WO2009093072A1

WO2009093072A1 - Vaccine composition

Info

Publication number: WO2009093072A1
Application number: PCT/GB2009/050050
Authority: WO
Inventors: Matthew Duchars; Alan Watkinson
Original assignee: Pharmathene UK Ltd
Current assignee: Pharmathene UK Ltd
Priority date: 2008-01-22
Filing date: 2009-01-22
Publication date: 2009-07-30
Anticipated expiration: 2010-07-22
Also published as: GB0801122D0

Abstract

A vaccine composition comprising a stabilized sub-unit vaccine, such as a sub-unit vaccine comprising recombinant anthrax Protective Antigen (rPA), and a pharmacologically-acceptable solid support, especially an adjuvant, in a pharmacologically-acceptable diluent is disclosed along with methods for producing such a vaccine composition by reconstitution from a freeze-dried material. Various types of packaging for such a vaccine composition are also described.

Description

VACCINE COMPOSITION

FIELD OF THE INVENTION The present invention concerns vaccine compositions.

BACKGROUND TO THE INVENTION

Sub-unit vaccines represent an effective alternative for the prevention of infectious diseases. In order to achieve maximum effectiveness, sub-unit vaccines are adsorbed onto a solid support, especially an adjuvant such as aluminium oxyhydroxide. In order to preserve their activity, the vaccines require refrigerated storage and transportation. An alternative method to avoid the need for refrigeration would be to stabilise the sub-unit vaccine, for example by freeze-drying the sub-unit vaccine. However, the need for stabilisation processes can restrict the nature of the solid supports employed, for example, freeze-drying is generally regarded as being incompatible with the use of aluminium oxyhydroxide.

SUMMARY OF THE INVENTION According to a first aspect of the present invention, there is provided a vaccine composition comprising a stabilised sub-unit vaccine and a separate suspension of a pharmacologically-acceptable solid support in a pharmacologically-acceptable diluent.

According to another aspect of the present invention, there is provided an article of commerce comprising a multi pack composition comprising a first pack comprising a stabilised sub-unit vaccine and a second pack comprising a suspension of a pharmacologically-acceptable solid support in a pharmacologically-acceptable diluent.

According to yet another aspect of the present invention, there is provided a method of preparing a medicament which comprises mixing a first composition comprising a stabilised sub-unit vaccine with a second composition comprising suspension of a pharmacologically-acceptable solid support in a pharmacologically-acceptable diluent.

According to a further aspect of the present invention, there is provided a twin pack vaccine composition comprising a) a first pack comprising a stabilised sub-unit vaccine: and b) a second pack comprising a suspension of a pharmacologically-acceptable solid support in a pharmaceutically-acceptable diluent.

According to a still further aspect of the present invention, there is provided a method of preparing a medicament which comprises: a) preparing a stabilised sub-unit vaccine; b) ^* preparing a suspension of a pharmacologically-acceptable solid support in a pharmacologically-acceptable diluent; and c) mixing the stabilised sub-unit vaccine prepared in a) with the diluent prepared in b). The sub-unit vaccine is most commonly stabilised by drying, for example by vacuum drying, spray drying or most preferably by freeze drying, including freeze spray drying. Methods of drying sub-unit vaccines, and apparatus therefore, are well known in the art. The sub-unit vaccine is stabilised in the substantial absence of solid support, such as aluminium oxyhydroxide. When freeze-drying is employed, the freeze-dried sub unit vaccine preferably comprises a pharmacologically-acceptable cryoprotectant or lyoprotectant. Examples of such cryoprotectants and lyoprotectants are well known in the art and include poiyhydroxy compounds such as sugars, and in particular mono-, di and polysaccharides, such as mannitol. Especially preferred lyoprotectants are sucrose and particularly trehalose.

Sub-unit vaccines which can be employed are usually recombinant protein-based antigens. Examples of such antigens include Hepatitis B protective antigens, Herpes Simplex Virus antigens, Influenza antigens, Congenital cytomegalovirus (CMV) antigens, Tuberculosis antigens, HIV antigens, Diphtheria antigens, Tetanus antigens, Pertussis antigens and

Yersinia pestis protective antigens, such as antigens comprising one, two or more antigenic proteins, for example those disclosed in patent application WO96/28551 , incorporated herein by reference. Most preferably, the antigen is an anthrax protective antigen.

Anthrax protective antigens which can be employed in the vaccine according to the present invention are well known in the art. Preferably, the anthrax protective antigen is a recombinant protective antigen as described in patent application WO02/04646, incorporated herein by reference, such as the antigen having the sequence given in Figure 1 (SEQ ID NO: 1 ).

Solid supports are preferably pharmacologically-acceptable particulate adjuvants, including synthetic polymers such as polyglycol-lactate polymers; other nano- or microparticulate structures such as microcrystals or glass microspheres. Preferred adjuvants are colloidal adjuvants, such as calcium phosphate, aluminium phosphate, such as the colloidal aluminium phosphate available under the trade name "AdjuPhos", and most preferably aluminium oxyhydroxide, most commonly known by the name "Alhydrogel". Aluminium oxyhydroxide is also known as aluminium hydroxide or alum.

Diluents which can be employed include saline buffers, preferably comprising a physiological buffering agent, such as acetate-buffered saline, histidine-buffered saline, succinate-buffered saline, citrate-buffered saline, glycine buffered saline and especially phosphate-buffered saline.

The concentration of buffer is commonly up to about 5OmM, such as from 0.25 to 2OmM. In many embodiments, when phosphate buffered saline is employed, the concentration of phosphate is in the range of from 1 to 1 OmM, and especially from 3 to 7 mM. In certain embodiments, good results have been achieved with a phosphate concentration of from 4 to 6 mM, and particularly

5mM. The buffer employed commonly has an approximately neutral pH, such as from 6.8 to 7.5.

The vaccine composition according to the present invention may comprise adjuvants additional to the solid support adjuvant. Examples of such adjuvants are well known in the art and include immunomodulatory oligonucleotides, especially so-called CpG immunomodulatory oligonucleotides, especially phosphorothioate oligonucleotides, and most preferably oligonucleotides which target Toll Like Receptor 9 receptors. Other additional adjuvants that may be present include saponins, such as ISCOM adjuvants based on quillaja saponins and the complex formed by the saponin molecules when mixed with cholesterol and phospholipid; poly[di(carboxylatophenoxy)phosphazene]; lipopolysacchahde derivatives; monophosphoryl lipids, including AS02, SB-AS2, muramyl di-peptide, threonyl muramyl di-peptide; synthetic Lipid A derivatives such as SB-AS4, OM-174, OM294 and OM-197; Leishmania elongation factor; chitosan; non-ionic block copolymers and Syntex Adjuvant Formulation.

When an additional adjuvant is employed, the additional adjuvant is preferably incorporated with the stabilised sub-unit vaccine although the additional adjuvant may alternatively be present in the separate solid support and diluent composition. In certain embodiments, the stabilised sub-unit vaccine composition may also incorporate an agent to reduce or prevent agglomeration during preservation, for example a small amount of a non-ionic surfactant, especially an ethoxylated sorbitan ester, for example polyoxyethylenesorbitan monolaurate, typically comprising about 20 ethyleneoxy units. Preferred additional adjuvants include oligonucleotide compounds of the formula:

5¹ Q¹CGQ² 3' wherein C is unmethylated, G may be unmodified or modified, for example base modified, such as incorporating a 7-deaza-modification, Q¹ and Q² are nucleotides selected such that the oligonucleotide comprises at least 8 nucleotides, such as up to 30 nucleotides. Preferably, the oligonucleotide is a phosphorothioate. Either or both of Q¹ and Q² may comprise one or more modifications, such as sugar modifications, including the presence of 2'-0Me groups, base modifications, or modifications to the phosphate linkage, such as the presence of linking moieties, such as glycerol linkers or poly alkylene groups, and especially hexamethylene groups. Examples of suitable oligonucleotide compounds which may be employed as additional adjuvants include those described in US6,207,646,

US6,589,940, US7,255,868 and US7,276,489 (the disclosures of which are incorporated herein by reference). Especially preferred oligonucleotides are compounds having the sequences:

TGACCGTGAACGTTCGAGATGA (SEQ ID NO: 2); TGACTGTGAACGTTCGAGATGA (SEQ ID NO: 3); TCATCTCGAACGTTCCACAGTCA (SEQ ID NO: 4); TGACTGTGAACGTTCCAGATGA (SEQ ID NO: 5); TCCATAACGTTCGCCTAACGTTCGTC (SEQ ID NO: 6);

TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 7); TCGTCGA-HEG-ACGTTCG-HEG-AGATGAT wherein HEG = hexaethylene glycol (SEQ ID NO: 8) δ'-TCTGTCRTTCT-X-TCTTRCTGTCT-δ' wherein R represents 2'- deoxy-7-deazaguanosine and X represents a glycerol linker (SEQ ID NO: 9); δ'-TCRTCRTTCTG-X-GTCTTRCTRCT-δ' wherein R represents 2'- deoxy-7-deazaguanosine and X represents a glycerol linker (SEQ ID NO: 10); and δ'-TCRAACRTTCR-X-RCTTRCAARCT-δ' wherein R represents 2'- deoxy-7-deazaguanosine and X represents a glycerol linker (SEQ ID NO: 1 1 ).

The vaccine compositions may comprise a pharmacologically-acceptable preservative, commonly formulated with the solid-support and diluent composition. Examples of such preservatives include 2-phenoxyethanol, optionally combined with formaldehyde, and phenol. The vaccine composition according to the present invention preferably comprises an amount of sub-unit vaccine equivalent to one or more, for example, ten or twenty, doses in a first container, preferably a vial, accompanied by a second container comprising the solid support suspended in diluent. The quantity of support and diluent in the second container is preferably chosen such that when mixed with the sub-unit vaccine, the concentration of the sub-unit vaccine in the composition formed is suitable for administration. In the case of anthrax protective antigen, the amount of antigen present in the first container is typically from 5 micrograms to 5 milligrams, such as from 10 micrograms to 1 milligram, depending on the number of doses to be administered, and the concentration of antigen in the composition suitable for administration is typically from 0.001 % w/v to 0.5% w/v, preferably from about

0.005% w/v to 0.05% w/v.

Certain preferred twin-pack vaccine compositions according to the present invention comprise a first pack containing freeze-dhed recombinant anthrax protective antigen which has been freeze-dried in the presence of trehalose, and which is free from aluminium oxyhydroxide, and a second pack comprising aluminium oxyhydroxide suspension in phosphate buffered saline solution. It is especially preferred that the weight ratio of anthrax protective antigen to trehalose is in the range of from 1 : 50 to 1 : 1000, preferably from 1 : 300 to 1 : 700. In many embodiments, the weight ratio of anthrax protective antigen in the first pack to aluminium oxyhydroxide in the second pack is from 1

: 5 to 1 : 30, such as from 1 : 10 to 1 : 15. In some embodiments, the concentration of alum in the phosphate buffered saline diluent is from 0.01 % w/v to 1 % w/v, and preferably from 0.1 % w/v to 0.5% w/v.

The vaccine compositions according to the present invention show excellent storage stability and do not require to be stored or transported under refrigerated conditions. When required, the vaccines of the present invention are employed to prepare a parenteral, ready for use composition by mixing the suspension of a pharmaceutically- acceptable solid support in a pharmaceutically-acceptable diluent with the freeze-dried vaccine composition and agitating the mixture. In many embodiments, an aliquot of diluent is injected into a container comprising the freeze-dried sub-unit vaccine and the mixture agitated to ensure mixing. The mixture is then left to stand until the sub-unit vaccine becomes adsorbed upon the solid support. In many instances, this adsorption process takes less than 5 minutes, and typically less than 1 minute. In the case of the antigen having the sequence given in Figure 1 (SEQ ID NO:

1 ) with alum as solid support, adsorption commonly takes less than 30 seconds, and most often less than 15 seconds. Once the adsorption has taken place, one or more doses can be removed from the container and administered to a patient or patients. In such embodiments, it will be recognised that the amount of sub- unit vaccine and diluent will be selected to be appropriate to the number of doses to be administered. Once prepared, the mixture is preferably used shortly after preparation, for example within one or two hours, but can be stored for relatively shod periods, such as up to 5 days, preferably under refrigeration.

In some embodiments, the freeze-dried sub-unit vaccine is stored in one compartment of a multi-compartment syringe, with the diluent being stored in another compartment. Preferably, a dual compartment syringe is employed. In such syringes, depressing the plunger results in mixing of the diluent and freeze-dried sub-unit vaccine, which is then followed by injection of the patient, after allowing the times indicated supra for adsorption to occur.

The present invention is illustrated without limitation by the following examples.

DESCRIPTION OF THE FIGURES

Figure 1 shows the sequence of anthrax protective antigen (SEQ ID NO:

1 ). Figure 2 shows a graph of immunoreactivity of a medicament according to an embodiment of the present invention containing rPA.

DETAILED DESCRIPTION OF THE INVENTION Example 1 1 ml_ of a solution comprising recombinant anthrax protective antigen

(rPA) having the sequence given in Figure 1 at 200 micrograms/ml in aqueous 0.5mM sodium phosphate buffered 1 OmM saline solution containing 0.04% w/v Tween 20 and 10% w/v trehalose was freeze dried in twelve 3ml glass vials using the freeze drying cycle detailed below. In order to determine the stability of the formulation, vials were stored at 70⁰C. rPA domain 4-immunoreactivity was used as a measure of crucial protein epitope integrity and indicator of potency. At weekly intervals over twelve weeks, a vial was removed from storage and mixed with 1 mL of a diluent containing 0.26% (w/v) alhydrogel in phosphate buffered saline, pH 7.4, with a phosphate concentration of 5mM, to form a medicament in administrable form. Analysis of unbound rPA indicated that adsorption of the rPA onto the alhydrogel was complete in less than 30 seconds. Domain 4 immunoreactivity of the rPA was determined using an assay method according to PCT patent application WO2007/122373. The activity was compared with that measured for a sample of the medicament prepared at the start of the storage trial. The results are shown in Figure 2. It can clearly be seen that the domain 4 epitope structure is retained during 12 weeks storage at 70⁰C, indicating excellent storage stability.

Freeze Drying Cycle

Freeze drying of the samples was accomplished using an LS40 freeze drier obtained from Severn Sciences Ltd, using the following freeze drying cycle:

Table 1

Product is stoppered under vacuum before introducing air into the chamber and aluminium seals used to cap the vials, by crimping, on removal from the freeze dryer. Example 2

Further development of the freeze drying cycle has been performed to improve the consistency of the process. This modified cycle (see Table 2) was performed using a Christ freeze-dryer.

Table 2

As with the previous cycle (Table 1 ), the product is stoppered under vacuum before air is introduced into the chamber. Aluminium seals are then applied to the vials and crimped, on removal from the freeze dryer.

Claims

What is Claimed:

1. A vaccine composition comprising a stabilized sub-unit vaccine and a pharmacologically-acceptable solid support in a pharmacologically-acceptable diluent.

2. An article of commerce comprising a multi-pack composition comprising a first pack comprising a stabilized sub-unit vaccine and a second pack comprising a suspension of a pharmacologically-acceptable solid support in a pharmacologically- acceptable diluent.

3. A twin pack vaccine composition comprising (a) a first pack comprising a stabilized subunit vaccine; and (b) a second pack comprising a suspension of a pharmacologically-acceptable solid support in a pharmacologically-acceptable diluent.

4. A method of preparing a medicament comprising mixing a first composition comprising a stabilized subunit vaccine with a second composition comprising a suspension of a pharmacologically-acceptable solid support in a pharmacologically- acceptable diluent.

5. A method of preparing a medicament comprising:

(a) preparing a stabilized subunit vaccine;

(b) preparing a suspension of a pharmacologically-acceptable solid support in a pharmacologically-acceptable diluent; and

(c) mixing the stabilized subunit vaccine prepared in (a) with the suspension prepared in (b).

6. The article, twin pack vaccine composition or method according to any one of claims 2 to 5, wherein the stabilized sub-unit vaccine is lyophilized in the presence of a lyoprotective or cryoprotective amount of a reducing sugar.

7. The article, twin pack vaccine composition or method of claim 6, wherein said reducing sugar is sucrose or trehalose.

8. The article, twin pack vaccine composition or method of claim 7, wherein said reducing sugar is trehalose.

9. The article, twin pack vaccine composition or method according to any one of claims 2 to 8, wherein the diluent comprises phosphate-buffered saline (PBS).

10. The article, twin pack, vaccine composition or method according to any one of claims 2 to 9, wherein the concentration of phosphate in the PBS is in the range of from 1 to 10 mM.

1 1 . The article, twin pack vaccine composition or method according to any one of claims 2 to 10, wherein the concentration of phosphate in the PBS is in the range of from 3 to 7 mM.

12. The article, twin pack vaccine composition or method according to any one of claims 2 to 1 1 , wherein the concentration of phosphate in the PBS is in the range of from 4 to 6 mM.

13. The article, twin pack vaccine composition or method according to any one of claims 2 to 12, wherein the concentration of phosphate in the PBS is about 5 mM.

14. The article, twin pack vaccine composition or method according to any one of claims 2 to 13, wherein the solid support is aluminum oxyhydroxide.

15. The article, twin pack vaccine composition or method according to any one of claims 2 to 14, wherein the sub-unit vaccine is rPA.

16. The article, twin pack vaccine composition or method according to any one of claims 2 to 15, further comprising an immunomodulatory oligonucleotide.

17. A twin pack vaccine composition, comprising a first pack containing freeze-dried recombinant anthrax protective antigen (rPA) that has been freeze- dried in the presence of trehalose, and which is free from aluminum oxyhydroxide, and a second pack comprising aluminum oxyhydroxide suspension in PBS.

18. The twin pack vaccine composition according to claim 17, wherein the weight ratio of rPA to trehalose is in the range of from 1 :50 to 1 :1000.

19. The twin pack vaccine composition according to claim 17, wherein the weight ratio of rPA to trehalose is in the range of from 1 :300 to 1 :700.

20. The twin pack vaccine composition according to claim 17, wherein the weight ratio of rPA in the first pack to aluminum oxyhydroxide in the second pack is in the range of from 1 :5 to 1 :30.

21. The twin pack vaccine composition according to claim 17, wherein the weight ratio of rPA in the first pack to aluminum oxyhydroxide in the second pack is in the range of from 1 :10 to 1 :15.

22. The twin pack vaccine composition according to claim 17, wherein the concentration of aluminum oxyhydroxide (alum) in the PBS is from 0.01% w/v to 1% w/v.

23. The twin pack vaccine composition according to claim 17, wherein the concentration of aluminum oxyhydroxide (alum) in the PBS is from 0.1 % w/v to 0.5% w/v.

24. The twin pack vaccine composition according to any of claims 17 to 23, wherein the amount of antigen present in the first pack is in the range of 5 micrograms to 5 milligrams.

25. The twin pack vaccine composition according to any of claims 17 to 23, wherein the amount of antigen present in the first pack is in the range of 10 micrograms to 1 milligram.

26. The twin pack vaccine composition according to one of claims 17 to 25, wherein the concentration of phosphate in the PBS is from 4 mM to 6 mM.

27. The twin pack vaccine composition according to one of claims 17 to 25, wherein the concentration of phosphate in the PBS is about 5 mM.