VACCINE COMPOSITION AND PREPARATION PROCESS

专利摘要:
vaccine composition, preparation process and stabilizing excipient for vaccine with whole inactivated virus. the present invention relates to a vaccine composition, comprising: a) inactivated whole virus and b) a stabilizing excipient comprising: i. a buffer solution; ii. a mix of essential and non-essential amino acids; iii. a disaccharide; iv. a polyol; v. a chelating agent; saw. urea or a urea derivative; and vii. a non-ionic surfactant.
公开号:BR112012008000B1
申请号:R112012008000-8
申请日:2010-10-07
公开日:2021-07-13
发明作者:Michel Chevalier；Nadège Moreno；Eric Calvosa；Sandrine Cigarini；Virginie Fabre；Alain Francon
申请人:Sanofi Pasteur；
IPC主号:

专利说明:

[0001] The present invention relates to a vaccine composition, comprising inactivated whole virus and an excipient that stabilizes the vaccine composition, the excipient composition comprising a buffer solution, a mixture of essential and non-essential amino acids, a disaccharide, a polyol, a chelating agent, urea or a urea derivative and a non-ionic surfactant. It also refers to a process for preparing this vaccine composition, as well as the composition of the excipient as such;
In the field of viral vaccines, three types of viral vaccines are classically distinguished: live virus vaccines, inactivated whole virus vaccines and subunit viral vaccines. These three types of vaccines are distinguished by characteristics that are unique to them and that affect the composition of the excipients that are used for their conservation. Vaccines are unitary, containing a restricted number of viral antigens and are generally easier to conserve than vaccines with inactivated whole viruses for which the aim is to preserve the structural integrity of the viruses, even if they are killed, or than vaccines with live viruses to which also seeks to preserve the infectious power of viruses. For each type of viral vaccines the adapted excipients were prepared. For viral vaccines with attenuated viruses, different stabilizing excipients were prepared depending on the preparation of attenuated viruses to be preserved. JP 57007423 describes a disaccharide and polyalcohol based excipient in a phosphate buffer for stabilizing an attenuated measles virus strain. EP 065905 describes an excipient comprising one or more amino acids chosen from a group of eleven amino acids, lactose and sorbitol in a phosphate buffer to stabilize an attenuated strain of dengue virus. Finally, EP 0869814 describes a vaccine composition comprising an attenuated strain of chickenpox virus and a stabilizing agent containing sorbitol, mannitol, sucrose, dextran, a mixture of amino acids, urea and EDTA.
In the field of vaccines with inactivated viruses, vaccine compositions ready to be administered often contain proteins of animal origin, such as bovine or human albumin, gelatin or casein. Proteins are known to improve the conservation (stabilization) of these vaccines, notably vaccines that contain viruses that are difficult to conserve. This is the case of vaccine compositions containing inactivated rabies virus. Frazatti-Gallina et Coll. In Vacina (2004), volume 23, pages 511-517 it underlines the important role of proteins in stabilizing the rabies vaccine, since the excipient contains albumin.
[0004] However, the presence of proteins in vaccines, in addition to the fact that they may represent a potential risk of disease transmission, if their origin is not strictly controlled, may also represent a potential allergic risk that is sought to be avoided, especially when the vaccines contain serum proteins such as albumin or albumin derivatives.
[0005] There is, therefore, a need to find a stabilizing excipient whose composition does not contain protein to stabilize viral vaccines with inactivated whole virus and, in particular, to stabilize vaccines containing inactivated whole virus difficult to preserve, such as rabies virus.
[0006] There is also a need to find a stabilizing excipient that is suitable also for stabilizing vaccines with lightly dosed inactivated viruses, that is, containing a small amount of total protein in an effective dose of vaccine. This need is even more important for highly purified vaccines containing very few residual protein impurities. Residual protein impurities, even if they pose a potential allergic risk, may contribute to some extent to stabilize lightly dosed inactivated viral vaccines. They can prevent the phenomenon of aggregation, structural degradation or adsorption of the virus, which can reduce the effectiveness of the vaccine.
[0007] For this purpose, the present invention aims to:
[0008] A vaccine composition comprising: a) an inactivated whole virus preparation, and b) a stabilizing excipient comprising: I. a buffer solution; II. a mixture of essential and non-essential amino acids; III. a disaccharide; IV. a polyol; V. a chelating agent; SAW. urea or a urea derivative, and VII. a non-ionic surfactant.
[0009] Preferably, the entire inactivated virus is the rabies virus.
[00010] According to one aspect of the invention, the vaccine composition is also devoid of any serum protein.
[00011] According to one aspect of the invention, the vaccine composition is also devoid of any exogenous protein of animal origin, and preferably devoid of any exogenous product of animal origin.
[00012] According to yet another aspect, the virus proteins represent at least 70% of the total proteins that are present in the vaccine composition.
[00013] According to yet another aspect, the concentration of total proteins in the vaccine composition is <100 µg/binding medium and preferably < 80 µg/binding medium.
[00014] In a particular aspect, the amount of total protein that is contained in an effective dose of the vaccine composition is < 100 µg.
[00015] In another particular aspect, the amount of total protein that is contained in an effective dose of the vaccine composition is comprised between 1 and 50 µg.
[00016] In another aspect of the invention, the stabilizing excipient is devoid of any protein and any other peptide, or, preferably, devoid of any protein, any peptide and any oligopeptide.
[00017] According to another aspect, the mixture of essential and non-essential amino acids contained in the vaccine composition comprises at least arginine or a salt of arginine and glutamic acid or a salt of glutamic acid.
[00018] According to a particular aspect, the amount of essential and non-essential amino acids contained in an effective dose of the vaccine composition is comprised between 0.5 and 2.5 mg.
[00019] According to another aspect, the disaccharide that is contained in the excipient is maltose.
[00020] According to another aspect, the polyol is contained in the vaccine composition is sorbitol.
[00021] According to a particular aspect, the total amount of disaccharide and polyol contained in an effective dose of the vaccine composition is comprised between 10 and 50 mg.
[00022] In another aspect, the chelating agent that is contained in the vaccine composition is EDTA or an EDTA salt.
[00023] According to a particular aspect, the amount of chelating agent contained in an effective dose of the vaccine composition is comprised between 0.01 and 0.1 mg.
[00024] According to another particular aspect, the amount of urea or urea derivative contained in an effective dose of the vaccine composition is comprised between 0.3 and 1.5 mg.
[00025] In another aspect, the non-ionic surfactant that is contained in the vaccine composition is a poloxamer.
[00026] Preferably, the poloxamer is poloxamer 188.
[00027] According to a particular aspect, the amount of non-ionic surfactant contained in an effective dose of the vaccine composition is comprised between 0.001 and 0.5 mg.
[00028] According to another aspect, the pH of the vaccine composition is comprised between 7.0 and 10.0 and preferably between 7.0 and 9.0.
[00029] In another aspect, the buffer solution that is contained in the vaccine solution is a phosphate buffer, a Tris buffer, a HEPES buffer or a mixture thereof.
[00030] In a particular aspect, the buffer solution is a phosphate buffer, whose molarity is comprised between 10 and 100 mM.
[00031] The invention also has for its object a process for the preparation of a vaccine composition, containing a preparation of purified and inactivated whole virus, according to which: a. produce a whole virus preparation by collecting the supernatant from a virus-infected cell culture; B. purify and inactivate the whole virus preparation or alternatively inactivate and purify the whole virus preparation and c. dilute the purified and inactivated whole virus preparation in a stabilizing excipient, the composition of which comprises: I. a buffer solution; II. a mixture of essential and non-essential amino acids; III. a disaccharide; IV. a polyol V. a chelating agent; SAW. urea or a urea derivative; and VII. a non-ionic surfactant.
[00032] According to a preferred mode, the process according to the invention is carried out without introducing exogenous product of animal origin.
[00033] In a preferred way of carrying out the process, according to the invention, the virus is the rabies virus.
[00034] In another aspect, the process according to the invention comprises, after having diluted the preparation of purified and inactivated whole virus, a step of sharing the vaccine composition thus obtained in packaging devices and optionally a step of lyophilization of the vaccine composition.
[00035] The subject of the invention is also a vaccine composition containing a preparation of purified and inactivated whole virus in a lyophilized form obtained according to one of the embodiments of the process of the invention.
[00036] The invention also has for its object a stabilizing excipient for a vaccine with inactivated whole virus whose composition comprises: I. a buffer solution; II. a mixture of essential and non-essential amino acids; III. a disaccharide; IV. a polyol V. a chelating agent; SAW. urea or a urea derivative; and VII. a non-ionic surfactant.
[00037] Preferably, the composition of the stabilizing excipient is also devoid of any protein, or of any protein and of any other peptide, or, preferably, devoid of any protein, of any peptide and of any oligopeptide.
[00038] Particularly preferably, the composition of the stabilizing excipient is also devoid of any product of animal origin. Detailed Description of the Invention
[00039] The vaccine composition, according to the invention, comprises inactivated whole virus (or a preparation of inactivated whole virus) and a stabilizing excipient, whose composition comprises a buffer, a mixture of essential and non-essential amino acids, a disaccharide, a polyol, a chelating agent, a urea or a urea derivative and a nonionic active bearing. The excipient composition advantageously replaces prior art excipients which contain a protein in their composition. Particularly interestingly, it stabilizes vaccine compositions which are difficult to preserve, and particularly vaccine compositions which contain as whole inactivated rabies virus virus, without the need to add a protein.
[00040] The vaccine composition according to the invention is stable at the same time in liquid form, in frozen liquid form or in lyophilized form, which offers a very great flexibility of use.
[00041] The stabilizing excipient, according to the invention, preserves the biological activity of the vaccine composition over time whether it is in frozen, liquid or lyophilized form. The storage temperature of frozen vaccine compositions is generally < -35°C; it ranges from +2°C to +8°C for compositions in liquid form and at a temperature of approximately a further 5°C for compositions in lyophilized form. Advantageously, it also preserves the biological activity and physical integrity of the viruses in vaccine compositions kept under unfavorable conditions, such as storage at + 37°C of the vaccine compositions in liquid form or when the vaccine compositions are subjected to a succession of thawing and of re-freezes.
[00042] The biological activity of the virus in the vaccine composition is evaluated by measuring over time the amount of a constitutive immunogen of the virus, which is essential to induce protective immunity against the virus and/or testing its effectiveness in a proof test on an officially recognized animal model. In the case of a vaccine composition containing inactivated rabies virus, the stability of the vaccine composition is evaluated by measuring over time (or during successive thawing), the virus titer that is estimated based on the measurement of the G glycoprotein concentration in undenatured form and/or testing the efficacy of the vaccine composition through the official NIH test. To assess the rate of glycoprotein G, a sandwich-type ELISA method can be applied which recognizes at least one, preferably two conformational epitopes of glycoprotein G, as described in example 1. When using an ELISA that recognizes two conformational epitopes of the G protein, an antibody that neutralizes the rabies virus that recognizes a conformational epitope located in the antigenic site II of the glycoprotein G is usually used as capture antibody (Journal of Clinical Investigation (1989) volume 84, page 971 a 975) and as revealing antibody a neutralizing antibody that recognizes a conformational epitope located at the antigenic site III of the G protein (Biologicals (2003), volume 31, pages 9 to 16). The results are expressed in international units, as a measurement that has been calibrated against the NIBSC international reference is usually used as a reference. It is also possible to apply the BIAcore technology, considered an interesting alternative to the ELISA method, which is based on the use of surface plasmon resonance to quantify the virus titer.
[00043] The stabilizing excipient, according to the invention, also acts, preserving the physical integrity of the viral particles. It notably prevents the formation of aggregates and/or the degradation of the structure of viral particles over time. This can be controlled by means of an apparatus such as the zetasizer Nano ZS (Malvern instrument) which detects the aggregates and profiles the size distribution of the viral particles in the vaccine composition.
[00044] The virus or virus preparation is in the form of whole viral particles that have been inactivated, usually by chemical treatment with formaldehyde, formaldehyde or β propiolactone. Other inactivation processes such as that described in WO 2005/093049 can also be used. The mostly used inactivated whole virus preparations contain involved viruses as they are often more difficult to preserve and require the use of specific excipient compositions to ensure their conservation. These are notably inactivated whole virus preparations from Japanese encephalitis. Particularly preferably, inactivated whole virus preparations contain rabies virus.
[00045] Virus preparations come from collections that are generally in the form of collections of the culture supernatant of cells infected by the virus. Classical media containing animal serum can be used to produce the cell stock and infect the cells. Advantageously, the media used for culturing and infecting cells do not contain serum protein or even animal product. The proteins that are eventually present in these media are generally low molecular weight proteins (< 10 KD) or above all peptides with very low concentrations, which greatly reduces the risk of allergy. This is the case, for example, of the media sold under the names VP SFM (In Vitrogen), Opti Pro TM serum-free (In Vitrogen), Ex-Cell ® MDCK (Sigma-Aldrich), Ex-Cell TM Vero (SAFC biosciences ) MP-BHK ® serum free (MP Biomedicals), SFC-10 BHK Express serum free (Promo Cell), SFC-20 BHK Express protein free (Promo Cell), HyQ PF Vero (Hyclone Ref. SH30352.02), Hyclone SFM4 Megavir, MDSS2 medium (Axcell biotechnology), Iscove modified DMEM medium (Hyclone), Ham's nutrient media (Ham-F10, Ham-F12), Leibovitz L-15 medium (Hyclone). For example, rabies virus collections are obtained from a stock of Vero cells that were produced, then infected, using culture and viral infection media, which are preferably devoid of any serum protein, of any protein of animal origin and even any product of animal origin, such as VP SFM medium.
[00046] Advantageously, the preparation of inactivated whole virus contained in the vaccine composition, according to the invention, is very purified. It usually purifies, then inactivates or conversely inactivates, then purifies the virus that comes from the collections. When all virus production and purification steps are carried out without using serum protein, without using exogenous animal protein, or even without using exogenous animal product, advantageously the vaccine composition, according to the invention, is devoid of any serum protein to reduce as much as possible the allergic risks, and devoid of any exogenous protein of animal origin or even any product of animal origin to reduce as much as possible the risks of treating disease transmission. By protein or product of animal origin, it is understood a protein or a product whose manufacturing process comprises at least one step in which a material originating from animals or man is used. By "exogenous protein or product" is meant a protein or a product that is introduced in one of the steps of the virus production and/or purification process. For example, proteins or products that are eventually contained in the composition of the culture media, enzymes such as trypsin or benzonase that are used during the virus production and/or purification steps are exogenous proteins or products. Proteins or exogenous products are of animal origin as long as their manufacturing process comprises at least one stage in which a material originating from the animal or man is used. Proteins or exogenous products are of non-animal origin when they are manufactured by other means, for example, using a plant material, by chemical synthesis or by genetic recombination, using yeast, bacteria or plants. The proteins or products from the cells from which the viruses are produced to obtain the vaccine composition, according to the invention, are, on the contrary, endogenous proteins or products, as they are produced (or amplified) at the same time as the viruses.
[00047] Within the scope of the present invention, purification processes can be advantageously applied, particularly with performances that lead to very pure viral preparations, citing, by way of example, the virus purification process which comprises a anion exchange chromatography followed by a cation exchange chromatography and completed by a metal chelation affinity chromatography which is described in WO 97/06243. To purify and inactivate the rabies virus from the infected cell culture supernatant, one method of choice is to use the process that is described in the patent application filed in France on April 14, 2009 under number 0952310 which comprises one step cation exchange chromatography on a support, comprising a polymethacrylate matrix onto which sulfoisobutyl groups are grafted, a benzonase treatment step, an ultracentrifugation step over a sucrose gradient and an inactivation step with β propiolactone. The whole and inactivated rabies virus preparations obtained are particularly pure, but are more difficult to preserve (stabilize) as there are very few residual protein impurities. This difficulty will be greater, the smaller the amounts of virus in the vaccine preparation.
[00048] Thanks to the composition of the excipient, the vaccine composition, according to the invention, remains stable even in cases where the concentration in total proteins is < 100 μg/mL, < 80 μg/mL or even < 20 μg/mL . Generally, the virus proteins represent at least 70% of the total proteins, preferably at least 80% of the total proteins and particularly preferably at least 90% of the total proteins which are present in the vaccine composition according to the invention. In an effective dose of vaccine this usually represents an amount in total protein < 100 μg/mL, < 50 μg/mL or even < 20 μg/mL. By way of indication, the amount of residual DNA is in terms of it < 100 pg, and preferably < 50 pg per effective dose of vaccine. By "effective dose of vaccine (or effective dose of a vaccine composition)" means the amount of inactivated virus contained in a dose that is necessary to induce protective immunity in man or animal after administration according to the route of immunization and protocol which are recommended in the context of a first vaccination or a command vaccination. In the case of rabies virus vaccination, the effectiveness of rabies vaccination is determined using the official WHO-accepted test, the NIH test (described in the WHO rabies monograph (WHO Technical Series Report 941 - janvier 2007). of inactivated rabies vaccine is effective when it contains at least 2.5 IU according to this test. The administration of this dose intramuscularly in humans, according to the vaccination or serum-vaccination protocols usually recommended induces the development of a protective immunity against anger.
[00049] The "total proteins" correspond to all the proteins that are present in the vaccine composition. They are represented by viral proteins, and residual proteins that were not removed during purification such as cellular proteins, proteins from cell culture media and viral infection, and proteins that were eventually introduced in the purification process (for example , benzonase in the case of the rabies virus purification process as mentioned above). The Bradford method is generally used to quantify total proteins. To quantify the proportion of viral proteins among the total proteins, a polyacrylamide gel electrophoresis is performed on a sample of vaccine composition under denaturing and reducing conditions, followed by a coomassie blue staining and a densitometric analysis of the electrophoretic profile. In the case of a vaccine composition containing the whole and inactivated rabies virus, the viral proteins represented by the envelope glycoprotein G, the nucleus protein N, the phosphoprotein P, the matrix protein M and the RNA polymerase RNA-dependent L are easily identifiable on an electrophoresis in polyacrylamide gel. The amount of total protein contained in an effective dose of the vaccine composition of the vaccine composition according to the invention is generally comprised between 1 µg and 50 µg, preferably between 2 µg and 20 µg. Particularly preferably at least 70%, at least 75%, at least 80%, at least 85%, or even at least 90% of the total proteins are viral proteins. The excipient according to the invention is particularly advantageous since it stabilizes vaccine compositions which are difficult to preserve, such as vaccine compositions containing whole and inactivated rabies viruses, and in which an effective vaccine dose is found: - an amount of viral proteins representing at least 70% total protein and/or - less than 100 μg of total protein, more often an amount of total protein between 1 μg and 50 μg, preferably between 2 μg and 20 μg.
[00050] The composition of the excipient, according to the invention, is preferably devoid of any protein, any peptide and even any oligopeptide and generally does not contain animal products, to reduce as much as possible the problems of biological safety and /or allergy sufferers that may be associated with its use.
[00051] In the context of the present invention, the expression "the stabilizing excipient is devoid of any protein" should be understood as meaning a stabilizing excipient, whose composition is devoid of any biological macromolecule, comprising a chain of more than 50 amino acids linked together through peptide bonds. The expression "the stabilizing excipient is devoid of any protein and any peptide" should be understood as meaning a stabilizing excipient, whose composition is devoid of any biological macromolecule comprising a chain of more than 20 amino acids linked together by means of peptide bonds . The expression "the stabilizing excipient is devoid of any protein, any peptide and any oligopeptide" is to be understood as meaning a stabilizing excipient whose composition is devoid of any biological molecule, comprising amino acids linked together by one or more peptide bonds. For example, a dipeptide that contains two amino acids linked together by a single peptide bond is excluded from the composition of a stabilizing excipient devoid of any protein, any peptide and any oligopeptide, but may be part of the composition of a stabilizing excipient devoid of any protein and any peptide.
[00052] The mixture of amino acids that is part of the excipient composition comprises at least one essential amino acid in the set of essential amino acids represented by sistine, by tyrosine by arginine, by histidine, by isoleucine, by leucine, by lysine, by methionine, by phenyl alanine, threonine, tryptophan and valine and at least one non-essential amino acid in the set of non-essential amino acids represented by aspartic acid, glutamic acid, alanine, asparagine, glutamine, glycine, proline and by the serine. Amino acids with an acid function can be in the form of acids or salts. The same is true for basic amino acids.
[00053] Preferably, the excipient according to the invention contains at least arginine or an arginine salt such as arginine hydrochloride and glutamic acid or a glutamic acid salt such as sodium glutamate. Definitely, the mixture of amino acids preferably comprises from one to twelve essential amino acids, including at least arginine or a salt thereof, and from one to eight non-essential amino acids, including at least glutamic acid. or a salt thereof. The total amino acid concentration in the vaccine composition is generally < 20 g/L, most often between 2 g/L and 10 g/L. In an effective dose of the vaccine composition according to the invention, this represents a total amount of essential and non-essential amino acids < 5 mg, preferably to an amount that is comprised between 0.5 and 2.5 mg and particularly between 0.8 and 1.8 mg is preferred. The ratio between the amount of arginine (in the form of hydrochloride) and the total amount of amino acids contained in the excipient is generally greater than 0.5, while the ratio between the amount of glutamic acid (in the form of sodium glutamate) and the total amount of amino acids contained in the excipient is less than 0.5.
[00054] The excipient, according to the invention, also contains a disaccharide. Disaccharides that are obtained from a raw material of animal origin, such as milk, are excluded. As disaccharide suitable for the object of the invention, mention is made of sucrose, maltose or trehalose, but preferably maltose is used alone or in combination with another disaccharide such as sucrose. The polyol which is also included in the excipient composition is of non-animal origin. It is mainly a hexol, such as sorbitol and/or mannitol. Preferably, sorbitol is used, as mannitol has the inconvenience of crystallizing when the vaccine composition according to the invention is in a lyophilized form. The total concentration of disaccharide and polyol in the vaccine composition is generally between 30 g/l and 100 g/l. Preferably the excipient according to the invention. Contains maltose and sorbitol. In an effective dose of the vaccine composition this usually corresponds to an amount of between 10 and 50 mg and preferably an amount of between 20 and 25 mg. The amount of maltose generally represents at least 70% of the total amount of maltose and sorbitol.
[00055] The chelating agent is also one of the excipient components, according to the invention. It is mainly EDTA (Ethylene diamine tetraacetic acid) or a salt thereof (Na+, K+, ...). The concentration of EDTA or EDTA salt in the vaccine composition is generally between 0.02 and 0.5 g/l and preferably between 0.02 and 0.2 g/l. In an effective dose of the vaccine composition this usually corresponds to an amount of between 0.01 and 0.1 mg, preferably between 0.01 and 0.05 mg and particularly preferably between 0.01 and 0.04 mg of EDTA or EDTA salt.
[00056] Urea or a urea derivative such as allylurea, acetamide, methyl carbamate or butyl carbamate is also part of the excipient composition. Preferably, the vaccine composition contains urea at a concentration generally comprised between 1 g and 10 g/L, preferably at a concentration comprised between 1 g and 5 g/L. In an effective dose of the vaccine composition this usually corresponds to an amount of between 0.3 and 3 mg, preferably between 0.3 and 1.5 mg and particularly preferably between 0.4 and 1.2 mg of urea.
[00057] The excipient, according to the invention, also comprising a non-ionic surfactant that contributes to the stabilization of the vaccine composition. Without being bound by theory, it contributes to the maintenance of the virus' biological activity and to the maintenance of the physical integrity of the viral particles, preventing the formation of aggregates or the degradation of the virus structure. It can also prevent virus adsorption on the walls of the package, particularly when the walls are made of glass or plastic. The nonionic surfactant suitable for the object of the invention is produced from a raw material of non-animal origin and is compatible on the pharmaceutical plan with a parenteral administration of the product. As a class of nonionic surfactant particularly convenient to the object of the invention, poloxamers are mentioned, which are "block copolymers" of ethylene oxide and propylene oxide having the chemical formula HO(C2H4O)a(C3H6O)b(C2H4O) aH whereby a designates the number of ethylene oxide units and b designates the number of propylene oxide units. Poloxamers generally have a molecular weight ranging from 1000 to 16000 daltons. Poloxamers of particular interest have a molecular weight of between 5000 and 15500 daltons. Poloxamers are marketed notably under the trade name Pluronic ® among which pluronic ® F68 or poloxamer 188 which designates a poloxamer in solid form at room temperature, whose molecular weight of the polyoxypropylene part is approximately 1750 daltons and whose polyoxyethylene part represents approximately 80% of the total weight of the molecule. Among the poloxamers, poloxamer 188 (pluronic ® F68) is particularly recommended. Optionally, a sorbitan ester can be used, in particular a polyoxyethylene ester of sorbitan sold under the trade name Tween ®, such as tween ® 20 (polyoxyethylene (20) sorbitan monolaurate or polysorbate 20) or tween ® 80 (polyoxyethylene (20) sorbitan monooleate or polysorbate 80). The nonionic surfactant suitable for the object of the invention is used at a very low concentration that preserves the structure and size of the inactivated viral particles, which must remain similar to that of live viruses. With very strong concentration, the active peptide can dissociate or modify the structure of the viral particles, in particular that of the viruses involved, which can affect the immunogenicity of the vaccine composition. When pluronic ® F68 is used as a surfactant, the total concentration in the composition is < 1g/L, generally between 0.005 g/L and 1 g/L, and particularly preferably between 0.01 g/L and 0.1 g/L. In this concentration range, poloxamer 188 has no adjuvant action on the immune system. In an effective dose of the vaccine composition, this usually corresponds to an amount comprised between 0.001 mg and 0.5 mg, and preferably between 0.03 and 0.3 mg. Most preferably, the vaccine composition contains poloxamer 188 or optionally a mixture of poloxamer 188 and polysorbate 20.
[00058] The excipient buffer, according to the invention, is chosen in such a way that the pH of the vaccine composition is comprised between 7.0 and 10.0, preferably between 7.0 and 9.0 and so particularly preferred, between 7.3 and 8.3. It is in this pH range that the physical thermostability of the inactivated virus particles is maximum, in particular that of the rabies virus particles. Phase diagram studies show that it is necessary to heat the rabies virus preparation to a temperature of at least 60°C to observe an aggregation of rabies virus particles when the pH is between 7.3 and 8.3, while that at a pH < 6.0, significant aggregation is observed at room temperature. The buffer solution is usually a phosphate buffer, a Tris buffer or HEPES buffer, or a mixture of these. Its concentration is generally between 10 and 100 mM. Preferably, the excipient composition comprises a phosphate buffer, the molarity of which is usually between 10 and 100 mM, or a phosphate buffer and a Tris buffer in admixture.
[00059] A particularly preferred vaccine composition, according to the invention, comprises: a preparation of whole and inactivated rabies virus, and a stabilizing excipient comprising: • a phosphate buffer; • a mixture of essential and non-essential amino acids containing at least arginine or a salt of arginine and glutamic acid or a salt of glutamic acid; • maltose; • sorbitol; • EDTA or an EDTA salt; • urea; • poloxamer 188; • the pH of the vaccine composition being between 7.3 and 8.3.
[00060] Eventually tween 20 can be incorporated as an additional nonionic surfactant to this vaccine composition. This vaccine composition is advantageously devoid of any serum protein and of any exogenous product of animal origin and generally contains a very purified rabies virus preparation. The concentration of poloxamer 188 in the vaccine composition is < 1 g/j and most preferably between 0.01 g/l and 0.1 g/l. The molarity of the phosphate buffer is generally between 10 and 100 mM. Arginine and glutamic acid or their respective salts are the very major components of the amino acid mixture, as they generally represent by weight at least 2/3 of the total amount of essential and non-essential amino acids. The mixture of essential and non-essential amino acids is usually at a concentration of between 2 and 10 g/l, the total concentration of maltose and sorbitol is usually between 50 and 100 g/l, the concentration of EDTA or EDTA salt it is usually between 0.02 and 0.2/l and urea is usually at a concentration of between 1 and 5 g/l. The total protein concentration of the vaccine composition is usually between 5 and 50 µg/ml. In an effective dose of rabies vaccine, this usually represents an amount of essential and non-essential amino acids between 0.5 mg and 2.5 mg, an amount of maltose and sorbitol between 10 and 50 mg, an amount of EDTA or EDTA salt comprised between 0.01 mg and 0.1 mg, an amount of urea comprised between 0.3 and 1.5 mg, an amount of poloxamer 188 comprised between 2 and 20 µg.
[00061] The process of preparing a vaccine composition, according to the invention, in addition to the steps of production, purification and inactivation of the virus preparation comprises a step in which the stabilizing excipient is introduced in the preparation of purified whole virus and inactivated and a stage of distribution of the composition thus obtained in packaging devices. In general, the virus preparation is diluted in the stabilizing excipient, according to the invention, before the vaccine composition is distributed in the packaging devices.
[00062] The vaccine composition, according to the invention, can be presented in bulk form: in this case, the concentration in whole inactivated virus is higher than that which exists in the vaccine composition a split, but the excipient composition it's the same. The bulk is kept frozen at a temperature < - 35°C. The vaccine composition distribution step is then carried out, thawing the bulk that is then diluted in the stabilizing excipient, in order to obtain the desired viral concentration. The final bulk product is then obtained, which is then distributed to the packaging devices. In order to guarantee the sterility of the vaccine composition, a sterilizing filtration step can also be incorporated, for example, on a 0.2 µm filter membrane, before the distribution step.
[00063] The bulk is usually obtained by diafiltering the purified and inactivated virus preparation obtained at the end of the application of the purification process in a buffer solution that corresponds, in general, to the excipient buffer. If, for example, the excipient buffer is a 50 mM phosphate buffer, a 50 mM phosphate buffer is used as the diafiltration buffer. If you want to increase the virus concentration, the diafiltration step will be preceded by an ultrafiltration step. An ultrafiltration membrane is used having a low cut-off, generally comprised between 5 kDa and 100 kDa, preferably between 8 kDa and 50 kDa, and particularly preferably around 10 kDa, in order to retain the virus as much as possible in the retentate and eliminate salts that can negatively impact the freeze-drying process. The retentate containing the virus preparation is then mixed with the stabilizing excipient to obtain a bulk, the composition of which corresponds to the vaccine composition, according to the invention. It is also possible to use as diafiltration buffer a buffer that has the same composition as the stabilizing excipient to obtain the bulk in a single step.
[00064] The process of preparing a vaccine composition, according to the invention, containing the whole and inactivated rabies virus, comprises the following steps: a preparation of whole virus is produced, collecting the supernatant of an infected Vero cell culture by viruses, using cell culture and viral infection media that preferably are devoid of any serum protein and any animal product, as, for example, using the VP SFM medium. The virus is purified and inactivated, notably using the process, as described in the patent application filed in France on April 14, 2009 under number 0952310, which comprises a chromatography step by cation exchange on a support which preferably comprises a matrix based on polymethacrylate onto which sulphoisobutyl groups are grafted, a treatment with a recombinant benzonase, an ultracentrifugation step over a sucrose gradient and an inactivation step with β propiolactone. The whole and inactivated rabies virus preparation obtained is very pure, and is devoid of any serum protein and any exogenous animal products; the residual DNA content is <100 pg/binding medium and viral proteins represent at least 70% of total proteins. A bulk is then prepared, according to the methods described in the preceding paragraph, which is generally preserved at a temperature < - 35°C. The bulk is then diluted to the desired viral concentration in the excipient, according to the invention, before being distributed in the packaging devices. Between 0.1 ml and 1 ml of the vaccine composition are usually introduced into each packaging device, such that the composition once dispensed contains at least one effective dose of vaccine. Packaging devices serving for the distribution of the final bulk product are usually in the form of vials (glass or plastic) or syringes, but any other packaging device compatible with the practice of vaccination can also be used.
[00065] The granulometric analysis of the vaccine compositions, according to the invention, containing rabies virus purified by means of the zétasizer Nano ZS (Malvern Instrument) device that measures the Braunian movement of particles on the basis of the "almost elastic" diffusion of light ( Dynamic Light scattering) shows the existence of a single homogeneous population of viral particles between 100 and 300 nm with an average value around 180 nm which corresponds to the average size of wild rabies virus. Neither aggregates nor changes in the size distribution profile of the viral particles are detected during the preservation of the vaccine compositions according to the invention.
The vaccine compositions are stable in liquid form for at least 3 months at a temperature of 2-8°C and for at least 1 month at 23-27°C (see example 3). They can also be stored for at least 12 months and preferably for at least 24 months in frozen form at a temperature < - 35°C. The vaccine compositions can also be preserved in lyophilized form, applying a classical lyophilization process. The composition of according to the invention does not cause significant loss of virus during the freeze-drying step. One method of lyophilization consists of freezing the composition at a temperature below -40°C, then drying the product in two steps. Primary desiccation taking place at a temperature close to -15°C under 80 μ arias and secondary desiccation taking place at a temperature close to + 40°C under 80 μ arias. Lyophilized vaccine doses have a residual moisture content < 3% and are stable at a temperature of 2 - 8°C for at least 18 months (see example 3). Definitely, the rabies vaccine composition as described in the invention is as stable as a vaccine composition of the prior art marketed under the name VeroraBTM which contains in a vaccine dose an amount of total protein that is at least 100 times greater than that which can be found in an effective dose of this vaccine composition which is usually < 50 μg. On the other hand, the composition of the excipient according to the invention advantageously makes it possible to preserve vaccine compositions in a liquid or frozen form, even if the preferred form of preservation is the lyophilized form.
[00067] The vaccine composition is administered directly to the subject to be vaccinated, when it is kept in liquid form or after thawing, if it is kept frozen. When it is in a lyophilized form, the lyophilizate is extemporaneously taken up in a diluent which is usually a saline solution, such as, for example, a hypotonic sodium chloride solution. The diluent may also contain a very low concentration surfactant, whose chemical structure is compatible in the pharmaceutical plan with parenteral use. uses at a very low concentration to exert an adjuvant action. The concentration of surfactant in the diluent is usually < 0.1% (w/w).
[00068] When the vaccine is in a lyophilized form, it is usually presented in the form of a kit with two packages, the first (usually in the form of a vial) contains the vaccine composition, lyophilized, the second (usually in the the form of a vial or a syringe) contains the diluent. It is also possible to use a syringe of the "by pass" type, in which the vaccine composition is found in the lower part of the syringe, while the upper part contains the diluent.
[00069] The invention finally has for its object a stabilizing excipient for a vaccine with inactivated whole virus, in particular for a vaccine with inactivated whole rabies virus which comprises: a. a buffer solution; B. a mixture of essential and non-essential amino acids; ç. a disaccharide; d. a polyol; and. a chelating agent f. urea or a urea derivative, and g. a non-ionic surfactant.
[00070] Preferably, the excipient is devoid of any protein, preferably of any peptide and of any protein and even more preferably of any protein, of any peptide and of any oligopeptide. It is also very preferably devoid of any animal products.
[00071] The present invention will be better understood based on the following examples that serve to illustrate the invention without limiting the content.
[00072] Figure 1 is an overlay of the size distribution profiles of viral particles obtained by light scattering ("dynamic light scattering" - DLS)" during a cycle of 3 successive thawing and refreezing of an anti-rabies vaccine composition in which composition of the stabilizing excipient is F04 + 0.001% poloxamer 188. • D0 (before freezing); ■ D1 (after the first thaw); ▲ D2 (after the second thaw) and + D3 (after the third thaw).
[00073] Figure 2 is an overlay of the viral particle size distribution profiles obtained by "dynamic light scattering (DLS)" over a cycle of 3 successive thawing and refreezing of a rabies vaccine composition in which the excipient composition stabilizer is F04 + 0.01% poloxamer 188. • D0 (before freezing); ■ D1 (after the first defrost); ▲ D2 (after the second defrost) and + D3 (after the third defrost). Example 1: Influence of the excipient on the stability of a vaccine composition containing inactivated purified rabies virus in bulk form. 1.1 Bulk Preparation
[00074] The production of rabies virus was carried out on Vero cells, using a serum-free viral infection medium. The Vero lineage cells were adapted to culture conditions in serum free medium as described in application WO 01/40443. They were then transferred into a biogenerator containing cytodex 1 microcarriers in VP SFM medium (Invitrogen). After a culture period of 3 to 4 days at 37°C maintaining a pH of approximately 7.2 + 0.2, an oxygen saturation of 25% + 10% and subjecting the medium to gentle agitation, the cells were infected with rabies virus (Pitmann - Moore strain) at a multiplicity of infection of 0.01, using a serum-free viral infection medium containing the VP SFM medium (Invitrogen). Culture supernatant was collected from infected cells on days D7 (R1), D11 (R2) and D15 (R3). After each collection, the new viral infection medium was reintroduced. The culture supernatant containing the infectious virus was clarified, using two successive filtrations: the first, using an 8 μm polypropylene pre-filter (Sartopure PP2. SARTORIUS) that eliminates some aspirated microcarriers, upon collection, the Vero cells detached from the supports and cell debris of considerable size; the second, using a PES filter, composed of a combination of two 0.8 μm and 0.45 μm filters (Sartopore 2, SARTORIUS) that eliminates the aggregates. The amount of rabies virus present in the clarified collections was determined based on the measurement of the amount of glycoprotein G (gpG) made by ELISA as follows:
[00075] Approximately 0.12 μg/100 μl of a solution of a monoclonal antibody 1112-1 anti-gpG (whose characteristics are described in the Journal of Clinical Investigation (1989), volume 84) is distributed in the wells of an ELISA microplate. pages 971 to 975) previously diluted in a buffer coating (0.2 M carbonate/bicarbonate buffer, pH 9.6). After an overnight incubation in a cold room, followed by several washes with a wash buffer (phosphate buffer added with Tween 20, 0.5%), 100 µl of a saturation buffer ( phosphate buffer added with 1% bovine albumin serum. After an incubation of one hour at 37°C, followed by several washings, a dilution range of each test sample was carried out in a dilution buffer (phosphate buffer added with 0.05% Tween 20 and serum albumin at 0.1%). In parallel, a range of dilution and reference measurement was carried out on each microplate, which was calibrated against the international reference of the NIBSC (eg PISRAV). After a further one-hour incubation at 37°C, followed by several washes, 100 µl of a solution of an anti-gpG mouse monoclonal antibody D1 (the characteristics of which are described in Biologicals (2003), volume) was dispensed into each well. 31, pages 9 to 16) which was biotinylated and used after dilution to 1/5000 in the dilution buffer. The plates were left for one hour at 37°C, then washed with several retakes before dispensing 100 µl of a peroxidase-coupled streptavidin solution (Southern Biotechnology Associates) previously diluted to 1/15000 in the dilution buffer into each well. . After a further one-hour incubation at 37°C followed by several washings, a dilution range of each sample to be tested was carried out in a dilution buffer (phosphate buffer added with 0.05% Tween 20 and serum albumin a 0.1%). In parallel, a dilution range of a reference measurement was performed on each microplate, which was calibrated against the NIBSC international reference (for example, PISRAV). After a further incubation of one hour at 37°C followed by several washings, 100 μl of a solution of a monoclonal antibody D1 mouse anti-gpG (whose characteristics are described in Biologicals (2003), volume 31, pages) were distributed to each well. 9 to 16) which was biotinylated and used after dilution to 1/5000 in the dilution buffer. The plates were left for one hour at 37°C, then washed in several times, before dispensing each well with 100 µl of a peroxidase-coupled streptavidin solution (Southern Biotechnology Associates) previously diluted to 1/5000 in the dilution buffer . After a further incubation of one hour at 37°C followed by several washings, 100 µl of a solution of a 0.05 M citrate buffer, pH5 containing the developing substrate (O-Phenylene diamine) were distributed in each well. After an incubation time of 30 minutes at room temperature in the dark, the development reaction was stopped by adding 50 µl/well of a 2N H2SO4 solution. The spectrophotometric reading of the microplates was performed with two wavelengths (492 nm and 620 nm). The measured optical density is the difference between the two readings to account for the plastic's absorption. The calculation of the relative activity was done by the method of parallel lines, according to the recommendations of the European Pharmacopoeia. The rabies virus titer of the sample is based on the determination of the concentration of rabies virus G glycoprotein which is expressed in IU/ml relative to the reference.
[00076] After also controlling the conductivity and pH of the clarified liquid, it was deposited on the chromatographic support Fractogel ® EMD SO3- (Merck) previously balanced with the aid of a 20 mM Tris, 150 mM NaCl, pH - 7 buffer .5, the equivalent of approximately 50 IU of glycoprotein G (measured by ELISA) per ml of support. The chromatographic support was then washed with the equilibration buffer, then the rabies virus was purified in a 20 mM Tris, 600 mM NaCl, pH - 7.5 buffer with viral peak recovery in an independent fraction. This was followed by an ultrafiltration step on a PES Medium Screen 100 KD membrane (PALL) followed by diafiltration in a 20 mM Tris buffer, 150 mM NaCl, pH - 7.5. A MgCl2 solution was added so that the concentration in the diafiltrate was 2 mM. The treatment with benzonase was carried out by adding 15 U/mL of crude collection to the reaction medium and leaving the reaction medium for two hours at laboratory temperature. The ultracentrifugation step was carried out on 34 - 60% sucrose pads with a 45 type Ti rotor at 21000 rpm for 2 hours at + 5°C. Gradient fractions containing purified virus were collected, pooled, then diluted in phosphate buffer 50 mM NaCl 150 mM, pH - 7.5, such that the final volume of the purified virus suspension was approximately 12.5 times smaller than the than the volume of the clarified collection. The purified virus suspension was then inactivated by a treatment with β propiolactone, followed by an inactivation of β propiolactone by heating at a temperature of approximately 37°C for 2 hours. The purified and inactivated virus preparation was concentrated six times by ultrafiltration on 10 kDa membrane (Omega medium screen PALL) followed by diafiltration in 50 mM phosphate buffer, 150 mM NaCl pH 7.5. The rabies virus preparation obtained is in the form of a concentrated bulk, whose titer based on the determination of the G glycoprotein concentration was approximately 80 IU/ml. Then, an aliquot of the bulk was dialyzed against one of the four excipients (F01 to F04) whose compositions were as follows. 1.2 Composition of tested excipients. F01: 50 mM phosphate buffer, 150 mM NaCl, Maltose (5% w/w), pH-8.0 F02: 50 mM phosphate buffer, Maltose (5% w/w), pH-8.0 F03: phosphate buffer 50 mM, Maltose (5% w/w), poloxamer 188 (BASF) (0.001% w/w), pH-8.0 F04: 489 PM buffer, pH-8.0, whose composition is as follows:

[00077] The essential amino acid solution was prepared by dissolving the contents of a vial containing 111.5 g of essential amino acids (Gibco, Reference 074-90680N, lot number 14773) in 5 liters of water for injectable preparation acidified per 100 mL of 12N HCl (Prolabo).
[00078] The non-essential amino acid solution was prepared by dissolving the contents of a vial containing 40.7 g of non-essential amino acids (Gibco, Reference 074-90681 N, lot number 14775) in 5 liters of water to injectable preparation. 1.3 Tests made on the 4 bulk formulations.
[00079] The bulk was formulated in the four tested excipient compositions. The stability of these four formulations (vaccine compositions) was evaluated over time at different storage temperatures: - 70°C, + 5°C and at + 37°C by measuring at regular intervals the titer in glycoprotein G (expressed in UI/ mL). The results obtained are shown in tables 1 to 3 and are expressed as UI/mL. Table I: stability of the 4 bulk formulations at + 37°C
Table II: stability of the 4 bulk formulations at +5°C
Table III: stability of the 4 bulk formulations at - 70°C
*: F01, F02. F03 and F04 refer to the composition of the excipients in which the bulk was formulated. ** : in IU/mL.
[00080] These results show that the excipient F04 is the one that best stabilizes the preparation of purified and inactivated rabies virus in particular, when the storage temperature increases. It also shows that an excipient comprising a sugar (maltose) and a nonionic surfactant such as poloxamer 188 in a buffered solution at pH 8.0 is not sufficient to effectively stabilize a rabies virus preparation. Example 2: Role of surfactant in stabilizing vaccine compositions containing purified rabies virus. 2.1 Preparation of vaccine compositions
[00081] A suspension of purified rabies virus was obtained using the same procedure as that used in example 1. After the centrifugation step, the purified virus preparation was in a very concentrated form. The total protein concentration was 7 mg/ml, using the Bradford method. This virus preparation was divided into three polypropylene containers, then diluted in three different excipients that were sought to be evaluated for their stabilizing role, so that the final concentration of total proteins in each of the tested excipients was reduced to 10 μg/mL ( dilution factor 1/700). The composition of the three excipients tested differed only in their content of poloxamer 188, but contained the entire 489 PM buffer, pH = 8.0, whose composition is described in example 1. The excipient without poloxamer 188 had the same composition as the excipient F04 described in example 1. The two other excipients were respectively supplemented with 0.01 g/L of poloxamer 188 (excipient named F04 + 0.01% poloxamer) and 0.1 g/L of poloxamer 188 (excipient named F04 + 0) .1% poloxamer). 2.2 Stability studies and results
[00082] The stability of the three vaccine compositions was evaluated, placing them in unfavorable conditions of conservation, that is, submitting them to a succession of freezing and thawing, according to the following protocol.
[00083] The three vaccine compositions were frozen at -70°C on the same day (day D0). Each of the three compositions was then subjected to three thawing at +5°C followed by refreezing at -70°C at 7 am to 2 pm, according to the thawing on days 1, 2 and 3 (respectively D1, D2 and D3) . The stability of the vaccine compositions after each thawing was evaluated by measuring the total viral protein concentration. The measurement was performed using BIAcore technology, which is based on the use of surface plasmonic resonance. The distribution profile of the total particles was also analyzed during the successive thawings, applying the quasi-elastic light scattering technique (Dynamic Light Scattering or even DLS technology).
[00084] In order to apply the BIAcore technology, the monoclonal antibody D1 anti-gpG mouse monoclonal antibody D1 is firstly coupled to a sensor chip, using the coupling kit provided by the manufacturer (coupling kit of reference amine, BR-1000-50). Then, 30 μl of the sample to be analyzed were automatically injected into the device within a period of 3 minutes. The interaction of the rabies virus present in the sample with the monoclonal antibody fixed on the sensor caused a change in the refractive index on the sensor chip surface, translating into a change in the registered signal (RU). The results obtained were registered in arbitrary units (ΔRU) and were compared with the results obtained with a measurement range of a reference that contained an amount such as purified rabies virus. The ΔRU measured for each sample was converted into total viral protein concentration based on the measurement curve obtained with the reference. For each sample tested, measurements were made four times (in quadruplicate). After each measurement, the sensor chip was cleaned, performing two successive injections of 10 μl of a Glycine buffer (10 mM, pH = 1.5). The results obtained for each vaccine composition tested are shown in table IV. Values are expressed in μg/ml of viral proteins. Table IV: Study of the stability of the 3 vaccine compositions during a cycle of 3 successive thawing and refreezing.
1 : mean titre in viral proteins obtained on the basis of four measurements made on the sample tested in μg/mL. 2 * : standard deviation calculated on the basis of the four measurements made on the tested sample is indicated in parentheses.
[00085] These results clearly show that the excipient F04, in the absence of poloxamer, is unable to effectively preserve the rabies virus preparation, after the loss in viral titer is 88% between the titer on D0 and the titer measured after the third thawing (D3). Conversely, since the excipient F04 is very lightly supplemented with poloxamer 188, the loss in viral titer is greatly diminished (it is 33% when F04 is supplemented with 0.001 g/L poloxamer 188 and 23% when F04 is supplemented with 0.01 g/L poloxamer 188). These results show that the excipient must contain, at the same time, the components that are present in the 489 PM buffer and the poloxamer 188 to effectively preserve a vaccine composition containing the rabies virus.
[00086] The particle size distribution profile of each of the three vaccine compositions was also analyzed after each thawing by DSL, using the Zetasizer Nano Zs device (Malvern Instrument). 450 μl of the sample to be analyzed are introduced into a single-use polystyrene tub, which was then subjected to monochromatic laser radiation (HeNe laser, À = 632.8 nm). The signal registered by the device corresponded to the fluctuations of scattered light resulting from the Brownian movement of the particles. The recorded data were processed by a program.
[00087] The results were finally represented in the form of a particle size distribution curve for each sample tested. The two figures represent a superposition of the 4 particle size distribution curves that were obtained respectively with a rabies virus composition stabilized with the excipient F04 + 0.001% poloxamer 188 on days D0 (before freezing), D1, D2 and D3 ( figure 1), and with a rabies virus composition stabilized with the excipient F04 + 0.01% poloxamer 188 (figure 2).
[00088] The two figures clearly show that: 1) the particle size distribution curves are unimodal and have a Gaussian profile, whose median is located at approximately 180 nm. This means that the population of viral particles in the vaccine composition stabilized with excipient F04 + 0.001% poloxamer 188 or excipient F04 + 0.01% poloxamer 188 is homogeneous, does not contain aggregates and comprises a population of whole rabies viruses similar to wild rabies virus population; and 2) the particle size distribution curves are not modified when rabies virus vaccine compositions are subjected to several successive thawing and refreezing.
[00089] The presence of a non-ionic surfactant such as poloxamer 188 in the excipient composition also preserves the physical integrity of the rabies virus population, even when the vaccine composition is placed in poor storage conditions. 2.3 Stability studies on freeze-dried vaccine compositions 2.3.1 Preparation of lyophilized vaccine compositions
[00090] A bulk was initially prepared from an inactivated whole purified virus preparation that was obtained using the process described in example 1. After the viral inactivation step, the inactivated purified rabies virus preparation was diafiltered over a 10 kDa membrane (Omega medium screen PALL), using an excipient called 488 TM which was prepared from a 488 medium whose composition is as follows (see table below).

[00091] The mixture of essential and non-essential amino acids was prepared by mixing two and a half liters of the essential amino acid solution, prepared according to example 1 with two and a half liters of the non-essential amino acid solution prepared according to the example 1 and adjusting the pH to approximately 7.2 using a 30% soda solution.
[00092] The composition of the 488 TM excipient is as follows:


[00093] The obtained vaccine preparation was in the form of a bulk in the excipient 488 TM whose viral titer based on the determination by ELISA of the concentration in glycoprotein G was approximately 12 IU/ml. The bulk was then preserved in frozen form at -70°C until the time of preparation of the lyophilized vaccine compositions.
[00094] Just before the lyophilization step a part of the bulk was thawed and diluted to bring the final titer in glycoprotein G to approximately 8 IU/mL using: - be the excipient F'04 whose composition contains a volume of the excipient 488 TM to three volumes of 50 mM phosphate buffer, maltose concentration being adjusted to 50 g/L, pH = 8.0; - is the excipient F'05 which is identical to the excipient F'04, but to which poloxamer 188 has been added at a final concentration of 0.01 g/L.
[00095] Then, the two vaccine compositions were divided into lyophilization vials at the rate of 0.4 mL/vial, after which a classic lyophilization cycle was carried out, comprising a freezing phase at a temperature < - 40°C followed by two successive desiccation phases, the first desiccation phase being carried out at a temperature of approximately -15°C under a pressure of approximately 80 μ baria and the second at a temperature of approximately + 40°C under a pressure of approximately 80 μ baria .
[00096] Each lyophilization vial contained a dose of the vaccine composition to be tested. The lyophilized vaccine compositions obtained differed only by the presence or absence of the surfactant, depending on whether the bulk had been diluted in the excipient F'04 or F'05.
[00097] The composition of the excipient contained in a dose of the vaccine composition in lyophilized form prepared from the excipient F'05 is indicated below.
2.3.2: Efficacy of freeze-dried vaccine compositions
[00098] The two lyophilized vaccine compositions that were prepared using two different stabilizing excipients were then tested for their effectiveness using the official NIH test. The "efficacy" of the two lyophilized vaccine compositions was analyzed, either immediately after lyophilization (D0), or after storage for one week at 45°C, or also after storage for one month at 37°C. A single officially recognized activity test was used, the NIH test, which makes it possible to determine the number of IUs contained in each of the doses of the tested vaccine compositions. If the number of IUs is > 2.5 IU the tested vaccine dose is effective. The NIH test was carried out each time in "double" for each of the conditions tested, collecting randomly from the lyophilized vials in the batch of vials prepared and kept under the same conditions and performing an NIH test on these collections. The protocol used corresponds to monograph 0216 of the European Pharmacopoeia. This test is a proof test carried out in mice, based on the comparison of the protection provided by the dose of vaccine tested with that provided by a known amount of a reference anti-rabies vaccine. The reference rabies vaccine is calibrated in international units (UI). The international unit (UI) is the activity contained in a specified quantity of an international standard. The UI equivalence of the international standard has been established by WHO. After verifying that the control parameters of the efficacy test were well respected, it was determined from the values of the protective dose 50% obtained for each tested collection and for the reference preparation (calibrated in UIs), the number of UIs that are contained in the dose of vaccine tested.
[00099] The results obtained are shown in table V below. Table V: Efficacy of the lyophilized vaccine compositions as a function of the composition of the lyophilization excipient used under storage conditions.
* : mean value expressed in IU obtained (all groups of rats that were tested contain 16 rats). ** : represents the extreme values obtained expressed in UI. NT: not tested
[000100] These results are in agreement with the results discussed in the preceding paragraph (2.2) and show that poloxamer 188 should be included in the composition of the excipient used to preserve the lyophilized vaccine compositions. In fact, only lyophilized vaccine compositions containing the excipient F'05 (which contains poloxamer 188) contain an effective dose of vaccine, since the average value in IU is greater than 2.5 IU. These vaccine compositions remain stable as their efficacy is not diminished over time under the storage conditions under which they were tested.
[000101] To summarize, the results that are presented in example 2 show that vaccine compositions containing rabies virus can be well preserved with a stabilizing excipient, which composition comprises a phosphate buffer a mixture of essential and non-essential amino acids, a sugar, such as maltose, a polyol such as sorbitol, EDTA, urea in combination with a nonionic surfactant such as poloxamer 188. In addition, and interestingly, "the stabilizing effect" of poloxamer 188 is exerted on a concentration too small that is too low for poloxamer 188 to activate the immune system or act as an adjuvant to the immune response. Example 3: Study of the stability of vaccine compositions containing purified and inactivated rabies virus.
[000102] Examples 1 and 2 having shown the importance of combining the compounds of an excipient comprising a mixture of essential and non-essential amino acids, maltose, sorbitol, EDTA, urea in a phosphate-based buffer and /or Tris with poloxamer 188, the purpose of the studies presented in the case was to confirm that this combination effectively stabilized rabies virus vaccine compositions in different forms (liquid, frozen or lyophilized) in a wide range of storage temperature (+ 37°C C, + 25°C, + 5°C, - 70°C) and over a long period of time. These studies were carried out on various rabies virus vaccine compositions which were preserved as a frozen bulk at a temperature < - 35°C, as a liquid bulk end product at +5°C or as a dose lyophilized units stored at + 5°C, + 25°C or + 37°C. 3.1 Stability studies of rabies vaccine batches in frozen bulk form.
[000103] The rabies virus that serves for the preparation of bulk batches was prepared according to the process as described in example 1. After the viral inactivation step, the purified and inactivated virus preparation was diafiltered on a 10 kDa membrane, using a diafiltration buffer is a 50 mM phosphate buffer as diafiltration buffer. In a second time, a volume of retentate was mixed with a volume of stabilizing excipient whose composition is that of the 489 PM buffer described in example 1 (apart from the fact that the concentration of each of the components is twice as important) to which poloxamer 188 was added at a concentration of 0.02 g/L, pH ~ 8.0. A bulk was thus obtained having a total protein concentration of 50 µg/binding medium of which more than 70% are rabies virus proteins and which contains less than 100 pg/ml of residual DNA. The stability of the bulk over time, after storage at -35°C and -70°C, was studied by measuring the titer in glycoprotein G every three months. The results obtained are shown in table VI below. Table VI: Stability of a bulk batch stored at - 35°C or at -70°C.
* : titre expressed in IU/mL NT : not tested
[000104] After 12 months of storage at - 35°C or at - 70°C there is no degradation of glycoprotein G, which is the major antigen of the rabies virus, which confirms the good stability of the vaccine composition in the form of frozen bulk. Furthermore, analysis of the size distribution of viral particles by DLS after a 12-month storage at -35°C or at -70°C showed that the profiles maintained a Gaussian elapse centered on a median value of 180 nm (similar profiles those of figures 1 and 2). There is no fragmentation of the profiles, indicating the presence of viral aggregates. The results also showed that the population of rabies virus particles did not change over time. No degradation or aggregation of viral particles is observed over time. 3.2: Stability studies of rabies vaccine batches in the form of liquid bulk final product (PFV).
[000105] 3 batches of vaccines were prepared in the form of liquid PFV from 3 batches of bulk, after dilution in buffer 489 PM (composition described in example 1) to which poloxamer 188 is added at a final concentration of 0.01 g/L, pH ~8.0. They contained less than 100 pg/ml of residual DNA (measured by quantitative PCR). The total protein concentration was approximately 15 μg/mL and more than 70% of the total proteins were represented by viral proteins after densitometric analysis of polyacrylamide gel electrophoresis. The stability of PFV batches stored at + 5°C and at + 25°C was studied by measuring at regular intervals the glycoprotein G rate by ELISA over a period of 3 months for PFV batches stored at + 5°C and within 30 days for batches of PFV stored at +25°C. The results obtained expressed in UI/mL are gathered in tables VII and VIII below: Table VII: Stability of 3 batches of PFV stored at + 5°C
Table VIII: Stability of 3 batches of PFV stored at + 25°C

[000106] These results show that there is no degradation of glycoprotein G during the conservation of batches of PFV in liquid form at + 5°C and at - 25°C during the time periods studied. This confirms that rabies vaccines can be stored in liquid form in the cold (+5°C) for at least 3 months and at room temperature (+25°C) for at least 30 days, without observing virus degradation. 3.3: Stability studies of rabies vaccine batches in lyophilized form
[000107] 2 batches of rabies vaccines in lyophilized form (LL1 and LL2) were prepared from the batches of PFV of example 3.2 which were distributed in lyophilization glass vials at the rate of 0.3 ml/vial before proceeding to a freeze-drying cycle during which freezing was carried out at a temperature < 40°C, the primary desiccation phase at a temperature close to -15°C under a pressure of approximately 80 μ baria until complete sublimation of the ice and the phase of secondary desiccation at a temperature close to + 40°C under a pressure of approximately 80 μ baria until the residual moisture content is < 3% in the lyophilisates. Losses in viral titer during the lyophilization step were < 5%.
[000108] The composition of the excipient contained in a vaccine dose in lyophilized form is as follows:

[000109] The lyophilized unit doses obtained had a white homogeneous appearance. The pH of the suspension of rabies virus after resuming the lyophilized with 0.5 mL of a 0.4% sodium chloride solution was in a range of 7.8 + 0.5. The stability of the two lyophilized batches was studied during the time at three storage temperatures: 35 - 39°C, 23 - 27°C and 2 - 8°C, checking the appearance of the lyophilized products before and after reconstitution, measuring the humidity residual, controlling the pH, the viral titer and testing the efficacy of the lyophilized doses in the NIH test. The first dilution of the dilution ranges that were performed to test the lyophilized doses in the NIH test, after resumption in sodium chloride, was performed in a 0.9% sodium chloride solution containing 2 g/L of human albumin.
[000110] Viral titers were measured based on the glycoprotein G rate by ELISA and expressed in IU/ml.
[000111] The test results regarding the appearance of the lyophilisates and measurement of the residual unit and pH were all in accordance with the specificities (i.e., a homogeneous white appearance for the lyophilisate, a clear colorless solution after reconstitution in hypotonic saline and a residual moisture rate (< 3%) irrespective of temperature and shelf life tested.The pH remained stable in a range of 7.8 ± 0.5 for the duration of the stability studies.
[000112] The results regarding the titles and efficacy tests (NIH) performed in unit doses are gathered in tables IX to XI below:
Table X: stability of the two batches of rabies vaccines preserved in lyophilized form at + 25°C
Table XI: stability of the two batches of rabies vaccines preserved in lyophilized form at + 37°C
M : means month ND: means not determined 1 : value expressed in IU/mL. 2 * : mean value expressed in number of IU supplemented by the observed extreme values indicated in parentheses.
[000113] The set of these results confirms that rabies vaccines preserved in lyophilized form remain very stable and do not lose effectiveness over time under the temperature conditions tested. G-glycoprotein titers and NIH test values (sharply greater than 2.5 IU) remain stable over time.
[000114] In conclusion, all stability tests used on batches of rabies vaccines preserved in frozen form, in liquid form or in lyophilized form show that these batches remain stable over time under the temperature conditions tested. This confirms that an excipient, whose composition comprises a buffer, a mixture of essential and non-essential amino acids, maltose, sorbitol, EDTA, urea and poloxamer 188 effectively stabilizes an anti-rabies vaccine composition, regardless of its presentation (frozen, liquid or lyophilized) and over a wide range of storage temperatures.

权利要求:
Claims (29)
[0001]
1. Vaccine composition, characterized in that it comprises: (a) a preparation of whole inactivated rabies virus, and (b) a stabilizing excipient comprising: (i) a buffer solution; (ii) a mixture of essential and non-essential amino acids; (iii) a disaccharide; (iv) a polyol; (v) a chelating agent; (vi) urea or a urea derivative, and (vii) a non-ionic surfactant.
[0002]
2. Composition according to claim 1, characterized in that it is devoid of any serum protein.
[0003]
3. Composition according to claim 1 or 2, characterized in that it is devoid of any exogenous protein of animal origin and, preferably, devoid of any exogenous product of animal origin.
[0004]
4. Composition according to any one of claims 1 to 3, characterized in that the virus proteins represent from 70% to 99.99% of the total proteins that are present in the vaccine composition.
[0005]
5. Composition according to any one of claims 1 to 4, characterized by the fact that the concentration in total proteins is < 100 μg/mL and, preferably, < 80 μg/mL.
[0006]
6. Composition according to any one of claims 1 to 5, characterized in that the amount of total protein that is contained in an effective dose of the vaccine composition is < 100 μg.
[0007]
7. Composition according to any one of claims 1 to 6, characterized in that the amount of total protein that is contained in an effective dose of the vaccine composition is between 1 and 50 μg.
[0008]
8. Composition according to any one of claims 1 to 7, characterized in that the stabilizing excipient is devoid of any protein and any other peptide or, preferably, devoid of any protein, any peptide and any oligopeptide.
[0009]
9. Composition according to any one of claims 1 to 8, characterized in that the mixture of essential and non-essential amino acids comprises at least arginine or a salt of arginine and glutamic acid or a salt of glutamic acid.
[0010]
10. Composition according to any one of claims 1 to 9, characterized in that the amount of essential and non-essential amino acids contained in an effective dose of the vaccine composition is between 0.5 and 2.5 mg.
[0011]
11. Composition according to any one of claims 1 to 10, characterized in that the disaccharide is maltose.
[0012]
12. Composition according to any one of claims 1 to 11, characterized in that the polyol is sorbitol.
[0013]
13. Composition according to any one of claims 1 to 12, characterized in that the total amount of disaccharide and polyol contained in an effective dose of the vaccine composition is between 10 and 50 mg.
[0014]
14. Composition according to any one of claims 1 to 13, characterized in that the chelating agent is EDTA or an EDTA salt.
[0015]
15. Composition according to any one of claims 1 to 14, characterized in that the amount of chelating agent contained in an effective dose of the vaccine composition is comprised between 0.01 and 0.1 mg.
[0016]
16. Composition according to any one of claims 1 to 15, characterized in that the amount of urea or urea derivative contained in an effective dose of the vaccine composition is between 0.3 and 1.5 mg.
[0017]
17. Composition according to any one of claims 1 to 16, characterized in that the non-ionic surfactant is a poloxamer.
[0018]
18. Composition according to any one of claims 1 to 17, characterized in that the non-ionic surfactant is poloxamer 188.
[0019]
19. Composition according to any one of claims 1 to 18, characterized in that the amount of nonionic surfactant contained in an effective dose of the vaccine composition is between 0.001 and 0.5 mg.
[0020]
20. Composition according to any one of claims 1 to 19, characterized in that the pH of the vaccine composition is between 7.0 and 10.0 and preferably between 7.0 and 9.0.
[0021]
21. Composition according to any one of claims 1 to 20, characterized in that the buffer solution is a phosphate buffer, a Tris buffer, a HEPES buffer or a mixture thereof.
[0022]
22. Composition according to any one of claims 1 to 21, characterized in that the buffer solution is a phosphate buffer, whose molarity is between 10 and 100 mM.
[0023]
23. Composition according to any one of claims 1 to 22, characterized in that the stabilizing excipient comprises: (i) a phosphate buffer, (ii) a mixture of essential and non-essential amino acids, containing at least arginine or a salt thereof and glutamic acid or a salt thereof, (iii) maltose, (iv) sorbitol, (v) EDTA or a salt thereof; (vi) urea, and (vii) poloxamer 188.
[0024]
24. Composition according to claim 23, characterized in that, in an effective dose of rabies vaccine: the amount of essential and non-essential amino acids is between 0.5 mg and 2.5 mg, the amount of maltose and sorbitol is between 10 mg and 50 mg, the amount of EDTA or a salt thereof is between 0.01 mg and 0.1 mg, the amount of urea is between 0.3 mg and 1.5 mg, and the amount of poloxamer 188 is between 0.001 mg and 0.5 mg.
[0025]
25. A process for preparing a vaccine composition containing a preparation of purified and inactivated whole rabies virus, characterized in that it comprises: (a) producing a whole virus preparation by collecting the supernatant of a virus-infected cell culture; (b) purifying and then inactivating the whole virus preparation or, alternatively, inactivating and purifying the whole virus preparation; and (c) diluting the purified and inactivated whole virus preparation in a stabilizing excipient, the composition of which comprises: (i) a buffer solution; (ii) a mixture of essential and non-essential amino acids; (iii) a disaccharide; (iv) a polyol (v) a chelating agent; (vi) urea or a urea derivative; and (vii) a non-ionic surfactant.
[0026]
26. Process according to claim 25, characterized in that it is carried out without introducing any exogenous product of animal origin.
[0027]
27. Process according to claim 25 or 26, characterized in that, after having diluted the preparation of purified and inactivated whole virus, the resulting vaccine composition is divided into packaging devices.
[0028]
28. Process according to claim 27, characterized in that the vaccine composition is lyophilized.
[0029]
29. Lyophilized vaccine composition, characterized in that it contains a preparation of purified and inactivated whole virus obtained from the process as defined in claim 28.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-13| B25G| Requested change of headquarter approved|Owner name: SANOFI PASTEUR (FR) |

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-01-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-11-24| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-05-04| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-07-13| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/10/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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FR09/04795|2009-10-07|

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FR0904795|2009-10-07|

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PCT/FR2010/052111|WO2011042663A1|2009-10-07|2010-10-07|Stabilising excipient for inactivated whole-virus vaccines|

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