HK1167604A - Stabilising excipient for inactivated whole-virus vaccines - Google Patents

Description

The present invention relates to a vaccine composition comprising inactivated whole rabies virus and an excipient that stabilizes the vaccine composition. The excipient composition includes 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 relates to a process for preparing this vaccine composition.

In the field of viral vaccines, three classic types of viral vaccines are distinguished: live virus vaccines, inactivated whole-virus vaccines, and subunit viral vaccines. These three types of vaccines differ by their specific characteristics, which influence the composition of the excipients used for their preservation. Subunit vaccines, which contain a limited number of viral antigens, are generally easier to preserve than inactivated whole-virus vaccines, for which the structural integrity of the virus is preserved even though it is killed, or than live virus vaccines, for which the infectious power of the virus is also preserved.For each type of viral vaccines, suitable excipients have been prepared. For attenuated virus vaccines, different stabilizing excipients have been prepared depending on the preparation of the attenuated virus to be preserved. JP 57007423 describes an excipient based on a disaccharide and a polyol in a phosphate buffer for stabilizing an attenuated strain of the measles virus. EP 065905 describes an excipient comprising one or more amino acids selected from a group of eleven amino acids, lactose, and sorbitol in a phosphate buffer for stabilizing an attenuated strain of the dengue virus. EP 0869814 and WO97/23238 describe a vaccine composition comprising an attenuated strain of the varicella virus and a stabilizing agent containing sorbitol.mannitol, sucrose, dextran, a mixture of amino acids, urea and EDTA, the dextran serving as a lyophilization bulking agent. Finally, Toriniwa et al., in Vaccine (2008), vol. 26, pp. 3680-3689, show that adding Tween 80 to an excipient to stabilize the Japanese encephalitis vaccine has a negative effect on the production of neutralizing antibodies against the virus.

In the field of inactivated virus vaccines, ready-to-administer vaccine formulations often contain animal-derived proteins, such as bovine or human albumin, gelatin, or casein. Proteins are known to improve the preservation (stabilization) of these vaccines, especially those containing viruses that are difficult to preserve. This is the case for vaccine formulations containing inactivated rabies virus. Frazatti-Gallina et al. in Vaccine (2004), vol. 23, pp. 511-517, emphasize the important role of proteins in the stabilization of the rabies vaccine since the excipient contains albumin.

Nevertheless, the presence of proteins in vaccines, aside from the potential risk of disease transmission if their origin is not strictly controlled, can also represent a potential allergic risk that should be avoided, particularly when vaccines contain serum proteins such as albumin or albumin derivatives.

Therefore, there is a need to find a stabilizing excipient whose composition does not contain protein, in order to stabilize inactivated whole-virus vaccines, and in particular to stabilize vaccines containing difficult-to-store inactivated whole viruses such as the rabies virus.

There is also a need to find a suitable stabilizing excipient that can stabilize weakly concentrated inactivated virus vaccines, meaning vaccines that contain a low amount of total proteins within an effective vaccine dose. This need is even more critical for highly purified vaccines containing very few residual protein impurities. Even though these residual protein impurities may represent a potential allergic risk, they can contribute to some extent to the stabilization of weakly concentrated inactivated viral vaccines. They can prevent aggregation, structural degradation, or virus adsorption phenomena that could reduce the vaccine's effectiveness.

To this end, the present invention relates to: A vaccine composition comprising: a) a preparation of inactivated whole virus, and b) a stabilizing excipient 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 selected from allylurea, acetamide, methyl carbamate, and butyl carbamate, and vii. a non-ionic surfactant, wherein the inactivated whole virus is the rabies virus, the buffer solution is a phosphate buffer, and the mixture of essential and non-essential amino acids includes at least arginine or an arginine salt and glutamic acid or a glutamic acid salt.The disaccharide is maltose, the polyol is sorbitol, the chelating agent is selected from EDTA or an EDTA salt, the non-ionic surfactant is poloxamer 188, and in which the pH of the vaccine formulation is between 7.3 and 8.3, and in an effective dose of the vaccine composition, 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 EDTA salt is between 0.01 mg and 0.1 mg, the amount of urea or a urea derivative selected from allylurea,The amount of acetamide, methyl carbamate, and butyl carbamate is between 0.3 mg and 1.5 mg, and the amount of poloxamer 188 is between 0.001 mg and 0.5 mg.

According to one aspect of the invention, the vaccine composition is also free of any serum protein.

According to another aspect, the vaccine composition is also free of any animal-derived exogenous protein and preferably free of any animal-derived exogenous product.

In another aspect, the viral proteins account for at least 70% of the total proteins present in the vaccine formulation.

According to another aspect, the total protein concentration in the vaccine formulation is ≤100 µg/ml and preferably ≤80 µg/ml.

In a particular aspect, the amount of total proteins contained in an effective dose of the vaccine formulation is ≤100 µg.

In another particular aspect, the amount of total proteins contained in an effective dose of the vaccine composition is between 1 and 50 µg.

In another aspect of the invention, the stabilizing excipient is free of any protein, or of any protein and any peptide, or preferably free of any protein, any peptide, and any oligopeptide.

In a particular aspect, the buffer solution is a phosphate buffer with a molarity ranging from 10 to 100 mM.

The invention also relates to a method for preparing a vaccine composition containing purified and inactivated whole rabies virus preparation, characterized by the following steps: a. producing a whole virus preparation by harvesting the supernatant of a cell culture infected with rabies virus; b. purifying and inactivating the whole rabies virus preparation, or alternatively inactivating and then purifying the whole rabies virus preparation, etc. The purified and inactivated whole rabies virus preparation is then diluted in a stabilizing excipient whose composition comprises: i. a phosphate buffer, ii. a mixture of essential and non-essential amino acids,comprising at least arginine or an arginine salt and glutamic acid or a glutamic acid salt, iii. maltose, iv. sorbitol, v. a chelating agent selected from EDTA or an EDTA salt, vi. urea or a urea derivative selected from allylurea, acetamide, methylcarbamate, and butylcarbamate, and vii. a nonionic surfactant, wherein said nonionic surfactant is poloxamer 188, wherein the pH of the vaccine composition is between 7.3 and 8.3, and wherein, in an effective dose of the vaccine composition, 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 EDTA salt is between 0.01 mg and 0.1 mg, the amount of urea or a chosen urea derivative among allylurea, acetamide, methyl carbamate, and butyl carbamate is between 0.3 mg and 1.5 mg, and the amount of poloxamer 188 is between 0.001 mg and 0.5 mg.

According to a preferred method, the process according to the invention is carried out without introducing an exogenous animal-derived product.

In another aspect, the process according to the invention comprises, after diluting the purified and inactivated whole rabies virus preparation, an step of distributing the resulting vaccine composition into packaging devices and optionally a step of lyophilizing the vaccine composition.

The invention also relates to a vaccine composition containing a preparation of purified and inactivated whole rabies viruses in a lyophilized form obtained according to one of the embodiments of the method of the invention.

The description also discloses a stabilizing excipient for an inactivated whole-virus vaccine 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, vi. urea or a urea derivative, and vii. a nonionic surfactant.

Preferably, the composition of the stabilizing excipient is also free of any protein, or of any protein and any peptide, or preferably free of any protein, any peptide, and any oligopeptide.

In particular, the composition of the stabilizing excipient is also free from any animal-derived product.

Detailed Description of the Invention

The vaccine composition according to the invention comprises inactivated whole rabies virus (or a preparation of inactivated whole viruses) and a stabilizing excipient whose composition includes a buffer, a mixture of essential and non-essential amino acids, a disaccharide, a polyol, a chelating agent, urea or a urea derivative selected from allylurea, acetamide, methyl carbamate, and butyl carbamate, and a non-ionic surfactant. More specifically, the stabilizing excipient comprises: i. a phosphate buffer, ii. a mixture of essential and non-essential amino acids, including at least arginine or an arginine salt and glutamic acid or a glutamic acid salt, iii. maltose,iv. sorbitol, v. a chelating agent selected from EDTA or an EDTA salt, vi. urea or a urea derivative selected from allylurea, acetamide, methylcarbamate and butylcarbamate, and vii. a nonionic surfactant, wherein said nonionic surfactant is poloxamer 188, in which the pH of the vaccine composition is between 7.3 and 8.3, and in an effective dose of the vaccine composition, 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 EDTA salt is between 0,0.1 mg and 0.01 mg, the amount of urea or urea derivative selected from allylurea, acetamide, methyl carbamate, and butyl carbamate, is between 0.3 mg and 1.5 mg, and the amount of poloxamer 188 is between 0.001 mg and 0.5 mg.

The composition of the excipient advantageously replaces excipients from the prior art that contain a protein in their composition. Particularly interestingly, it stabilizes difficult-to-store vaccine formulations, and especially vaccine formulations containing the inactivated whole rabies virus, without the need to add a protein.

The vaccine composition according to the invention is stable in both liquid form, frozen liquid form, or as a lyophilizate, which offers great flexibility in use.

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 for frozen vaccine compositions is generally ≤ -35°C; for liquid formulations, it ranges from +2°C to +8°C, and for lyophilized formulations, it is approximately +5°C. Advantageously, it also preserves the biological activity and physical integrity of rabies viruses in vaccine compositions stored under unfavorable conditions, such as storage at +37°C for liquid vaccine formulations, or when the vaccine compositions are subjected to repeated cycles of thawing and refreezing.

The biological activity of the rabies virus in the vaccine formulation is assessed by measuring over time the amount of a constitutive antigen of the virus, which is essential for inducing protective immunity against the virus, or by testing its efficacy in a challenge test using an officially recognized animal model. In the case of a vaccine formulation containing inactivated rabies virus, the stability of the vaccine formulation is evaluated by measuring over time (or after successive thawing) the virus titer, which is estimated based on the measurement of the concentration of the G glycoprotein in its non-denatured form and/or by testing the efficacy of the vaccine formulation using the official NIH test.To evaluate the glycoprotein G level, one can use a sandwich ELISA method that recognizes at least one, preferably two, conformational epitopes of the glycoprotein G, as described in Example 1. When implementing an ELISA that recognizes two conformational epitopes of the G protein, a rabies virus-neutralizing antibody is usually used as the capture antibody, which recognizes a conformational epitope located on antigenic site II of the glycoprotein G (Journal of Clinical Investigation (1989), vol. 84, p. 971 to 975), and a second rabies virus-neutralizing antibody is used as the detection antibody, which recognizes a conformational epitope located on antigenic site III of the G protein (Biologicals (2003),Volume 31, pages 9 to 16). The results are expressed in international units since it is common practice to use a reference standard that has been calibrated against the international reference of the NIBSC. The BIAcore technology can also be implemented, which is considered an interesting alternative to the ELISA method, and which relies on the use of surface plasmon resonance to quantify the virus titer.

The stabilizing excipient according to the invention also acts by preserving the physical integrity of the viral particles. It particularly prevents the formation of aggregates and/or the degradation of the structure of the viral particles over time. This can be monitored using a device such as the Zetasizer Nano ZS (Malvern Instruments), which detects aggregates and determines the particle size distribution profile of the viral particles in the vaccine formulation.

The virus or viral preparation is in the form of whole viral particles that have been inactivated, usually by chemical treatment with formalin, formaldehyde, or β-propiolactone. Other inactivation methods, such as those described in WO 2005/093049, may also be implemented.

Virus preparations come from harvests that are generally in the form of supernatants from cell cultures infected with the virus. Classic media containing animal serum can be used to produce the cell stock and infect the cells. Advantageously, the media used for cell culture and infection do not contain serum proteins or even any animal-derived products. The proteins that may be present in these media are generally low molecular weight proteins (≤ 10 kD) or rather peptides at very low concentrations, which reduces the risk of allergic reactions accordingly.This is the case, for example, with media marketed under the names VP SFM (InVitrogen), Opti Pro™ serum-free (InVitrogen), Episerf (InVitrogen), Ex-Cell® MDCK (Sigma-Aldrich), Ex-Cell™ 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, the MDSS2 medium (Axcell biotechnology), the modified Iscove's DMEM medium (Hyclone), Ham's nutrient media (Ham-F10, Ham-F12), and Leibovitz L-15 medium (Hyclone). For example, rabies virus harvests are obtained from a Vero cell stock that has been produced and then infected using culture and viral infection media that are preferably free of any serum proteins.from any animal-derived protein and even from any animal-derived product, such as the VP SFM medium.

Advantageously, the preparation of whole inactivated rabies viruses contained in the vaccine composition according to the invention is highly purified. Usually, the virus is first purified and then inactivated, or alternatively, it is first inactivated and then purified. When all production and purification steps of the virus are carried out without using serum proteins, without using exogenous animal-derived proteins, or even without using any exogenous animal-derived products, advantageously the vaccine composition according to the invention is free from any serum proteins, thus minimizing allergic risks as much as possible, and also free from any exogenous animal-derived proteins or even any animal-derived products, thereby minimizing as much as possible the risk of disease transmission.By "protein or animal-derived product," we mean a protein or product whose manufacturing process includes at least one step in which a material from an animal or human is used. By "exogenous protein or product," we mean a protein or product that is introduced during one of the steps in the production and/or purification process of the virus. For example, proteins or products that may be present in the composition of culture media, or enzymes such as trypsin or benzonase used during the production and/or purification steps of the virus, are considered exogenous proteins or products.Proteins or exogenous products are of animal origin when their manufacturing process includes at least one step where a material from an animal or human is used. Proteins or exogenous products are of non-animal origin when they are produced by other means, for example using plant materials, through chemical synthesis, or by genetic recombination using yeasts, bacteria, or plants. On the contrary, proteins or products derived from cells from which viruses are produced to obtain the vaccine formulation according to the invention are considered endogenous proteins or products because they are produced (or released) at the same time as the viruses.

In the context of the present invention, it is possible to implement particularly efficient purification processes that lead to the production of very pure viral preparations. For example, the virus purification process comprising an anion exchange chromatography followed by a cation exchange chromatography and completed by a metal chelate affinity chromatography, as described in WO 97/06243, can be advantageously applied. To purify and inactivate the rabies virus from the supernatant of infected cell cultures, a preferred method consists of using the process described in the French patent application filed on April 14, 2009, under number 0952310, which includes a cation exchange chromatography step on a support comprising a polymethacrylate matrix grafted with sulfoisobutyl groups.A benzonase treatment step, an ultracentrifugation step on a sucrose gradient, and an inactivation step with beta-propiolactone. The preparations of whole and inactivated rabies viruses obtained are particularly pure, but are more difficult to store (stabilize) because there are very few residual protein impurities.

Thanks to the excipient composition, the vaccine composition according to the invention remains stable even in cases where the total protein concentration is ≤100 µg/ml, ≤80 µg/ml, ≤50 µg/ml, or even ≤20 µg/ml. Generally, the viral 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 present in the vaccine composition according to the invention. In an effective vaccine dose, this usually corresponds to a total protein amount of ≤100 µg, ≤50 µg, or even ≤20 µg.As an indication, the residual DNA quantity is itself ≤100 pg, and preferably <50 pg per effective vaccine dose. By "effective vaccine dose (or effective dose of a vaccine formulation)" is meant the amount of inactivated virus contained in a dose that is necessary to induce protective immunity in humans or animals after administration according to the recommended route of immunization and immunization protocol for primary vaccination or booster vaccination. In the case of rabies vaccination, the efficacy of the rabies vaccine is determined by means of the official test recognized by the WHO.The NIH test (described in the WHO monograph on rabies (WHO Technical Series Report 941 - January 2007)). An inactivated rabies vaccine dose is considered effective when it contains at least 2.5 IU according to this test. Administration of this dose by the intramuscular route in humans, according to standard vaccination or post-exposure prophylaxis protocols, induces the development of protective immunity against rabies.

"Total proteins" refer to all the proteins present in the vaccine formulation. They include viral proteins and residual proteins that were not removed during purification, such as cellular proteins, proteins from cell culture media and viral infection media, and proteins that may have been introduced during the purification process (for example, benzonase in the case of the rabies virus purification process mentioned above). The Bradford method is generally used to quantify total proteins. To determine the proportion of viral proteins among the total proteins, an electrophoresis on a polyacrylamide gel under denaturing and reducing conditions is performed on a sample of the vaccine formulation, followed by staining with Coomassie blue and densitometric analysis of the electrophoretic profile. In the case of a vaccine formulation containing inactivated whole rabies virus,The viral proteins represented by the envelope glycoprotein G, the nucleoprotein N, the phosphoprotein P, the matrix protein M, and the RNA-dependent RNA polymerase L are easily identifiable on a polyacrylamide gel electrophoresis. The amount of total proteins contained in an effective dose of the vaccine composition according to the invention is generally 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 difficult-to-store vaccine compositions.such as vaccine formulations containing inactivated whole rabies virus, in which an effective vaccine dose contains: a quantity of viral proteins representing at least 70% of the total proteins and/or less than 100 µg of total proteins, usually a total protein content ranging between 1 µg and 50 µg, preferably between 2 µg and 20 µg.

The excipient composition according to the invention is preferably free of any protein, any peptide, and even any oligopeptide, and generally does not contain any animal-derived product in order to minimize as much as possible the biological safety and/or allergic problems that may be associated with their use.

In the context of the present invention, the expression "the stabilizing excipient is free of any protein" should be understood as meaning a stabilizing excipient whose composition is free of any biological macromolecule comprising a chain of more than 50 amino acids linked together by peptide bonds. The expression "the stabilizing excipient is free of any protein and any peptide" should be understood as meaning a stabilizing excipient whose composition is free of any biological macromolecule comprising a chain of more than 20 amino acids linked together by peptide bonds. The expression "the stabilizing excipient is free of any protein,""any peptide and any oligopeptide" must be understood as referring to a stabilizing excipient whose composition does not contain any biological molecule consisting of amino acids linked together by one or more peptide bonds. For example, a dipeptide containing two amino acids linked by a single peptide bond is excluded from the composition of a stabilizing excipient that is free of any protein, peptide, and oligopeptide, but may be part of the composition of a "stabilizing excipient free of any protein and any peptide."

The mixture of amino acids that is part of the excipient composition comprises at least one essential amino acid among the essential amino acids represented by cystine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, and at least one non-essential amino acid among the non-essential amino acids represented by aspartic acid, glutamic acid, alanine, asparagine, glutamine, glycine, proline, and serine. Acidic amino acids can be in the form of acids or salts. The same applies to basic amino acids.It contains at least arginine or an arginine salt such as arginine hydrochloride and glutamic acid or a glutamic acid salt such as sodium glutamate. In summary, the amino acid mixture preferably includes from 1 to 12 essential amino acids, among which at least arginine or a salt thereof is present, and from 1 to 8 non-essential amino acids, among which at least glutamic acid or a salt thereof is present. The total concentration of amino acids in the vaccine formulation is generally 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 ranging from 0.5 to 2.5 mg, and particularly preferably from 0.8 to 1.8 mg. 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 higher 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 lower than 0.5.

The excipient according to the invention also contains a disaccharide, which is maltose. Disaccharides obtained from animal-derived raw materials such as milk are excluded. The polyol also included in the excipient composition is of non-animal origin. It is sorbitol. The excipient according to the invention contains maltose and sorbitol. In an effective dose of the vaccine composition, the amount of maltose and sorbitol is between 10 and 50 mg, preferably between 20 and 25 mg. The amount of maltose generally represents at least 70% of the total amount of maltose and sorbitol.

The chelating agent is also one of the excipient components according to the invention. It is EDTA (ethylenediaminetetraacetic acid) or a salt thereof (Na+, K+, ...). The concentration of EDTA or its 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 corresponds to an amount ranging from 0.01 to 0.1 mg, preferably from 0.01 to 0.05 mg, and particularly preferably from 0.01 to 0.04 mg of EDTA or its salt.

Urea or a urea derivative selected from allylurea, acetamide, methyl carbamate, and butyl carbamate is also part of the excipient composition. The vaccine composition contains urea at a concentration generally ranging from 1 g to 5 g per liter. In an effective dose of the vaccine composition, this corresponds to an amount ranging from 0.3 to 1.5 mg, and particularly preferably from 0.4 to 1.2 mg of urea.

The excipient according to the invention also includes a non-ionic surfactant that contributes to the stabilization of the vaccine formulation. Without being bound by theory, it helps maintain the biological activity of the virus and the physical integrity of the viral particles by preventing the formation of aggregates or the degradation of the virus structure. It may also prevent the virus from adhering to the walls of the packaging, particularly when the walls are made of glass or plastic. The suitable non-ionic surfactant for the purpose of the invention is produced from a non-animal origin raw material and is pharmaceutically compatible with parenteral administration of the product.Nonionic surfactants particularly include poloxamers, which are "block copolymers" of ethylene oxide and propylene oxide, with the chemical formula HO(C2H4O)a(C3H6O)b(C2H4O)aH, where 'a' represents the number of ethylene oxide units and 'b' represents the number of propylene oxide units. Poloxamers are commercially available, for example under the trade name Pluronic®; the specific poloxamer used is Pluronic® F68, also known as poloxamer 188. This is a solid poloxamer at room temperature, with a polyoxypropylene molecular weight of approximately 1750 Daltons, and the polyoxyethylene portion accounts for about 80% of the total molecular weight.The nonionic surfactant poloxamer 188 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 those of live viruses. At too high a concentration, the surfactant can dissociate or modify the structure of the viral particles, particularly that of enveloped viruses, which may affect the immunogenicity of the vaccine formulation. Pluronic® F68 is used as a surfactant at a total concentration in the vaccine formulation which is ≤1 g/l, generally between 0.005 g/l and 1 g/l, and particularly preferably between 0.0.1 g/L and 0.1 g/L. In this concentration range, poloxamer 188 does not exert an adjuvant effect on the immune system. At an effective dose of the vaccine formulation, this corresponds to an amount ranging from 0.001 mg to 0.5 mg, preferably from 0.003 mg to 0.3 mg. The vaccine composition may optionally contain a mixture of poloxamer 188 and polysorbate 20.

The buffer of the excipient according to the invention is selected such that the pH of the vaccine formulation is between 7.3 and 8.3. It is within this pH range that the physical thermostability of the inactivated virus particles is maximal, particularly 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 aggregation of rabies virus particles when the pH is between 7.3 and 8.3, whereas at a pH < 6.0, significant aggregation occurs at ambient temperature. The buffer solution is a phosphate buffer. Preferably, the excipient composition includes a phosphate buffer whose molarity is usually between 10 and 100 mM, or a mixture of a phosphate buffer and a Tris buffer.

Possibly, Tween 20 may be incorporated as an additional non-ionic surfactant into this vaccine formulation. This vaccine formulation is advantageously free of any serum protein and any animal-derived exogenous product, and generally contains a highly purified preparation of rabies virus. The concentration of poloxamer 188 in the vaccine formulation is less than 1 g/l and preferably between 0.01 g/l and 0.1 g/l. The phosphate buffer molarity is generally between 10 and 100 mM. Arginine and glutamic acid or their respective salts are the major components of the amino acid mixture, as they generally account for at least two-thirds of the total weight of essential and non-essential amino acids.The mixture of essential and non-essential amino acids is usually at a concentration ranging from 2 to 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 is usually between 0.02 and 0.2 g/l, and urea is usually at a concentration between 1 and 5 g/l. The total protein concentration in the vaccine formulation is usually between 5 and 50 µg/ml. In an effective dose of rabies vaccine, this corresponds to an amount of essential and non-essential amino acids ranging from 0,5 mg and 2.5 mg, an amount of maltose and sorbitol ranging from 10 to 50 mg, an amount of EDTA or EDTA salt ranging from 0.01 to 0.1 mg, an amount of urea ranging from 0.3 to 1.5 mg, an amount of poloxamer 188 ranging from 0.001 to 0.5 mg, and an amount of total proteins ranging from 2 to 20 µg.

The method for preparing a vaccine composition according to the invention, in addition to the steps of production, purification, and inactivation of the rabies virus preparation, includes a step where a stabilizing excipient is introduced into the purified and inactivated whole rabies virus preparation, and a step of filling the resulting composition into packaging devices. Generally, the virus preparation is diluted with the stabilizing excipient according to the invention before filling the vaccine composition into the packaging devices.

The vaccine composition can be presented in bulk form: in this case, the concentration of inactivated whole virus is higher than that in the vaccine composition once it has been aliquoted, but the excipient composition remains the same. The bulk is generally stored frozen at a temperature below -35°C. The step of aliquoting the vaccine composition then involves thawing the bulk and subsequently diluting it in the stabilizing excipient to obtain the desired viral concentration. The resulting final bulk product is then filled into the packaging devices. To ensure the sterility of the vaccine composition, a sterilizing filtration step, for example using a 0.2 µm filter membrane, can also be incorporated before the aliquoting step.

Bulk material is generally obtained by diafiltration of the purified and inactivated virus preparation obtained at the end of the purification process, in a buffer solution that usually corresponds to the excipient's buffer. For example, if the excipient's buffer is a 50 mM phosphate buffer, a 50 mM phosphate buffer is used as the diafiltration buffer. If it is desired to increase the virus concentration, the diafiltration step is preceded by an ultrafiltration step. An ultrafiltration membrane with a low cut-off value, generally between 5 kDa and 100 kDa, preferably between 8 kDa and 50 kDa, and particularly preferably around 10 kDa, is used in order to retain the virus as much as possible in the retentate and remove salts that may have a negative impact on the lyophilization process. The retentate containing the virus preparation is then mixed with the stabilizing excipient to obtain a bulk material whose composition corresponds to the vaccine composition according to the invention. It is also possible to use a diafiltration buffer having the same composition as the stabilizing excipient in order to obtain the bulk material in a single step.

The method for preparing a vaccine composition according to the invention containing whole and inactivated rabies virus comprises the following steps: a preparation of whole virus is produced by collecting the supernatant of a Vero cell culture infected with virus, using cell culture and viral infection media which preferably do not contain any serum protein or any animal-derived product, such as for example using VP SFM medium. The virus is then purified and inactivated using a process, notably as described in the French patent application filed on April 14, 2009, under number 0952310, which includes an ion exchange chromatography step on a support comprising preferably a polymethylacrylate-based matrix grafted with sulfobutyl groups.A treatment with recombinant benzonase, an ultracentrifugation step on a sucrose gradient, and an inactivation step using beta-propiolactone. The preparation of whole and inactivated rabies virus obtained is very pure, and is free from any serum protein and any exogenous animal-derived product; the residual DNA content is less than 100 pg/ml, and viral proteins represent at least 70% of the total proteins. A bulk is then prepared according to the procedures described in the previous paragraph, which is generally stored at a temperature below -35°C. The bulk is then diluted to the desired viral concentration in the excipient according to the invention, before being filled into the packaging devices.Usually, between 0.1 ml and 1 ml of the vaccine formulation is introduced into each packaging unit in such a way that the formulation, once distributed, contains at least one effective dose of the vaccine. Packaging units used for the distribution of the final bulk product are usually in the form of vials (glass or plastic) or syringes, but any other compatible packaging device can also be used for the practice of vaccination.

The particle size analysis of the vaccine compositions according to the invention, containing purified rabies virus using the Zetasizer Nano ZS apparatus (Malvern Instruments), which measures the Brownian motion of particles based on "quasi-elastic" light scattering (Dynamic Light Scattering), shows the existence of a single homogeneous population of viral particles ranging between 100 and 300 nm, with an average value around 180 nm, corresponding to the average size of the wild-type rabies virus. No aggregates or changes in the particle size distribution profile are detected during the storage of the vaccine compositions according to the invention.

Vaccine formulations 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, preferably for at least 24 months, in frozen form at a temperature ≤-35°C. Vaccine formulations can also be stored in lyophilized form using a conventional lyophilization process. The excipient composition according to the invention does not cause any significant loss of virus during the lyophilization step. A lyophilization method consists of freezing the formulation at a temperature below -40°C and then drying the product in two steps,Primary drying occurs at a temperature close to -15°C under 80 µbar, and secondary drying occurs at a temperature close to +40°C under 80 µbar. The lyophilized vaccine doses have a residual moisture content of ≤3% and are stable at a temperature of 2-8°C for at least 18 months (see Example 3). In conclusion, the rabies vaccine formulation as described in the invention proves to be as stable as a prior art commercial vaccine called VeroraB™, which contains in a single dose an amount of total proteins that is at least 100 times higher than that found in an effective dose of this vaccine, which is generally ≤50 µg.Moreover, the composition of the excipient according to the invention advantageously allows for the long-term preservation of vaccine formulations in liquid or frozen form, even though the preferred form of preservation is the lyophilized form.

The vaccine formulation is administered directly to the person being vaccinated when it is stored in liquid form or after thawing if it is stored frozen. When it is in a lyophilized form, the lyophilized powder is reconstituted extemporaneously in a diluent, which is usually a saline solution, such as a hypotonic sodium chloride solution. The diluent may further contain a surfactant at a very low concentration whose chemical structure is pharmaceutically compatible with parenteral use. This is Pluronic® F68, which is used at a concentration too low to exert an adjuvant effect. The surfactant concentration in the diluent is generally ≤ 0.1% (w/w).

When the vaccine is in a lyophilized form, it is usually provided as a kit with two packages: the first (usually in the form of a vial) contains the lyophilized vaccine formulation, and the second (usually in the form of a vial or syringe) contains the diluent. A "bypass" type syringe can also be used, in which the vaccine formulation is located in the lower part of the syringe, while the upper part contains the diluent.

The description finally reveals a stabilizing excipient for an inactivated whole-virus vaccine, particularly for an inactivated whole-rabies virus vaccine, which comprises: a. a buffer solution, b. a mixture of essential and non-essential amino acids, c. a disaccharide, d. a polyol, e. a chelating agent, f. urea or a urea derivative, and g. a nonionic surfactant.

Preferably, the excipient is free of any protein, more preferably free of any peptide and any protein, and even more preferably free of any protein, any peptide, and any oligopeptide. It is also very preferably free of any animal-derived product.

The present invention will be better understood in the light of the following examples, which serve to illustrate the invention without limiting its scope.

Figure 1 shows the overlay of particle size distribution profiles obtained by "dynamic light scattering (DLS)" during a cycle of three successive thawing and freezing steps of a rabies vaccine formulation in which the stabilizing excipient composition is F04 + 0.001% poloxamer 188. ● D0 (before freezing), ■ D1 (after first thawing); ▲ D2 (after second thawing) and + D3 (after third thawing).

Figure 2 shows the overlay of particle size distribution profiles obtained by "dynamic light scattering (DLS)" during a cycle of three successive thawing and refreezing steps of a rabies vaccine formulation in which the stabilizing excipient composition is F04 + 0.01% poloxamer 188. ● D0 (before freezing), ■ D1 (after first thawing); ▲ D2 (after second thawing) and + D3 (after third thawing).

Example 1: Influence of the excipient on the stability of a vaccine formulation containing purified and inactivated rabies virus in bulk form. 1.1 Bulk preparation

Rabies virus production was carried out on Vero cells using a serum-free viral infection medium. The Vero cell line was adapted to serum-free culture conditions as described in WO 01/40443. They were then transferred into a biogenerator containing Cytodex 1 microcarriers in VP SFM medium (Invitrogen). After a 3- to 4-day cultivation period at 37°C, maintaining a pH of approximately 7.2 ± 0.2, an oxygen saturation of 25% ± 10%, and subjecting the medium to low agitation,The cells were infected with rabies virus (Pitmann-Moore strain) at an infection multiplicity of 0.01 using a serum-free viral infection medium containing VP SFM medium (Invitrogen). The supernatants from the infected cell cultures were collected on days J7 (R1), J11 (R2), and J15 (R3). After each collection, fresh viral infection medium was reintroduced. The culture supernatants containing infectious virus were clarified by two successive filtrations: the first one using an 8 µm polypropylene pre-filter (Sartopure PP2, SARTORIUS) which removes the few microcarriers that were aspirated during collection,Detached Vero cells and large cellular debris; the second one using a PES filter, composed of a combination of two filters 0.8 µm and 0.45 µm (Sartopore 2, SARTORIUS) which removes aggregates.

About 0.12 µg/100 µl of a previously diluted solution of the monoclonal antibody 1112-1 anti-gpG (whose characteristics are described in the Journal of Clinical Investigation (1989), volume 84, pages 971 to 975) was distributed into the wells of an ELISA microplate. After an overnight incubation at 4°C, followed by several washes with a washing buffer (phosphate buffer supplemented with 0.05% Tween 20), 100 µl of a blocking buffer (phosphate buffer supplemented with 1% bovine serum albumin) were added to each well. After an incubation for one hour at 37°C,Following several washes, a dilution series of each sample to be tested was prepared in a dilution buffer (phosphate buffer supplemented with 0.05% Tween 20 and 0.1% serum albumin). At the same time, a dilution series of a reference standard was prepared in each microplate, which had been calibrated against the international reference material from NIBSC (for example, PISRAV). After another one-hour incubation at 37°C, followed by several washes, 100 µl of a solution of a mouse monoclonal antibody D1 against gpG (whose characteristics are described in Biologicals (2003)) were added to each well.Volume 31, pages 9 to 16) was biotinylated and used after being diluted 1/5000 in the dilution buffer. The plates were incubated for 1 hour at 37°C, then washed several times, and 100 µl of a streptavidin-peroxidase conjugate solution (Southern Biotechnology Associates) previously diluted 1/15000 in the dilution buffer was added to each well. After another 1-hour incubation at 37°C followed by several washes, 100 µl of a 0.05 M citrate buffer, pH 5, containing the development substrate (o-phenylenediamine) was added to each well. After an incubation period of 30 minutes at room temperature, away from light, the development reaction was stopped by adding 50 µl/well of a 2N H2SO4 solution.Spectrophotometric reading of the microplates was performed at two wavelengths (492 nm and 620 nm). The measured optical density is the difference between the two readings to account for plastic absorption. The calculation of relative activity was carried out using the parallel line method 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 glycoprotein G, expressed in IU/ml relative to the reference.

After also checking the conductivity and pH of the clarified liquid, approximately 50 UI of glycoprotein G (measured by ELISA) per ml of support was loaded onto the pre-equilibrated Fractogel® EMD SO3- (Merck) chromatography support, which had been equilibrated with Tris buffer 20 mM, NaCl 150 mM, pH = 7.5. The chromatography support was then washed with the equilibration buffer, and the rabies virus was eluted using Tris buffer 20 mM, NaCl 600 mM, pH = 7.5, with the viral peak being collected in an independent fraction. Subsequently, an ultrafiltration step was performed using a PES Medium Screen 100 kD membrane (PALL), followed by diafiltration in Tris buffer 20 mM,NaCl 150 mM, pH = 7.5. A solution of MgCl2 was added so that the concentration in the diafiltrate was 2 mM. The benzonase treatment was carried out by adding 15 U/ml of crude harvest to the reaction mixture and allowing the reaction mixture to stand at room temperature for 2 hours. The ultracentrifugation step was performed on 34-60% sucrose cushions using a 45Ti type rotor at 21,000 rpm for 2 hours at +5°C. The fractions containing the purified virus from the gradient were collected, pooled, and then diluted in 50 mM phosphate buffer with 150 mM NaCl,pH = 7.5, so that the final volume of the purified virus suspension was approximately 12.5 times smaller than the volume of the clarified harvest. The purified virus suspension was then inactivated by treatment with beta-propiolactone followed by inactivation of the beta-propiolactone by heating at about 37°C for 2 hours. The preparation of purified and inactivated virus was concentrated sixfold by ultrafiltration through a 10 kDa membrane (Omega medium screen PALL), followed by diafiltration in 50 mM phosphate buffer, 150 mM NaCl, pH 7.5. The resulting rabies virus preparation is in the form of a concentrated bulk, with a titer based on the determination of G glycoprotein concentration of approximately 80 UI/ml.Subsequently, 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 the tested excipients

F01: Phosphate buffer 50 mM, NaCl 150 mM, Maltose (5% w/w), pH=8.0 F02: Phosphate buffer 50 mM, 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: Buffer 489 PM, pH=8.0, whose composition is as follows:

Composants	Volume/Quantité
Acides Aminés essentiels	40 ml
Acides Aminés non essentiels	40 ml
Sorbitol (Roquette)	20 g
Arginine chlorhydrate (Jerafrance)	4 g
EDTA (Prolabo)	0,148 g
Glutamate de sodium (SAFC)	1,6 g
	8,428 g
	0,413 g
Maltose (Hayashibara)	50 g
Urée	4 g
Ajustement du pH à pH=8,0 avec de la soude 10 N
Eau déminéralisée QSP	1000ml

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, batch number 14773) in 5 liters of injection-grade water acidified with 100 ml of 12 N HCl (Prolabo).

The solution of non-essential amino acids was prepared by dissolving the contents of a vial containing 40.7 g of non-essential amino acids (Gibco, Reference 074-90681N, batch number 14775) in 5 liters of water for injection.

1.3 Tests conducted on the four bulk formulations

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 +37°C, by regularly measuring the glycoprotein G titer (expressed in IU/ml). The results obtained are shown in Tables I to III and are expressed in IU/ml. Tableau I : stabilité des 4 formulations du vrac à +37°C

Temps (semaines)	0	1	2
F01*	41,25*	30,21	17,51
F02	60,41	23,15	5,12
F03	60,29	23,16	5,2
F04	57,26	52,37	52,68

Tableau II : stabilité des 4 formulations du vrac à +5°C

Temps (semaines)	0	3	12	24
F01	41,25**	39,71	45,56	38,55
F02	60,41	53,29	42,27	38,92
F03	60,29	52,64	47,75	41,33
F04	57,26	62,19	61,06	61,32

Tableau III : stabilité des 4 formulations du vrac à -70°C

Temps (semaines)	0	3	12	24
F01	41,25	33,81	37,61	27,59
F02	60,41	53,52	53,06	53,62
F03	60,29	57,87	60,15	56,46
F04	57,26	57,48	59,92	56,56

Tableau III : stabilité des 4 formulations du vrac à -70°C

* : F01, F02, F03 et F04 font référence à la composition des excipients dans lesquels ont été formulés le vrac. **: en UI/ml

These results show that the excipient F04 is the one that best stabilizes the preparation of purified and inactivated rabies virus, especially when the storage temperature increases. This also shows that an excipient containing a sugar (maltose) and a non-ionic surfactant such as poloxamer 188 in a buffer solution at a pH of 8.0 is not sufficient to effectively stabilize a rabies virus preparation.

Example 2: Role of the surfactant in the stabilization of vaccine formulations containing purified rabies virus 2.1 Preparation of vaccine formulations

A suspension of purified rabies virus was obtained using the same procedure as in Example 1. After the ultracentrifugation step, the purified virus preparation was in a highly concentrated form. The total protein concentration was 7 mg/ml using the Bradford method. This virus preparation was divided into 3 polypropylene containers and diluted in 3 different excipients that were to be evaluated for their stabilizing role, so that the final total protein concentration in each of the tested excipients was reduced to 10 µg/ml (dilution factor 1/700). The composition of the three tested excipients differed only by their poloxamer 188 content, but all contained the 489 PM buffer at 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 other two excipients were supplemented respectively with 0.01 g/l of poloxamer 188 (excipient named F04 + 0.01% poloxamer) and with 0.1 g/l of poloxamer 188 (excipient named F04 + 0.1% poloxamer).

2.2 Stability studies and results

The stability of the three vaccine formulations was evaluated by placing them under unfavorable storage conditions, that is, by subjecting them to a series of freeze-thaw cycles according to the following protocol: The three vaccine formulations were frozen at -70°C on the same day (Day J0). Each of the three formulations was then subjected to three thawing steps at +5°C followed by refreezing at -70°C within 7 to 14 hours after thawing on days 1, 2, and 3 (respectively Day J1, J2, and J3). The stability of the vaccine formulations after each thawing step was assessed by measuring the total viral protein concentration. This measurement was performed using BIAcore technology, which is based on the use of surface plasmon resonance. Additionally, the particle size distribution profile was analyzed during successive thawing steps using the technique of quasi-elastic light scattering (Dynamic Light Scattering or DLS technology).

To implement the BIAcore technology, we first covalently immobilized the mouse monoclonal antibody D1 against gpG onto a sensor chip using the coupling kit provided by the manufacturer (Amine Coupling Kit, Reference BR-1000-50). Subsequently, 30 µl of the sample to be analyzed was automatically injected into the instrument over a period of 3 minutes. The interaction of the rabies virus present in the sample with the monoclonal antibody immobilized on the sensor chip caused a change in the refractive index at the surface of the sensor chip, resulting in a change in the recorded signal (RU).The results obtained were recorded in arbitrary units (ΔRU) and compared with the results obtained using a calibration range of a reference containing a known quantity of purified rabies virus. The measured ΔRU for each sample was converted into total viral protein concentration based on the calibration curve obtained with the reference. For each tested sample, measurements were performed four times (in quadruplicate). After each measurement, the sensor chip was cleaned by performing two successive injections of 10 µl of Glycine buffer (10 mM, pH=1.5). The results obtained for each tested vaccine formulation are presented in Table IV.The values are expressed in µg/ml of viral proteins. Table IV : Etude de la stabilité des 3 compositions vaccinales au cours d'un cycle de 3 décongélations et recongélations successives

Excipient stabilisant	D0	D1	D2	D3	Perte en titre viral
F04	11* (0.5)**	3.4 (0.4)	2.1 (0.3)	1.4 (0.3)	88%
F04 +0.001% poloxamère 188	14.8 (0.3)	11.2 (0.5)	10.8 (0.5)	9.9 (0.6)	33%
F04 + 0.01% Poloxamère 188	18.9 (0.0)	15.7 (0.3)	15.2 (0.4)	14.7 (0.4)	23%

Table IV : Etude de la stabilité des 3 compositions vaccinales au cours d'un cycle de 3 décongélations et recongélations successives

* : titre moyen en protéines virales obtenu sur la base de 4 mesures réalisées sur l'échantillon testé en µg/ml ** : écart type calculé sur la base des 4 mesures réalisées sur l'échantillon testé est indiqué entre parenthèse.

These results clearly show that the excipient F04, in the absence of poloxamer, is unable to effectively preserve the rabies virus preparation since the viral titer loss is 88% between the titer at D0 and the titer measured after the third thawing (D3). On the contrary, as soon as the excipient F04 is slightly supplemented with poloxamer 188, the viral titer loss is significantly reduced (it is 33% when F04 is supplemented with 0.001 g/l of poloxamer 188 and 23% when F04 is supplemented with 0.01 g/l of poloxamer 188). These results show that the excipient must contain both the components present in the 489 PM buffer and poloxamer 188 in order to effectively preserve a rabies virus-containing vaccine formulation.

The particle size distribution profile of each of the three vaccine formulations was also analyzed after each thawing by DLS using the Zetasizer Nano ZS instrument (Malvern Instruments). A 450 µl sample was introduced into a disposable polystyrene cuvette and then exposed to a monochromatic laser beam (HeNe laser, λ = 632.8 nm). The signal recorded by the instrument corresponded to fluctuations in scattered light resulting from the Brownian motion of the particles. The recorded data were processed by software. The results were finally presented as a particle size distribution curve for each tested sample. The two figures show an overlay of the four particle size distribution curves obtained respectively with a rabies virus formulation stabilized with excipient F04 + 0.001% poloxamer 188 on days J0 (before freezing), J1, J2, and J3 (Figure 1), and with a rabies virus formulation stabilized with excipient F04 + 0.01% poloxamer 188 (Figure 2).

The two figures clearly show that: 1) the particle size distribution curves are unimodal and have a Gaussian profile, with the median located around 180 nm. This means that the viral particle population in the stabilized vaccine formulation with excipient F04 + 0.001% poloxamer 188 or with excipient F04 + 0.01% poloxamer 188 is homogeneous, does not contain aggregates, and includes a population of intact rabies viruses similar to that of wild-type rabies viruses; and 2) the particle size distribution curves are not modified when the rabies virus vaccine formulations are subjected to multiple successive freeze-thaw cycles.

The presence of a non-ionic surfactant such as poloxamer 188 in the excipient formulation also preserves the physical integrity of the rabies virus population even when the vaccine composition is placed under poor storage conditions.

2.3 Stability studies on freeze-dried vaccine formulations 2.3.1: Preparation of freeze-dried vaccine formulations

A bulk was first prepared from a preparation of inactivated whole purified viruses obtained by using the process described in Example 1. After the viral inactivation step, the preparation of purified and inactivated rabies viruses was dialyzed through a 10 kDa membrane (Omega medium screen PALL) using an excipient called 488 TM, which was prepared from a 488 medium having the following composition (see table below):

Composants	Volume/Quantité
Mélange d'Acides Aminés essentiels et d'acides aminés non essentiels	200 ml
Sorbitol (Roquette)	50 g
Arginine chlorhydrate (Jerafrance)	10 g
EDTA (Prolabo)	0,37 g
Glutamate de sodium (SAFC)	4 g
Urée	10 g
	2,42g
Eau déminéralisée QSP	1000ml
Acide chlorhydrique 6N (Sanofi) QSP	pH=8,0

The mixture of essential and non-essential amino acids was prepared by mixing 2.5 liters of the essential amino acid solution prepared according to Example 1 with 2.5 liters of the non-essential amino acid solution prepared according to Example 1, and adjusting the pH to approximately 7.2 using a 30% sodium hydroxide solution.

The composition of excipient 488 TM is as follows:

Composants	Volume/Quantité
Milieu 488	400 ml
Maltose (Hayashibara)	50 g
	1.45g
Eau déminéralisée QSP	1000ml
Acide chlorhydrique 6N (sanofi) QSP	pH=8,0

The obtained vaccine preparation was in the form of a bulk in excipient 488 TM, with a viral titer around 12 UI/ml based on the determination by ELISA of the glycoprotein G concentration. The bulk was then stored frozen at -70°C until the time of preparation of the lyophilized vaccine formulations.

Just before the lyophilization step, part of the bulk was thawed and diluted to bring the final titer of glycoprotein G to approximately 8 UI/ml using: either excipient F'04, whose composition contains 1 volume of excipient 488 TM per 3 volumes of 50 mM phosphate buffer, with the maltose concentration adjusted to 50 g/l, pH = 8.0; or excipient F'05, which is identical to excipient F'04 but with the addition of poloxamer 188 at a final concentration of 0.01 g/l.

The two vaccine formulations were then distributed into vials for lyophilization at a rate of 0.4 ml/vial. A conventional freeze-drying cycle was subsequently carried out, consisting of a freezing phase at a temperature lower than -40°C, followed by two successive drying phases. The first drying phase was performed at approximately -15°C under a pressure of about 80 µbar, and the second drying phase at approximately +40°C under a pressure of about 80 µbar.

Each lyophilized vial contained a dose of the vaccine formulation being tested. The obtained lyophilized vaccine formulations differed only in the presence or absence of the surfactant, depending on whether the bulk was diluted in excipient F'04 or F'05.

The composition of the excipient contained in a dose of the lyophilized vaccine formulation prepared from excipient F'05 is indicated below.

Composant	Quantité en mg/dose
Mélange d'acides aminés essentiels et non essentiels	0,12
Poloxamère 188	0,004
Maltose	20,00
Chlorhydrate d'Arginine	0,41
Sorbitol	2,05
EDTA disodique	0,01
Urée	0,41
Glutamate de sodium	0,16
Na2HPO4, 2H20	2,53
NaH2PO4, 2H20	0,12
Tris	0,24
HCl	QSP
NaOH	QSP

2.3.2: Efficiency of freeze-dried vaccine formulations

The two freeze-dried vaccine formulations prepared using two different stabilizing excipients were subsequently tested for their effectiveness using the official NIH test. The "effectiveness" of the two freeze-dried vaccine formulations was analyzed immediately after freeze-drying (Day 0), after one week of storage at 45°C, and also after one month of storage at 37°C. Only the officially recognized activity test, the NIH test, was used. This test allows determining the number of IU (International Units) contained in each dose of the tested vaccine formulations. If the number of IU is ≥2.5 IU, the tested vaccine dose is considered effective.The NIH test was performed in "duplicate" for each tested condition by randomly selecting two lyophilized vials from the batch of prepared and stored vials under the same conditions, and performing the NIH test on these samples. The procedure used corresponds to the European Pharmacopoeia monograph 0216. This is a challenge test conducted in mice, based on the comparison of the protection provided by the tested vaccine dose with that provided by a known quantity of a reference rabies vaccine. The reference rabies vaccine is calibrated in international units (IU).The International Unit (IU) is the activity contained in a defined amount of an international standard. The equivalence in IUs of the international standard was established by the WHO. After verifying that the control parameters of the potency test were properly met, the number of IUs contained in the tested vaccine dose was determined from the protective dose 50% values obtained for each tested sample and for the reference preparation (calibrated in IUs).

The results obtained are shown in Table V below. Tableau V : efficacité des compositions vaccinales lyophilisées en fonction de la composition de l'excipient de lyophilisation utilisé et des conditions de conservation.

Durée (en jours) et température de conservation de la composition lyophilisée	J0/ + 4°C	J7/ +45°C	J30/ +37°C
F'04	0,8* (0,4 ; 1,8)**	0,9 (0,4 ; 2,1)	NT
F'05	6,1 (1,6 ; 41,3)	2,8 (0,9 ; 7,9)	3,5 (1,4 ; 8,7)

Tableau V : efficacité des compositions vaccinales lyophilisées en fonction de la composition de l'excipient de lyophilisation utilisé et des conditions de conservation.

*: valeur moyenne exprimée en UI obtenues (tous les groupes de souris qui ont été éprouvés contiennent 16 souris) ** : représente les valeurs extrêmes obtenues exprimées en UI NT : Non testé

These results are consistent with the results discussed in the previous paragraph (2.2) and show that poloxamer 188 must be included in the composition of the excipient used to preserve the lyophilized vaccine formulations. Indeed, only the lyophilized vaccine formulations containing excipient F'05 (which contains poloxamer 188) contain an effective dose of vaccine, since the average value in IU is higher than 2.5 IU. These vaccine formulations remain stable because their efficacy is not reduced over time under the storage conditions that have been tested.

To summarize, the results presented in Example 2 show that vaccine formulations containing rabies virus can be well preserved using a stabilizing excipient whose composition includes 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 non-ionic surfactant such as Poloxamer 188. Moreover, and interestingly, the "stabilizing effect" of Poloxamer 188 is exerted at a very low concentration that is too low for Poloxamer 188 to activate the immune system or act as an immune response adjuvant.

Example 3: Study of the stability of vaccine formulations containing purified and inactivated rabies virus

Examples 1 and 2 have demonstrated the importance of combining the components of an excipient that includes a mixture of essential and non-essential amino acids, maltose, sorbitol, EDTA, and urea in a phosphate and/or Tris buffer with Poloxamer 188. The aim of the studies presented here was to confirm that this combination effectively stabilizes rabies virus vaccine formulations in different forms (liquid, frozen, or lyophilized) over a wide range of storage temperatures (+37°C, +25°C, +5°C, -70°C) and over a long period of time. These studies were conducted on several rabies virus vaccine formulations that were stored as frozen bulk at a temperature ≤-35°C, as a liquid bulk final product at +5°C, or as individual lyophilized doses stored at +5°C, +25°C, or +37°C.

3.1 Stability studies of batches of frozen bulk rabies vaccines.

The rabies virus used for the preparation of bulk batches was prepared according to the procedure described in Example 1. After the viral inactivation step, the purified and inactivated virus preparation was dialyzed using a 10 kDa membrane, with a 50 mM phosphate buffer as the dialysis buffer. Subsequently, one volume of retentate was mixed with one volume of stabilizing excipient, whose composition is that of Buffer 489 PM described in Example 1 (except that the concentration of each component is twice as high), to which Poloxamer 188 was added at a concentration of 0.02 g/L, with a pH of approximately 8.0. A bulk product was obtained with a total protein concentration of 50 µg/mL, among which more than 70% are rabies virus proteins, and containing less than 100 pg/mL of residual DNA. The stability of the bulk was studied over time after storage at -35°C and -70°C, by measuring the glycoprotein G titer every three months. The results obtained are presented in Table VI below. Tableau VI : Stabilité d'un lot de vrac conservé à -35°C ou à -70°C

Température de conservation	T0	T0 + 3 mois	T0 + 6 mois	T0 + 9 mois	T0 + 12 mois
- 35°C	20,4*	20,84	24,4	24,3	22,4
- 70°C	21,3	NT	22,4	22,8	21,8

Tableau VI : Stabilité d'un lot de vrac conservé à -35°C ou à -70°C

* : titre exprimé en UI/ml NT : non testé

After 12 months of storage at -35°C or -70°C, no degradation of the glycoprotein G, which is the major antigen of the rabies virus, was observed. This confirms the good stability of the vaccine formulation in the frozen bulk form. Moreover, analysis of the viral particle size distribution by DLS after 12 months of storage at -35°C or -70°C showed that the profiles retained a Gaussian shape centered around a median value of 180 nm (similar profiles to those shown in 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 was observed over time.

3.2: Stability studies of batches of rabies vaccines in the form of bulk liquid final product (PFV).

Three batches of liquid PFV vaccines were prepared from three bulk lots after dilution in 489 PM buffer (composition described in Example 1), to which Poloxamer 188 was 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 as determined by densitometric analysis of polyacrylamide gel electrophoresis. The stability of the PFV batches stored at +5°C and +25°C was studied by regularly measuring the glycoprotein G content by ELISA over a period of 3 months for the batches stored at +5°C and over a period of 30 days for the batches stored at +25°C. The results expressed in UI/ml are summarized in the following tables VII and VIII: Table VII: Stabilité des 3 lots de PFV conservés à +5°C

N° de lot	T0	T0+ 1 mois	T0 + 2 mois	T0 + 3 mois
N°1	10.5	12.0	8.6	10.8
N°2	11.1	10.7	9.3	11.9
N°3	13.9	11.1	11.6	11.2

Table VIII: Stabilité des 3 lots de PFV conservés à +25°C

N° de lot	T0	T0 +15 jours	T0 +30 jours
N°1	12.0	10.9	10.9
N°2	10.7	11.4	10.1
N°3	11.1	12.0	11.5

These results show that there is no degradation of the glycoprotein G during the storage of liquid PFV batches at +5°C and +25°C over the time periods studied. This confirms that rabies vaccines can be stored in liquid form at cold temperature (+5°C) for at least 3 months and at room temperature (+25°C) for at least 30 days without observing any viral degradation.

3.3: Stability Studies of Lyophilized Rabies Vaccine Lots

Two batches of lyophilized rabies vaccines (LL1 and LL2) were prepared from the PFV batches of Example 3.2, which were distributed into glass vials at a dose of 0.3 ml/vial before undergoing a lyophilization cycle. Freezing was carried out at a temperature ≤-40°C, primary drying was performed at approximately -15°C under a pressure of about 80 µbar until complete sublimation of ice, and secondary drying was conducted at approximately +40°C under a pressure of about 80 µbar until the residual moisture content in the lyophilizates was ≤3%. The loss of viral titer during the lyophilization step was less than 5%.

The composition of the excipient contained in a lyophilized vaccine dose is as follows:

Composant	Quantité en mg/dose
Mélange d'acides aminés essentiels et non essentiels	0,37
Poloxamère 188	0,003
Maltose	15,00
Chlorhydrate d'Arginine	1,20
Sorbitol	6,00
EDTA disodique	0,04
Urée	1,2
Glutamate de sodium	0,15
Na2HPO4, 2H20	2,53
NaH2PO4, 2H20	0,12
HCl	QSP
NaOH	QSP

The obtained freeze-dried single doses had a homogeneous white appearance. The pH of the rabies virus suspension after reconstitution of the lyophilized material with 0.5 ml of a 0.4% sodium chloride solution was in the range of 7.8 ± 0.5. The stability of the two lyophilized batches was studied over time at three storage temperatures: 35–39°C, 23–27°C, and 2–8°C, by checking the appearance of the lyophilizates before and after reconstitution, measuring residual moisture, controlling pH, viral titer, and testing the efficacy of the lyophilized doses in the NIH test. The first dilution of the dilution series performed to test the lyophilized doses in the NIH test after reconstitution in sodium chloride was carried out in a 0.9% sodium chloride solution containing 2 g/L of human albumin. Viral titers were measured based on the glycoprotein G rate by ELISA and expressed in IU/ml.

The test results regarding the appearance of the lyophilized products, residual moisture content, and pH were all in compliance with the specifications (i.e., a homogeneous white appearance for the lyophilizate, a clear, colorless solution after reconstitution in hypotonic physiological serum, and a residual moisture content <3%), regardless of the temperature and storage duration tested. The pH remained stable within a range of 7.8 ± 0.5 throughout the entire stability study period. The results concerning viral titers and efficacy tests (NIH) performed on individual doses are summarized in tables IX to XI below. Tableau IX : stabilité des deux lots de vaccins antirabiques conservés sous forme lyophilisée à +5°C

lot	Test	T0	1 M	3 M	6 M	9 M	12 M	18 M
LL1	Titre viral	7.1*	7.7	7.8	7.9	7.3	8.8	7.8
	NIH		ND	ND		ND
LL2	Titre viral	6.1	6.6	6.7	6.5	6.9	6.4	ND
	NIH		ND	ND		ND		ND

Tableau X : stabilité des deux lots de vaccins antirabiques conservés sous forme lyophilisée à +25°C

Lot	Test	T0	1 M	3 M	6 M
LL1	Titre viral	7.7	7.1	7.8	6.9
	NIH	ND	ND	ND
LL2	Titre viral	6.6	6.1	6.0	6.2
	NIH	ND		ND

Tableau XI : stabilité des deux lots de vaccins antirabiques conservés sous forme lyophilisée à +37°C

Lot	Test	T0	1 M	3 M	6 M
LL1	Titre viral	7.7	7.5	10.1	10.3
	NIH	ND		ND
LL2	Titre viral	6.6	7.2	7.5	7.7
	NIH	ND		ND

Tableau XI : stabilité des deux lots de vaccins antirabiques conservés sous forme lyophilisée à +37°C

M : signifie mois ND : signifie non déterminé * : valeur exprimée en UI/ml ** : valeur moyenne exprimée en nombre d'UI complétée par les valeurs extrêmes observées indiquées entre parenthèse.

All of these results confirm that the freeze-dried rabies vaccines remain very stable and do not lose efficacy over time under the tested temperature conditions. The titers of glycoprotein G and the NIH test values (clearly above 2.5 IU) remain stable over time.

In conclusion, all the stability tests performed on batches of rabies vaccines stored in frozen, liquid, or lyophilized forms show that these batches remain stable over time under the tested temperature conditions. This confirms that an excipient whose composition includes a buffer, a mixture of essential and non-essential amino acids, maltose, sorbitol, EDTA, urea, and poloxamer 188 effectively stabilizes a rabies vaccine formulation regardless of its presentation (frozen, liquid, or lyophilized) and across a wide range of storage temperatures.

Claims (15)

1. A vaccine composition comprising:

   a) a preparation of inactivated whole rabies virus, and

   b) a stabilizing excipient which comprises:

   i. a phosphate buffer,

   ii. a mixture of essential and nonessential amino acids, comprising at least arginine or an arginine salt and glutamic acid or a glutamic acid salt,

   iii. maltose,

   iv. sorbitol,

   v. a chelating agent, chosen from EDTA or an EDTA salt,

   vi. urea or a urea derivative chosen from allylurea, acetamide, methyl carbamate and butyl carbamate, and

   vii. a nonionic surfactant,

   wherein said nonionic surfactant is poloxamer 188, wherein the pH of the vaccine composition is between 7.3 and 8.3, and wherein, in one effective dose of the vaccine composition, the amount of essential and nonessential amino acids is between 0.5 and 2.5 mg, the amount of maltose and of sorbitol is between 10 and 50 mg, the amount of EDTA or EDTA salt is between 0.01 and 0.1 mg, the amount of urea or of urea derivative chosen from allylurea, acetamide, methyl carbamate and butyl carbamate is between 0.3 and 1.5 mg and the amount of poloxamer 188 is between 0.001 and 0.5 mg.
2. A composition as claimed in claim 1, characterized in that it is also free of any serum protein.
3. A composition as claimed in one of claims 1 or 2, characterized in that it is also free of any exogenous protein of animal origin, and preferably free of any exogenous product of animal origin.
4. A composition as claimed in one of claims 1 to 3, characterized in that the virus proteins represent at least 70% of the total proteins present in the vaccine composition.
5. A composition as claimed in one of claims 1 to 4, characterized in that the total protein concentration is ≤ 100 µg/ml and preferably ≤ 80 µg/ml.
6. A composition as claimed in one of claims 1 to 5, characterized in that the amount of total proteins that is contained in one effective dose of the vaccine composition is ≤ 100 µg.
7. A composition as claimed in one of claims 1 to 6, characterized in that the amount of total proteins that is contained in one effective dose of the vaccine composition is between 1 and 50 µg.
8. A composition as claimed in one of claims 1 to 7, characterized in that the stabilizing excipient is free of any protein, or of any protein and of any peptide, or preferably free of any protein, of any peptide and of any oligopeptide.
9. A composition as claimed in one of claims 1 to 8, characterized in that the chelating agent is EDTA.
10. A composition as claimed in one of claims 1 to 9, characterized in that the amount of poloxamer 188 contained in one effective dose of the vaccine composition is between 0.003 and 0.3 mg.
11. A composition as claimed in one of claims 1 to 10, characterized in that the phosphate buffer has a molarity between 10 and 100 mM.
12. A process for preparing a vaccine composition containing a preparation of purified and inactivated whole rabies virus, comprising:

    a. producing a whole rabies virus preparation by harvesting the supernatant of a rabies virus-infected cell culture,

    b. purifying and inactivating the whole rabies virus preparation or, alternatively, inactivating then purifying the whole rabies virus preparation, and

    c. diluting the preparation of purified and inactivated whole rabies virus in a stabilizing excipient, the composition comprising:

    i. a phosphate buffer,

    ii. a mixture of essential and nonessential amino acids, containing at least arginine or an arginine salt and glutamic acid or a glutamic acid salt,

    iii. maltose,

    iv. sorbitol,

    v. EDTA or an EDTA salt,

    vi. urea or a urea derivative chosen from allylurea, acetamide, methyl carbamate and butyl carbamate, and

    vii. a nonionic surfactant,

    wherein said nonionic surfactant is poloxamer 188, wherein the pH of the vaccine composition is between 7.3 and 8.3, and wherein, in one effective dose of the vaccine composition, the amount of essential and nonessential amino acids is between 0.5 and 2.5 mg, the amount of maltose and of sorbitol is between 10 and 50 mg, the amount of EDTA or EDTA salt is between 0.01 and 0.1 mg, the amount of urea or of urea derivative chosen from allylurea, acetamide, methyl carbamate and butyl carbamate is between 0.3 and 1.5 mg and the amount of poloxamer 188 is between 0.001 and 0.5 mg.
13. The process as claimed in claim 12, characterized in that it is carried out without introducing any exogenous product of animal origin.
14. The process as claimed in one of claims 12 to 13, wherein, after having diluted the preparation of purified and inactivated whole rabies virus the resulting vaccine composition is divided up into packaging devices, and, optionally, the vaccine composition is freeze-dried.
15. A vaccine composition containing a preparation of purified and inactivated whole rabies virus in a freeze-dried form obtained according to the process of claim 14.