bound, the consumption of route of administration and immunizing agent to be applied.Above-mentioned factor is that the vaccine field is known, and carries out this decision to be entirely immunologist's technical ability and need not undo experimentation.
Material can exist by variable concentrations in immunogenic composition of the present invention.Generally speaking, the Cmin of material is the quantity that must reach its desired use, and Cmax is can be retained in the solution or evenly be suspended in maximum quantity in the initial mixture.For example, the minimum number of therapeutic agent preferably can provide the quantity of single therapy effective dose.If before crystallization, form foamed glass, also can use supersaturated solution.For bioactive substance, for the necessary quantity of biological activity, Cmax was even suspension residing concentration can't keep the time when Cmin was reprovision.Under the single dosage unit situation, described quantity is the quantity that single therapy is used.As a rule, estimate that each dosage can comprise the proteantigen of 1-100ug, preferably 5-50ug and more preferably 5-25ug.Preferred bacterial polysaccharides dosage is 10-20ug, 10-5ug, 5-2.5ug or 2.5-lug.But the preferred amount of different material is to be easily those skilled in the art to determine between the various materials.
Method of the present invention
Method of the present invention is to be used for preserving comprising IPV and stabiliser compositions, produced the compositions that the antigenicity of IPV wherein obtains keeping.Preferably, the bacterial polysaccharides that mixes in described sample should be exsiccant.
In one embodiment, method of the present invention relates to dry IPV and comprises step:
By in stabiliser solution, suspending or dissolving IPV preparation preservation sample; There are bacterial polysaccharides and/or glass shape polyol in the preferably described preservation sample;
Make described preservation sample stand following temperature and pressure condition, i.e. the condition from preserve sample, lost of solvent; With
Removing desolvates forms solid or high viscosity liquid until described preservation sample drying, and wherein the antigenicity of IPV and/or immunogenicity obtain keeping.
In a preferred embodiment, described preservation sample is loaded to and carries out drying in the container with waterproof inner surface.
It is dry that another kind of method of the present invention relates to foaming, comprises step:
By in stabiliser solution, suspending or dissolving IPV preparation preservation sample; There are bacterial polysaccharides and/or glass shape polyol in the preferably described preservation sample;
Make described preservation sample stand following temperature and pressure condition, promptly described preservation sample forms foamy condition; With
Removing desolvates forms solid until described foam-drying, and wherein the antigenicity of IPV and/or immunogenicity obtain keeping.
A kind of preferred foaming drying means of the present invention has used the container with waterproof inner surface, comprises step:
By in stabiliser solution, suspending or dissolving IPV and bacterial polysaccharides preparation preservation sample preferably;
Described preservation sample packed into have in the container of waterproof inner surface;
Make the described container of preserving sample that contains stand following temperature and pressure condition, make described preservation sample form foam by described temperature and pressure condition;
Removing desolvates forms solid until described foam-drying, and wherein the antigenicity of IPV and/or immunogenicity obtain keeping.
The foaming drying means of the invention described above randomly comprises freezing step.Described preservation sample can be all or part of freezing.Therefore some method of the present invention comprises step:
By in stabiliser solution, suspend or dissolving IPV and preferably bacterial polysaccharides prepare to the refrigerated preservation sample of small part and freezing described mixture;
Make and describedly stand following temperature and pressure condition to the refrigerated preservation sample of small part, promptly described preservation sample forms foamy condition; With
Removing desolvates forms solid until described foam-drying, and wherein the antigenicity of IPV and/or immunogenicity obtain keeping.
The freezing step of said method is preferably used the refrigerated processing of chilling, is to cause refrigerated reason by the evaporation decompression wherein.It causes the sample quick freezing, makes antigen losses less.Therefore a kind of method of the present invention comprises step:
By in stabiliser solution, suspending or dissolving IPV and bacterial polysaccharides preparation preservation sample preferably;
Make described preservation sample stand decompression, make described preservation sample become to small part refrigerated;
Make and describedly stand following temperature and pressure condition to the refrigerated preservation sample of small part, promptly described preservation sample forms foamy condition; With
Removing desolvates forms solid until described foam-drying, and wherein the antigenicity of IPV and/or immunogenicity obtain keeping.
The another kind of preferable methods of the present invention is to be used to preserve IPV, comprises step:
By in stabiliser solution, suspending or dissolving IPV preparation preservation sample:
Make described preservation sample stand following temperature and pressure condition, promptly described preservation sample is by the evaporation release solvent, and non-foaming formation foam and preferably need not freezing;
Removing desolvates forms high viscosity liquid until described sample drying, and wherein the antigenicity of IPV and/or immunogenicity obtain keeping.
The IPV preparation that method of the present invention is produced can stand Long-term Storage, and the antigenicity of IPV and/or immunogenicity obtain keeping in storage.Preferably, storage is after at least 3,6,9,12,24 months down at 4 ℃, and IPV has kept at least 40,50,60,70, preferably initial antigenicity of its of 80,90,95% and/or immunogenicity.Behind the IPV reprovision, in suitable aqueous solution and use suitable method, for example those above-mentioned methods are measured antigenicity and immunogenicity.
Need not drying means freezing or foaming and be specially adapted to the activating agent that will be dried, wherein said activating agent is in dry run, owing to being exposed to freezing or foam forms and is easy to lose activity and/or antigenicity in the relevant foaming.It also is specially adapted to the glass shape polyol of low concentration wherein is favourable and/or preferred shorter dry run wherein.
Stabilizing agent
The stabilizing agent that is used for the inventive method can preferably comprise glass shape polyol.Suitable material includes but not limited to, all polyols comprise saccharide and non-saccharide polyol.Preferably, described stabilizing agent can make activating agent obtain storage, does not lose activity basically by degeneration, gathering or other method.Specially suitable raw material comprises saccharide, sugar alcohols and carbohydrate derivative.Preferably, described glass shape polyol is the saccharide or derivatives thereof, comprise glucose, maltulose, isomaltulose, lactulose, sucrose, maltose, lactose, dextrinose, maltose alcohol, lactose, isomalt (palatinit), trehalose, Raffinose, stachyose, melezitose or glucosan, most preferably trehalose, sucrose, Sorbitol, Raffinose, mannitol, lactose, lactose or isomalt.
Bacterial polysaccharides is as stabilizing agent, and the preferred embodiment of the invention is mixed the composition of bacterial polysaccharides as described stabilizing agent.Described in this embodiment bacterial polysaccharides is being played the part of stabilizing agent and immunogenic dual role.
Saccharide includes but not limited to, monosaccharide, disaccharide, trisaccharide, oligosaccharide and their corresponding sugar alcohols, such as polyol, hetastarch and the saccharide copolymer of the saccharide of carbohydrate derivative and chemical modification.Natural and synthetic saccharide all is suitable for using.Synthetic saccharide includes but not limited to, has those of the glycosidic bond that replaces for mercaptan or carbon bond.D and L type saccharide all can use.Described saccharide can be non-reducing or reductive.When wherein using recuding sugars, preferably add the inhibitor of maillard reaction (Mailard reaction).
Be suitable for recuding sugars that the present invention uses and be well known in the art those, include but not limited to glucose, maltose, lactose, fructose, galactose, mannose, maltulose and lactulose.Non-reducing saccharide includes but not limited to, is selected from the non-reduced glycoside of the polyol of sugar alcohols and other straight chain polyol.Other useful saccharide comprises Raffinose, stachyose, melezitose, glucosan sucrose, cellobiose, mannobiose and sugar alcohols.The preferred monosaccharide glycoside of described sugar alcohol glycoside (monoglycosides) particularly is the chemical compound that obtains by reduction such as the disaccharides of lactose, maltose, lactulose and maltulose.
Particularly preferred saccharide is trehalose, sucrose, Sorbitol, maltose alcohol, lactose, isomalt and glucopyranosyl-1 → 6-mannitol.
Aminoacid can be used as stabilizing agent and can use separately and preferably unite use with polyol.Though all aminoacid or amino acid whose combination, peptide, hydrolyzed protein or can be used as stabilizing agent such as sero-abluminous albumen, preferred amino acids comprises glycine, alanine, arginine, lysine and glutamine.
Preferably, described preservation sample can contain and can suppress the composition that crystallization forms in drying solid of the present invention or the high viscosity liquid.Salt and comprise aminoacid and phenol red other molecules in inhibiting crystallization forms.
The concentration of the stabilizing agent of Shi Yonging can be at 1%-50%w/v, preferably at 1-5%, 5-10%, 5-10%, 15-20%, 20-25% or 25-50%, most preferably less than 25% (w/v) in the methods of the invention.The quantity of the quantity of required stabilizing agent and the salt of existence is proportional.Therefore, although the stabilizing agent level of 3%-10% is preferred, the drying sample with high content of salt may need the stabilizing agent level of 10%-25% (w/v) higher concentration.
Container
Difference and mixture and various container profile and size can be processed simultaneously.Ideally, employed container dimensional is enough to be used to hold initial mixture and can to provide capacity to the drying agent of its formation.In general, the surface area of its quality, described container and determined that the temperature and the pressure condition that whether foam are determined by glass shape material.The quality that glass forms material should be enough to form the viscosity slurry, randomly foams, and in fact changes the minimum mass of every vessel surface per unit area into.This ratio changes with mixture and the different of employed container, but it is easy to can determine by rule of thumb by following listed herein method for those skilled in the art.Any above-mentioned spendable container comprises the phial of Wheaton crystallizer and pipe truncate.
Method of the present invention is preferably used the container with waterproof inner surface.Can reach this purpose by lining hydrophobic composition on inner surface, for example pass through silicification.Can finish silicification by the well-known method of those skilled in the art.In one approach, described container obtains silicidation by be full of (rising) internal tank with the silicones emulsion, then by the baking oven high-temperature process, usually at 350 ℃.Alternatively, described waterproof inner surface can be by being realized by the container of water-proofing composition preparation.
The waterproof inner surface of described container can be more suitable for taking place foaming and the more repeatability of tool.It allows the polyol that uses low concentration in preserving sample, can reduce the required time span of drying sample successively, reduce maillard reaction (Mailard rections) influence or with other interaction of the polyol of infringement activating agent.Comprise vaccine if preserve sample, because the polyol of low quantity exists, the foamed glass that is produced can reach reprovision easily fast, and the lower viscosity of vaccine solution tool that is produced, and is more convenient for using.Described waterproof inner surface is allowed more easily reprovision drying solid or high viscosity liquid, makes it be easier to effective reprovision because it impels sample to remain on container bottom.
Although single type can be used for herein, can there be more than one stabilizing agent, more than one additive and more than one material.The effective dose of these compositions is determined by those skilled in the art easily.
Solvent
Obtain described preservation sample by dissolving/suspension IPV and stabilizing agent in water with the preparation aqueous solution.Preferably, water is present in the 5-98% volume level to be preserved in the sample, more preferably 80-98% volume, most preferably 85-98% volume.
The volume of solvent can change, and depends on stabilizing agent and the material that will mix and all additives.Required minimum volume is the quantity that must be used to dissolve various compositions.But, also can use the homodisperse suspension of described material.The suitable quantity of composition is determined according to the embodiment that this paper provided by those skilled in the art easily in specific embodiments.
Various additives can be put into described preservation sample.In general, additive enhanced foaming and/or dried and/or help the dissolving of material.Alternatively, additive helps the Stability of Substance of mixing in solid.Can there be one or more additives.
As an example, in foam glass, add volatility/foaming salt and allow bigger initial volume and produce higher surface area, therefore can reach and bubble preferably with dry more quickly.The volatility salt of Shi Yonging is to be used to produce evaporable salt under the condition of foam glass in this article.The example of suitable volatility salt includes but not limited to, ammonium acetate, ammonium bicarbonate and ammonium carbonate.Decompose the salt that produces gaseous product and also can reach enhanced foaming and more quick-drying purpose.The example of above-mentioned salt is sodium bicarbonate and sodium metabisulfite.Preferably, described volatility salt exists with the quantity of about 0.01-5M.Concentration up to 5M is adapted at using herein.The foam glass that is produced has identical foam conformation, and apparent in view drier with the foam glass that does not wherein use volatility/foaming salt.
Another kind of suitable additive is a foam stabiliser, and it can be used in combination with volatile salts or salt decomposition.It can be the reagent that maybe can increase the viscosity of foaming slurry such as the surface active ingredient of amphipathic molecule (that is, for example phospholipid and surfactant), for example such as guar gum and the derivant thickening agent.
Other additive is the inhibitor of maillard reaction (Maillard reaction).Preferably, if described material and/or glass matrix moulding material comprise carbonyl and amino, imino group or guanidine radicals, described compositions further comprises the inhibitor that at least a physiology goes up acceptable maillard reaction (Maillard reaction), and quantity amino and the active carbonyl group condensation exists in described compositions effectively to stop substantially.The inhibitor of described maillard reaction (Maillard reaction) can be any known inhibitor in this area.Described inhibitor is with enough preventions or stop the quantity of amino and active carbonyl group condensation to exist basically.In general, described material provides amino and glass matrix moulding material that carbonyl is provided, or opposite.Yet amino and carbonyl can be present in described material and the carbohydrate molecule in described material or saccharide.
Known polytype compound exhibits plays inhibitory action to maillard reaction (Maillard reaction), therefore can be used in the compositions as herein described.These chemical compounds are the competitiveness or the noncompetitive inhibitor of maillard reaction (Maillard reaction) normally.Competitive inhibitor includes but not limited to, the combination of amino acid residue (D and L), amino acid residue and peptide.Particularly preferably be lysine, arginine, histidine and tryptophan.Lysine and arginine are the most effective.Exist many known noncompetitive inhibitors.These include but not limited to, aminoguanidine and derivant and amphotericin B.EP-A-0 433 679 has also described suitable wheat rad (Maillard) inhibitor, and it comprises 4-hydroxyl-5,8-dioxy quinoline.
Activating agent
Method of the present invention is used to preserve inactivated poliovirus (IPV-is preferably included in the vaccine field 1,2 and 3 types as standard, more preferably Salk poliomyelitis vaccine).IPV comprises the 20-80, preferred 40 or 8D-antigen unit (Mahoney) of 1 type, the 4-20 of 2 types, preferred 8 or the 20-64 of 16D-antigen unit (MEF-1) and 3 types, preferred 32 or 64D-antigen unit (Saukett).Described IPV bacterin preparation is suitable for injection behind the reprovision in aqueous solution, it preferably contains extra vaccine composition.
The bacterial polysaccharides that mixes in the inventive method is the capsular polysaccharide that for example is derived from one or more Neisseria meningitidiss (Neisseria meningitidis), b type Haemophilus influenzae (Haemophilusirafluenzae b), streptococcus pneumoniae (Streptococcus pneumoniae), A group B streptococcus, B group B streptococcus, staphylococcus aureus (Staphylococcus aureus) or staphylococcus epidermidis (Staphylococcus epidermidis), the PRP capsular polysaccharide of preferred Haemophilus influenzae (Haemophilus irafluenzae).Preferred capsular polysaccharide also comprises those of serum group A, the C, W-135 and the Y that are derived from one or more Neisseria meningitidiss (Neisseria meningitidis).Another kind of preferred capsular polysaccharide is derived from streptococcus pneumoniae (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 from serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F).Another embodiment preferred comprises 5 types, the 8 or 336 type capsular polysaccharides of staphylococcus aureus (Staphylococcus aureus).Another kind of preferred polysaccharide comprises I type, II type or the III type capsular polysaccharide of staphylococcus epidermidis (Staphylococcusepidermidis), the Ia type of B group B streptococcus, Ic type, II type or III type capsular polysaccharide.Another kind of preferred polysaccharide comprises the capsular polysaccharide of A group B streptococcus, preferably further comprises at least a M albumen and more preferably comprises polytype M albumen.
Use the preservable preferred activating agent combination of the inventive method to comprise IPV.Preferably, IPV combines with bacterial polysaccharides, and described bacterial polysaccharides comprises one or more Hib PRP polysaccharide and/or meningococcus A, C, W and/or Y polysaccharide and/or streptococcus pneumoniae polysaccharides.Preferred combination comprises IPV and Hib; IPV and MenC; IPV and MenA and C; IPV and Hib and MenC or IPV, Hib, MenA and C.Every kind of bacterial polysaccharides can 1-5 μ g, the dosage of 5-10 μ g, 10-20 μ g or 20-40 μ g exists.
Bacterial polysaccharides is unconjugated or is incorporated into such as tetanus toxoid, tetanus toxoid fragment C, diphtheria toxin, diphtherotoxin, CRM197, pneumolysin or the albumen (carrier protein of US6342224.
Described polysaccharide conjugate can be by any known coupling technology preparation in this area.Preferred associated methods depends on the activation of polysaccharide and Tetrafluoroboric acid 1-cyano group-4-dimethylaminopyridine (CDAP) to form cyanate.Activatory polysaccharide can be directly or by the amino coupled on base at interval and the carrier protein.Preferably, use to relate to the xenogenesis connection chemical reaction that forms thioether bond, cyanate is combined with carrier protein with the hexamethylene diamine coupling and with the deutero-polysaccharide of amino.The PCT that above-mentioned conjugate is described in Uniformed Services University openly applies among the WO93/15760.
Described conjugate can be randomly by direct reductive amination method preparation, and described method is described among US 4365170 (Jennings) and the US 4673574 (Anderson).Other method is described among EP-0-161-188, EP-208375 and the EP-0-477508.
Another kind method relates to the polysaccharide of the deutero-cyanogen bromide-activated of adipic acid hydrazides (ADH) by carbodiimide condensation and protein carrier coupling Infect.Immunity such as (, 1,983,245 256) Chu C..
Dried
In one embodiment, method of the present invention relates to dry IPV in the presence of stabilizing agent, preferably in the presence of bacterial polysaccharides.In the method, preserve the temperature and pressure condition that sample stands to reduce.Described temperature is reduced to less than 20 ℃ or 0 ℃, preferably less than-10 ℃ or-20 ℃, more preferably-40 ℃ or-60 ℃.Described pressure is reduced to less than 1 millibar, or preferably is more preferably 0.05 or 0.01 millibar less than 0.5,0.1 millibar pressure.The temperature and pressure condition of described reduction was kept 10,12,16,20 hours at least, and preferred 24,36 hours, more preferably 48 or 72 hours.Removing desolvates forms solid until described preservation sample drying.
In the application's full text, solid comprises glass, rubber and the crystal that forms drying sample.Above-mentioned solid preferably keeps the water content of 10-20% or 5-10%, the water content of preferred 5-6%, 4-5% or 3-4% or 2-3%, the more preferably water content of 1-2% or 0-1% (w/w).
Foaming is dry
A kind of preferable methods of the present invention relates to makes the preservation sample stand such pressure and temperature condition, so that described sample begins to bubble, forms foam.
Because the heat absorptivity of evaporation process, the temperature of described preservation sample interior can be different from the temperature of this sample outside.Reference temperature is the temperature of preserving the sample external environment condition, and for example, the place using big industrial freeze dryer is the temperature of shelf.It is usually corresponding to the freeze dryer desired temperature.
Embodiment preferred of the present invention is by reducing pressure while holding temperature condition to obtain foaming.Described pressure is adjusted to or is lower than 8,7,6, preliminary election 5,4,3, and more preferably 2,1.5,1, most preferably 0.8 or 0.5 millibar, holding temperature sets value in the temperature that is higher than 0 ℃ simultaneously, preferably at 10 ℃-15 ℃; 15 ℃-20 ℃; 20 ℃-25 ℃; 25 ℃-30 ℃ or 30 ℃-37 ℃.Keep these conditions at least 1,2,3,4,5,8,10,12,16 or 24 hours.
Another embodiment of the invention is kept pressure condition to obtain foaming simultaneously by changing temperature.Described desired temperature is increased to more than 20 ℃, preferably at 20 ℃-30 ℃; 30 ℃-40 ℃; 40 ℃-50 ℃ or 50 ℃-70 ℃; Or described desired temperature is in 10-50 ℃ of scope, preferably at 20-40 ℃, more preferably at 25-35 ℃.Pressure condition maintains pressure reducing horizontal or is lower than 8,7,6, and is preferred 5,4,3, and more preferably 2,1.5,1, most preferably 0.8 or 0.5 millibar.Keep these conditions at least 1,2,3,4,5,8,10,12,16 or 24 hours.
Remove the formation foam glass that desolvates
The foaming drying means of subsequent stage of the present invention relates to except that desolvating until described foam-drying formation solid.In one embodiment of the invention, by keeping the pressure and temperature condition in foaming applied those conditions to reach this purpose in order to obtain.For example, described pressure is maintained at or is lower than 8,7,6, and is preferred 5,4,3, and more preferably 2,1.5,1, most preferably 0.8 or 0.5 millibar, the holding temperature setting value is higher than 0 ℃ simultaneously, preferably at 2 ℃-10 ℃, 10 ℃-20 ℃; 20 ℃-30 ℃; 30 ℃-35 ℃, 35 ℃-40 ℃, most preferably at 5 ℃-25 ℃.Kept these temperature and pressure conditions 1,2,3,4,5,6,8,10,12,18 hours or more, be less than or equal to 10,8,5,4, preferred 3,2 or the solid of most preferably 1% (w/w) so that obtain to have solvent.
Another embodiment of the invention is increased to desired temperature more in the method than the higher desired temperature of early being kept of temperature value in solvent removal process.Its favourable part is to allow that solvent leaves sample with very fast speed, but so that method of the present invention finish within a short period of time.For instance, described desired temperature is increased to more than 0 ℃, preferably at 2 ℃-10 ℃; 10 ℃-20 ℃; 20 ℃-30 ℃; 30 ℃-40 ℃; 40 ℃-50 ℃; 50 ℃-60 ℃, keep simultaneously pressure in or be lower than 8,7,6, preferred 5,4,3, more preferably 2,1.5,1, most preferably 0.8 or 0.5 millibar.Kept these temperature and pressure conditions 1,2,3,4,5,6,8,10,12,18 hours or more of a specified duration, so that obtain to have less than 10,8,5,4, preferred 3,2 or the solid of most preferably 1% (w/w) water content.
Another embodiment of the invention is reduced to pressure set points than the lower pressure set points of employed pressure set points in foaming process in solvent removal process.Its favourable part is to allow that solvent leaves sample with very fast speed, but so that method of the present invention finish within a short period of time.For instance, described pressure set points reduces to or is lower than 5,4,3, and is preferred 2,1,0.8, more preferably 0.5,0.1, most preferably 0.05 or 0.01 millibar, simultaneously holding temperature in or be higher than 0 ℃, preferably at 10 ℃-20 ℃; 20 ℃-30 ℃; 30 ℃-35 ℃ or be higher than 40 ℃.Kept these temperature and pressure conditions 1,2,3,4,5,6,8,10,12,18 hours or more of a specified duration, be less than or equal to 5,4, preferred 3 or 2 or the solid of more preferably 1% (w/w) so that obtain to have solvent.
The foaming drying that comprises freezing step
Method of the present invention randomly comprises freezing described sample.Freezing sample helps increasing the same batch of repeatability between the sample before foaming is dry.This is because all samples begins described processing from identical freezing physical state.Preserving sample can be all or part of freezing.
Freezing choose wantonly by with described preservation sample in being lower than 0 ℃ of temperature, place to allow refrigerated suitable a period of time of described sample, carry out before making described sample decompression.Preferred employed temperature is or is lower than-10 ℃ ,-15 ℃ ,-20 ℃ ,-30 ℃ ,-40 ℃ ,-70 ℃ or-140 ℃.Sample can continue 1,2,3,4,5,8,16 or took out under with the condition of allowing freezing generation in more hours being lower than 0 ℃ of temperature.
For some sample, particularly, sample is to be easy to solvent crystallization damages, for example cellular preparations or other biology system, preferably to be less than or equal to 0.1,0.5,1,2,3,4,5 ℃ of freezing lentamente described sample of speed hourly.Other compositions is by instantaneous freezing can more effectively the preservation, for example by quick freezing in liquid nitrogen.This method is particularly useful for protein or virion.By the freezing quick freezing that also causes sample of evaporation.
Alternatively, described preservation sample is by making described sample that all or part of freezing being frozen be taken place the sample decompression.In high capacity freeze dryer equipment, shelf temperature for or be higher than and carry out above-mentioned quenching under 0 ℃, 10 ℃, 15 ℃, 20 ℃, 30 ℃, 37 ℃ and freeze.Preferably, described shelf temperature is at 5-35 ℃, more preferably at 10-20 ℃, most preferably at 15 ℃.Described pressure randomly from reducing to 200 millibars at first, continues 5,10,20,30,60 minutes or more of a specified duration, and tolerable is bled.For freezing sample, described pressure further reduces to the pressure that is equal to or less than 2,1,0.5,0.2,0.1 millibars.It is all or part of freezing until sample to keep this pressure at least 5,10,20 or 30 minutes.
Form solid step description as above with post-foaming with except that desolvating.
In embodiment preferred of the present invention, the freezing and blistered step of sample is carried out under constant temperature in exsiccator, preferably changes pressure condition.
In another preferred embodiment, freezing, the foaming of sample and remove is desolvated and to form solid step and carry out under constant temperature in exsiccator, preferably changes pressure condition.
In another embodiment of the invention, at freezing sample, foaming with remove to desolvate and form in the solid process, the pressure and temperature condition is all inequality.
Method of the present invention is preferably used the container with waterproof inner surface.Can reach this purpose by lining hydrophobic composition on inner surface, for example pass through silicification.Can finish silicification by the well-known method of those skilled in the art.Alternatively, described waterproof inner surface can be by being realized by the container of water-proofing composition preparation.
Exist the waterproof inner surface of container can be more suitable for taking place foaming and the more repeatability of tool.It allows the polyol that uses low concentration in preserving sample, can reduce the required time span of drying sample successively, reduce maillard reaction (Mailard rections) influence or with other interaction of the polyol of infringement activating agent.Comprise vaccine if preserve sample, because the polyol of low quantity exists, the foamed glass that is produced can reach reprovision easily fast, and the lower viscosity of vaccine solution tool that is produced, and is more convenient for using.
Need not drying freezing or foaming
A kind of particularly preferred method of the present invention is included in stabilizing agent, there is dry IPV down with the preferred bacterium polysaccharide, its used gentle method with avoid IPV and be exposed to freezing or foaming process in, make IPV in dry run, stand lower pressure and keep high-caliber antigenicity.
This method is specially adapted under low concentration glass shape polyol condition, and for example in the concentration conditions that is lower than 10% (w/v), more preferably less than 5% (w/v), it is favourable and preferred shorter drying time.
By evaporation forfeiture solvent (evaporation drying-step b)
Need not drying means freezing or foaming and comprise sample is stood such pressure and temperature condition, so that described preservation sample is lost solvent by evaporation, it is freezing or bubble and form foam to need not sample.
Because the heat absorptivity of evaporation process, the temperature of described preservation sample interior can be different from the temperature of sample outside sometimes.Mentioned temperature is the temperature of preserving the sample external environment condition, for example, using under the big industrial freeze dryer situation, is the temperature of shelf.It is usually corresponding to the freeze dryer desired temperature.
Randomly, the described preservation sample preliminary step of bleeding is present in the method for the present invention.Described pressure reduces to or is lower than 200 millibars, preferably at the 200-35 millibar, is further continuing at least 5 minutes before the decompression.
Embodiment preferred of the present invention is controlled temperature conditions simultaneously by decompression and is realized evaporation drying.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 1 millibars, the holding temperature setting value is in the temperature that is higher than 0 ℃, preferably at 4 ℃-37 ℃, 4 ℃-10 ℃, 10 ℃-15 ℃ simultaneously; 15 ℃-20 ℃; 20 ℃-25 ℃; 25 ℃-30 ℃; Or 30 ℃-37 ℃; Or 37 ℃-45 ℃.Keep these conditions at least 1,2,3,4,5,8,10,12,16 or 24 hours, preferably continue 2-4 hour, 4-6 hour, 6-8 hour, 8-12 hour or 12-18 hour.In an especially preferred embodiment, described pressure is kept and is higher than 2 millibars, in this desired temperature be 15 ℃ to prevent the freezing of sample.In a preferred embodiment, temperature maintenance is in 15 ℃, and pressure setting is at the 5-10 millibar, more preferably 6-9 millibar, most preferably from about 8 millibars.Using under the higher temperature setting value situation, pressure that may be lower can avoid sample freezing, is using under the lower temperature setting value situation, and pressure should maintain higher level to avoid freezing.Preferably, keep the enough time of described condition, make that the temperature of sample is approximate identical with the temperature of sample outside so that evaporation rate can slow down.
Preferably, preserve sample and do not answer freezing or boiling formation foam, can lose solvent and form viscous liquid or high viscosity liquid.
Remove the formation high viscosity liquid that desolvates
Method comprised that removing desolvates and formed high viscosity liquid until described preservation sample drying next stage of the present invention, need not foaming and preferably need not freezing.
In one embodiment of the invention, by keep the pressure and temperature condition in those conditions that are applied to the initial evaporation drying stage to achieve the goal.For instance, pressure is maintained at or is lower than 30,25,20, and is preferred 15,12, and more preferably 10,8,7,6,5,4,3,2 or 1 millibars, the holding temperature setting value is higher than 0 ℃ simultaneously, preferably at 5 ℃-37 ℃, 5 ℃-10 ℃, 10 ℃-15 ℃; 15 ℃-20 ℃; 20 ℃-25 ℃; 25 ℃-30 ℃; Or 30 ℃-37 ℃.For 15 ℃ desired temperature, pressure is the 5-10 millibar, and preferred 6-9 millibar, was kept preferred 1-4,4-8,8-12 or 12-16 hour 4-24 hour by most preferably from about 8 millibars.Kept these temperature and pressure conditions 1,2,3,4,5,6,8,10,12,18 hours or be less than or equal to 15,12, the high viscosity liquid of preferred 10,8,5,4,3,2 or 1% (w/w) solvent more for a long time so that obtain to have.
Another embodiment of the invention is in solvent removal process, and the temperature of being kept at this method initial stage is increased to higher desired temperature with desired temperature.It is allowed that solvent leaves with very fast speed and leaves sample, makes method of the present invention to finish in shorter time.For example, desired temperature is increased to is higher than 0 ℃, more preferably be higher than 20 ℃, preferably at 5 ℃-37 ℃, 5 ℃-10 ℃, 10 ℃-20 ℃; 20 ℃-30 ℃; More preferably at 30 ℃-40 ℃; More preferably at 40 ℃-50 ℃; Most preferably at 50 ℃-60 ℃, keep simultaneously pressure in or be lower than 30,25,20, preferred 15,12, most preferably 10,8,7,6,5,4,3,2 or 1 millibars.Kept these temperature and pressure conditions 1,2,3,4,5,6,8,10,12,18 hours or more of a specified duration, be less than or equal to 15,12, preferred 10,8,5,4,3,2 or 1% solid so that obtain to have.For method completes successfully, this embodiment need be used heat-staple activating agent under described temperature.
Embodiment preferred of the present invention is removed at solvent (in the step c) process, than (employed pressure of step b) initial stage reduces to lower pressure set points with pressure set points in this method.It allows that solvent leaves sample with very fast speed, makes method of the present invention to finish in shorter time.Also can make more a high proportion of solvent forfeiture.For instance, pressure set points is set in or is lower than 7,6, preferred 5,4,3, more preferably 2,1.5,1, most preferably 0.8,0.5,0.2,0.1,0.05,0.02,0.01 or 0.005 millibar, simultaneously holding temperature in or be higher than 0 ℃, preferably at 10 ℃-20 ℃; 20 ℃-30 ℃; 30 ℃-35 ℃ or be higher than 40 ℃.Kept these temperature and pressure conditions 1,2,3,4,5,6,8,10,12 or 18 hours or more of a specified duration, be less than or equal to 15,12 so that obtain to have, the solid of preferred 10,8,5,4,3,2 or 1% (w/w) solvent, preferably (Eur.J.Pharm.Biopharm. (2000) 50 by Karl Fischer electric weight moisture analyser mensuration; 277-284).
Preferably, step b) and c) should be equal to or less than 18 hours, more preferably 16,14,12, most preferably finish in 10,8,6 or 4 hours time.
Dry compositions is the compositions of having removed by evaporation, boiling or distillation already from solvent, and the solvent that stays is less than or equal to 15,12,10, and more preferably 8,5,4,3,2 or 1% (w/w) preferably measures by Karl Fischer method.The preferable range of solvent is 1-3%, 3-5%, 5-10% or 10-15% (w/w).Described term comprises high viscosity liquid and exsiccant foam glass and cryodesiccated solid.
High viscosity liquid is defined as to have removed by evaporation already from solvent and need not ebullient material, and left solvent is less than or equal to 15,12,10, and preferred 8,5,4,3,2 or 1% (w/w) preferably measures by Karl Fischer method.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, make activating agent under 4 ℃, preserve at least 3,6,9,12 or 24 months with stable state, after during this period of time, allow that activating agent keeps at least 40,50,60, preferred 70,80,90,95% its antigenicity and/or immunogenicity.Preferably, described high viscosity liquid has the solid appearance except glass, after 2,4 or 6 days, more preferably 1,2,3,4,6,7,10 or 12 months after, can flow very lentamente.Described flow very lentamente to contain the container of this high viscosity liquid and to place under the room temperature by inversion until observing high viscosity liquid flow and obtain measuring.In a preferred embodiment, described high viscosity liquid after 2,4 or 6 days, preferably after 2,3 or 4 weeks, more preferably 2,4,6,8,10 or 12 months after as if do not flow at upside down position.
Viscous liquid is defined as the initial phase product that solvent is removed, and loses from sample already at the terminal most of solvents of this initial phase.Can discern this point, because when the heat absorption influence of volume evaporation loses, evaporation rate reduces makes sample temperature get back to ambient temperature.
Foam glass is the dry compositions that contains the forming of glass polyol, it forms by the method that wherein will preserve sample and stand following temperature and pressure condition, and described condition is that sample bubbles tempestuously or seethes with excitement and makes formation foam when sample drying.
All lists of references or the patent application of quoting in patent specification are incorporated herein by reference document in this article.
Embodiment
The following embodiment that we are adopted has used standard techniques, unless have a detailed description in addition, well-known and be routine techniques for those skilled in the art.Embodiment is illustrative, but does not limit the present invention.
Embodiment 1. evaporative freezings are handled
Heto Drywinner 8-85 freeze dryer has been used in the processing of being carried out, and wherein shelf temperature can be regulated in 1 ℃ of scope, and the outlet temperature of condenser is-85 ℃, and pressure is regulated with vent valve, and 6 thermocouples can be used for measuring the product temperature.
Preserve sample by in aqueous solution, adding the preparation of stabilizing agent (10% trehalose or 3.5% sucrose) and activating agent.Sample is placed freeze dryer, and shelf temperature maintains 15 ℃ fixed temperature setting value in overall process.Pressure reduces to 200 millibars at first, and is further keeping on this level 10 minutes before the decompression.At 1.5 millibars of places and since evaporative cooling solution begin freezing, as shown in Figure 1.Pressure further reduce to 0.1 millibar freezing fully to allow that sample becomes.Then pressure is increased to 0.8 millibar-3.5 millibars, forms at this place's foam, moisture is lost from sample.Under this experiment condition, in only containing the reference sample of water, do not see boiling.Sample can be by evaporation forfeiture moisture by boiling.After 18 hours, sample is dried and frothing solution becomes foam glass under these conditions.
Shelf temperature is remained on other desired temperature up to 37 ℃, successfully carried out similar processing.
Determining of embodiment 2. freezing conditions
Provide 1%, 5%, 10% and 20% solution by dissolving saccharose in water and prepare sample.Sample is placed freeze dryer, and shelf temperature maintains 15 ℃ in overall process.Pressure reduces to 200 millibars at first, and keeps on this level 10 minutes before further being decompressed to 50 millibars, 5 millibars, 2.5 millibars, 0.75 millibar, 0.4 millibar and 0.2 millibar.On each stress level, keep and reached balance with allowable temperature in 20 minutes, the temperature of using thermocouple to read sample.Thermocouple is attached on the sample with different sucrose, and the temperature that is recorded in the table 1 is the meansigma methods of temperature.
The result
All samples is freezing between the 1.66-1.1 millibar, and is irrelevant with the sucrose concentration that exists.Very approaching those that predicted from the three phase point curve of the temperature of under different pressures, measuring.Therefore as if for the sample temperature under the different pressures, the existence of sucrose does not have big influence.
In a preferred method of the present invention, the freezing of sample must to be avoided.Can be higher than 2 millibars by keeping pressure, use 15 ℃ shelf temperature to realize.At a lower temperature, pressure should maintain higher level, otherwise when using higher temperature, answers allowable pressure further to reduce, and avoids sample freezing.
Table 1
Pressure Measure temperature Theoretical temperatures Liquid/freezing
1000 millibars 15℃ Liquid
50 millibars 15℃ Liquid
5 millibars 1℃ 1℃ Liquid
2.5 millibar -5℃ -7℃ Liquid
0.75 millibar -21℃ -21℃ Freezing
0.4 millibar -22℃ -27℃ Freezing
0.2 millibar -27℃ -32℃ Freezing
Embodiment 3. has the foaming condition of the sample of different sugar concentration
Preparation contains the preservation sample of 0%, 5%, 10%, 15%, 20%, 25% and 50% sucrose.Sample is placed freeze dryer, and shelf temperature maintains 15 ℃ in overall process.Pressure reduces to 200 millibars at first, and is further keeping on this level 10 minutes before the decompression.With pressure further reduce to 0.1 millibar freezing fully to allow that sample becomes.Then in experiment subsequently, pressure is increased to 0.788 millibar, 0.812 millibar or 3.5 millibars.Keep these conditions, kept 3 hours, kept 6 hours for 0.788 millibar experiment for the experiment of 3.5 millibars and 0.812 millibar.Assess the physical characteristic of every kind of sample.
The result
As shown in table 2, under 3.5 millibars of pressure, for reliable foaming, need 50% high-sucrose concentration.In contrast, under the low sucrose concentration of 10-25%, 0.8 millibar lower pressure is allowed reliable foaming.Use low sucrose concentration can be beneficial to the sample that for example will be used for the preservation of vaccine.Therefore it is preferred using 0.8 millibar of method with the 10-25% cane sugar content.
Table 2
Pressure % sucrose Physical characteristic
3.5 millibar 20 4/5 foaming, 1/5 viscous liquid
3.5 millibar 25 2/5 foaming, 3/5 viscous liquid
3.5 millibar 50 5/5 foaming
0.812 millibar 5 Ring crystalline solid and foam
0.812 millibar 10 All all foam
0.812 millibar 15 All all foam
0.812 millibar 20 All all foam
0.812 millibar 25 All all foam
0.788 millibar 5 Ring crystalline solid and foam
0.788 millibar 20 All all foam
0.788 millibar 25 All all foam
0.788 millibar 50 Foam and slurry
Embodiment 4. uses the influence of silicator
Preparation contains the preservation sample of 5%, 10%, 15% and 25% sucrose, and in the phial of packing into, some phial is by silication.In once testing, sample is placed freeze dryer, shelf temperature maintains 15 ℃ in overall process.Pressure reduces to 200 millibars at first, and is further keeping on this level 10 minutes before the decompression.Pressure is further reduced to 2.8 millibars, continue 3 hours.During this period of time, when existing steam reduced, pressure reduced to 2.00 millibars.Assess the physical characteristic of every kind of sample.
In experiment for the second time, sample is placed freeze dryer, shelf temperature maintains 37 ℃ in overall process.Pressure reduces to 200 millibars at first, and is further keeping on this level 10 minutes before the decompression.Pressure is further reduced to 2.4 millibars, continue 3 hours.During this period of time, when existing steam reduced, pressure reduced to 1.06 millibars.Assess the physical characteristic of every kind of sample.
The result
Silication is influential to bleeding of sample.In the phial of silication sample being bled causes pressure to reduce to 200 millibars, does not then have this result in the phial of not silication.Observe by the foaming of sample and to bleed.
The silication of phial also makes foaming more may produce, and the more repeatability of tool (table 3).The silication of phial is allowed to foam under lower polyol concentration and is repeated.Lower polyol concentration has reduced the length of drying sample required time, and reduced maillard reaction (Mailard rections) influence or with other interaction of polyol of infringement activating agent.At related sample is under the vaccine situation, and it has reduced the viscosity of sample and has been more convenient for using.
Table 3
Temperature and pressure % sucrose Characteristic is the phial of silication not The phial of characteristic silication
15 ℃, 2.8 millibars 5％ Viscous liquid
15 ℃, 2.8 millibars 10％ Viscous liquid Foaming
15 ℃, 2.8 millibars 15％ Viscous liquid
15 ℃, 2.8 millibars 25％ Viscous liquid
37 ℃, 2.4 millibars 5％ 2 foaming of 3 viscous liquids
37 ℃, 2.4 millibars 10％ Promising viscous liquid 5 foaming, 1 viscous liquid
37 ℃, 2.4 millibars 15％ All all foam
37 ℃, 2.4 millibars 25％ All all foam
Embodiment 5. is by normal freeze-drying or by the dry relatively preservation of Hib-IPV of foaming
The activating agent that is saved is the mixture of three kinds of strains of the poliovirus (IPV) of the PRP polysaccharide of b type Haemophilus influenzae (Hib) and deactivation.Preserve sample by Hib-IPV is dissolved preparation in 3.15% sucrose solution or 10% aqueous trehalose.
By using conventional freeze-drying method, sample need be in big freeze dryer lyophilizing 3 days, or by using embodiment 1 described foaming drying means to come the lyophilizing sample.
Sample is reprovision in water, uses ELISA to assess three kinds of antigenic conservation rates of poliovirus strain.Three kinds of polyclonal antibodies and three kinds of monoclonal antibodies, every kind of anti-a kind of strain is used for different ELISAs.The result is to represent without the percent of the given reading of sample that is subjected to lyophilization or foaming drying program.
By with the IPV-Hib of the reprovision mouse inoculation to 10 group, blood drawing and monitor the antibody horizontal of anti-IPV and Hib polysaccharide from mice for example by the method for ELISA or Western blotting, is assessed and is preserved sample their immunogenicity in vivo.The degree of assessment protection in the mouse model of challenge.
The result
Use sucrose or trehalose as polyol, compare, use the antigenicity of foaming dry technology IPV to keep better with using normal freeze-drying.
Table 4
*Repeat 5 lyophilizations experiment in the presence of 3.15% sucrose, result displayed is from once representative experiment.
There is the protection effect of lyophilization IPV under the Hib polysaccharide in embodiment 6.
Preparation contains the preservation sample of 3.15% sucrose and IPV or IPV and Hib polysaccharide mixture.Sample is placed Heto Drywinner 8-85 freeze dryer, and lyophilization is 40 hours under-32 ℃ of desired temperatures, then continues dry 16 hours down at 4 ℃.
Sample is reprovision in water, uses ELISA to assess three kinds of antigenic conservation rates of poliovirus strain.Three kinds of monoclonal antibodies, every kind of anti-a kind of strain is used for different ELISAs, with the reference sample comparison that is not frozen to be evaluated at antigen conservation rate level in reprovision, the freeze drying example.The result is to represent without the percent of the given reading of sample that is subjected to lyophilization or foaming drying program.
The result
As shown in table 5, after lyophilization, the effect that when the independent lyophilization of IPV, is reached, the existence that contains Hib in the preservation sample of IPV can cause the antigenic higher conservation rate of IPV.Except having the effect that sucrose reached that exists as stabilizing agent, the Hib polysaccharide to IPV antigenic reservation work.
Table 5
The compositions lyophilization Polyol content ELISA-1/2/3 type %
IPV 3.15% sucrose 26/25/0
IPV-Hib 3.15% sucrose 52/68/0
Embodiment 7. different stabilizers are to the cryodesiccated influence of IPV-Hib
Preparation contains the preservation sample of IPV-Hib, and uses 3.15% sucrose; 2.5% Sorbitol, 0.8% glutamine and 0.01%HSA; MMR stabilizing agent and lactose; 3% glycine, 2% arginine and 4% sucrose; Or 4% sucrose and 2% glycine as stabilizing agent.Comprise and have 3.15% sucrose uses double strength as the experiment of the sample of stabilizing agent IPV-Hib.Using 3 days conventional lyophilization cycles that sample is carried out lyophilization in the freeze dryer in batches.
With sample reprovision in water, use ELISA to assess three kinds of antigenic conservation rates of poliovirus strain.Three kinds of polyclonal antibodies and three kinds of monoclonal antibodies, every kind of anti-strain separately is used for different ELISAs.The result is to represent without the percent of the given reading of sample that is subjected to lyophilization or foaming drying program.
By with the IPV-Hib of the reprovision mouse inoculation to 10 group, blood drawing and monitor the antibody horizontal of anti-IPV and Hib polysaccharide from mice for example by the method for ELISA or Western blotting, is assessed and is preserved sample immunogenicity in vivo.The degree of assessment protection in the mouse model of challenge.
The result
IPV dosage is increased to the increase that the 80/16/64DU/ agent causes IPV antigenicity conservation rate from the 40/8/32DU/ agent, and is as shown in table 6.The variation of stabilizing agent also influences antigenic conservation rate, and 4% sucrose/2% glycine and 2.5% Sorbitol/0.8% glutamine/0.01%HAS have produced higher antigen conservation rate, shown in the ELISA data.
Table 6
Stabilizing agent Polyclone ELISA result Monoclonal ELISA result
3.15% sucrose 50/50/70 ?25/0/0
2.5% Sorbitol, 0.8% glutamine 0.01%HSA 55/72/72 ?33/50/0
MMR stabilizing agent lactose 59/62/65 ?28/25/0
The IPV-Hib of 3.15% sucrose, two multiple doses 84/92/120 ?102/138/0
3% glycine, 2% arginine, 4% sucrose
4% sucrose, 2% glycine 46/62/78 ?25/50/15
Embodiment 8. lyophilization, foaming is dry or contain the foaming drying of freezing step after the repeatability of sample characteristics of for example.
Sample is preserved in preparation, it comprises IPV, mumps virus, Measles virus, rubella virus, varicella zoster virus, CMV, hepatitis virus, HSV1, HSV2, respiratory syncytial virus, dengue virus, such as the paramyxovirus of parainfluenza virus, togavirus and influenza virus, and/or Hib is as activating agent.Activating agent is dissolved in containing the aqueous solution of polyol.By lyophilization, follow the foaming kept dry several samples that does not have freezing step that the rules that are described in embodiment 1 are used the foaming drying of freezing step or used the rules be described in embodiment 4.Sample is at aqueous solution kind reprovision and assess its activity.Use ELISA algoscopy as described in Example 5 to finish active assessment, this algoscopy is used the antibody that is specific to native antigen.With regard to live virus, the titre of every kind of sample is by using virus and infect proper host cell and forming or assess infectivity by immunocytochemistry and determined by plaque.Under immunogenic composition or vaccine foaming drying regime, in animal model, pass through dry or several treated animals of cryodesiccated vaccine immunity with foaming, and after the immunity first time, for example reply the 14th day and 28 days enhance immunity, can test immunogenic conservation rate.When immunization schedule ends up from animal separation of serum, and the titre of using the standard test method to test its anti-vaccine is for example measured by ELISA, immunocytochemistry, Western blotting, immunoprecipitation, serum sterilizing or agglutination is measured.It is as follows that the result abides by: at first by lyophilization, use the foaming of freezing step dry or need not the foaming drying of freezing step after comparison activating agent activity.Secondly, by comparative sample stand three kinds of store methods each after field of activity assess the repeated degree of preservation technology.
Embodiment 9. is by the Long-term Storage of the activating agent of lyophilization and foaming kept dry
Sample is preserved in preparation, it comprises IPV, mumps virus, Measles virus, rubella virus, varicella zoster virus, CMV, hepatitis virus, HSV1, HSV2, respiratory syncytial virus, dengue virus, such as the paramyxovirus of parainfluenza virus, togavirus and influenza virus, and/or Hib is as activating agent.Activating agent is dissolved in containing the aqueous solution of polyol.By lyophilization, follow the foaming kept dry several samples that does not have freezing step that the rules that are described in embodiment 1 are used the foaming drying of freezing step or used the rules be described in embodiment 4.Sample is preserved down at 37 ℃ or 23 ℃ and was worn out in 7 days, and relatively more active with the sample that remained on already under 4 ℃.Reprovision sample and assess its activity in aqueous solution.Use ELISA algoscopy as described in Example 5 to finish active assessment, this algoscopy is used the antibody that is specific to native antigen.With regard to live virus, the titre of every kind of sample is by using virus and infect proper host cell and forming or assess infectivity by immunocytochemistry and determined by plaque.The result abides by as follows: at first by preserving in the temperature that improves and preserving and compare the activating agent activity after 4 ℃.Secondly, assess the repeated degree of preservation technology by the field of activity of comparative sample after standing every set condition.
Embodiment 10. need not drying means freezing or foaming
Preparation contains the preservation sample of 5%, 10%, 15% and 25% sucrose, and in the phial of packing into.Sample is placed freeze dryer, and temperature is set in 15 ℃ in overall process.Pressure reduces to 200 millibars at first, and is further bleeding before the decompression to allow in 10 minutes keeping on this level.Pressure further reduces to 8 millibars, continues 2-3 hour, and owing to evaporative cooling, the thermocouple show sample temperature of inserting sample is reduced to 4 ℃ in this time period.After 2-3 hour, sample temperature is got back to 15 ℃, shows that evaporation is near finishing under these temperature and pressure conditions.In this operation stage, sample does not seethe with excitement and forms foam or freezing, makes that the activating agent in the sample is exposed in the as far as possible little pressure.Sample has solid appearance, is similar to end-product.
By further minimizing pressure to 0.1 millibar, keep shelf temperature to set value in 15 ℃ simultaneously to realize further drying to sample.Keep these conditions and reach 10-16 hour.In this stage, sample temperature remains in 15 ℃, because evaporation rate has slowed down.Carry out further drying, the sample that is generated has solid appearance.If sample is placed on the shelf limit, it is very slow that sample size reduces, and shows that just this sample is the liquid glass of high viscosity behind several days or some months.Fig. 2 has shown that the container that has high viscosity liquid can be squeezed and do not cause flowing immediately of high viscosity liquid.
Embodiment 11. need not the immunogenic conservation rate of dry back IPV of freezing or foaming
According to the method drying sample of embodiment 10, it need not to stand and foaming or the relevant pressure of the freezing foaming of following already.When being used for dry IPV, experimentize to determine whether that this method produces high-caliber antigen conservation rate.
Carry out three independently experiments, wherein IPV is resuspended in and contains in 10% sucrose or the aqueous solution of 10% trehalose as stabilizing agent.Sample is placed the phial of silication, phial is positioned in the Heto Drywinner 8-85 freeze dryer, temperature is set in 15 ℃.Pressure reduces to 35 millibars at first so that sample is outgased.After 10 minutes, pressure further reduces to 8 millibars, and keeps 2 hours on this level.During this period of time, desired temperature remains on 15 ℃, and the temperature in the test sample.When moisture evaporated from sample, temperature dropped to 4 ℃ except last two hours, and temperature is got back to 15 ℃ when evaporation rate slows down.Do not occur under these conditions bubbling or foaming.Then pressure is further reduced to 0.1 millibar, in the first two times experiment, keeps these conditions and surpass 16 hours, in experiment for the third time above 10 hours.
Sample is reprovision in water, uses ELISA to assess three kinds of antigenic conservation rates of poliovirus strain.In ELISA, use the monoclonal antibody of anti-3 type IPV, with the reference sample that is not frozen relatively, with the assessment reprovision, antigen conservation rate level in the cryodesiccated sample.The result is to represent without the percent of the given reading of sample that is subjected to lyophilization or foaming drying program.
The result
As if drying sample has solid appearance, but they exist with the form of high viscosity liquid/glass, because after several days, if in the room temperature inverted container, then drying solid can flow.
The 3 type IPV antigen conservation rates that table 7 is measured by the ELISA that uses monoclonal antibody (need not foaming or refrigerated drying)
Preparation Experiment (18 hours period) for the first time Experiment (18 hours period) for the second time Experiment (12 hours period) for the third time
10% sucrose 75％ 78％ 91％
10% trehalose 82％ 79％ 93％
It is very favourable that 3 type IPV antigen conservation rates of these levels are compared with the lyophilization result of the very low numerical value of following demonstration, and when using the monoclonal antibody of anti-3 types, described very low numerical value derives from identical ELISA experiment usually.
Table 8: measure 1,2 and 3 type IPV by the ELISA that uses monoclonal and polyclonal antibody Antigenic conservation rate (lyophilization)
Figure G038A8184319960327D000341
*Repeat 5 lyophilizations experiment in the presence of 3.15% sucrose, result displayed is from once representative experiment.
Embodiment 12. is with the Long-term Storage stability of the exsiccant IPV of high viscosity liquid/glass storage
Use the exsiccant IPV of method that describes among the embodiment 11 in 4 ℃ of storages 9 months.In water,, assess three kinds of antigenic conservation rates of poliovirus strain with ELISA with 150mM NaCl reprovision sample.Three kinds of monoclonal antibodies, every kind of anti-strain separately is used for antigen conservation rate level in the storage sample that different ELISAs assesses reprovision.Reprovision sample from same batch before the storage had been carried out similar ELISA already.All results compare with the reference sample that is not dried.The result represents with the percent of the given reading of sample that do not stand drying program.
The result
Table 9. is the antigenic conservation rate of IPV after preserving 9 months with high viscosity liquid
Handle 1 type ELISA 2 type ELISA 3 type ELISA
The not storage of exsiccant/reprovision 72％ 75％ 88％
4 ℃ of exsiccant/reprovision, 9 months 70％ 94％ 90％
Therefore can be by the exsiccant already IPV of the method that is described in embodiment 11 in 4 ℃ of storages at least 9 months, and do not lose antigenicity.
Embodiment 13. is than undried IPV, immunogenic comparison in the IPV body after drying forms high viscosity liquid and reprovision
With the Wistar rat of 10 one group of the dilution IPV inoculation of difference, used embodiment 10 disclosed methods, described IPV was dry already in the presence of 10% sucrose to form high viscosity liquid, and obtains reprovision.10 one group Wistar rat in addition inoculates with IPV reference sample, and described reference sample prepares in the same way, but is not dried.
After 21 days, from all rats, collect serum, the above-mentioned serum of test in the different immune precipitation determination that uses 1 type, 2 types and 3 type polioviruses.
The results are shown in the table 10, comprise :-a) reply the quantity of rat for every kind of IPV dilution factor, b) ED50, it is to guarantee that 50% rat blood serum transforms necessary dosage, by immunoprecipitation assay assessment and c) compare the relative potency of the IPV of exsiccant and reprovision with undried reference IPV.
Table 10. is compared with undried reference IPV (JLE097), forms high viscosity in drying The immunogenicity of IPV behind liquid (JLE017/05) and the reprovision
Sample Quantity Reply ED50 RP is relative
Undiluted 1/1.25 1/3.125 1/7.81 Effect
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 dry high viscosity liquid and IPV batch of reprovision subsequently of forming.JLE097 is undried reference sample.
Table 10 has shown that between the IPV of drying and reprovision two batch samples and undried reference sample the quantity of inoculating with every kind of dilution IPV of replying is similar.In general, 1 type IPV causes best immunne response, and 2 types cause immunne response in few slightly rat.3 types cause the most weak immunne response.
The dry process that forms high viscosity liquid is not damaged the ability that IPV causes the immunoprecipitation of internal antibody.Relative potency (RP) reading 1.0 expression samples cause replying of equating with the reference sample.For all three kinds of polioviruses, two kinds of RP readings near 1.0 that drying sample produced show that dry run does not influence the ability that sample causes immunne response.
Embodiment 14. uses sucrose or trehalose as the dry influence that forms high viscosity liquid to the immunne response of immunoprecipitation in the IPV primosome of stabilizing agent
With the Wistar rat of 10 one group of IPV inoculation, described IPV drying already in the presence of 10% sucrose or 10% trehalose, as described in embodiment 2, reprovision then.Use not being dried of equal amount, inoculate other 10 one group Wistar rat as the IPV of reference sample.
After 21 days, collect the serum of all rats, carry out the quantity that immunity neutralization mensuration is used to assess the immune neutralizing antibody that resists every kind 1 type, 2 types and 3 type polioviruses as described in embodiment 5, the quantity of described immune neutralizing antibody improved already.
By comparing, every kind of sample is calculated relative potency with the immunne response that not dry reference sample is caused.
The results are shown in the table 11.
Table 11. is exsiccant comparison in sucrose and trehalose
Lot number The sugar that exists Relative effect 1 type/2 types/3 types in the body Humidity % Karl Fischer Persistent period (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 using sucrose as stabilizing agent, moisture quantity residual in the sample is lower, has approximately kept 5% humidity, and Comparatively speaking, when trehalose during as stabilizing agent, the high humidity of being measured by the KarlFischer method is about 10%.
In dry run, sucrose and trehalose can both be stablized IPV effectively, make the IPV of reprovision provide relative potency reading near 1.0 for most of dissimilar polioviruses.For 3 type polioviruses, relative potency is good especially, can relatively easily discharge its immunogenicity.
Embodiment 15: measure humidity with Karl Fischer
In Karl Fischer electric weight moisture analyser (Aqua 30.00-ElektrochemieHalle), analyze.To samples weighing and place the baking oven that is set in 80 ℃.Use the nitrogen wash sample, then add hydranal reagent (Riedel de Hahn) so that analyze by coulometric analysis.

Claims (28)

1. one kind comprises the capsular polysaccharide of IPV, b type Haemophilus influenzae or the immunogenic composition of oligosaccharide antigen and stabilizing agent, all is formulated as dry compositions, behind reprovision, can produce the immunne response of poliomyelitis virus.
2. the described immunogenic composition of claim 1, wherein said polysaccharide or oligosaccharide are incorporated into carrier protein.
3. the described immunogenic composition of claim 2, wherein said polysaccharide or oligosaccharide are incorporated into tetanus toxoid.
4. the described immunogenic composition of claim 1, wherein said polysaccharide or oligosaccharide are adsorbed on the aluminum phosphate.
5. the described immunogenic composition of claim 1 comprises the capsular polysaccharide or the oligosaccharide that are derived from C type Neisseria meningitidis.
6. the described immunogenic composition of claim 1 also comprises the capsular polysaccharide or the oligosaccharide that are derived from arbitrary A, C, Y or W type Neisseria meningitidis or its combination.
7. the described immunogenic composition of claim 5, capsular polysaccharide or the oligosaccharide of the wherein said C of being derived from type Neisseria meningitidis are incorporated into carrier protein.
8. the described immunogenic composition of claim 7 comprises Hib polysaccharide or oligosaccharide and at least a meningococcal polysacharide or the oligosaccharide that is incorporated into the same type carrier protein.
9. the described immunogenic composition of claim 7 comprises Hib polysaccharide or oligosaccharide and at least a proteic meningococcal polysacharide of different carriers or the oligosaccharide of being incorporated into.
10. the described immunogenic composition of claim 1 further comprises phenol red.
11. the described immunogenic composition of claim 1, wherein said dry compositions is cryodesiccated.
12. the described immunogenic composition of claim 1, wherein said dry compositions is a foam glass.
13. the described immunogenic composition of claim 1, wherein said dry compositions is a high viscosity liquid.
14. the described immunogenic composition of claim 13, wherein said high viscosity liquid is not frozen.
15. a medicine box is included in each described immunogenic composition and liquid acellular in second kind of container or full cell diph-tet pertussis vaccine among the claim 1-14 in a kind of container.
16. the described medicine box of claim 15 further is included in second hepatitis B virus surface antigen in the container.
17. a vaccine comprises each described immunogenic composition among the claim 1-14.
18. the described vaccine of claim 17, its before use reprovision become aqueous solution.
19. the container with waterproof inner surface, it comprises claim 17 or 18 described vaccines.
20. a method for preparing vaccine is included in the aqueous solution step of each described immunogenic composition among the reprovision claim 1-14.
21. the described method of claim 20, wherein said aqueous solution comprise acellular or full cell diphtheria toxin, diphtherotoxin, tetanus toxoid and pertussis antigen.
22. the described method of claim 21, wherein diph-tet pertussis vaccine to small part is used aluminum hydroxide adjuvant.
23. claim 21 or 22 described methods, wherein said aqueous solution comprises hbs antigen.
24. a preservation comprises the capsular polysaccharide of IPV, b type Haemophilus influenzae or the method for compositions of oligosaccharide antigen and stabilizing agent, comprises step:
A) by in stabiliser solution, suspending or dissolving capsular polysaccharide or the oligosaccharide antigen preparation preservation sample of IPV and b type Haemophilus influenzae;
B) described preservation sample is stood following temperature and pressure condition, promptly solvent is lost from described preservation sample under the described conditions; With
C) form solid or high viscosity liquid except that desolvating until described preservation sample drying, wherein the antigenicity of IPV obtains keeping.
25. the described method of claim 24, wherein said preservation sample is exsiccant, is positioned at the container with waterproof inner surface.
26. the described method of claim 24, wherein the temperature of the step b) pressure that is higher than 0 ℃ and step b) is lower than 20mbar.
27. the described method of claim 24, wherein in step b), described preservation sample is stood following temperature and pressure condition, promptly described under the described conditions preservation sample is by the evaporation release solvent, need not freezing or relate to the foaming that foam forms, with the formation viscous liquid, and in step c), remove and desolvate until described preservation sample drying formation high viscosity liquid.
28. claim 24 or 25 described methods, wherein said preservation sample comprises polysaccharide or the oligosaccharide that is derived from arbitrary A, C, Y or W type Neisseria meningitidis or its combination.
CN2003801081843A 2002-11-01 2003-10-30 Immunogenic composition Expired - Fee Related CN1735430B (en)

Applications Claiming Priority (13)

Application Number Priority Date Filing Date Title
GB0225520.6 2002-11-01
GB0225532.1 2002-11-01
GB0225532A GB0225532D0 (en) 2002-11-01 2002-11-01 Drying process
GB0225543.8 2002-11-01
GBGB0225520.6A GB0225520D0 (en) 2002-11-01 2002-11-01 Drying process
GB0225543A GB0225543D0 (en) 2002-11-01 2002-11-01 Immunogenic composition
GB0317371A GB0317371D0 (en) 2003-07-24 2003-07-24 Immunogenic composition
GB0317381A GB0317381D0 (en) 2003-07-24 2003-07-24 Drying method
GB0317380A GB0317380D0 (en) 2003-07-24 2003-07-24 Drying method
GB0317380.4 2003-07-24
GB0317371.3 2003-07-24
GB0317381.2 2003-07-24
PCT/EP2003/012160 WO2004039399A1 (en) 2002-11-01 2003-10-30 Immunogenic composition

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BRPI0909820A2 (en) * 2008-03-05 2015-08-11 Sanofi Pasteur Process for stabilizing influenza vaccine composition, for stabilizing adjuvanted-containing vaccine composition and for preparing vaccine, vaccine compositions, vaccine kit and stockpiling method
WO2015050177A1 (en) * 2013-10-03 2015-04-09 日東電工株式会社 Dried influenza vaccine preparation and method for producing dried influenza vaccine preparation

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