WO2009056651A1

WO2009056651A1 - Angiotensin-carrier formulation

Info

Publication number: WO2009056651A1
Application number: PCT/EP2008/064894
Authority: WO
Inventors: Stefan Landolt; Lorenz Vogt; Alexander Zuercher
Original assignee: Cytos Biotechnology Ag
Priority date: 2007-11-02
Filing date: 2008-11-03
Publication date: 2009-05-07

Abstract

The present invention is in the fields of medicine, vaccine and pharmaceutical formulation. The invention provides formulations comprising an angiotensin-virus-like particle conjugate and a stabilizer, wherein said stabilizer comprises a non-reducing disaccharide and a non-ionic surfactant. The lyophilized formulations are stable after a long time of storage at room temperature.

Description

ANGIOTENSIN-CARRIER FORMULATION

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention is in the fields of medicine, vaccine and pharmaceutical formulation. The invention provides formulations comprising an angiotensin- virus- like particle conjugate and a stabilizer, wherein said stabilizer comprises a non-reducing disaccharide and a non-ionic surfactant. The lyophilized formulations are stable after a long time of storage at room temperature.

Related Art

[0002] Vaccines for the treatment of hypertension are under development. These vaccines typically contain an angiotensin moiety, such as angiotensin I or angiotensin II, which is covalently bound to a carrier.

[0003] US 7,115,266 describes the coupling of angiotensin molecules to a virus-like particle (VLP) that forms an ordered and repetitive angiotensin-carrier conjugate and leads to the production of high titer of angiotensin- specific antibodies. It has been shown that a vaccine comprising a virus-like particle of an RNA-bacteriophage Qβ to which angiotensin II are covalently bound is efficacious in the reduction of blood pressure in hypertensive patients

(Lancet: VoI 371, 821-827).

SUMMARY OF THE INVENTION

[0004] We have found a lyophilized formulation that stabilizes angiotensin- virus- like particle conjugates which contain angiotensin molecules covalently bound to the virus-like particle. Moreover, we have surprisingly found that this lyophilized formulation is very stable for at least 13 weeks, preferably at least 15 weeks, more preferably for at least 25 weeks, and still more preferably for at least 40 weeks, when stored at room temperature or even at accelerated temperature (40 ⁰C). In addition, the lyophilized formulation of the present invention comprises a simple and economic stabilizer composition due to a minimum number of excipients included therein.

[0005] Thus, in one aspect, the invention provides a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus- like particle with at least one first attachment site; (b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and (ii) a stabilizer composition comprising: (c) at least one, preferably only one, non- reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 12% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization.

[0006] In another aspect, the invention provides a process for making the lyophilized formulation of the invention.

[0007] In still another aspect, the invention provides a reconstituted formulation comprising the lyophilized formulation of the invention dissolved and/or suspended in a physiological acceptable solution or medium or in sterile water, preferably in water for injection (WFI). In a further preferred embodiment, the reconstituted formulation further comprises an adjuvant, preferably comprises aluminium hydroxide.

FIGURES

[0008] FIG. 1 shows the stability of AngQβ under different pH in liquid solutions using SE-HPLC. Y-axis represents relative area of AngQβ main peak [%] and x-axis represents days of storage. The starting materials for FIG. IA and FIG. IB are from different batches.

[0009] FIG 2 shows the stability of AngQβ in different buffers using SE-HPLC. Y- axis represents relative changes of fronting (white bars), tailing (black bars) and main peak areas (striped bars) of formulations F020 (containing 20 mM sodium maleate pH 7.2; FIG. 2A), F023 (containing 20 mM sodium phosphate; pH 7.2; FIG. 2B) and F024 (containing 20 mM L-histidine pH 7.2; FIG. 2C) after storage of seven days at different temperatures when compared to the respective values at the beginning.

[0010] FIG 3A shows the stability of AngQβ in different NaCl concentrations after buffer exchange dialysis/tangential field flow fractionation by overlay of the SE-HPLC chromatograms. Y-axis represents absorbance [milli absorbance units] and x-axis represents retention time [minutes]. [0011] FIG. 3B: LDS-PAGE and subsequent silver staining showing the chemical stability of AngQβ in buffer containing different amounts of NaCl. Lane 1 : Ix reducing buffer, Lane 2: Qb standard 10 ng, Lane 3: Qb standard 2.50ng, Lane 4: Qb standard 1 ng, 0.1 %, Lane 5: LMW-Marker, Lane 6: QAN003 (containing 50 mM NaCl), Lane 7: QbAngFBOl (containing 0 mM NaCl), Lane 8: QbAngFB04 (containing 25 mM NaCl), Lane 9: QbAngFB05 (containing 75 mM NaCl).

[0012] FIG. 4: Bioassay analysis of formulation F067.040 (containing 0.6 mg/ml

CYT006-AngQb, 6.5% [w/v] trehalose dehydrate, 0.005% [w/v] polysorbate 20, 2OmM L- Histidine pH 7.2, 5OmM NaCl) stored at different temperatures. The p-values obtained by comparison with the reference stored at -80⁰C (QbAngFB07 containing 3.4 mg/ml AngQb, 2OmM L-Histidine pH 7.2, 5OmM NaCl) using an unpaired two-tailed t-test with a confidence interval of 95% are shown on top of each bar. All differences in the mean antibody titers elicited by the respective samples were identified as statistically not significant.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0014] Adjuvant: The term "adjuvant" as used herein refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine and pharmaceutical composition, respectively, of the present invention may provide for an even more enhanced immune response. A variety of adjuvants can be used. Examples include complete and incomplete Freund's adjuvant, aluminum hydroxide and modified muramyldipeptide. Further adjuvants are mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. Further adjuvants that can be administered with the compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts (Alum), MF-59, OM- 174, OM- 197, OM-294, and Virosomal adjuvant technology. The adjuvants can also comprise a mixture of these substances. VLP has been generally described as an adjuvant. However, the term "adjuvant", as used within the context of this application, refers to an adjuvant not being the VLP used for the inventive formulation, but in addition to said VLP.

[0015] Coat protein: The term "coat protein" and the interchangeably used term

"capsid protein" within this application, refers to a viral protein, preferably a subunit of a natural capsid of a virus, preferably of an RNA-bacteriophage, which is capable of being incorporated into a virus capsid or a VLP.

[0016] Formulation prior to lyophilization: The term "formulation prior to lyophilization" refers to the liquid formulation of the present invention, which is subject to lyophilization process, typically and preferably within 24 hours, and further typically and preferably within 8 hours, and even more typically and preferably within 2 to 4 hours. The term "lyophilization process" and the term "freeze-drying process" are interchangably used herein and shall be regarded as synonyms.

[0017] Lyophilized formulation: the term "lyophilized formulation" refers to the composition that is obtained or obtainable by the process of freeze drying of a liquid formulation. Typically and preferably it is a solid composition having a water content of less than 5%, preferably of less than 3%. Preferably, the term "lyophilized formulation" refers to the composition obtained or obtainable by the process for making the lyophilized formulation of the present invention.

[0018] Reconstituted formulation: the term "reconstituted formulation" refers to the liquid formulation resulted from the dissolving and/or suspension of the lyophilized formulation in a physiologically acceptable solution or medium, wherein preferably said physiologically acceptable medium is water for injection. Optionally and preferably said physiologically acceptable medium may also further include adjuvants containing media such as, preferably, an aluminium hydroxide suspension.

[0019] Room temperature: the term "room temperature" as used herein, refers to a temperature from 15°C to 30 ⁰C, preferably from 23°C to 27°C, more preferably 25°C. [0020] Stable: the term "stable" as used herein, refers to the state of the lyophilized formulation of the invention comprising angiotensin- VLP conjugates, preferably comprising angiotensin- VLP of an RNA-bacteriophage, and even further preferably comprising AngQβ, in which, up to 13 weeks, preferably up to 15 weeks, more preferably up to 20 weeks, and still more preferably up to 25 weeks of storage at room temperature or at accelerated temperature (40⁰C), the total amount of angiotensin- VLP degradation products, preferably the total amount of angiotensin- VLP of an RNA-bacteriophage degradation products, further preferably the total amount of AngQβ degradation products, increases not more than 8%, preferably not more than 5%, more preferably not more than 3%, even more preferably not more than 2%, even more preferable not more than 1%, as compared to the respective total amount of degradation products prior to lyophilization. The amount of the sum of degradation products after storage after subtracting the amount of the sum of degradation products prior to lyophilization gives the percentage of increase, as used herein. For example, if in the formulation prior to lyophilization there are 1% of AngQβ degradation products and after lyophilization and 15 weeks of storage, there are 3% of AngQβ degradation products in the reconstituted formulation, then the percentage of increase is 2%. Furthermore the amount of the sum of angiotensin- VLP oligomers and aggregates in the formulation after storage subtracts the amount of the sum of angiotensin- VLP oligomers and aggregates prior to lyophilization gives the percentage of increase, as used herein. For example, if in the formulation prior to lyophilization there is 1% of AngQβ oligomers and aggregates and after lyophilization according to the present invention and 15 weeks of storage, there is 4% of AngQβ oligomers and aggregates in the reconstituted formulation, then the percentage of increase is 3%. The method to determine the amount of the sum of angiotensin- VLP oligomers and aggregates, preferably the sum of angiotensin- VLP of RNA-bacteriophage Qβ oligomers and aggregates, preferably the sum of AngQβ oligomers and aggregates, is preferably done by asymmetrical flow field flow fractionation (AF4) assay as described in EXAMPLE 1 herein, in which fractions containing particles larger than angiotensin- VLP monomers and dimers, preferably larger than angiotensin- VLP of RNA-bacteriophage Qβ monomers and dimers, preferably larger than AngQβ monomers and dimers, are combined in calculation.

[0021] The term "degradation products", as used herein, refers to substances resulting from the degradation of the linkage between the angiotensin and the virus-like particle, the disassociation between coat proteins of the virus-like particle, the leakage of nucleic acids, particularly RNA content from the virus-like particle as well as degradation of peptide bonds in the conjugate. Methods to determine the amount of degradation products are known to a skilled person. Preferred methods are the asymmetrical flow field flow fractionation (AF4) assay, LDS-PAGE and size exclusion HPLC (SE-HPLC) assay as described in EXAMPLE 1 herein. By way of example, in SE-HPLC chromatogram, the main peak normally corresponds to the intact angiotensin- VLP conjugate monomers. The area of the main peak is calculated from the lowest point of the left valley to the lowest point of the right valley. The total area left to the lowest point of the left valley normally represents the aggregations and oligomers of the angiotensin- VLP conjugate, whereas the total area right to the lowest point of the right valley normally represents the degradation products. The percentage of the total area of the degradation products relative to the total area of all three areas (which corresponds to 100 %) is defined as the total amount of degradation products.

[0022] Oligomer: The term "oligomer", as used in the term "angiotensin-

VLP oligomer", "angiotensin- VLP of an RNA-bacteriophage oligomer" and "AngQβ oligomer" refers to the aggregation of at least three and up to ten VLPs, VLPs of an RNA-bacteriophage or VLPs of Qβ, respectively.

[0023] Aggregate: The term "aggregate", as used in the term "angiotensin- VLP aggregate", "angiotensin- VLP of an RNA-bacteriophage aggregate" and "AngQβ aggregate" refers to the aggregation of at least ten VLPs, VLPs of an RNA-bacteriophage or VLPs of Qβ, respectively.

[0024] Virus particle: The term "virus particle" as used herein refers to the morphological form of a virus. In some virus types it comprises a genome surrounded by a protein capsid; others have additional structures (e.g., envelopes, tails, etc.). [0025] Virus-like particle (VLP), as used herein, refers to a non-replicative and noninfectious virus particle, or refers to a non-replicative and non-infectious structure resembling a virus particle, preferably a capsid of a virus. The term "non-replicative", as used herein, refers to being incapable of replicating the genome comprised by the VLP. The term "noninfectious", as used herein, refers to being incapable of entering the host cell. Preferably a virus-like particle in accordance with the invention is non-replicative and non-infectious since it lacks all or part of the viral genome or genome function. In one embodiment, a virus-like particle is a virus particle, in which the viral genome has been physically or chemically inactivated. Typically and more preferably a virus-like particle lacks all or part of the replicative and infectious components of the viral genome. A virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome. A preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus, bacteriophage, preferably RNA- bacteriophage. The terms "viral capsid" or "capsid", refer to a macromolecular assembly composed of viral protein subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits. Typically and preferably, the interactions of these subunits lead to the formation of viral capsid or viral-capsid like structure with an inherent repetitive organization, wherein said structure is, typically, spherical or tubular. For example, the capsids of RNA-bacteriophages or HBcAgs have a spherical form of icosahedral symmetry. The term "capsid-like structure" as used herein, refers to a macromolecular assembly composed of viral protein subunits resembling the capsid morphology in the above defined sense but deviating from the typical symmetrical assembly while maintaining a sufficient degree of order and repetitiveness. One common feature of virus particle and virus-like particle is its highly ordered and repetitive arrangement of its subunits.

[0026] Virus-like particle of an RNA-bacteriophage: As used herein, the term "virus-like particle of an RNA-bacteriophage" refers to a virus-like particle comprising, or preferably consisting essentially of or consisting of coat proteins, mutants or fragments thereof, of an RNA-bacteriophage. In addition, virus-like particle of an RNA-bacteriophage resembling the structure of an RNA-bacteriophage, being non replicative and non-infectious, and lacking at least the gene or genes encoding for the replication machinery of the RNA-bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host. This definition should, however, also encompass virus- like particles of RNA-bacteriophages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and non-infectious virus- like particles of a RNA-bacteriophage. Preferred VLPs derived from RNA-bacteriophages exhibit icosahedral symmetry and consist of 180 subunits. Within this present disclosure the term "subunit" and "monomer" are interexchangeably and equivalently used within this context. Preferred methods to render a virus-like particle of an RNA-bacteriophage non- replicative and non-infectious is by physical, chemical inactivation, such as UV irradiation, formaldehyde treatment, typically and preferably by genetic manipulation. [0027] One, a, or an: when the terms "one", "a", or "an" are used in this disclosure, they mean "at least one" or "one or more" unless otherwise indicated.

[0028] Attachment Site, First: As used herein, the phrase "first attachment site" refers to an element which is naturally occurring with the VLP and the VLP of an RNA- bacteriophage, respectively, or which is artificially added to the VLP and the VLP of an RNA-bacteriophage, respectively, and to which the second attachment site may be linked. The first attachment site may be a protein, a polypeptide, an amino acid, a peptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxy 1 group, a guanidinyl group, histidinyl group, or a combination thereof. A preferred embodiment of a chemically reactive group being the first attachment site is the amino group of an amino acid such as lysine. A preferred embodiment of said first attachment site is the amino group of an amino acid such as lysine. The first attachment site is located, typically on the surface, and preferably on the outer surface of the VLP and the VLP of an RNA- bacteriophage, respectively. Multiple first attachment sites are present on the surface, preferably on the outer surface of virus-like particle and the VLP of an RNA-bacteriophage, respectively, typically in a repetitive configuration. In a preferred embodiment the first attachment site is associated with the VLP and the VLP of an RNA-bacteriophage, respectively, through at least one covalent bond, preferably through at least one peptide bond. In a further preferred embodiment the first attachment site is naturally occurring with the VLP and the VLP of an RNA-bacteriophage, respectively. Alternatively, in a preferred embodiment the first attachment site is artificially added to the VLP and the VLP of an RNA- bacteriophage, respectively. In further preferred embodiment, the at least one first attachment site is the amino group of a lysine residue, wherein said lysine residue is comprised by a recombinant protein of the VLP of an RNA-bacteriophage, preferably of the VLP of RNA bacteriophage Qβ. In a further very preferred embodiment, the at least one first attachment site is the amino group of a lysine residue, wherein said lysine residue is comprised by a coat protein of the VLP of an RNA-bacteriophage, wherein preferably said lysine residue is comprised by a coat protein of the VLP of RNA-bacteriophage Qβ, wherein further preferably said coat protein has the amino acid sequence of SEQ ID NO:7.

[0029] Attachment Site, Second: As used herein, the phrase "second attachment site" refers to an element which is naturally occurring with or which is artificially added to the angiotensin molecule and to which the first attachment site may be linked. The second attachment site may be a protein, a polypeptide, a peptide, an amino acid, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxy 1 group, a guanidinyl group, histidinyl group, or a combination thereof. A preferred embodiment of a chemically reactive group being the second attachment site is the sulfhydryl group, preferably of an amino acid cysteine. A preferred embodiment of said second attachment site is the sulfhydryl group, preferably of an amino acid cysteine. The terms "angiotensin molecule with at least one second attachment site" refers, therefore, to a construct comprising the angiotensin molecule and at least one second attachment site. However, in particular for a second attachment site, which is not naturally occurring within the angiotensin molecule such a construct typically and preferably further comprises a "linker". In another preferred embodiment the second attachment site is associated with the angiotensin molecule through at least one covalent bond, preferably through at least one peptide bond. In a further embodiment, the second attachment site is naturally occurring within the angiotensin molecule. In another further preferred embodiment, the second attachment site is artificially added to the angiotensin molecule through a linker, wherein said linker comprises or alternatively consists of a cysteine. Preferably the linker is fused to the angiotensin molecule by a peptide bond.

[0030] Linker: A "linker", as used herein, either associates the second attachment site with angiotensin molecule or already comprises, essentially consists of, or consists of the second attachment site. Preferably, a "linker", as used herein, already comprises the second attachment site, typically and preferably - but not necessarily - as one amino acid residue, preferably as a cysteine residue. However, this does not imply that such a linker consists exclusively of amino acid residues, even if a linker consisting of amino acid residues is a preferred embodiment of the present invention. The amino acid residues of the linker are, preferably, composed of naturally occurring amino acids. Association of the linker with the angiotensin molecule is preferably by way of at least one covalent bond, more preferably by way of at least one peptide bond.

[0031] In one aspect the invention provides a lyophilized formulation comprising, consisting essentially of, or consisting of: a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising or consisting of: (a) a virus-like particle with at least one first attachment site; (b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and (ii) a stabilizer composition comprising or consisting of: (c) at least one, preferably only one, non-reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 1 to 12% (w/v), preferably from 3 to 12% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non- ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization. In contrast to the corresponding liquid formulation, in which degradation and aggregation can be readily detected within two weeks of storage, the lyophilized formulation is surprisingly so stable that almost no changes in VLP integrity after 3 months of storage at 40 ⁰C was detected. Furthermore, the lyophilized formulation has an enhanced stability of the RNA component packaged inside the virus-like particle, preferably packaged inside the virus-like particle of an RNA-bacteriophage, more preferably a virus-like particle of an RNA-bacteriophage Qβ. [0032] Alternatively in another aspect, the invention provides a liquid formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus- like particle with at least one first attachment site; (b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and (ii) a stabilizer composition comprising: (c) at least one, preferably only one, non- reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 1 to 12% (w/v), preferably from 3 to 12% (w/v) in terms of the concentration in said formulation, (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non- ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in said formulation; and wherein said stabilizer composition has a pH value from 6.6 to 7.8. Furthermore, the invention provides a formulation obtainable by a method of lyophilization comprising the step of freezing said liquid formulation and drying said liquid formulation. [0033] In one preferred embodiment, the liquid or lyophilized formulation of the invention comprises only one carbohydrate, preferably only one sugar, the sugar is preferably a non-reducing disaccharide.

[0034] In one preferred embodiment, the liquid or lyophilized formulation of the invention does not comprise a bovine serum albumin or a human serum albumin. In one further preferred embodiment, the formulation of the invention does not comprise any kind of a serum protein. The exclusion of serum advantageously avoids the potential serum contamination problem.

[0035] In one preferred embodiment, the at least one, preferably one single, non- reducing disaccharide is sucrose or trehalose. In one further preferred embodiment, the non- reducing disaccharide is trehalose.

[0036] In one preferred embodiment, the concentration of the at least one, preferably one single, non-reducing disaccharide is from 4% to 10% (w/v), preferably from 3% to 8%, preferably from 5% to 8% (w/v), preferably 6.5% (w/v), in terms of concentration in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. The concentration of trehalose expressed in the whole application, unless otherwise explicitly indicated, refers to the concentration of trehalose dihydrate (2H₂O) (w/v). It is general knowledge for a skilled person to convert between the concentration of trehalose dihydrate and the concentration of water-free trehalose. For example, 10% trehalose dihydrate equals to approximately 9% water- free trehalose. [0037] In one preferred embodiment, the stabilizer composition of the liquid or lyophilized formulation of the invention, preferably of the lyophilized formulation, further comprises at least one, preferably one single, bulking agent. In one further preferred embodiment, the total concentration of said non-reducing disaccharide and said bulking agent is from 1 to 12% (w/v), with the proviso that the concentration of said non-reducing disaccharide is at least 0.5% (w/v), preferably at least 0.75% (w/v), in terms of the concentration in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. In one still further preferred embodiment, the total concentration of said at least one, preferably one single, non-reducing disaccharide and said at least one, preferably one single, bulking agent is from 3% to 10%, preferably from 3% to 8% (w/v), preferably from 3% to 7%, preferably from 3% to 5%, more preferably from 3% to 4% (w/v) in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. [0038] In one preferred embodiment, the total concentration of said at least one, preferably one single, non-reducing disaccharide and said at least one, preferably one single, bulking agent is from 3% to 10% (w/v), preferably from 3% to 7% (w/v) in the formulation prior to lyophilization, wherein the concentration of said non-reducing disaccharide is at least 0.5% (w/v). Preferably the osmolarity of the formulation is from 250 to 350 mosm/Kg, more preferably about 300mosm/Kg.

[0039] In one preferred embodiment, the total concentration of said at least one, preferably one single, non-reducing disaccharide and said at least one, preferably one single, bulking agent is from 3% to 10% (w/v), preferably from 3% to 7% (w/v) in the formulation prior to lyophilization, wherein the concentration of said bulking agent is at least 1% (w/v). Preferably the osmolarity of the formulation is from 250 to 350 mosm/Kg, more preferably about 300mosm/Kg.

[0040] In one preferred embodiment, the total concentration of said at least one, preferably one single, non-reducing disaccharide and said at least one, preferably one single, bulking agent is from 3% to 10% (w/v), preferably from 3% to 7% (w/v) in formulation prior to lyophilization, wherein the concentration of said bulking agent is at least 1% (w/v) and the concentration of said non-reducing disaccharide is at least 0.5% (w/v). Preferably the osmolarity of the formulation is from 250 to 350 mosm/Kg, more preferably about 300mosm/Kg.

[0041] In one preferred embodiment, the non-reducing disaccharide is trehalose and the bulking agent is mannitol.

[0042] In one very preferred embodiment, the total concentration of said at least one, preferably one single, non-reducing disaccharide and said at least one, preferably one single, bulking agent is from 3.0 to 5.0%(w/v) in the formulation prior to lyophilization. In one further preferred embodiment, the ratio between the bulking agent and the non-reducing disaccharide is from 3.5:1 to 4.5:1, preferably 4:1. In one still further preferred embodiment, the non-reducing disaccharide is trehalose and the bulking agent is mannitol. In one very preferred embodiment, the concentration of trehalose is 0.8% (w/v) and the concentration of mannitol is 3.2% (w/v), in the formulation prior to lyophilization.

[0043] In one preferred embodiment, the bulking agent is mannitol or glycine. In one further preferred embodiment, the bulking agent is mannitol. The inclusion of the bulking agent, preferably mannitol, contributes to the obtaining of a stable cake structure and may allow higher primary drying temperature in the lyophilization process, which advantageously reduces the production cost.

[0044] In one preferred embodiment, the pH of the stabilizer composition is from 6.6 to 7.8, preferably from 6.8 to 7.6, preferably from 7.0 to 7.4. In one further preferred embodiment, the pH is at 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 and 7.8. In one preferred embodiment, the pH of the stabilizer is 7.2.

[0045] In one preferred embodiment, the non-ionic surfactant is from 0.0025% to

0.02% (w/v), preferably 0.0025%-0.01% (w/v), preferably 0.005% (w/v), in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization.

[0046] In one preferred embodiment, the non- ionic surfactant is polysorbate 20 or polysorbate 80. In one preferred embodiment, the non-ionic surfactant is polysorbate 20. [0047] Virus-like particles may be of any virus known in the art having an ordered and repetitive structure. Illustrative DNA or RNA viruses, the coat or capsid protein of which can be used for the preparation of VLPs have been disclosed in WO 2004/009124 on page 25, line 10-21, on page 26, line 11-28, and on page 28, line 4 to page 31, line 4. These disclosures are incorporated herein by way of reference.

[0048] In one further preferred embodiment, the virus-like particle is of a virus selected from a group consisting of: a) RNA bacteriophages; b) bacteriophages; c) Hepatitis B virus, preferably its capsid protein (Ulrich, et al., Virus Res. 50:141-182 (1998)) or its surface protein (WO 92/11291); d) measles virus (Warnes, et al., Gene 160:173-178 (1995)); e) Sindbis virus; f) rotavirus (US 5,071,651 and US 5,374,426); g) foot-and-mouth-disease virus (Twomey, et al., Vaccine 13:1603 1610, (1995)); h) Norwalk virus (Jiang, X., et al., Science 250:1580 1583 (1990); Matsui, S.M., et al., J. Clin. Invest. 87:1456 1461 (1991)); i) Alphavirus; j) retrovirus, preferably its GAG protein (WO 96/30523); k) retrotransposon Ty, preferably the protein pi; 1) human Papilloma virus (WO 98/15631); m) Polyoma virus; n) Tobacco mosaic virus; o) cowpea mosaic virus; and p) Flock House Virus; q) Cowpea Chlorotic Mottle Virus; and r) an Alfalfa Mosaic Virus. Methods to produce VLP of Cowpea Chlorotic Mottle Virus, Alfalfa Mosaic Virus and cowpea mosaic virus have been described in US 2005/0260758 and in WO 05/067478.

[0049] In one preferred embodiment, the virus-like particle is a virus-like particle of an RNA-bacteriophage. In one further preferred embodiment, the RNA-bacteriophage is selected from the group consisting of a) bacteriophage Qβ; b) bacteriophage Rl 7; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage Mi l; h) bacteriophage MXl; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205. In one preferred embodiment, the virus-like particle is a virus-like particle of an RNA-bacteriophage Qβ. Methods for the production of VLP of an RNA-bacteriophage, in particular VLP of bacteriophage Qβ and VLP bacteriophage AP205 have been described at page 37-47 of WO 04/009124 and in EXAMPLES 1 and 21 thereof.

[0050] In another preferred embodiment of the present invention, the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant proteins, or fragments thereof, of the RNA-bacteriophage Qβ, of the RNA- bacteriophage fr, or of the RNA-bacteriophage AP205.

[0051] In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant coat proteins, or fragments thereof, of the RNA-bacteriophage Qβ, of the RNA- bacteriophage fr, or of the RNA-bacteriophage AP205.

[0052] Specific examples of bacteriophage coat proteins which can be used to prepare the angiotensin- virus- like particle conjugates of the invention include the coat proteins of RNA bacteriophages such as RNA-bacteriophage Qβ (SEQ ID NO:7), and RNA- bacteriophage AP205 (SEQ ID NO:8 and SEQ ID NO:9).

[0053] In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or alternatively consists of one or more, preferably 180, recombinant coat proteins of RNA-bacteriophage Qβ, wherein preferably said recombinant coat protein of RNA-bacteriophage Qβ comprises, or alternatively essentially consists of, or alternatively consists of the amino acid sequence as set forth in SEQ ID NO.7.

[0054] In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or alternatively consists of recombinant coat proteins of RNA-bacteriophage AP205, wherein preferably said recombinant coat protein of RNA-bacteriophage AP205 comprises, or alternatively essentially consists of, or alternatively consists of one or more coat proteins having an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO:9.

[0055] In one preferred embodiment, the virus-like particle is a virus-like particle of

RNA-bacteriophage Qβ. In one further preferred embodiment, the virus-like particle is a virus-like particle of RNA-bacteriophage Qβ recombinantly expressed in E. coli. In a further very preferred embodiment of the present invention, the virus-like particle is a virus-like particle of RNA-bacteriophage Qβ, wherein said virus-like particle of said RNA- bacteriophage Qβ comprises, or alternatively essentially consists of, or alternatively consists of one or more, preferably 180, recombinant coat proteins of RNA-bacteriophage Qβ, wherein said recombinant coat protein comprises, or alternatively essentially consists of, or alternatively consists of the amino acid sequence as set forth in SEQ ID NO:7. [0056] In one preferred embodiment, the angiotensin molecule is selected from the group consisting of: (a) angiotensinogen (SEQ ID NO:1); (b) angiotensin I (SEQ ID NO:2); and (c) angiotensin II (SEQ ID NO:3). In one further preferred embodiment, the angiotensin molecule is angiotensin II (SEQ ID NO:3).

[0057] In one preferred embodiment, the at least one angiotensin with at least one second attachment site comprises a linker, wherein preferably said linker comprises a cysteine residue. Preferably the linker is fused, either to the C-terminus, or preferably to the N- terminus of the angiotensin molecule by a peptide bond.

[0058] In one further preferred embodiment, the angiotensin molecule with at least one second attachment site has an amino acid sequence selected from a group consisting of: (a) CGG angiotensinogen (SEQ ID NO:6); (b) CGG angiotensin I (SEQ ID NO:5) and (c) CGG angiotensin II (SEQ ID NO:4).

[0059] In one further preferred embodiment, the angiotensin molecule with at least one second attachment site has the amino acid sequence as set forth in SEQ ID NO:4. [0060] In one preferred embodiment, the first attachment site comprises or preferably is an amino group. In one further preferred embodiment, the first attachment site comprises or preferably is an amino group of a lysine residue. In further preferred embodiment, the at least one first attachment site is the amino group of a lysine residue, wherein said lysine residue is comprised by and is part of, respectively, a recombinant protein of a RNA-bacteriophage, and further preferably of RNA-bacteriophage Qβ. In a further very preferred embodiment, the at least one first attachment site is the amino group of a lysine residue, wherein said lysine residue is comprised by and is part of, respectively, a coat protein of an RNA-bacteriophage, preferably wherein said lysine residue is comprised by and is part of, respectively, a coat protein of the RNA-bacteriophage Qβ, wherein further preferably said coat protein has the amino acid sequence of SEQ ID NO:7. In a further very preferred embodiment, the at least one first attachment site is the amino group of a lysine residue, wherein said lysine residue is comprised by and is part of, respectively, a coat protein of an RNA-bacteriophage Qβ, wherein said coat protein has the amino acid sequence of SEQ ID NO:7. [0061] In one preferred embodiment, the second attachment site comprises or preferably is a sulfhydryl group. In one further preferred embodiment, the second attachment site comprises or preferably is a sulfhydryl group of a cysteine residue. In another preferred embodiment, the second attachment site comprises or preferably is a sulfhydryl group of a cysteine residue, wherein the cysteine residue is comprised by and is part of, respectively, an linker, wherein said linker is fused to the N-terminus of the angiotensin molecule. [0062] In one preferred embodiment, association between said first and second attachment site is through a hetero-bifunctional linker. Several hetero-bifunctional cross- linkers are known in the art. These include the cross-linkers SMPH, Sulfo- MBS, Sulfo- EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross- linkers available, for example from the Pierce Chemical Company (Rockford, 111., USA), and having one functional group reactive towards amino groups and one functional group reactive towards SH residues. The above mentioned cross-linkers all lead to formation of a thioether linkage. Another class of cross-linkers suitable in the practice of the invention is characterized by the introduction of a disulfide linkage between the angiotensin molecule and the virus-like particle upon coupling. Cross-linkers belonging to this class include, for example SPDP and Sulfo-LC-SPDP (Pierce). In one very preferred embodiment, the hetero-bifunctional linker is SMPH (succinimidyl-6-β-maleimidoprpionamido hexanoate).

[0063] In one preferred embodiment, the concentration of the angiotensin- VLP conjugate is from 0.1mg/ml to 2.5 mg/ml in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. In one preferred embodiment, the the concentration of angiotensin- VLP conjugate is from 0.2mg/ml to 2mg/ml in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. In another preferred embodiment of the present invention, the concentration of the angiotensin- VLP conjugate in the liquid formulation, typically and preferably in terms of concentration in the formulation prior to lyophilization, is 0.2mg/ml, 0.6mg/ml, 1.0mg/ml, 1.2mg/ml, 1.8mg/ml or 2mg/ml. In again another preferred embodiment of the present invention, the concentration of the angiotensin- VLP conjugate in the liquid formulation, typically and preferably in terms of the concentration in the formulation prior to lyophilization is 0.6mg/ml. [0064] In one preferred embodiment, the stabilizer composition comprises a buffering agent. Examples of such buffer agent include Succinate, Acetate, Maleate, Citrate, Lactate, Tartrate, Tris, Bis-tris, Triethanolamine, Tricine, Bicine, Histidine, Glycinate, Glutamate, Lysine, Alanine, Phenylalanine, Arginine, phosphate, iso-leucine and Proline. In one further preferred embodiment, the stabilizer composition comprises a buffering agent selected from the group consisting of: Maleate, Tris, Bis-tris, Triethanolamine, Tricine, Bicine, Histidine, phosphate, Glycinate. In one further preferred embodiment, the stabilizer composition comprises a buffering agent selected from the group consisting of: Tris, Bis-tris, Triethanolamine, Tricine, Histidine, phosphate, Glycinate. In one preferred embodiment, the stabilizer composition comprises a buffering agent, wherein said buffer agent is a phosphate buffer. In one preferred embodiment, the stabilizer composition comprises a buffering agent, wherein said buffer agent is Glycinate buffer

[0065] In one further preferred embodiment, the concentration of the buffering agent is from 10-6OmM in terms of concentration in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. In one preferred embodiment, the concentration of the buffering agent is 10-4OmM. In one preferred embodiment, the concentration of the buffering agent is 2OmM. In one further preferred embodiment, the buffering agent is sodium phosphate or potassium phosphate, preferably sodium phosphate. In one preferred embodiment, the buffering agent is histidine/histidine HCl. In one preferred embodiment, the buffering agent is Glycinate buffer. [0066] Normally a lyophilized formulation does not comprise sodium chloride. The exclusion of NaCl avoids unnecessary high osmolarity in the formulation. Moreover the exclusion of salt further eliminates the possible adverse effect of salt on protein stability during lyophilization. Furthermore, as stated in Chapter 5 (author Carpenter, J.F: et al) of "Rational Design of Stable Protein Formulations: Theory and Practice", Pharmaceutical Biotechnology, Volume 13, 2002), "during lyophilization, NaCl can greatly reduce the collapse temperature of a formulation, if a fraction of the salt does not crystallize. Crystallization of NaCl during freezing and annealing can be inhibited by other excipents (e,g, bulking agents and stabilizing sugars). Because of the low collapse temperature, a low temperature cycle must be used, which increases production time and costs. Thus, if at all possible, NaCl should not be used in lyophilized formulations.

[0067] However, a liquid formulation comprising the angiotensin- VLP conjugates of the invention in a buffer agent within optimal pH range, preferably at pH 7.2 is not sufficiently stable. While no detectable increase of oligomers and aggregates of angiotensin- VLP conjugates have been detected, there is a detectable increase of angiotensin- VLP conjugates degradation products by the SE-HPLC method. This problem is more pronounced if the buffer agent is Histidine/HistidineHCl or other non-salt buffer agent, particularly amino acid buffer agent.

[0068] It has been surprisingly found that the addition of salt, preferably sodium chloride, effectively prevented the degradation of the angiotensin- VLP conjugates. More surprisingly we have found that the presence of sodium chloride in the lyophilized formulation did not exert any adverse effect on the stability of the angiotensin- VLP conjugates. In addition, despite the presence of sodium chloride, the lyophilized formulation was still obtainable through an efficient lyophilizing process disclosed herein. [0069] In one preferred embodiment, the liquid or lyophilized formulation of the invention further comprises a salt, preferably only one salt. Examples of suitable salts include NaCl, MgCl₂, KCl, CaCl₂, Na₂SO₄, CaSO₄, MgSO₄ and K₂SO₄. In one preferred embodiment, the salt is sodium chloride, wherein the concentration of sodium chloride is at least 2OmM, preferably at least 3OmM. In one preferred embodiment, the concentration of sodium chloride is from 25mM to 75mM, preferably 5OmM, in terms of the concentration in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. The concentration of sodium chloride, normally and preferably, does not exceed 9OmM.

[0070] In one preferred embodiment, the stabilizer composition comprises only one buffering agent, wherein said buffer agent is Histidine/HistidineHCl. Preferably the concentration of said Histidine/HistidineHCl is 1OmM to 4OmM, preferably 2OmM, in terms of the concentration in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization.

[0071] In one further preferred embodiment, the stabilizer composition further comprises a salt, preferably only one salt, wherein the salt is preferably selected from the group consisting of: NaCl, MgCl₂, KCl, CaCl₂, Na₂SO₄, CaSO₄, MgSO₄ and K₂SO₄, further preferably the salt is sodium chloride, and wherein the buffer agent is Glycinate, or preferably is Histidine/HistidineHCl, and wherein the concentration of sodium chloride is at least 2OmM, preferably at least 3OmM. In one preferred embodiment, the concentration of sodium chloride is 25mM to 75mM, preferably 5OmM, in terms of the concentration in the liquid formulation, or with respect to the lyophilized formulation, in terms of concentration in the formulation prior to lyophilization. The concentration of sodium chloride, normally and preferably, does not exceed 9OmM. [0072] In one aspect the invention provides a lyophilized formulation comprising: a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA-bacteriophage Qβ with at least one first attachment; and (b) at least one angiotensin molecule with at least one second attachment site having, preferably consisting of, the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; and (ii) a stabilizer composition comprising: (c) at least one, preferably only one, non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, wherein the concentration of said non-reducing disaccharide is from 3 to 8%, preferably 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v), preferably 0.005% (w/v), in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 7.0 to 7.4, preferably 7.2, prior to lyophilization. [0073] In one aspect the invention provides a lyophilized formulation comprising: a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA-bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA-bacteriophage Qβ comprises one or more, preferably 180, recombinant coat proteins having, preferably consisting of, the amino acid sequence as set forth in SEQ ID NO:7, and wherein said at least one first attachment is comprised by and is part of, respectively, of said recombinant coat protein; and (b) at least one angiotensin molecule with at least one second attachment site, wherein said angiotensin molecule with said second attachment site has, preferably consists of, the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH and through at least one non-peptide covalent bond; and (ii) a stabilizer composition comprising: (c) at least one, preferably only one, non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, wherein the concentration of said non-reducing disaccharide is from 3 to 8%, preferably 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v), preferably 0.005% (w/v), in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 7.0 to 7.4, preferably 7.2, prior to lyophilization. [0074] In a further aspect the invention provides a lyophilized formulation comprising: a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA-bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA-bacteriophage Qβ comprises one or more, preferably 180, recombinant coat proteins having, preferably consisting of, the amino acid sequence as set forth in SEQ ID NO: 7; and (b) at least one angiotensin molecule with at least one second attachment site, wherein said angiotensin molecule with said second attachment site has, preferably consists of, the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH and through at least one non-peptide covalent bond; and (ii) a stabilizer composition comprising: (c) at least one, preferably only one, non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, wherein the concentration of said non- reducing disaccharide is from 3 to 8%, preferably 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v), preferably 0.005% (w/v), in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 7.0 to 7.4, preferably 7.2, prior to lyophilization.

[0075] In one aspect the invention provides a lyophilized formulation comprising, consisting essentially of, or preferably consisting of (i) at least one angiotensin- virus- like particle conjugate comprising or preferably consisting of: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment; and (b) at least one angiotensin molecule with at least one second attachment site having, preferably consisting of, the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; and (ii) a stabilizer composition comprising or preferably consisting of: (c) at least one, preferably only one, non- reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 8%, preferably 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent, wherein said buffering agent is Histidine/HistidineHCl, and wherein preferably the concentration of said

Histidine/HistidineHCl is 2OmM; (e) 25-75 mM, preferably 5OmM, sodium chloride, in terms of the concentration in the formulation prior to lyophilization (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non- ionic surfactant is from 0.0025% to 0.1% (w/v), preferably 0.005% (w/v), in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 7.0 to 7.4, preferably 7.2, prior to lyophilization. [0076] In one aspect the invention provides a lyophilized formulation comprising, consisting essentially of, or preferably consisting of (i) at least one angiotensin- virus- like particle conjugate comprising or preferably consisting of: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more, preferably 180, recombinant coat proteins having, preferably consisting of, the amino acid sequence as set forth in SEQ ID NO:7, and wherein said at least one first attachment is comprised by and is part of, respectively, of said recombinant coat protein; and (b) at least one angiotensin molecule with at least one second attachment site having, preferably consisting of, the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH and through at least one non-peptide covalent bond; and (ii) a stabilizer composition comprising or preferably consisting of: (c) at least one, preferably only one, non-reducing disaccharide, wherein the concentration of said non- reducing disaccharide is from 3 to 8%, preferably 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent, wherein said buffering agent is Histidine/HistidineHCl, and wherein preferably the concentration of said Histidine/HistidineHCl is 2OmM; (e) 25-75 mM, preferably 5OmM, sodium chloride, in terms of the concentration in the formulation prior to lyophilization (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v), preferably 0.005% (w/v), in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 7.0 to 7.4, preferably 7.2, prior to lyophilization.

[0077] In one aspect the invention provides a lyophilized formulation comprising, consisting essentially of, or preferably consisting of (i) at least one angiotensin- virus- like particle conjugate comprising or preferably consisting of: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more, preferably 180, recombinant coat proteins having, preferably consisting of, the amino acid sequence as set forth in SEQ ID NO:7; and (b) at least one angiotensin molecule with at least one second attachment site having, preferably consisting of, the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH and through at least one non-peptide covalent bond; and (ii) a stabilizer composition comprising or preferably consisting of: (c) at least one, preferably only one, non-reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 8%, preferably 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent, wherein said buffering agent is Histidine/HistidineHCl, and wherein preferably the concentration of said Histidine/HistidineHCl is 2OmM; (e) 25-75 mM, preferably 5OmM, sodium chloride, in terms of the concentration in the formulation prior to lyophilization (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non- ionic surfactant is from 0.0025% to 0.1% (w/v), preferably 0.005% (w/v), in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 7.0 to 7.4, preferably 7.2, prior to lyophilization.

[0078] In one aspect, the present invention provides a reconstituted formulation comprising the lyophilized formulation of the invention dissolved and/or suspended in a physiologically acceptable solution or medium or in sterile water, preferably in water for injection. In another preferred embodiment, the solution is NaCl solution. Preferably the reconstituted formulation has a physiologically acceptable osomolarity value. [0079] In one preferred embodiment, the reconstituted formulation further comprises an adjuvant. In one further preferred embodiment, the adjuvant is aluminium hydroxide hydrated gels or aluminium phosphate hydrated gels.

[0080] In one aspect, the present invention provides for a process for manufacturing a lyophilized formulation comprising the steps of (i) freezing a liquid formulation to a temperature in the range of -20⁰C to -80⁰C preferably in the range of -40⁰C to -50⁰C, more preferably -42°C to -48°C, most preferably -45°C, wherein said liquid formulation comprises at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle with at least one first attachment site; (b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and a stabilizer composition comprising: (c) at least one non-reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 12% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non- ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization; (ii) applying a vacuum in the range of 0.2 to O.OOOlmbar, preferably 0.01 to O.lmbar, more prerably 0.03 to 0.07mbar, most preferably 0.045mbar to said freezed liquid formulation; (iii) performing at least one drying step at a temperature in the range of -80⁰C to 40⁰C and, wherein said drying step includes a step of raising the initial temperature which is defined as the temperature of the frozen liquid formulation prior to step (ii) and said initial temperature is preferably in the range of -40⁰C to -50⁰C, more preferably -42°C to -48°C, most preferably -45°C, to a higher temperature present in the range of 1°C to 40⁰C, preferably 10⁰C to 35°C, more preferably 20⁰C to 30⁰C and most preferably 25°C wherein as further optional step, the temperature of the shelf is maintained at a fixed temperature in the range of -80⁰C to 40⁰C, preferably - 60⁰C to 30⁰C, more preferably -50⁰C to 27.5°C, most preferably from -45°C to 25°C at any given period of time. Preferably, said liquid formulation further contains a salt, further preferably sodium chloride.

[0081] In one aspect, the present invention provides a process for manufacturing a lyophilized formulation comprising the steps of: (i) loading a liquid formulation into a lyophilisation chamber of the freeze dryer and maintaining the temperature at 20⁰C and holding for 5 minutes at pressure of 1013 mbar, wherein said liquid composition comprises at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle with at least one first attachment site; (b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and a stabilizer composition comprising: (c) at least one non-reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 12% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non- ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization, and wherein preferably said liquid formulation further contains a salt, further preferably sodium chloride; (ii) freezing the said formulation by lowering the shelf temperature by l°C/min to -40⁰C and reducing further by 0.5°C/min to -45°C and holding for 120 minutes at pressure of 1013mbar (iii) performing an annealing step wherein temperature is raised by 1.0°C/min to -15°C and holding for 120 minutes at pressure of 1013mbar followed by lowering the temperature by l°C/min to -45⁰C and holding for 120 minutes at a pressure of 1013mbar and reducing the pressure to 0.045mbar and holding for 10 minutes (iv) performing a drying step by raising the shelf temperature by 0.025°C/min to -20⁰C and holding for 180 minutes (v) performing secondary drying step by raising the shelf temperature by 0.25°C/min to 25°C and holding for 600 minutes, the chamber is aerated with filtered dry nitrogen and (vi) sealing the obtained composition in air tight container using a lyophilization stopper, preferably a coated bromobutyl stopper.

[0082] In another aspect, the present invention also provides a process for manufacturing a lyophilized formulation comprising the steps of: (i) loading a liquid formulation in to a lyophilisation chamber of the freeze dryer and maintaining the temperature at 20⁰C and holding for 5 minutes at a pressure of 1013 mbar, wherein said liquid formulation comprises at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle with at least one first attachment site; (b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and a stabilizer composition comprising: (c) at least one non- reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 12% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization, and wherein preferably said liquid formulation further contains a salt, further preferably sodium chloride; (ii) freezing the said formulation by lowering the shelf temperature by l°C/min to -40⁰C and reducing further by 0.5°C/min to -45°C and holding for 120 minutes at pressure of 1013mbar followed by reducing the pressure to 0.045mbar and holding for 10 minutes (iii) performing a drying step by raising the shelf temperature by 0.025°C/min to -20⁰C and holding for 180 minutes (iv) performing secondary drying step by raising the shelf temperature by 0.25°C/min to 25°C and holding for 600 minutes, the chamber is aerated with filtered dry nitrogen and (v) sealing the obtained composition in air tight container using a lyophilization stopper preferably a coated bromobutyl stopper. [0083] In one aspect, the present invention provides a process for manufacturing the lyophilized formulation of the invention comprising, consisting essentially of, or consisting of the steps of: (i) load the liquid formulation of the invention into a lyophilization chamber; (ii) Shelf temperature was lowered at 1.0°C/min to -40⁰C and further at 0.5°C/min to -45°C. The temperature was held at -45°C for 2 hours. Chamber pressure was reduced to 0.045 mbar. (iii) The shelf temperature was ramped/raised to -37°C at 0.25°C/min and held for 22 hours, (iv) The shelf temperature was raised to -20⁰C at 0.1°C/min and held for 4 hours, (iv) The shelf temperature was raised to 20⁰C at 0.25°C/min and held for 10 hours. The lyophilization chamber was then aerated with filtered dry nitrogen to 800 mbar and the vials were capped in the lyophilization chamber. The vials were removed from the chamber and sealed with Flip- Off^® seals. After freeze drying stable lyophilizates were achieved, sufficient cake structure was given.

[0084] In another aspect, the present invention provides a lyophilized formulation comprising :(i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus- like particle of RNA-bacteriophage Qβ with at least one first attachment, wherein said virus- like particle of RNA-bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) at least one angiotensin molecule with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; (ii) a stabilizer composition comprising: (c) only one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 3-8 % (w/v) in terms of the concentration in the formulation prior to lyophilization; and (d) only one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.0025-0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) only one buffer agent; and wherein said stabilizer composition has a pH value from 7 ^'.0-7.4 prior to lyophilization is obtainable by a process for manufacturing said lyophilized formulation comprising the steps of (i) freezing a liquid formulation to a temperature in the range of -20⁰C to -80⁰C preferably in the range of -40⁰C to -50⁰C, more preferably -42°C to -48°C, most preferably -45°C, wherein said liquid formulation comprises at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle with at least one first attachment site; (b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and a stabilizer composition comprising: (c) at least one non-reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 12% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) at least one, preferably only one, buffer agent preferably L-histidine; (e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization; (ii) applying a vacuum in the range of 0.2 to 0.000 lmbar, preferably 0.01 to O.lmbar, more prerably 0.03 to 0.07mbar, most preferably 0.045mbar to said freezed liquid formulation; (iii) performing at least one drying step at a temperature in the range of -80⁰C to 40⁰C and, wherein said drying step includes a step of raising the initial temperature which is defined as the temperature of the frozen liquid formulation prior to step (ii) and said initial temperature is preferably in the range of -40⁰C to -50⁰C, more preferably -42°C to -48°C, most preferably -45°C, to a higher temperature present in the range of FC to 40⁰C, preferably 10⁰C to 35°C, more preferably 20⁰C to 30⁰C and most preferably 25°C wherein as further optional step, the temperature of the shelf is maintained at a fixed temperature in the range of -80⁰C to 40⁰C, preferably - 60⁰C to 30⁰C, more preferably -50⁰C to 27.5°C, most preferably from -45°C to 25°C at any given period of time. Preferably, said liquid formulation further contains a salt, further preferably sodium chloride.

[0085] In yet another aspect, the present invention provides a lyophilized formulation comprising :(i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus- like particle of RNA-bacteriophage Qβ with at least one first attachment, wherein said virus- like particle of RNA-bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) at least one angiotensin molecule with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; (ii) a stabilizer composition comprising: (c) only one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 3-8 % (w/v) in terms of the concentration in the formulation prior to lyophilization; and (d) only one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.0025-0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) only one buffer agent; and wherein said stabilizer composition has a pH value from 7 ^'.0-7.4 prior to lyophilization is obtainable by a process for manufacturing said lyophilized formulation comprising

[0086] In another aspect, the present invention provides a lyophilized product obtainable or obtained from the processes described above.

[0087] The invention thus provides lyophilized formulations which may be used for preventing and/or attenuating diseases or conditions associated with one or more components of the rennin-activated angiotensin system (RAS). The invention further provides vaccination methods for preventing and/or attenuating diseases or conditions in individuals, particularly in animals such as mammals, and particularly humans. In one embodiment, the formulations of the invention stimulate an immune response leading to the production of immune molecules, including antibodies that bind to one or more angiotensin molecules. The invention further provides vaccination methods for preventing and/or attenuating diseases or conditions associated with the RAS in individuals.

[0088] In one embodiment of the present invention, the lyophilized formulation comprises:(i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA-bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA-bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) at least one angiotensin molecule with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; (ii) a stabilizer composition comprising: (c) only one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 3-8 % (w/v) in terms of the concentration in the formulation prior to lyophilization; and (d) only one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.0025-0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) only one buffer agent; and wherein said stabilizer composition has a pH value from 7 ^'.0-7.4 prior to lyophilization may be available as such in the form of an orally consumable tablet form or in a reconstituted liquid formulation form wherein the lyophilized formulation of the present invention can be dissolved and/or suspended in a physiologically acceptable solution or medium, wherein preferably said physiologically acceptable medium is water for injection or may also further include adjuvants containing media preferably such adjuvants containing media is an aluminium hydroxide suspension.

[0089] In another embodiment of the present invention, a lyophilized formulation comprises: (i) at least one angiotensin- virus- like particle conjugate comprises: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) angiotensin II with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; and (ii) a stabilizer composition consisting of: (c)one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.005% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) 5OmM sodium chloride in terms of the concentration in the formulation prior to lyophilization; (f) one buffering agent, wherein said buffering agent is Histidine/HistidineHCl, and wherein the concentration of said Histidine/HistidineHCl is 2OmM; and wherein said stabilizer composition has a pH value of 7.2 prior to lyophilization may be available as such in the form of an orally consumable tablet form or in a reconstituted liquid formulation form wherein the lyophilized formulation of the present invention can be dissolved and/or suspended in a physiologically acceptable solution or medium, wherein preferably said physiologically acceptable medium is water for injection or may also further include adjuvants containing media preferably such adjuvants containing media is an aluminium hydroxide suspension.

[0090] Formulations of the invention are said to be "pharmacologically acceptable" if their administration can be tolerated by a recipient individual. Further, the lyophilized formulation of the invention will be administered in a "therapeutically effective amount" (i.e., an amount that produces a desired physiological effect).

[0091] In another embodiment, the present invention relates to a use of a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) at least one angiotensin molecule with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; (ii) a stabilizer composition comprising: (c) only one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 3-8 % (w/v) in terms of the concentration in the formulation prior to lyophilization; and (d) only one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.0025-0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) only one buffer agent; and wherein said stabilizer composition has a pH value from 7.0-7.4 prior to lyophilization may be available as such in the form of an orally consumable tablet form or in a reconstituted liquid formulation form wherein said lyophilized formulation can be dissolved and/or suspended in a physiologically acceptable solution or medium, wherein preferably said physiologically acceptable medium is water for injection or may also further include adjuvants containing media preferably such adjuvants containing media is an aluminium hydroxide suspension for the manufacture of a medicament for treatment and/or prevention of a physical disorder associated with the renin-activated angiotensin, wherein said physical disorder is associated with the renin-activated angiotensin system is selected from the group consisting of hypertension, stroke, infarction, congestive heart failure, kidney failure and retinal hemorrhage.

[0092] In another embodiment, the present invention relates to a use of a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) angiotensin II with at least one second attachment site having the sequence as set forth in SEQ ID NO:4,wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; and (ii) a stabilizer composition consisting of: (c) one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.005% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) 5OmM sodium chloride in terms of the concentration in the formulation prior to lyophilization; (f) one buffering agent, wherein said buffering agent is Histidine/HistidineHCl, and wherein the concentration of said Histidine/HistidineHCl is 2OmM; and wherein said stabilizer composition has a pH value of 7.2 prior to lyophilization, may be available as such in the form of an orally consumable tablet form or in a reconstituted liquid formulation form wherein said lyophilized formulation can be dissolved and/or suspended in a physiologically acceptable solution or medium, wherein preferably said physiologically acceptable medium is water for injection or may also further include adjuvants containing media preferably such adjuvants containing media is an aluminium hydroxide suspension for the manufacture of a medicament for treatment or prevention of a physical disorder associated with the renin- activated angiotensin, wherein said physical disorder is associated with the renin-activated angiotensin system is selected from the group consisting of hypertension, stroke, infarction, congestive heart failure, kidney failure and retinal hemorrhage. To induce an immune response, the said lyophilized formulation may be administered to an animal, suitably a mammal such as a human, by various methods known in the art, but will normally be administered by injection, infusion, inhalation, oral administration, or other suitable physical methods. The formulations may alternatively be administered intramuscularly, intravenously, transmucosally, transdermally or subcutaneously. Components of conjugates for administration include sterile aqueous (e.g., physiological saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption.

[0093] In one embodiment, the present invention relates to a kit comprising a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) at least one angiotensin molecule with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; (ii) a stabilizer composition comprising: (c) only one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 3-8 % (w/v) in terms of the concentration in the formulation prior to lyophilization; and (d) only one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.0025-0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) only one buffer agent; and wherein said stabilizer composition has a pH value from 7.0-7.4 prior to lyophilization a physiologically acceptable medium to reconstitute the lyophilized formulation wherein preferably said physiologically acceptable medium is water for injection or may also further include adjuvants containing media preferably such adjuvants containing media is an aluminium hydroxide suspension and a pharmaceutically acceptable device for administration of such a reconstituted formulation to a mammal. In one preferred embodiment the pharmaceutically acceptable device is a syringe and may already contain the said lyophilized formulation.

[0094] In another embodiment, the present invention relates to a kit comprising a lyophilized formulation comprising: (i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and (b) angiotensin II with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH; and (ii) a stabilizer composition consisting of: (c)one non-reducing disaccharide, wherein said non-reducing disaccharide is trehalose, and wherein the concentration of trehalose is 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization; (d) one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.005% (w/v) in terms of the concentration in the formulation prior to lyophilization; (e) 5OmM sodium chloride in terms of the concentration in the formulation prior to lyophilization; (f) one buffering agent, wherein said buffering agent is Histidine/HistidineHCl, and wherein the concentration of said Histidine/HistidineHCl is 2OmM; and wherein said stabilizer composition has a pH value of 7.2 prior to lyophilization; a physiologically acceptable medium to reconstitute the lyophilized formulation wherein preferably said physiologically acceptable medium is water for injection or may also further include adjuvants containing media preferably such adjuvants containing media is an aluminium hydroxide suspensionand a pharmaceutically acceptable device for administration of such a reconstituted formulation to a mammal. In one preferred embodiment the pharmaceutically acceptable device is a syringe and may already contain said lyophilized formulation.

[0095] It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are readily apparent and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

EXAMPLES

EXAMPLE 1

[0096] "AngQβ" - The term "AngQβ", as used herein should refer to at least one angiotensin- virus- like particle conjugate comprising (a) a virus-like particle of RNA- bacteriophage Qβ with at least one first attachment site; and (b) at least one angiotensin II with at least one second attachment site (SEQ ID NO:4), wherein the first attachment site is lysine and wherein the second attachment site is cysteine, and wherein said virus-like particle of RNA- bacteriophage Qβ comprises coat proteins of RNA- bacteriophage Qβ consisting of the amino acid sequence of SEQ ID NO:7, and wherein said lysine is part of said coat protein, and wherein the first attachment site associates covalently with the second attachment site via a hetero-bifunctional linker SMPH. The method of producing AngQβ is described briefly below.

[0097] A solution of 2028 ml with 2.2 mg/ml Qβ capsid protein in 20 mM NaP, 50 mM NaCl pH 7.2 was reacted for 120 ± 10 minutes with 97 ml of a solution of 65 mM SMPH (succinimidyl-6-β-maleimidoprpionamido hexanoate) in DMSO at 24°C in a 51 flask with a magnetic stirrer. The reaction mixture was diluted to a Qβ concentration of 1.5 mg/ml with 20 mM NaP, 50 mM NaCl pH 7.2. The solution was subsequently dialyzed against 10-times the volume for 100 minutes with 20 mM NaP, 50 mM NaCl pH 7.2. The 2962 ml of the dialyzed reaction mixture was then reacted with 50.5ml of the 25 mM Angiotensin-peptide stock solution (in purified water) for 120 ± 10 minutes at 24°C in a 51 flask with a magnetic stirrer. The reaction mixture was subsequently dialyzed against 10-times the volume for 100 minutes with 20 mM NaP, 50 mM NaCl pH 7.2. The solution was diluted to a Qβ concentration 1.4 mg/ml. This product is named QAN003, which is stored at -80 degree and thawed at room temperature before test or further processing.

[0098] AngQβ was lyophilized in the stabilizer formulation of the present invention through the following preferred freeze drying protocols:

Table 1: Preferred Freeze Drying Process 1

Table 2: Freeze Drying Process 2

Sample preparation

[0099] The AngQβ bulk material was thawed at room temperature. The liquid formulations were produced by pipetting the AngQβ bulk material into excipient stock solutions, subsequent stirring and sterile- filtering prior to filling into sterile 2R glass vials and subsequent freeze drying. The filling volume was 0.7 ml. The vials were capped with bromobutyl or chlorobutyl stoppers. After the rubber stoppers were placed onto the vials, the vials were transferred into the lyophilisation chamber of the freeze dryer and freeze dried. After the freeze drying process was finished, the lyophilisation chamber was aerated with filtered dry nitrogen and the vials were directly capped in this environment. The vials were removed from the chamber and sealed.

Water content analysis

[00100] Moisture content measurements of the lyophilizates were conducted with a coulo metric Karl Fischer titrator with a head- space oven (Analytic Jena AG). The lyophilizates were measured right in the 2R glass vial at a head-space temperature of 80⁰C.

The samples were heated in the oven chamber for at least 5 minutes.

Sample moisture contents were determined using a GRS 2000 Coulometric Karl Fischer moisture titrator (GRS Instruments, UK). The GRS2000 titrator contains a regenerating cell, which electrolyses the hydrogen iodide formed in the reaction back to free iodine. In addition to improving titrator sensitivity, the regenerating cell ensures that both titrator and reagent are maintained in a dry condition.

A weighed quantity of dry Karl Fischer reagent was added to the sample within the vial and moisture extracted from the freeze-dried powder. After a stabilizing period, a sub-sample of the Karl Fischer reagent/sample mix was injected into the titrator and the water content was determined. The amount of moisture measured by the titrator was then expressed as a percentage (w/w) of the dried sample content.

Appearance lyophilized formulations

[00101] The cakes are evaluated based on series of features - color, shape, porosity, density and cohesiveness, adherence to the vial, collapse and skin formation. Depending on the subjective overall impression of the assessor, a score between one and five is allocated, with five being the best.

Reconstitution of the lyophilized formulations

[00102] As known to a skilled person, for some of the analyses described below, the lyophilized formulations and lyophilizates, respectively, need to be brought into aqueous solution. Thus, the lyophilizates were reconstituted with sterile filtrated water, typically and preferably with sterile filtrated water of a volume to adjust to the total volume of the formulation prior to lyophilization. By way of example, if the formulation prior to the lyophilization process consisted of 0.7 ml per vial, then the preferably formed cake resulting from the lyophilization process is reconstituted in such a volume of sterile water such as the final composition again consists of 0.7 ml. Asymmetrical flow field flow fractionation (AF4) measurements to determine VLP aggregates [00103] AF4 measurements were conducted using a Wyatt separation channel with a

350 μm spacer, Eclipse2 separation system (Wyatt Technology Corporation), Agilent 1100 G1310A isocratic pump, G1379A degasser,G1329A autosampler, G1330B thermostat for autosampler, G1314A UV detector, G1362A RI detector and Wyatt DAWN EOS MALS detector.

[00104] The channel flow was 1.2 ml/min. The cross flow was 1.6 ml/min for 18 minutes, subsequently reduced to 0.15 ml/min in 15 minutes and held for 5 minutes at 0.15 ml/min. In a final step the cross flow was 0.0 ml/min for 10 minutes.

[00105] The concentration of VLP was determined at 260 nm with the UV detector.

The Wyatt DAWN HELEOS MALS detector was used for the determination of the hydrodynamic radius and the molecular weight of VLP species. An amount of around 20 μg VLP from the starting material QAN003 and reconstituted lyophilizates (reconstituted with water as described above) were injected into the AF4, respectively.

Differential scanning calorimetry (DSC) measurements to determine glass transistion temprature

[00106] DSC measurements were conducted with a Perkin Elmer Diamond Differential

Scanning Calorimeter which was calibrated using high purity indium. The samples were lightly crushed to a powder within the vial under a nitrogen atmosphere and then weighed directly into an encapsulating type aluminum pan, which was then sealed with an aluminum lid.

The samples were run under the following conditions:

Sample mass: 7-11 mg

Temperature range: 20- 100⁰C

StepScan Parameters: 1°C increment, 1 min isothermal holds,

Heating rate: 10⁰C min^"1, criterion: ± 0.02 mW

Crucible Type: Encapsulated 50 μL Al.

Atmosphere: N₂ at 20 cm³ min^"1

[00107] The glass transition temperature, Tg, of the lyophilizates was determined by means of single DSC scans. Exertion of mechanical stress to determine precipitation

[00108] In front of a black background approx. 700 μl of a particular liquid formulation under investigation was filled in a semi-micro cuvette and 100 μl thereof was pipetted up and down (with a 200 μl pipette tip) 20 times. After every up and down cycle the sample was visually observed and possible precipitation was noted.

SE-HPLC - VLP integrity

[00109] SE-HPLC is an analytical method to separate different compounds in a sample according to their size. Thus large Qβ particles can be separated from smaller molecules, e.g. the Qβ coat protein monomers or nucleic acid fragments and therefore the method was used to confirm the integrity of the VLP. The method was also used to confirm purity of the drug substance. As a control a Qβ VLP standard was analyzed with the sample in the same series.

Detection was performed at 260 nm. Product-related impurities may be protein aggregates, smaller cleavage products and/or nucleic acids.

Column: TSKgel G5000PWXL 7.8 mm * 30 cm(Lot: 5PWX02HH3554, Art:

08023, Tosoh Bioscience)

Eluent: 20 mM sodium phosphate buffer containing 50 mM NaCl, pH 7.2)

Injection volume: 40 μl

Flow rate: 0.8 ml/min

Gradient: Isocratic

Run time: 20 min

Wavelength: 215, 260 and 280 nm, data evaluation at 260 nm

Column oven temp.: 25°C Autosampler temp.: 8°C

[00110] The VLP integrity was determined by valley to valley integration. Prior to analysis an overlay of the chromatograms of all the individual samples was performed. According to this overlay, different areas were determined as AngQβ aggregates area (area from start until appearance of the first valley left of maximum peak height), AngQβ main peak area (area from first valley left of maximum peak height until second valley right of maximum peak height) and AngQβ degradation area (area from first valley right of maximum peak height until end of recording). The AngQβ integrity is defined as the area in % of the AngQβ main peak relative to the total area of all three areas (which corresponds to 100 %). If no valleys are observed, the whole area is attributed to AngQβ main peak area. Bioanalyzer - RNA integrity

[00111] The integrity analysis of RNA extracted from virus-like particles was performed using Agilent 2100 RNA 6000 Nano kits. The RNA was diluted to RNA concentrations of 0.1 - 1.0 mg/ml with Diethylpyrocarbonate-H2O, homogenized in TRI- Reagent (a combination of phenol and guanidine thiocyanate in a mono-phase solution to inhibit RNase activity) followed by RNA extraction with l-bromo-3-chloropropane - phase separation reagent. Extracted RNA was precipitated with isopropanol and the pellet washed with ethanol. The RNA was then dissolved in diethylpyrocarbonate-lHtO, heat denatured and the content determined by spectroscopy. RNA was diluted to 0.5 μg/μl with diethylpyrocarbonate-H2θ and analyzed according to the Agilent RNA 6000 nano kit instruction guide. (Quick Start Guide Edition April 2007). The resulting electropherogram was transformed to a unique numeric qualifier for the RNA integrity by means of a weighted average approach.

EXAMPLE 2

Effect of pH on the stability of AngQβ

[00112] The chemical and physical stability of AngQβ in liquid formulations was investigated at different pH values using SE-HPLC. Physical instability of AngQβ results in the aggregation of the VLP monomers to VLP dimers, trimers and multimers. Chemical instability of the VLP results in the degradation of the VLP into monomers or multimers of the Q β coat protein and in the degradation and release of the RNA contained inside the virus- like particle. A further result of the chemical instability of the AngQβ may be the disassociation between the angiotensin II peptide and the VLP of Qβ. The stability of AngQβ was first analyzed at four different pH's in the range of 6.0 to 7.8. The bulkware was supplied in sodium phosphate buffer. The pH of the bulkware was adjusted by using either a 0.1 N NaOH solution or a 0.1 N H₃PO₄ solution. All samples were diluted to a concentration of 0.5 mg/ml AngQβ using water and contained 50 mM NaCl. The samples were stored at room temperature up to 7 days. Analyses were carried out at day 0, day 2 and day 7 of storage, respectively. The results are shown in Figure IA.

[00113] Best results were achieved for samples having a pH of 7.2. Results obtained for samples having a pH of 6.6 and 7.8 were comparable. AngQβ was not stable in the sample having a pH of 6.0.

[00114] In the second set of experiments, the influence of pH on stability was further studied in the range from 6.8 to 7.4. All samples contained 50 mM NaCl and were diluted to a concentration of 1.0 mg/ml AngQβ. Samples were stored at 40 ⁰C for 14 days. VLP integrity was assessed at day 0, 1, 2, 4, 7 and 14 of storage. The results are shown in Figure IB. [00115] Best results were achieved for samples having a pH of 7.2. Results obtained for samples having a pH of 7.0 and 7.4 were comparable. The sample having a pH of 6.8 was less stable.

EXAMPLE 3

Effect of buffer on the stability of AngQβ

[00116] The stability of AngQβ was analyzed in three different buffer systems, all having a pH of 7.2. The liquid samples produced for this investigation were manufactured by buffer exchange dialysis. All formulations contained the buffering agents at 20 mM, 50 mM NaCl, 0.005 % polysorbate 20 and 6.5 % trehalose. The concentration of AngQβ was 1.2 mg/ml. In order to address differences with regard to the stability of the different formulations, corresponding samples were stored at 5°C, 25°C and 40⁰C for one week. The VLP integrity was analyzed at day 0, day 1, 3 and 7 of storage using SE-HPLC. In order to assess qualitative differences in the stability profile of the individual formulations, all AngQβ main peaks were integrated at a peak height of 30% of the maximum peak height. The relative changes in fronting, tailing and main peak areas compared to start values were assessed. Changes in the relative peak area of the fraction eluting until 30 % height left of the main peak are considered to be attributable to changes in aggregation characteristics. On the other hand, changes in the fraction eluting until 30% height right of the main peak (tailing) are considered to be attributable to changes in degradation products. The results of day 7 of storage at the respective temperatures in comparison to the values at start are shown in FIG 2.

[00117] The most prominent qualitative difference became apparent after 7 days of storage at 40 ⁰C. Whereas a slight decrease with respect to fronting is observed for all formulations, differences with respect to the increase in tailing can be observed. Tailing accounts for most of the decrease of the main peak area percentage. Tailing is the least pronounced in the formulation comprising L-histidine as the buffering agent. Based on the 40 ⁰C data, L-histidine displays the lowest increase in degradation products and the lowest decrease of the main peak area.

EXAMPLE 4

Effect of NaCl on the stability of AngQβ [00118] QAN003 as prepared according to Example 1 was thawed and concentrated to a concentration of approx. 3.0 mg/ml AngQβ. The aliquots were dialyzed against 10-times the volume 20 mM L-Histidine pH 7.2 with different sodium chloride concentrations. QbAngFBOl (20 mM L-Histidine pH 7.2), QbAngFB03 (20 mM L-Histidine, 50 mM NaCl pH 7.2), QbAngFB04 (20 mM L-Histidine, 25 mM NaCl pH 7.2) and QbAngFB05 (20 mM L-Histidine, 75 mM NaCl pH 7.2). The resulting dialyzed products were assessed using SE- HPLC. The VLP integrity was determined essentially as described in EXAMPLE 1 by valley to valley integration. Prior to analysis an overlay of the chromatograms of all the individual samples was performed. According to this overlay, two different areas were determined: AngQβ main peak area and AngQβ degradation area, respectively. The AngQβ integrity is defined as the area in % of the AngQβ main peak relative to the total area of the two areas (which corresponds to 100 %).

[00119] As shown in FIG. 3A and Table 3, a considerable amount of degradation product was detected in QβAngFBOl, which contains no sodium chloride. [00120] Surprisingly, the addition of NaCl in the dialysis buffer was beneficial in preventing further degradation of AngQβ in comparison to the starting material. While 25 mM NaCl already showed to have a positive effect on the particle integrity, a concentration of 50 mM NaCl was capable of preventing further degradation almost completely.

Table 3: composition of batches and results from SE-HPLC analysis

[00121] LDS-Page with subsequent silver staining analysis was performed to further assess the nature of the degradation products.

[00122] Reducing PAGE combined with silver staining is employed for the detection of proteins / peptides with a molecular weight smaller than the Qβ coat protein. All bands below the Qβ coat protein band are regarded as impurities or degradation products of AngQβ. Their intensities are classified by visual comparison on the wet gel to three different dilutions of Qβ coat protein (1 ng, 2.5 ng and 10 ng) loaded on the same gel. Samples are denatured with sample buffer containing LDS in the presence of reducing agent (1,4-dithiothreitol) and separated by size on a 12% polyacrylamide Bis-Tris gel. The samples of the VLP standard and a molecular weight marker are run in parallel on each gel. Staining occurs through a fixation steps (with 30 ml 10% Methanol / 7% Acetic Acid for > 5 minutes), two pretreating steps (first with 30 ml 50% Acetone for 5 minutes and then with 30 ml purified water 50 μl 10 % sodium thiosulfate for 2 minutes) followed by impregnation with a silver nitrate solution (30 ml purified water, 400 μl 20% silver nitrate, 75 μl 37% formaldehyde for 8 minutes) and finally development of the staining (with 60 ml purified water, 1.2 g sodium carbonate, 25 μl 10% sodium tiosulfate, 25 μl 37% formaldehyde for 0.5 to 2 minutes). The development is stopped by the addition of acetic acid as soon as the desired stain intensity is achieved (30 ml 2% acetic acid for 2 minutes).

[00123] As shown in FIG. 3B, sample QbAngFBOl containing no sodium chloride did not show more coat protein degradation products as compared to the other formulations (bands below the Qβ monomer). Furthermore as the degradation product showed a strong absorption at 260nm and weak absorbtion as 280nm or 215 nm, it is suspected that the degradation product observed by means of SE-HPLC (Chromatograms were recorded at a wavelength of 260 nm) were not of proteinaceous origin, but of nucleic nature. Most likely it is the leakage of host RNA, which is normally packaged into the virus-like particle during expression of the viral coat protein and assembly of the virus-like particle within the cytosol of the host, in most cases, E.coli.

EXAMPLE 5

Effect of mechanical stress on the precipitation behaviour of AngQβ

[00124] A total of 12 different formulations of AngQβ were subjected to mechanical stress. In front of a black background approx. 700 μl of each formulation was filled in a semi- micro cuvette and 100 μl thereof was pipetted up and down (with a 200 μl pipette tip) 20 times. After every up and down cycle the sample was visually observed and possible precipitation was noted.

[00125] Effect of trehalose and the addition of polysorbate 20 - Formulations comprised 1.2mg/ml or 1.8 mg/ml AngQβ in 20 mM L-histidine pH 7.2, 50 mM NaCl, 5 or 10% w/v trehalose and various amounts of polysorbate 20. The formulations under investigation are presented in the Table below:

Table 4: Composition of the formulations subjected to shear force stress. All formulations contained 50 mM NaCl in a 20 mM L-histidine buffer, pH 7.2.

[00126] As shown in Table 5, a minimal concentration of 0.0025 % polysorbate 20 is required to prevent precipitation when the AngQβ is provided at a concentration of 1.2 mg/ml. At a concentration of 0.0025 % or higher, polysorbate 20 completely prevented the formation of precipitates with AngQβ at a concentration of 1.2 mg/ml. At a concentration of

1.8 mg/ml of AngQβ, 0.005% polysorbate 20 is required to fully prevent precipitation of

AngQβ.

Table 5: Results of shear force analyses.

"-" represents no precipitation, "(+)" very faint precipitation, "+" faint precipitation, "++"precipitation and "+++" pronounced precipitation.

EXAMPLE 6

Influence of lyophilization on the stability of AngQβ during freeze drying

[00127] The AngQβ was thawed at room temperature. The formulations with varying concentrations of AngQβ (0.0 mg/ml, 0.2mg/ml, 0.6 mg/ml, 1.2 mg/ml and 1.8 mg/ml), 6.5% trehalose, 0.005 % polysorbate 20 and 50 mM NaCl in 20 mM L-histidine buffer pH 7.2 were produced by pipetting the AngQβ into excipient stock solutions. The solutions were stirred on a magnetic stirrer for 5 - 10 minutes. The final drug solutions were sterile filtrated (0.22 μm membrane filter) and then lyophilized.

[00128] Briefly, the drug solutions were filled into sterile 2R glass vials. 0.7 ml was filled per vial. 13 mm bromobutyl or chlorobutyl lyophilization stoppers (West

Pharmaceutical Services), were placed onto the vials. The vials were transferred into the lyophilization chamber of a Christ Epsilon 2-12 D freeze drier (Martin Christ

Gefriertrocknungsanlagen GmbH) or a VirTis Genesis freeze-dryer equipped with an Encore

PC control system. Shelf temperature was lowered at 1.0°C/min to -40⁰C and further at

0.5°C/min to -45°C. The temperature was held at -45°C for 2 hours. Chamber pressure was reduced to 0.045 mbar. The shelf temperature was ramped to -35°C at 0.25°C/min and held for 25 hours. Afterwards the shelf temperature was raised to -20⁰C at 0.1°C/min and held for

4 hours. Subsequently the shelf temperature was raised to 20⁰C at 0.25°C/min and held for 10 hours. The lyophilization chamber was then aerated with filtered dry nitrogen to 800 mbar and the vials were capped in the lyophilization chamber. The vials were removed from the chamber and sealed with Flip-Off^® seals. After freeze drying stable lyophilizates were achieved, sufficient cake structure was given.

[00129] All of the resulting lyophilizates with varying AngQβ concentrations described above had a moisture content of around 1 %.

[00130] The SE-HPLC (VLP integrity) measurements showed that the relative content of AngQβ was higher than 97 % for all formulations.

[00131] LDS-Page (protein degradation), Bioanalyzer (RNA integrity) and mass spectrometry (peptide degradation) measurements showed no significant changes for all formulations in comparison to the respective starting material. This indicates that no significant degree of degradation occurred during freeze drying. [00132] The glass transition temperature of the lyophilizates was typically between

60⁰C tO l IO⁰C.

[00133] The performed analytical measurements led to the conclusion that the above mentioned formulation was capable of stabilizing AngQβ in the concentration from 0.20 mg/ml to 1.8 mg/ml.

EXAMPLE 7

Appearance analysis

[00134] The preferred liquid formulation F067.040 comprising 0.6 mg/ml AngQβ,

6.5% w/v trehalose dihydrate, 0.005% w/v polysorbate 20, 5OmM NaCl in 2OmM L-Histidine buffer at a pH of 7.2 was freeze dried according to the preferred freeze drying process 2 described in Table 2.

[00135] The SE-HPLC (VLP integrity) measurements showed that the relative content of AngQβ was higher than 97 % as compared to the respective starting material. The LDS-

Page (protein degradation), Bioanalyzer (RNA integrity) and mass spectrometry (peptide degradation) measurements showed no significant changes for the lyophilized formulation as compared to the respective starting material. This indicates that no significant degree of degradation occurred during freeze drying.

[00136] The appearance analysis and the residual moisture content analysis of the lyophilized formulation based on the liquid formulation F067.040 and obtained by the freeze drying process 2 of Table 2 reveals that after freeze drying, the lyophilized formulation displayed an excellent cake structure (with a score 5/5) with favourable residual moisture content of 0.72 %. Furthermore, no evidence of collapse of the cake (at the bottom of the vial) was observed.

EXAMPLE 8

Stability studies of freeze dried AngQβ formulations

[00137] Lyophilizates were produced essentially as described in EXAMPLE 6. The filling volume of the vials was 0.7ml. The concentration of AngQβ was 0.6 mg/ml.

[00138] The liquid formulation samples F042.039 (containing 0.60 mg/ml AngQβ in

10% trehalose, 0.005 % polysorbate 20 and 50 mM NaCl, in 20 mM L-histidine buffer pH

7.2) and F043.040 (containing 0.60 mg/ml AngQβ in 6.5% trehalose, 0.005 % polysorbate 20 and 50 mM NaCl, in 20 mM L-histidine buffer pH 7.2) were prepared as described in

Example 1 based on the reference QbAngFB06 (containing 3.5 mg/ml AngQβ, 20 mM L- histidine buffer pH 7.2 and 50 rnM NaCl) and lyophilized essentially the same as described in

Example 6 except the primary drying was performed for 21 hours. After lyophilization, stable lyophilisates were achieved and sufficient cake structure was obtained.

[00139] Samples were stored at 40⁰C up to 13 weeks. The cake structure of the lyophilizates was stable during storage. This was observed for all time points. The glass transition temperature of the lyophilizates was > 60⁰C and was not altered significantly during storage.

[00140] LDS-Page (protein degradation), Bioanalyzer (RNA integrity) and mass spectrometry (peptide degradation) measurements showed no significant changes for all formulations upon storage at 40⁰C. This indicates that no significant degree of degradation occurred upon freeze drying and subsequent storage.

[00141] Compared to the starting material, the amounts of AngQβ-oligomers and

AngQβ-aggregates did not relevantly increase during storage, as analyzed by AF4 measurements. After storage of the lyophilized formulations for thirteen weeks at 40⁰C, no relevant change in aggregation behaviour compared to the starting material was observed as analyzed by AF4.

Table 6: Monomers (M), Dimers (D), trimers (T) and aggregates(A) regions from

AF4 analysis of formulations F042.039 and F043.040 after 13 weeks of storage at

40⁰C, compared to their reference material (QbAngFB06).

[00142] The SE-HPLC (VLP integrity) measurements showed that the relative content of AngQβ was higher than 97 % throughout all time points.

[00143] The performed analytical measurements led to the conclusion that the formulation described in EXAMPLE 6 is capable for stabilizing AngQβ during lyophilization and storage, even at an accelerated temperature of 40⁰C. EXAMPLE 9

Antibody titers - Bioassav

[00144] The determination of antibody titers is based on a combined in vivo I in vitro assay. 10 female balb/c mice per group were immunised subcutaneously using a reconstituted formulation based on the lyophilized formulation F067.040 as described in Example 7, whereas 10 μg of AngQb per dose was applied. Mice were boosted after fourteen days with the same amount of that reconstituted formulation. Sera were collected after another seven days and analyzed by ELISA. Maxisorp microtiter plates were coated with 10 μg/ml RNase- CAng II. Serial dilutions of sera from mice bled at day 0 and at day 21 were analyzed by ELISA. As an internal standard, a commercially available mouse anti-human Angiotensin II antibody (subclass IgG2a) was used. As secondary antibody, a goat anti-mouse IgG HRP conjugate (AbD Serotec) was used in 1 :500 dilution. The binding reaction as well as the incubation step with the secondary antibody were performed for 2 hours. The IgG antibody titer was determined as the serum dilution giving half-maximal binding (EC50). Data fitting was performed using the Four-Parameter-Logistic model (4PL). The antibody titers resulting from immunization with stored lyophilized samples and the respective starting material were compared. The results of this investigation are shown in Figure 4.

Claims

1. A lyophilized formulation comprising:

(i) at least one angiotensin-virus-like particle conjugate comprising:

(a) a virus-like particle with at least one first attachment site;

(b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and (U) a stabilizer composition comprising:

(c) at least one non-reducing disaccharide, wherein the concentration of said non-reducing disaccharide is from 3 to 12% (w/v) in terms of the concentration in the formulation prior to lyophilization;

(d) at least One, preferably only one, buffer agent;

(e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization.

2. The lyophilized formulation of claim 1, wherein said non-reducing disaccharide is sucrose or trehalose.

3. The lyophilized formulation of claim 1 or 2, wherein said non-reducing disaccharide is trehalose.

4. The lyophilized formulation of any one of the preceding claims, wherein the concentration of said at least one non-reducing disaccharide is 5-8% (w/v), more preferably 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization.

5. The lyophilized formulation of any one of the preceding claims, wherein the concentration of said non-ionic surfactant is from 0.0025% to 0.01% (w/v), preferably 0.005% (Wv), in terms of concentration in the formulation prior to lyophilization.

6. The lyophilized formulation of any one of the preceding claims, wherein said non-ionic surfactant is polysorbate 20 or polysorbate 80, preferably polysorbate 20.

7. The lyophilized formulation of any one of the preceding claims, wherein said pH value is from 7.0 to 7.4, preferably is 7.2 prior to lyophilization.

8. The lyophilized formulation of any one of the preceding claims, wherein said virus-like particle is a virus-like particle of an RNA-bacteriophage, wherein preferably said virus-like particle is a virus-like particle of RNA bacteriophage Qβ.

9. The lyophilized formulation of any one of the preceding claims, wherein said angiotensin molecule is selected from the group consisting of:

(a) angiotensinogen (SEQ ID NO:1)

(b) angiotensin I (SEQ ID NO:2); and

(c) angiotensin II (SEQ ID NO:3).

10. The lyophilized formulation of any one of the preceding claims, wherein said angiotensin molecule is angiotensin II (SEQ ID NO:3).

11. The lyophilized formulation of any one of the preceding claims, wherein said at least one angiotensin molecule with at least one second attachment site comprises a linker, wherein preferably said linker comprises a cysteine residue.

12. The lyophilized formulation of any one of the preceding claims, wherein said at least one angiotensin molecule with at least one second attachment site has an amino acid sequence as set forth in SEQ ID NO:4.

13. The .lyophilized formulation of any one of the preceding claims, wherein said first attachment site is a lysine residue.

14. The lyopfailized formulation of any one of the preceding claims, wherein said second attachment site is a cysteine residue.

15. The lyophilized formulation of any one of the preceding claims, wherein said association between said first and second attachment site is through a hetero- bifunctional linker, wherein preferably said hetero-bifunctional linker is SMPH.

16. The lyophilized formulation of any one of the preceding claims, wherein said buffer agent is Histidine/HistidiπeHCl.

17. The lyophilized formulation of any one of the preceding claims, wherein the concentration of said Histidine/HistidineHCl is 1OmM to 4OmM, preferably 2OmM in terms of the concentration in the formulation prior to lyopbilization.

18. The lyophilized formulation of claim 17, wherein said composition further comprises a salt, wherein preferably said salt is sodium chloride.

19. The lyophilized formulation of claim 18, wherein the concentration of said sodium chloride is 25mM to 75mM, preferably 5OmM in terms of the concentration in the formulation prior to lyophilization.

20. A lyophilized formulation comprising:

(i) at least one angiotensin-virus-like particle conjugate comprising:

(a) a virus-like particle of RNA-bacteriophage Qp with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and

(b) at least one angiotensin molecule with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifunctional molecule SMPH;

(ii) a stabilizer composition comprising: (c) only one non-reducing disaccharide, wherein said non- reducing disaccharide is trehalose, and wherein the concentration of trehalose is 3-8 % (w/v) in terms of the concentration in the formulation prior to lyophilization; and

(d) only one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.0025-0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization;

(e) only one buffer agent; and wherein said stabilizer composition has a pH value from 7.0-7.4 prior to lyophilization.

21. A lyophilized formulation comprising:

(i) at least one angiotensin- virus-like particle conjugate comprising:

(a) a virus-like particle of RNA-bacteriophage Qβ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Qβ comprises one or more recombinant coat proteins having the amino acid sequence as set forth in SEQ ID NO:7; and

(b) angiotensin II with at least one second attachment site having the sequence as set forth in SEQ ID NO:4, wherein said first attachment site is a lysine residue and said second attachment site is a cysteine residue, and wherein said lysine residue is part of said recombinant coat protein; wherein said first attachment site associates with said second attachment site through a hetero-bifuαctional molecule SMPH; and

(U) a stabilizer composition consisting of:

(c) one non-reducing disaccharide, wherein said non- reducing disaccharide is trehalose, and wherein the concentration of trehalose is 6.5% (w/v) in terms of the concentration in the formulation prior to lyophilization;

(d) one non-ionic surfactant, wherein said non-ionic surfactant is polysorbate 20, and wherein the concentration of polysorbate 20 is 0.005% (w/v) in terms of the concentration in the formulation prior to lyophilization;

(e) 5OmM sodium chloride in terms of the concentration in the formulation prior to lyophilization; (f) one buffering agent, wherein said buffering agent is Histidine/HistidineHCl, and wherein the concentration of said Histidine/HistidineHCl is 2OmM; and herein said stabilizer composition has a pH value of 7.2 prior to lyophilization.

22. The lyophilized formulation of any one of the preceding claims, wherein said lyophilized formulation is stable at room temperature for at least 13 weeks, preferably at least IS weeks, and most preferably for at least 25 weeks.

23. A process for manufacturing a lyophilized formulation comprising the steps of

(i) freezing a liquid formulation to a temperature in the range of -20⁰C to -80⁰C wherein said liquid formulation comprises at least one angiotensin-virus-like particle conjugate comprising:

(a) a virus-like particle with at least one first attachment site;

(b) at least one angiotensin molecule with at least one second attachment site, wherein said at least one first attachment site associates with said at least one second attachment site through at least one non-peptide covalent bond; and a stabilizer composition comprising:

(d) at least one, preferably only one, buffer agent;

(e) at least one non-ionic surfactant, preferably only one non-ionic surfactant, wherein the concentration of said non-ionic surfactant is from 0.0023% to 0.1% (w/v) in terms of the concentration in the formulation prior to lyophilization; and wherein said stabilizer composition has a pH value from 6.6 to 7.8 prior to lyophilization

(ii) applying a vacuum in the range of 0.2 to O.OOOlmbar to said freezed liquid formulation;

(iii) performing at least one drying step at a temperature in the range of -80⁰C to 40⁰C.

24. A reconstituted formulation comprising the Iyophilized formulation of any one of the claims 1 to 21, preferably the Iyophilized formulation of claim 20 or claim 21. dissolved and/or suspended in a physiologically acceptable medium.

25. A Iyophilized formulation of any one of claims 1 to 21, preferably the Iyophilized formulation of claim 20 or claim 21, or a reconstituted formulation of claim 24 for use in treatment and/or prevention of a physical disorder associated with the renin-activated angiotensin,

26. Use of a Iyophilized formulation of any one of claims 1 to 21, preferably the Iyophilized formulation of claim 20 or claim 21, or a reconstituted formulation of claim 24 for the manufacture of a medicament for treatment and/or prevention of a physical disorder associated with the renin-activated angiotensin.

27. A kit comprising:

(a) a Iyophilized formulation of any one of the claims 1 to 21, preferably the Iyophilized formulation of claim 20 or claim 21;

(b) a diluent or an excipient; and optionally

(c) a pharmaceutically acceptable device.