WO2009056651A1 - Formulation de support de l'angiotensine - Google Patents

Formulation de support de l'angiotensine Download PDF

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Publication number
WO2009056651A1
WO2009056651A1 PCT/EP2008/064894 EP2008064894W WO2009056651A1 WO 2009056651 A1 WO2009056651 A1 WO 2009056651A1 EP 2008064894 W EP2008064894 W EP 2008064894W WO 2009056651 A1 WO2009056651 A1 WO 2009056651A1
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WIPO (PCT)
Prior art keywords
formulation
concentration
attachment site
angiotensin
virus
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PCT/EP2008/064894
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English (en)
Inventor
Stefan Landolt
Lorenz Vogt
Alexander Zuercher
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Cytos Biotechnology Ag
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Publication of WO2009056651A1 publication Critical patent/WO2009056651A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5184—Virus capsids or envelopes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • 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.
  • angiotensin moiety such as angiotensin I or angiotensin II
  • 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 Q ⁇ to which angiotensin II are covalently bound is efficacious in the reduction of blood pressure in hypertensive patients
  • 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 0 C).
  • the lyophilized formulation of the present invention comprises a simple and economic stabilizer composition due to a minimum number of excipients included therein.
  • 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
  • the invention provides a process for making the lyophilized formulation of the invention.
  • 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).
  • the reconstituted formulation further comprises an adjuvant, preferably comprises aluminium hydroxide.
  • FIG. 1 shows the stability of AngQ ⁇ under different pH in liquid solutions using SE-HPLC.
  • Y-axis represents relative area of AngQ ⁇ main peak [%] and x-axis represents days of storage.
  • the starting materials for FIG. IA and FIG. IB are from different batches.
  • FIG 2 shows the stability of AngQ ⁇ 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.
  • FIG 3A shows the stability of AngQ ⁇ 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].
  • FIG. 3B LDS-PAGE and subsequent silver staining showing the chemical stability of AngQ ⁇ 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).
  • 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 -80 0 C (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.
  • Adjuvant 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.
  • 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.
  • 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.
  • the term "adjuvant” refers to an adjuvant not being the VLP used for the inventive formulation, but in addition to said VLP.
  • 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.
  • 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.
  • lyophilization process and the term “freeze-drying process” are interchangably used herein and shall be regarded as synonyms.
  • 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%.
  • lyophilized formulation refers to the composition obtained or obtainable by the process for making the lyophilized formulation of the present invention.
  • 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.
  • physiologically acceptable medium may also further include adjuvants containing media such as, preferably, an aluminium hydroxide suspension.
  • Room temperature refers to a temperature from 15°C to 30 0 C, preferably from 23°C to 27°C, more preferably 25°C.
  • Stable 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 AngQ ⁇ , 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 (40 0 C), 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 AngQ ⁇ degradation products, increases not more than 8%, preferably not more than 5%, more preferably not more
  • 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 AngQ ⁇ degradation products and after lyophilization and 15 weeks of storage, there are 3% of AngQ ⁇ 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.
  • the method to determine the amount of the sum of angiotensin- VLP 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 Q ⁇ monomers and dimers, preferably larger than AngQ ⁇ monomers and dimers, are combined in calculation.
  • AF4 assay asymmetrical flow field flow fractionation
  • degradation products 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.
  • 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.
  • Oligomer The term "oligomer”, as used in the term “angiotensin-
  • VLP oligomer refers to the aggregation of at least three and up to ten VLPs, VLPs of an RNA-bacteriophage or VLPs of Q ⁇ , respectively.
  • Aggregate refers to the aggregation of at least ten VLPs, VLPs of an RNA-bacteriophage or VLPs of Q ⁇ , respectively.
  • virus particle 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.).
  • virus-like particle 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.
  • non-replicative refers to being incapable of replicating the genome comprised by the VLP.
  • 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.
  • a virus-like particle is a virus particle, in which the viral genome has been physically or chemically inactivated.
  • 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.
  • 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.
  • capsids of RNA-bacteriophages or HBcAgs have a spherical form of icosahedral symmetry.
  • capsid-like structure 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.
  • virus particle and virus-like particle is its highly ordered and repetitive arrangement of its subunits.
  • 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.
  • 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.
  • 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.
  • 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.
  • physical, chemical inactivation such as UV irradiation, formaldehyde treatment, typically and preferably by genetic manipulation.
  • 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.
  • 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.
  • the first attachment site is naturally occurring with the VLP and the VLP of an RNA-bacteriophage, respectively.
  • the first attachment site is artificially added to the VLP and the VLP of an RNA- bacteriophage, respectively.
  • 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 Q ⁇ .
  • 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 Q ⁇ , wherein further preferably said coat protein has the amino acid sequence of SEQ ID NO:7.
  • 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".
  • the second attachment site is associated with the angiotensin molecule through at least one covalent bond, preferably through at least one peptide bond.
  • the second attachment site is naturally occurring within the angiotensin molecule.
  • the second attachment site is artificially added to the angiotensin molecule through a linker, wherein said linker comprises or alternatively consists of a cysteine.
  • the linker is fused to the angiotensin molecule by a peptide bond.
  • Linker either associates the second attachment site with angiotensin molecule or already comprises, essentially consists of, or consists of the second attachment site.
  • 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.
  • 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.
  • 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
  • the lyophilized formulation is surprisingly so stable that almost no changes in VLP integrity after 3 months of storage at 40 0 C 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 Q ⁇ .
  • 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
  • the invention provides a formulation obtainable by a method of lyophilization comprising the step of freezing said liquid formulation and drying said liquid formulation.
  • the liquid or lyophilized formulation of the invention comprises only one carbohydrate, preferably only one sugar, the sugar is preferably a non-reducing disaccharide.
  • the liquid or lyophilized formulation of the invention does not comprise a bovine serum albumin or a human serum albumin.
  • 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.
  • 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.
  • 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.
  • concentration of trehalose expressed in the whole application refers to the concentration of trehalose dihydrate (2H 2 O) (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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the osmolarity of the formulation is from 250 to 350 mosm/Kg, more preferably about 300mosm/Kg.
  • 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).
  • the osmolarity of the formulation is from 250 to 350 mosm/Kg, more preferably about 300mosm/Kg.
  • 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).
  • the osmolarity of the formulation is from 250 to 350 mosm/Kg, more preferably about 300mosm/Kg.
  • the non-reducing disaccharide is trehalose and the bulking agent is mannitol.
  • 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.
  • the ratio between the bulking agent and the non-reducing disaccharide is from 3.5:1 to 4.5:1, preferably 4:1.
  • the non-reducing disaccharide is trehalose and the bulking agent is mannitol.
  • 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.
  • 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.
  • 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.
  • the non-ionic surfactant is from 0.0025% to
  • the non- ionic surfactant is polysorbate 20 or polysorbate 80. In one preferred embodiment, the non-ionic surfactant is polysorbate 20.
  • 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.
  • 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.
  • the virus-like particle is a virus-like particle of an RNA-bacteriophage.
  • the RNA-bacteriophage is selected from the group consisting of a) bacteriophage Q ⁇ ; 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.
  • the virus-like particle is a virus-like particle of an RNA-bacteriophage Q ⁇ .
  • Methods for the production of VLP of an RNA-bacteriophage, in particular VLP of bacteriophage Q ⁇ and VLP bacteriophage AP205 have been described at page 37-47 of WO 04/009124 and in EXAMPLES 1 and 21 thereof.
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant proteins, or fragments thereof, of the RNA-bacteriophage Q ⁇ , of the RNA- bacteriophage fr, or of the RNA-bacteriophage AP205.
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant coat proteins, or fragments thereof, of the RNA-bacteriophage Q ⁇ , of the RNA- bacteriophage fr, or of the RNA-bacteriophage AP205.
  • 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 Q ⁇ (SEQ ID NO:7), and RNA- bacteriophage AP205 (SEQ ID NO:8 and SEQ ID NO:9).
  • 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 Q ⁇ , wherein preferably said recombinant coat protein of RNA-bacteriophage Q ⁇ comprises, or alternatively essentially consists of, or alternatively consists of the amino acid sequence as set forth in SEQ ID NO.7.
  • 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.
  • the virus-like particle is a virus-like particle of
  • the virus-like particle is a virus-like particle of RNA-bacteriophage Q ⁇ recombinantly expressed in E. coli.
  • the virus-like particle is a virus-like particle of RNA-bacteriophage Q ⁇ , wherein said virus-like particle of said RNA- bacteriophage Q ⁇ comprises, or alternatively essentially consists of, or alternatively consists of one or more, preferably 180, recombinant coat proteins of RNA-bacteriophage Q ⁇ , 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.
  • 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).
  • the at least one angiotensin with at least one second attachment site comprises a linker, wherein preferably said linker comprises a cysteine residue.
  • the linker is fused, either to the C-terminus, or preferably to the N- terminus of the angiotensin molecule by a peptide bond.
  • 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).
  • the angiotensin molecule with at least one second attachment site has the amino acid sequence as set forth in SEQ ID NO:4.
  • the first attachment site comprises or preferably is an amino group.
  • the first attachment site comprises or preferably is an amino group of a lysine residue.
  • 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 Q ⁇ .
  • 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 Q ⁇ , wherein further preferably said coat protein has the amino acid sequence of SEQ ID NO:7.
  • 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 Q ⁇ , wherein said coat protein has the amino acid sequence of SEQ ID NO:7.
  • 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.
  • 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.
  • association between said first and second attachment site is through a hetero-bifunctional linker. Several hetero-bifunctional cross- linkers are known in the art.
  • 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).
  • the hetero-bifunctional linker is SMPH (succinimidyl-6- ⁇ -maleimidoprpionamido hexanoate).
  • 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 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.
  • the concentration of the angiotensin- VLP conjugate in the liquid formulation is 0.2mg/ml, 0.6mg/ml, 1.0mg/ml, 1.2mg/ml, 1.8mg/ml or 2mg/ml.
  • the concentration of the angiotensin- VLP conjugate in the liquid formulation is 0.6mg/ml.
  • the stabilizer composition comprises a buffering agent.
  • the stabilizer composition comprises a buffering agent selected from the group consisting of: Maleate, Tris, Bis-tris, Triethanolamine, Tricine, Bicine, Histidine, phosphate, Glycinate.
  • the stabilizer composition comprises a buffering agent selected from the group consisting of: Tris, Bis-tris, Triethanolamine, Tricine, Histidine, phosphate, Glycinate.
  • the stabilizer composition comprises a buffering agent, wherein said buffer agent is a phosphate buffer.
  • the stabilizer composition comprises a buffering agent, wherein said buffer agent is Glycinate buffer
  • 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.
  • 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.
  • 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.
  • the liquid or lyophilized formulation of the invention further comprises a salt, preferably only one salt.
  • 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.
  • the stabilizer composition comprises only one buffering agent, wherein said buffer agent is Histidine/HistidineHCl.
  • said buffer agent is Histidine/HistidineHCl.
  • 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.
  • the stabilizer composition further comprises a salt, preferably only one salt, wherein the salt is preferably selected from the group consisting of: NaCl, MgCl 2 , KCl, CaCl 2 , Na 2 SO 4 , CaSO 4 , MgSO 4 and K 2 SO 4 , 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.
  • the salt is preferably selected from the group consisting of: NaCl, MgCl 2 , KCl, CaCl 2 , Na 2 SO 4 , CaSO 4 , MgSO 4 and K 2 SO 4 , 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 3Om
  • 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.
  • 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 Q ⁇ 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
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA-bacteriophage Q ⁇ 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
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA-bacteriophage Q ⁇ 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
  • 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 Q ⁇ 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
  • 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.
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Q ⁇ 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
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Q ⁇ 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
  • 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.
  • the solution is NaCl solution.
  • the reconstituted formulation has a physiologically acceptable osomolarity value.
  • the reconstituted formulation further comprises an adjuvant.
  • the adjuvant is aluminium hydroxide hydrated gels or aluminium phosphate hydrated gels.
  • 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 -20 0 C to -80 0 C preferably in the range of -40 0 C to -50 0 C, 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;
  • 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 20 0 C 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 surfact
  • 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 20 0 C 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-reducing disaccharide,
  • 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 -40 0 C 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/raised to -37°C at 0.25°C/min and held for 22 hours, (iv) The shelf temperature was raised to -20 0 C at 0.1°C/min and held for 4 hours, (iv) The shelf temperature was raised to 20 0 C 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.
  • 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 Q ⁇ with at least one first attachment, wherein said virus- like particle of RNA-bacteriophage Q ⁇ 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 dis
  • 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 Q ⁇ with at least one first attachment, wherein said virus- like particle of RNA-bacteriophage Q ⁇ 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
  • the present invention provides a lyophilized product obtainable or obtained from the processes described above.
  • 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.
  • 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.
  • the lyophilized formulation comprises:(i) at least one angiotensin- virus- like particle conjugate comprising: (a) a virus-like particle of RNA-bacteriophage Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA-bacteriophage Q ⁇ 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
  • a lyophilized formulation comprises: (i) at least one angiotensin- virus- like particle conjugate comprises: (a) a virus-like particle of RNA- bacteriophage Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Q ⁇ 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
  • 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).
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Q ⁇ 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,
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Q ⁇ 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
  • 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.
  • 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.
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Q ⁇ 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
  • 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 Q ⁇ with at least one first attachment, wherein said virus-like particle of RNA- bacteriophage Q ⁇ 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
  • AngQ ⁇ should refer to at least one angiotensin- virus- like particle conjugate comprising (a) a virus-like particle of RNA- bacteriophage Q ⁇ 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 Q ⁇ comprises coat proteins of RNA- bacteriophage Q ⁇ 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.
  • a solution of 2028 ml with 2.2 mg/ml Q ⁇ 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- ⁇ -maleimidoprpionamido hexanoate) in DMSO at 24°C in a 51 flask with a magnetic stirrer.
  • the reaction mixture was diluted to a Q ⁇ 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 Q ⁇ 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.
  • AngQ ⁇ was lyophilized in the stabilizer formulation of the present invention through the following preferred freeze drying protocols:
  • the AngQ ⁇ bulk material was thawed at room temperature.
  • the liquid formulations were produced by pipetting the AngQ ⁇ 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.
  • 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 80 0 C.
  • the samples were heated in the oven chamber for at least 5 minutes.
  • 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.
  • 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.
  • 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.
  • the lyophilized formulations and lyophilizates need to be brought into aqueous solution.
  • 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.
  • the formulation prior to the lyophilization process consisted of 0.7 ml per vial
  • 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
  • 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.
  • the cross flow was 0.0 ml/min for 10 minutes.
  • 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 ⁇ g VLP from the starting material QAN003 and reconstituted lyophilizates (reconstituted with water as described above) were injected into the AF4, respectively.
  • DSC Differential scanning calorimetry
  • StepScan Parameters 1°C increment, 1 min isothermal holds,
  • Heating rate 10 0 C min "1 , criterion: ⁇ 0.02 mW
  • Tg glass transition temperature
  • SE-HPLC is an analytical method to separate different compounds in a sample according to their size.
  • large Q ⁇ particles can be separated from smaller molecules, e.g. the Q ⁇ 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 Q ⁇ VLP standard was analyzed with the sample in the same series.
  • Product-related impurities may be protein aggregates, smaller cleavage products and/or nucleic acids.
  • Wavelength 215, 260 and 280 nm, data evaluation at 260 nm
  • 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 AngQ ⁇ aggregates area (area from start until appearance of the first valley left of maximum peak height), AngQ ⁇ main peak area (area from first valley left of maximum peak height until second valley right of maximum peak height) and AngQ ⁇ degradation area (area from first valley right of maximum peak height until end of recording).
  • the AngQ ⁇ integrity is defined as the area in % of the AngQ ⁇ 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 AngQ ⁇ main peak area. Bioanalyzer - RNA integrity
  • 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.
  • TRI- Reagent a combination of phenol and guanidine thiocyanate in a mono-phase solution to inhibit RNase activity
  • Extracted RNA was precipitated with isopropanol and the pellet washed with ethanol.
  • RNA was then dissolved in diethylpyrocarbonate-lHtO, heat denatured and the content determined by spectroscopy. RNA was diluted to 0.5 ⁇ g/ ⁇ l with diethylpyrocarbonate-H2 ⁇ 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.
  • the pH of the bulkware was adjusted by using either a 0.1 N NaOH solution or a 0.1 N H 3 PO 4 solution. All samples were diluted to a concentration of 0.5 mg/ml AngQ ⁇ 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.
  • the stability of AngQ ⁇ 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 AngQ ⁇ was 1.2 mg/ml.
  • corresponding samples were stored at 5°C, 25°C and 40 0 C for one week.
  • the VLP integrity was analyzed at day 0, day 1, 3 and 7 of storage using SE-HPLC.
  • 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 0 C data, L-histidine displays the lowest increase in degradation products and the lowest decrease of the main peak area.
  • QAN003 as prepared according to Example 1 was thawed and concentrated to a concentration of approx. 3.0 mg/ml AngQ ⁇ . 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
  • 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: AngQ ⁇ main peak area and AngQ ⁇ degradation area, respectively.
  • the AngQ ⁇ integrity is defined as the area in % of the AngQ ⁇ main peak relative to the total area of the two areas (which corresponds to 100 %).
  • Reducing PAGE combined with silver staining is employed for the detection of proteins / peptides with a molecular weight smaller than the Q ⁇ coat protein. All bands below the Q ⁇ coat protein band are regarded as impurities or degradation products of AngQ ⁇ . Their intensities are classified by visual comparison on the wet gel to three different dilutions of Q ⁇ 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 ⁇ l 10 % sodium thiosulfate for 2 minutes) followed by impregnation with a silver nitrate solution (30 ml purified water, 400 ⁇ l 20% silver nitrate, 75 ⁇ l 37% formaldehyde for 8 minutes) and finally development of the staining (with 60 ml purified water, 1.2 g sodium carbonate, 25 ⁇ l 10% sodium tiosulfate, 25 ⁇ l 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).
  • sample QbAngFBOl containing no sodium chloride did not show more coat protein degradation products as compared to the other formulations (bands below the Q ⁇ monomer).
  • 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.
  • a total of 12 different formulations of AngQ ⁇ were subjected to mechanical stress.
  • 700 ⁇ l of each formulation was filled in a semi- micro cuvette and 100 ⁇ l thereof was pipetted up and down (with a 200 ⁇ l pipette tip) 20 times. After every up and down cycle the sample was visually observed and possible precipitation was noted.
  • 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.
  • polysorbate 20 As shown in Table 5, a minimal concentration of 0.0025 % polysorbate 20 is required to prevent precipitation when the AngQ ⁇ 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 AngQ ⁇ at a concentration of 1.2 mg/ml. At a concentration of
  • the AngQ ⁇ was thawed at room temperature.
  • the formulations with varying concentrations of AngQ ⁇ (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 AngQ ⁇ 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 ⁇ m membrane filter) and then lyophilized.
  • the vials were transferred into the lyophilization chamber of a Christ Epsilon 2-12 D freeze drier (Martin Christ
  • LDS-Page protein degradation
  • Bioanalyzer RNA integrity
  • mass spectrometry peptide degradation
  • the preferred liquid formulation F067.040 comprising 0.6 mg/ml AngQ ⁇ ,
  • Lyophilizates were produced essentially as described in EXAMPLE 6.
  • the filling volume of the vials was 0.7ml.
  • the concentration of AngQ ⁇ was 0.6 mg/ml.
  • F043.040 containing 0.60 mg/ml AngQ ⁇ in 6.5% trehalose, 0.005 % polysorbate 20 and 50 mM NaCl, in 20 mM L-histidine buffer pH 7.2
  • Example 1 based on the reference QbAngFB06 (containing 3.5 mg/ml AngQ ⁇ , 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.
  • Samples were stored at 40 0 C 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 > 60 0 C and was not altered significantly during storage.
  • AngQ ⁇ -aggregates did not relevantly increase during storage, as analyzed by AF4 measurements. After storage of the lyophilized formulations for thirteen weeks at 40 0 C, no relevant change in aggregation behaviour compared to the starting material was observed as analyzed by AF4.
  • a commercially available mouse anti-human Angiotensin II antibody (subclass IgG2a) was used.
  • 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.

Abstract

La présente invention appartient au domaine des médicaments, vaccins et des formulations pharmaceutiques. L'invention porte sur des formulations comprenant un conjugué angiotensine-particule de type virus et un stabilisant, ledit stabilisant comprenant un disaccharide non réducteur et un agent tensioactif non ionique. Les formulations lyophilisées sont stables après une longue période de stockage à température ambiante.
PCT/EP2008/064894 2007-11-02 2008-11-03 Formulation de support de l'angiotensine WO2009056651A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2829285A1 (fr) * 2012-03-22 2015-01-28 Beijing SoloBio Genetechnology Company Ltd. Formulation de vecteur lentiviral recombinant
US9045728B2 (en) 2010-12-02 2015-06-02 Oncolytics Biotech Inc. Liquid viral formulations
US9044498B2 (en) 2010-12-02 2015-06-02 Oncolytics Biotech Inc. Lyophilized viral formulations
US20160256532A1 (en) * 2009-06-25 2016-09-08 Revance Therapeutics, Inc. Albumin-Free Botulinum Toxin Formulations
EP3283125B1 (fr) 2015-04-17 2021-12-29 CureVac Real Estate GmbH Lyophilisation de l'arn
CN113891725A (zh) * 2019-10-31 2022-01-04 南京海维医药科技有限公司 包含血管紧张素ii的固态组合物及其制备方法、使用方法、用途
US11471708B2 (en) 2008-12-31 2022-10-18 Revance Therapeutics, Inc. Injectable botulinum toxin formulations
US11534405B2 (en) 2015-05-20 2022-12-27 Curevac Ag Dry powder composition comprising long-chain RNA

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031466A2 (fr) * 2001-10-05 2003-04-17 Cytos Biotechnology Ag Conugues porteur-peptide angiotensine et utilisations de ceux-ci

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031466A2 (fr) * 2001-10-05 2003-04-17 Cytos Biotechnology Ag Conugues porteur-peptide angiotensine et utilisations de ceux-ci

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AMBUEHL PATRICE M ET AL: "A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity", JOURNAL OF HYPERTENSION, vol. 25, no. 1, January 2007 (2007-01-01), pages 63 - 72, XP009110644, ISSN: 0263-6352 *
FRANKS F: "Freeze-drying of bioproducts: putting principles into practice", EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, ELSEVIER SCIENCE PUBLISHERS B.V., AMSTERDAM, NL, vol. 45, no. 3, 1 May 1998 (1998-05-01), pages 221 - 229, XP004256976, ISSN: 0939-6411 *
SCHWEMANN J J ET AL: "PRACTICAL FORMULATION AND PROCESS DEVELOPMENT OF FREEZE-DRIED PRODUCTS", PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY, NEW YORK, NY, US, vol. 10, no. 2, 1 January 2005 (2005-01-01), pages 151 - 173, XP008048993, ISSN: 1083-7450 *
SHI LI ET AL: "Stabilization of human papillomavirus virus-like particles by non-ionic surfactants", JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 94, no. 7, July 2005 (2005-07-01), pages 1538 - 1551, XP002510329, ISSN: 0022-3549 *
WANG WEI: "Lyophilization and development of solid protein pharmaceuticals", INTERNATIONAL JOURNAL OF PHARMACEUTICS, ELSEVIER BV, NL, vol. 203, no. 1-2, 10 August 2000 (2000-08-10), pages 1 - 60, XP002428586, ISSN: 0378-5173 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11471708B2 (en) 2008-12-31 2022-10-18 Revance Therapeutics, Inc. Injectable botulinum toxin formulations
US20160256532A1 (en) * 2009-06-25 2016-09-08 Revance Therapeutics, Inc. Albumin-Free Botulinum Toxin Formulations
US10111939B2 (en) * 2009-06-25 2018-10-30 Revance Therapeutics, Inc. Albumin-free botulinum toxin formulations
US11911449B2 (en) 2009-06-25 2024-02-27 Revance Therapeutics, Inc. Albumin-free botulinum toxin formulations
US11351232B2 (en) 2009-06-25 2022-06-07 Revance Therapeutics, Inc. Albumin-free botulinum toxin formulations
US9610352B2 (en) 2010-12-02 2017-04-04 Oncolytics Biotech Inc. Lyophilized viral formulations
US9610309B2 (en) 2010-12-02 2017-04-04 Oncolytics Biotech Inc. Liquid viral formulations
US9044498B2 (en) 2010-12-02 2015-06-02 Oncolytics Biotech Inc. Lyophilized viral formulations
US9045728B2 (en) 2010-12-02 2015-06-02 Oncolytics Biotech Inc. Liquid viral formulations
US9969984B2 (en) 2012-03-22 2018-05-15 Beijing Solobio Genetechnology Company Ltd. Storage stable recombinant lentiviral vector preparation
EP2829285A1 (fr) * 2012-03-22 2015-01-28 Beijing SoloBio Genetechnology Company Ltd. Formulation de vecteur lentiviral recombinant
EP2829285A4 (fr) * 2012-03-22 2015-11-04 Beijing Solobio Genetechnology Company Ltd Formulation de vecteur lentiviral recombinant
EP3283125B1 (fr) 2015-04-17 2021-12-29 CureVac Real Estate GmbH Lyophilisation de l'arn
US11491112B2 (en) 2015-04-17 2022-11-08 CureVac Manufacturing GmbH Lyophilization of RNA
US11534405B2 (en) 2015-05-20 2022-12-27 Curevac Ag Dry powder composition comprising long-chain RNA
CN113891725A (zh) * 2019-10-31 2022-01-04 南京海维医药科技有限公司 包含血管紧张素ii的固态组合物及其制备方法、使用方法、用途

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