NEW FORMULATION

专利摘要:
The present invention relates to the formulation of adenoviral vectors in compositions containing sorbitol in combination with an additional amorphous sugar, its formulation and a process for obtaining a dried composition.
公开号:BE1025187B1
申请号:E2018/5040
申请日:2018-01-25
公开日:2018-12-03
发明作者:Erwan Bourles；Olivier Despas；Delphine Guillaume；Frédéric Mathot；Mathieu Vasselle
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

NEW FORMULATION
The present invention relates to the formulation of adenoviral vectors in lyophilized compositions, their formulations as well as methods of obtaining the lyophilized composition.
BACKGROUND
Adenoviral vectors represent a prophylactic or therapeutic protein delivery platform, where the nucleic acid sequence encoding the prophylactic or therapeutic protein is incorporated into the adenoviral genome, which is caused to be expressed when the adenoviral particle is administered to the subject being treated. The development of stabilizing formulations for adenoviral vectors which allow storage at acceptable storage temperatures with considerable shelf life has been a challenge in the art.
SUMMARY OF THE INVENTION
Stabilizing formulations have been reported for human adenoviral vectors as described by R. K Evans et al. (‘Development of stable Liquid Formulations for Adenovirus-Based Vaccines' Journal of Pharmaceutical Sciences (2004) Vol. 93, No. 10, 2458-2475). However, there remains a need in the art for formulations preserving the stability of adenoviral vectors.
The inventors have surprisingly found that the use of sorbitol in the formulation of simian adenoviral vectors appreciably improves the stability throughout freeze-drying, in particular in combination with the amorphous sugar trehalose as an additional cryoprotective. The invention therefore provides an aqueous mixture for lyophilization and a lyophilized composition obtained from said aqueous mixture by lyophilization (hereinafter referred to as the “desiccated composition”) comprising sorbitol in combination with an additional amorphous sugar acting as a cryoprotective, such than trehalose.
In addition, it has been found that having a low salt content has additional favorable effects on the stability of the particles of simian adenoviral vector, in particular on the stability during cryodessiccation and during a reconstitution of the lyophilized cake. Thus, the invention provides adenoviral compositions containing sorbitol and an additional amorphous sugar also comprising a small amount of NaCl. The invention also provides a method of using the lyophilized composition, where the composition is reconstituted with an aqueous liquid low in salt, for example water for injection or an aqueous solution of a nonionic isotonic agent. The invention further provides a method for lyophilizing the adenoviral vector compositions described.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 - illustration of the freeze-drying cycle as used in Example 1.
Figure 2 - glass transition temperatures (Tg) as determined for the composition tested in Example 1: (1) composition comprising 23% trehalose, (2) composition comprising 23% sucrose, ( 3) composition comprising 23% trehalose + 2% sorbitol, (4) composition comprising 23% sucrose + 2% sorbitol.
Figure 3 - infectivity of the adenoparticles contained in the compositions as obtained in Example 1: (1) trehalose at 23% (designated by (+), (2) sucrose at 23% (designated by X), (3) 23% trehalose + 2% sorbitol (designated by (Y), (4) 23% sucrose + 2% sorbitol (designated by (Z) and (5) fresh purified raw control (designated by an erected triangle) and (6) degraded purified raw witness (designated by an inverted triangle).
Figure 4 - illustration of the freeze-drying cycle as used in Example 2.
Figure 5 - Tg as determined in Example 2.
Figure 6 - moisture content as determined in Example 2.
Figure 7 - osmolality as determined in Example 2.
Figure 8 - PicoGreen data as determined in Example 2.
Figure 9 - infectivity as determined in Example 2.
Figure 10 - Recovery by ultra-performance liquid chromatography (UPLC for "UltraPerformance Liquid Chromatography") as determined in Example 2.
Figure 11 - illustration of the statistical analysis of data in relation to different parameters as determined in Example 2 to identify optimized combinations (experimental design (PE) diagram 1).
Figure 12 - illustration of the statistical analysis of data in relation to different parameters as determined in Example 2 to identify optimized combinations in which higher trehalose concentrations are authorized (PE diagram 2).
Figure 13 - freeze drying cycle 1 as used in Example 3.
Figure 14 - freeze drying cycle 2 as used in Example 3.
Figure 15 - PicoGreen data as determined in Example 3: (x) sample data points obtained using a freeze-drying condition with secondary drying at +10 ° C (lyo 2 cycle), ( +) sample data points obtained using a freeze-drying condition with secondary drying at +25 ° C (lyo cycle (1)), (erect triangle) - control adenoviral load, (inverted triangle) - load degraded adenoviral negative control.
Figure 16 - infectivity data as determined in Example 3: (x) sample data points obtained using a freeze-drying condition with secondary drying at +10 ° C, (+) sample data obtained using a freeze-drying condition with secondary drying at +25 ° C, triangle (upright) - control adenoviral load, (inverted triangle) - negative control degraded adenoviral load.
Figure 17 - UPLC data as determined in Example 3: (x) sample data points obtained using a freeze-drying condition with secondary drying at +10 ° C, (+) data points d 'samples obtained using a freeze-drying condition with secondary drying at +25 ° C, (upright triangle) - control adenoviral load, (inverted triangle) - degraded adenoviral load negative control.
Figure 18 - stability of simian adenovirus as determined by estimates of losses due to cryodessiccation; comparison of 18% trehalose + 3.5% sorbitol with 23% trehalose at secondary drying temperatures of 15 ° C and 25 ° C.
Figure 19 - dispersion diagram showing the stability of 18% trehalose + 3.5% sorbitol and 23% trehalose at drying temperatures of 15 ° C and 25 ° C for 200 days.
Figure 20 - appearance of freeze-dried adenovirus formulated with 18% trehalose + 3.5% sorbitol (left boards) or 23% trehalose (right boards) at secondary drying temperatures of 15 ° C or 25 ° C after simulating two hours of road transport and two hours of air transport. Vials were either silicone (S +) or non-silicone (S-). Silicone bottles were loaded with an average of 50% and non-silicone bottles were loaded with an average of either 50% or 100%. (O) = intact; (+) = cracked; (X) = fragmented.
Figure 21 - consistency of lyophilized adenovirus formulated with 18% trehalose + 3.5% sorbitol (left boards) or 23% trehalose (right boards) at secondary drying temperatures of 15 ° C or 25 ° C after simulating two hours of road transport and two hours of air transport. Vials were either silicone (S +) or non-silicone (S-). Silicone bottles were loaded with an average of 50% and non-silicone bottles were loaded with an average of either 50% or 100%. (O) = not powdery; (+) = slightly powdery; (X) = powdery.
DETAILED DESCRIPTION OF THE INVENTION
Contrary to reports in the art on the formulation of adenoviral vectors, the inventors have found that the stabilizing formulations developed for, for example, human adenoviral vectors, could not be successfully applied to all adenoviral vectors, for example. example of simian adenoviral vectors. The present invention now describes adenoviral vector compositions in which the structural integrity and functionality of the adenoviral particles are better protected or maintained.
The inventors have found that the addition of sorbitol in combination with an additional amorphous sugar when formulating adenoviral vectors, and in particular simian adenoviral vectors, for cryodessication, increases the stability of this adenoviral vector during cryodessiccation and / or during further storage.
Both sorbitol and additional amorphous sugar are considered to be cryoprotectants. The term "cryoprotective" refers to a class of excipients which prevents freezing damage to what is frozen, in this case, the adenoviral vector.
An amorphous sugar suitable for use according to the present invention in combination with sorbitol can be chosen from sucrose, trehalose, mannose, mannitol, raffinose, lactitol, lactobionic acid, glucose, maltulose, l iso-maltulose, lactulose, maltose, lactose, isomaltose, maltitol, palatinite, stachyose, melezitose, dextran, or one of their combinations. In one embodiment, the amorphous sugar is chosen from sucrose, trehalose, lactose, raffinose, dextran and their combinations.
In a specific embodiment, the additional amorphous sugar in combination with sorbitol is trehalose or sucrose, or trehalose in combination with a second amorphous sugar as chosen from sucrose, lactose, raffinose, dextran and mannitol. Alternatively, the amorphous sugar is trehalose, sucrose, or a combination of sucrose and trehalose. In another embodiment, the amorphous sugar is trehalose or trehalose in combination with sucrose. In yet another embodiment, the amorphous sugar is trehalose.
The sorbitol and the selected amorphous sugar, for example trehalose, may be present in a defined ratio. In one embodiment, the sorbitol to amorphous sugar ratio is 4/10 or less, 4/12 or less, 4/13 or less, 4/14 or less. In another embodiment, the sorbitol to amorphous sugar ratio is between 4/10 and 3/23, between 4/12 and 4/23, between 4/13 and 4/20, between 4/14 and 4/18 , between 4/14 and 3.5 / 16, or between 4/14 and 4/16. In a specific embodiment, the ratio is between 4/14 and 4/16. In a further specific embodiment, the amorphous sugar is trehalose and the sorbitol to trehalose ratio is between 4/14 and 4/16.
Sorbitol can be present in defined amounts in the aqueous mixture from which the composition is lyophilized. In one embodiment, the aqueous mixture contains between 2 and 4% (w / v), between 2.5 and 4% (w / v) or between 3 and 4% (w / v) of sorbitol. In a specific embodiment, sorbitol is present in an amount between 3 and 4% (m / v).
The amorphous sugar as chosen according to the present embodiments can be present in defined quantities. In one embodiment, the aqueous mixture contains at least 3% (w / v), at least 5% (w / v), at least 10% (w / v), at least 11% (w / v), at least 12% (w / v), at least 13% (w / v), or at least 14% (w / v) of the amorphous sugar as selected above. In another embodiment, the selected amorphous sugar is present in the aqueous mixture in a total amount of less than 23% (w / v), such as less than 20% (w / v), less than 18% (w / v), less than 17% (w / v), less than 16% (w / v), or less than 15% (w / v). Alternately, the amorphous sugar is present in the aqueous mixture in a total amount of 23% or less (w / v), such as 20% or less (w / v), 18% or less (w / v), 17 % or less (w / v), 16% or less (w / v), or 15% or less (w / v). Alternatively, the amorphous sugar may be present in the aqueous mixture in a total amount of at least 12%, at least 13% or at least 14% (w / v), but less than 18%, less 17%, or less than 16% (w / v).
In a specific embodiment, the amorphous sugar is trehalose and is present in an amount between 12% and 18% (w / v), or, between 14% and 16.5% (w / v).
The inventors have found that adenoviral vectors can be significantly impacted by the presence of a salt, such as sodium chloride, either when it is in dry form or when it is in liquid form. The invention therefore also relates to formulations, that is to say aqueous mixtures for lyophilization and desiccated compositions as described herein, taking into account the sensitivity of the adenoviral vectors to salt, such as sodium chloride. In one embodiment, simian adenoviral vectors are formulated using the aqueous mixtures and desiccated compositions described herein.
The term "salt" as used herein refers to ionic compounds which result from the neutralization reaction of an acid and a base, composed of a related number of cations and anions such that the product is free of charge clear, for example sodium chloride. The constituent ions can be either inorganic or organic, and can be monoatomic or polyatomic.
According to one embodiment, the amount of salt, in particular the amount of NaCl, present in the aqueous mixture is defined as less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 15 mM, less than 10 mM, or, less than 7.5 mM. Alternatively, the amount of NaCl present in the aqueous mixture can be defined as 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, or, 7.5 mM or less. Preferably, the composition is not completely free of salt or is not completely free of NaCl. To dispel the doubt concerning each of the embodiments relating to the salt and NaCl content in particular, it is understood that a salt, respectively NaCl, is present in a measurable amount. Consequently, according to one embodiment of the invention, a salt, in particular sodium chloride, is present in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, or, at least 4 mM. As a variant, the sodium chloride is present in an amount between 1 and 50 mM, between 2.5 and 25 mM, between 2.5 and 15 mM, between 2.5 and 10 mM or between 2.5 and 7, 5 mM. According to a particular embodiment, the sodium chloride is present in an amount of approximately 5 mM, for example 5 +/- 0.5 mM.
In the sense of defining ranges, the term "between" as used here is considered to include the limits of the range. For example, when sodium chloride is said to be present in an amount between 2.5 and 10 mM, those formulations in which NaCl is present at a concentration of 2.5 mM or 10 mM are included.
According to additional embodiments, the salt content, such as sodium chloride in the aqueous liquid or diluent for reconstituting the desiccated composition is also defined. By reconstitution of a lyophilized composition, it is meant rehydrating the desiccated composition to obtain a liquid mixture again. According to one embodiment, the amount of salt, for example sodium chloride, present in the aqueous liquid for reconstitution is less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 15 mM, less than 10 mM, or, less than 7.5 mM, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 15 mM or less, 10 mM or less or 7.5 mM or less.
The aqueous liquid for reconstituting the lyophilized composition may be essentially free of salt such as essentially free of sodium chloride. By essentially free, it is meant that the salt or sodium chloride concentration is at zero mM or very close to this. In a particular embodiment, the lyophilized composition can be reconstituted with water for injection (PPE).
In a further embodiment, the aqueous liquid for reconstituting the composition is not completely free of salt or sodium chloride. Consequently, a salt, such as sodium chloride, may be present in the aqueous liquid used to reconstitute the desiccated composition in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, or, at least 4 mM. As a variant, a salt, such as sodium chloride, is present in the aqueous liquid used to reconstitute the composition in an amount between 1 and 50 mM, between 2.5 and 25 mM, between 2.5 and 15 mM, between 2.5 and 10 mM or between 2.5 and 7.5 mM. According to a particular embodiment, a salt, such as sodium chloride, is present in the aqueous liquid used to reconstitute the composition in an amount of 5 mM. The invention therefore also provides a method of using the desiccated composition as described here, in which the desiccated composition is reconstituted with an aqueous liquid to reconstitute the composition as defined here.
The aqueous mixture or the desiccated composition may also include a surfactant chosen from poloxamer surfactants (for example poloxamer 188), polysorbate surfactants (for example polysorbate 80 and / or polysorbate 20), octoxinal surfactants, polidocanol surfactants, surfactants polyoxyl stearate, polyoxyl castor oil surfactants, N-octyl glucoside surfactants, macrogol hydroxy stearate, and combinations thereof. In one embodiment, the surfactant is chosen from poloxamer surfactants (for example poloxamer 188), polysorbate surfactants (for example polysorbate 80 and / or polysorbate 20), in particular polysorbate surfactants such as polysorbate 80.
In one embodiment, the surfactant is present in an amount of at least 0.001%, at least 0.005%, at least 0.01% (w / v), and / or up to 0.5 % (w / v) as calculated with respect to the aqueous mixture. The surfactant can be present in an amount of less than 0.25% or less than 0.1% (w / v). In another embodiment, the surfactant is present in an amount of 0.02% (w / v).
According to specific embodiments, the surfactant is polysorbate 80 or poloxamer 188 present in the aqueous mixture in an amount between 0.005% and 0.5% (w / v), such as about 0.02% (w / v).
In a further embodiment, a buffer is added to the aqueous mixture or to the desiccated composition. The pH is typically adjusted in consideration of the therapeutic components of the composition. Suitably, the pH of the aqueous mixture is at least 6, at least 6.5, at least 7 or at least 7.5. Alternately stated, the pH of the aqueous mixture may be less than 10, less than 9.5, less than 9 or less than 8.5. In other embodiments, the pH of the aqueous mixture is between 6 and 10, between 7 and 9.5, between 7.5 and 9.5, or, between 7.5, for example 7.5 + / - 0.5, or, 8.5 +/- 0.5. The optimal pH is also partly determined by the specific adenoviral vector formulated and / or the transgene incorporated therein.
A suitable buffer can be chosen from Tris, succinate, borate, Tris-maleate, lysine, histidine, glycine, glycylglycine, citrate, carbonate, phosphate or combinations thereof. In one embodiment, the buffer is Tris, succinate or borate. In a further embodiment, the buffer is Tris.
The buffer can be present in the aqueous mixture in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM or at least 5 mM. Alternatively, the buffer may be present in the aqueous mixture in an amount of less than 50 mM, less than 40 mM, less than 30 mM or less than 20 mM. For example, the buffer can be present in an amount of 0.5 mM to 50 mM, 1 mM to 50 mM or 2 mM to 20 mM. In one embodiment, the buffer is present in an amount of about 10 mM.
According to specific embodiments, the buffer is Tris, present in the aqueous mixture in an amount between 2 and 20 mM, such as at about 10 mM.
In one embodiment, the composition also includes histidine in an amount of up to about 20 mM, such as at a concentration of about 10 mM.
According to additional embodiments, the composition also comprises bivalent metal ions, such as Mg2 +, Ca2 +, or Mg2 + or Ca2 + in the form of a salt, such as MgCl2, CaCl2 or MgSO4. In one embodiment, the bivalent metal ion is Mg2 +. Typical amounts in which the bivalent metal ions are present in the aqueous mixture are between 0.5 and 10 mM, such as 1 or 2 mM, or 1 mM in particular.
For the purpose of describing embodiments of the invention and in the absence of any indication to the contrary, the specified amounts of excipients considered for inclusion in the composition (i.e. salt, sodium chloride, cryoprotective , buffer, surfactant and others described here) are typically (and unless otherwise indicated) expressed in w / v% calculated relative to the volume of the aqueous mixture. As a variant, in the case where the aqueous mixture is freeze-dried and reconstituted, the amount of excipients can be expressed in w / v% calculated relative to the volume of the reconstituted composition.
In addition to the increased stability during the freeze-drying process, the new formulation can also increase the stability of the adenoviral vector during storage of the lyophilized composition. The new formulation allows storage of the composition, liquid or dried, at 4 ° C, 25 ° C or 37 ° C, for up to 1 month, 3 months, 6 months, 1 year, 2 years or 3 years. In one embodiment, the desiccated composition can be stored at 4 ° C for 3 years, at 25 ° C for 3 months or at 37 ° C for 1 month. It is understood that the storage is adequate if at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the infectivity is preserved in comparison with the infectivity of the starting material.
The mixtures, compositions and methods described here allow storage of the adenoviral vector for at least 1 month at 37 ° C, or at least 3 months at 25 ° C or at least 3 years at 4 ° C while retaining at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the infectivity compared to the infectivity of the starting material.
The stability of the adenoviral vectors can, among other methods, be determined by measuring the infectivity of the vector, for example the retention of infectivity during manipulation (for example cryodessiccation) or storage of the viral vector. The term "infectivity" refers to the ability of the vector to enter a predisposed host, that is, cells, and to deliver its genetic material for expression by the host. Infectivity can be expressed as the "50% cell culture infectious dose" (CCID50 for "50% cell culture infectious dose"), which is the amount of adenoviral vector that is required to infect 50% of cells in a culture given cell. Infectivity can be measured by measuring the proportion of cells in which an adenoviral transgene is expressed. For example, a green fluorescent protein can be used as an infectivity marker, where the number of cells expressing a green fluorescent protein after 24 hours of incubation with the vector is determined. Alternatively, the infectivity can be measured by determining the number of cells expressing the adenovirus hexon capsid protein after 24 hours of incubation with the vector. The adenovirus has been widely used for gene transfer applications due to its ability to accomplish highly efficient gene transfer in a variety of target tissues and high transgenic capacity. The adenoviral vectors for use in the present invention can be derived from a range of mammalian hosts. Over 100 distinct adenovirus serotypes have been isolated which infect various mammalian species. These adenoviral serotypes have been categorized into six subgenera (A to F; B is subdivided into B1 and B2) according to a sequence homology and their capacity to agglutinate red blood cells (Tatsis and Ertl, Molecular Therapy (2004) 10 : 616-629).
In one embodiment, the adenoviral vector of the present invention is derived from a human adenovirus. Examples of such adenoviruses derived from humans are Ad1, Ad2, Ad4, Ad5, Ad6, Ad11, Ad24, Ad34, Ad35, particularly Ad5, Ad11 and Ad35. Although Ad5-based vectors have been widely used in a number of gene therapy trials, the use of Ad5 and other human group C adenoviral vectors has limitations due to preexisting immunity in the general population due to natural infection. Ad5 and other members of the human C group tend to be among the most seroprevalent serotypes. In addition, immunity to existing vectors may develop as a result of exposure to the vector during treatment. These types of immunity preexisting or developed to seroprevalent vectors can limit the effectiveness of gene therapy or efforts in terms of vaccination. Alternative adenovirus serotypes therefore constitute very important targets in the search for gene delivery systems capable of evading the immune response of the host.
Accordingly, in another embodiment, the adenoviral vector of the present invention is derived from a non-human simian adenovirus, also referred to simply as simian adenovirus. Many adenoviruses have been isolated from non-human simians such as chimpanzees, bonobos, rhesus macaques and gorillas, and vectors derived from these adenoviruses induce strong immune responses to transgenes encoded by these vectors (Colloca et al. ( 2012) Sci. Transl. Med. 4: 1-9; Roy et al. (2004) Virol. 324: 361-372; Roy et al. (2010) J. Gene Med. 13: 1725). Some advantages of vectors based on non-human simian adenoviruses include the relative lack of inter-neutralizing antibodies to these adenoviruses in the target population. For example, a cross-reaction of certain chimpanzee adenoviruses with preexisting neutralizing antibody responses is only present in 2% of the target population compared to 35% in the case of certain candidate human adenovirus vectors.
In specific embodiments, the adenoviral vector is derived from a non-human adenovirus, such as a simian adenovirus and in particular a chimpanzee adenovirus such as ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6,
Pan 7 (also designated by C7) or Pan 9. Examples of such strains are described in documents WO03 / 000283, WO2010 / 086189 and GB1510357.5 and are also available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and other sources. Alternatively, adenoviral vectors can be derived from non-human simian adenoviruses isolated from bonobos, such as PanAd1, PanAd2 or PanAd3. Examples of such vectors can be found described here for example in documents WO2005 / 071093 and WO2010 / 086189. Adenoviral vectors can also be derived from adenoviruses isolated from gorillas as described in documents WO2013 / 52799, WO2013 / 52811 and WO2013 / 52832.
Adenoviruses have a characteristic morphology with an icosahedral capsid comprising three major proteins, hexon (II), penton base (III) and head fiber (IV), together with a number of other minor proteins, VI, VIII, IX , IIIa and IVa2. Hexon constitutes the majority of the structural components of the capsid, which consists of 240 trimeric hexon capsomers and 12 penton bases. The hexon has three double barrels kept, while the top has three towers, each tower containing a loop from each subunit which forms the majority of the capsid. The base of the hexon is highly conserved between adenoviral serotypes, while the surface loops are variable (Tatsis and Ertl, Molecular Therapy (2004) 10: 616-629). Penton is another protein in the adenoviral capsid that forms a pentameric base to which fibers attach. The trimeric fiber protein protrudes from the base of penton at each of the 12 apexes of the capsid and constitutes a rod-to-head structure. The main role of the fiber protein is the knotting of the viral capsid on the cell surface via the interaction of the head region with a cellular receptor, and variations in the flexible stem as well as regions of the head fiber are characteristic. different serotypes (Nicklin et al. Molecular Therapy 2005 12: 384-393).
Adenoviral vectors can be used to deliver desired RNA or protein sequences, for example heterologous sequences, for expression in vivo. A vector can include any genetic element including naked DNA, a phage, a transposon, a cosmid, an episome, a plasmid, or a virus. By “expression cassette” (or “minigene”) is meant the combination of a selected heterologous gene (transgene) and the other regulatory elements necessary to drive the translation, transcription and / or expression of the gene product in a host cell.
Typically, an adenoviral vector is designed so that the expression cassette is located in a nucleic acid molecule which contains other adenoviral sequences in the region native to a selected adenoviral gene. The expression cassette can be inserted into an existing gene region to interrupt the function of this region, if necessary. Alternatively, the expression cassette can be inserted into the site of a partially or fully deleted adenoviral gene. For example, the expression cassette can be located in the site of a mutation, insertion or deletion which renders at least one gene of a genomic region chosen from the group consisting of E1A, E1B, E2A, E2B, E3 and E4. The expression "renders non-functional" means that a sufficient quantity of the gene region is eliminated or otherwise interrupted, so that the gene region is no longer capable of producing functional products of gene expression. If desired, the entire gene region can be eliminated (and adequately replaced with the expression cassette). Adequately, E1 genes of adenovirus are deleted and replaced by an expression cassette consisting of the promoter of choice, a cDNA sequence of the gene of interest and a poly A signal, resulting in a recombinant virus with deficient replication.
In one embodiment, the transgene encoded by the adenoviral vector is a sequence encoding a product which is useful in biology and medicine, such as therapeutic or immunogenic proteins, RNA, enzymes, or catalytic RNA. Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNA, RNA aptamers, and antisense RNA. An example of a useful RNA sequence is a sequence which quenches the expression of a targeted nucleic acid sequence in the treated animal.
Thus, in one embodiment, the mixture or composition as described here is intended for use in a prophylactic (therefore immunogenic or preventive) or therapeutic treatment of a subject, such as 'a mammalian or human subject, according to the transgene encoded by the adenoviral vector.
The transgene may encode a polypeptide or protein used for the treatment, for example, of genetic deficiencies, such as a therapeutic product or cancer vaccine, for induction of an immune response, and / or to prophylactic vaccine purposes. As used herein, the induction of an immune response refers to the ability of a protein, also known as an "antigen" or "immunogen", to induce an immune response to T cells and / or protein humoral.
Immunogens expressed by adenoviral vectors formulated as described herein and which are useful for immunizing a human or non-human animal against other pathogens include, for example, bacteria, fungi, parasitic microorganisms or multicellular parasites that infect vertebrates human and non-human, or from a cancer cell or a tumor cell. For example, immunogens can be selected from a variety of viral families.
In one embodiment, the immunogen comes from a filovirus, for example Ebola (species from Zaire, Sudan, Reston, Budibugyo and Ivory Coast) or Marburg. Such antigens can be derived from viral glycoprotein (transmembrane and / or secreted forms) and / or from viral nucleoprotein. Examples of such vectors can be found, among others, in document WO2011 / 130627.
In another embodiment, immunogens can be selected from respiratory viruses such as respiratory syncytial virus (RSV) and other paramyxoviruses such as human metapneumovirus, MPVh and parainfluenza virus (IPV). Adequate VSR antigens which are useful as immunogens for immunizing a human or non-human animal can be selected from: fusion protein (F), attachment protein (G), matrix protein (M2) and nucleoprotein (NOT). Such vectors are described in documents WO2012 / 089833 and PCT / EP2016 / 063297. In one embodiment, the construction product ChAd155-VSR as disclosed in document PCT / EP2016 / 063297 is considered for the disclosed compositions and methods.
In another embodiment, the immunogen can be derived from a retrovirus, for example a lentivirus such as the human immunodeficiency virus (HIV). In such an embodiment, immunogens can be derived from HIV-1 or HIV-2 sequences, such as Gag, Pol, Nef, Env, and others. Such vectors are described, among others, in documents GB1510357.5 and WO2008 / 107370.
In another embodiment, the immunogen can be from the human papilloma virus (HPV). In such an embodiment, immunogens can be derived from any type of HPV and in particular from types of HPV known to cause ailments or disease, for example types of high-risk HPV causing urogenital cancers HPV16, HPV18 and Similar.
Alternatively or additionally, a transgenic sequence may include a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (PFV), chloramphenicol acetyltransferase (CAT), luciferase, related proteins to the membrane including, for example, CD2, CD4, CD8, the hemagglutinin protein of influenza, and others well known in the art, to which high affinity antibodies are directed exist or can be produced by conventional means , and fusion proteins comprising a membrane-bound protein adequately fused to an antigen tag domain among, among others, hemagglutinin or Myc. These coding sequences, when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, cell sorting assays marked by fluorescence and immunoassays, including the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
In addition to the transgene, the expression cassette can also include conventional control elements which are operably linked to the transgene in a manner which allows its transcription, translation and / or expression in a cell transfected with the adenoviral vector. As used herein, "operably linked" sequences include both expression control sequences that are contiguous to the gene of interest and expression control sequences that act trans or remotely to control the gene of interest.
Expression control sequences include appropriate initiation, termination, promoter and transcription activator sequences; effective RNA processing signals such as splicing and polyadenylation signals (poly A) including poly A of rabbit beta-globin; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (for example, a Kozak consensus sequence); sequences which enhance the stability of proteins; and where desired, sequences which enhance the secretion of the encoded product. Among other sequences, chimeric introns can be used.
A "promoter" is a nucleotide sequence that allows RNA polymerase to bind and directs the transcription of a gene. Typically, a promoter is located in the 5 'non-coding region of a gene, near the transcriptional starting site of the gene. Sequence elements within promoters that function in initiating transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans). A large number of expression control sequences, including promoters which are internal, native, constitutive, inductive and / or tissue specific, are known in the art and can be used.
Adenoviral vectors are generated by modification of the wild-type adenovirus to express heterologous genes (transgenes) and / or to delete or inactivate undesirable adenoviral sequences. Adenoviral vectors may also have an affected replication competence. For example, the vector may be defective replication or have limited replication so that it has a reduced ability to replicate in non-complementary cells, compared to the wild type virus. This can be caused by a mutation of the virus, for example by a deletion of the gene involved in replication, for example a deletion of the E1a, E1b, E3 or E4 gene. Such modifications are known to those skilled in the art and described in the art, for example by Roy et al., Human Gene Therapy 15: 519-530, 2004; Colloca et al. (2012) Sci. Transl. Med. 4: 1-9; Roy et al. (2004) Virol. 324: 361-372; or WO 03/000283.
These vectors are generated using techniques known to those skilled in the art. Such techniques include conventional cDNA cloning techniques such as those described in texts, the use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the sequence of nucleotides desired. Particularly suitable methods include typical homologous recombination methods such as those provided in Colloca et al. (2012) Sci. Transl. Med. 4: 1-9; Roy et al. (2004) Virol. 324: 361-372; Roy et al. (2010) J. Gene Med. 13: 17-25; and WO2010 / 085984 or recombination methods as described in Warming et al. Nuc. Acids Res. (2005) 33: e36.
Adenoviral vectors can be produced on any suitable cell line in which the virus is capable of replicating. In particular, complementary cell lines which provide the missing factors in the viral vector which result in its altered replication characteristics (such as E1) can be used. Without limitation, such a cell line can be HeLa cells (ATCC access number CCL 2), A549 (ATCC access number CCL 185), HEK 293, KB (CCL 17), Detroit (e.g. Detroit 510, CCL 72) and WI-38 (CCL 75), among others. These cell lines are all available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209. Other suitable parent cell lines can be obtained from other sources, such as PER.C6 ™ cells, as represented by cells deposited with ECACC No. 96022940 at the European Collection of Animal Cell Cultures ( ECACC) and the Center for Applied Microbiology and Research (CAMR, UK) or Her 96 cells (Crucell).
A particularly suitable complementation cell line is the cell line
Procell92. The Procell92 cell line is based on HEK 293 cells which express adenoviral E1 genes, transfected with the Tet repressor under the control of the human phosphoglycerate kinase-1 (PGK) promoter, and the resistance gene G418 (Vitelli et al. PLOS One (2013) 8 (e55435): 1-9). Procell92.S is suitable for growth in suspension conditions and is also useful for producing adenoviral vectors expressing toxic proteins (www.okairos.com/e/inners.php m=00084, the last access dated 13 April 2015).
Adenoviral delivery methods and dosage A mixture or composition as described herein may comprise one or more recombinant vectors capable of inducing an immune response, e.g. a humoral response (e.g. antibody) and / or mediated by cell (for example, a cytotoxic T cell), against a transgenic product delivered by the vector at the end of the delivery to a mammal, suitably a human. A recombinant adenovirus can comprise (suitably in any of its gene deletions) a gene encoding a desired immunogen and therefore can be used in a vaccine. Recombinant adenoviruses can be used as prophylactic or therapeutic vaccines against any pathogen for which the antigen (s) crucial (crucial) for inducing an immune response and capable (s) of limiting the spread of the pathogen a (have) been identified and for which (which) the cDNA is available.
Thus, in one embodiment, the mixture and / or the composition described here are intended for the immunization of a subject, such as a human subject. The immunity levels of the selected gene can be monitored to determine the need, if any, for booster injections. After an estimate of serum antibody titers, optional booster immunizations may be desired.
In one embodiment, the aqueous mixture and / or (lyophilized) compositions described herein can be administered to a mammal, for example to a human subject. In particular, these mixtures or compositions comprising an adenoviral vector encoding a transgene (that is to say a recombinant adenoviral vector) which is a therapeutic or immunogenic protein are considered for a formulation in the aqueous mixture or lyophilized compositions described here.
Optionally, a mixture or composition of the invention may be formulated to contain other components, including, for example, one or more additional immunogens, for example one or more polypeptide antigen (s), and / or adjuvants. Such an adjuvant can be administered with a sensitizing DNA vaccine encoding an antigen to enhance the specific immune response of the antigen or in comparison to the immune response generated upon sensitization with a DNA vaccine encoding the antigen only. Alternatively, such an adjuvant can be administered with a polypeptide antigen which is administered in an administration regimen involving the adenoviral vectors of the invention.
In certain embodiments, the mixture or composition as described here is administered to a subject by intramuscular injection, intravaginal administration, intravenous injection, intraperitoneal injection, subcutaneous injection, epicutaneous administration, intradermal, nasal or oral administration.
If the therapeutic regime involves co-administration of one or more adenoviral vectors and / or an additional component, these can be co-formulated (that is to say in the same mixture or the same composition) or each formulated in different compositions. When formulated separately, they are favorably administered in the same place or at the same site or near it. For example, the components can be administered (for example via a route of administration chosen from intramuscular, transdermal, intradermal, subcutaneous) on the same side or at the same end (“co-lateral” administration) or on sides or opposite extremities (“contralateral” administration).
The dosages of the viral vector will depend mainly on factors such as the condition treated, the age, the weight and the health of the patient, and may thus vary between patients. For example, a therapeutically effective human or veterinary adult dosage of the viral vector generally contains 1 x 105 to 1 x 1015 viral particles, such as 1 x 108 to 1 x 1012 (for example, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 2.5 x 1010, 5 x 1010, 1 x 1011 5 x 1011, 1 x 1012 particles). Alternatively, a viral vector may be administered at a dose which is typically 1 x 105 to 1 x 1010 plaque forming units (UFP), such as 1 x 105 UFP, 5 x 105 UFP, 1 x 106 UFP, 5 x 106 UFP 1 x 107 UFP, 5 x 107 UFP, 1 x 108 UFP, 5 x 108 UFP, 1 x 109 UFP, 5 x 109 UFP, or 1 x 1010 UFP. The dosages will vary depending on the size of the animal and the route of administration. For example, an adequate human or veterinary dosage (for an animal of about 80 kg) for intramuscular injection is in the range of about 1 x 109 to about 5 x 1012 particles per mL, for a single site. Optionally, multiple administration sites can be used. In another example, an adequate human or veterinary dosage may be in the range of about 1 x 1011 to about 1 x 1015 particles for an oral formulation.
The adenoviral vector can be quantified by quantitative PCR analysis (Q-PCR), for example with primers and a probe designed on a CMV promoter region using as standard curve a serial dilution of plasmid DNA containing the genome of the vector with an expression cassette including the HCMV promoter. The number of copies in the test sample is determined by the parallel line analysis process. Alternative methods of quantifying vector particles can be an analytical HPLC or spectrophotometric method based on A260 nm.
An immunologically effective amount of a nucleic acid can be suitably between 1 ng and 100 mg. For example, an adequate amount may be 1 μg to 100 mg. An appropriate amount of the particular nucleic acid (e.g., the vector) can be readily determined by those of skill in the art. Examples of effective amounts of a nucleic acid component may be between 1 ng and 100 pg, such as between 1 ng and 1 μg (for example, 100 ng to 1 pg), or between 1 μg and 100 pg, such as 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 250 ng, 500 ng, 750 ng, or 1 pg. Effective amounts of a nucleic acid can also include from 1 pg to 500 pg, such as between 1 pg and 200 pg, such as between 10 and 100 pg, for example 1 pg, 2 pg, 5 pg, 10 pg , 20 pg, 50 pg, 75 pg, 100 pg, 150 pg, or 200 pg. As a variant, an effective amount by way of example of a nucleic acid may be between 100 pg and 1 mg, such as from 100 pg to 500 pg, for example, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg or 1 mg. Generally, a human dose will be contained in a volume between 0.3 mL and 2 mL. Thus, the mixture and / or the composition described here can be formulated so that during a reconstitution of the desiccated composition, a volume of, for example 0.3, 0.4, 0, 5, 0.6, 1.0, 1.5 or 2.0 mL of human dose per individual or combined immunogenic components is administered. Those skilled in the art can adjust these doses, depending on the route of administration and the therapeutic or vaccine application for which the recombinant vector is used. Expression levels of the transgene, or for an adjuvant, the level of circulating antibody, can be monitored to determine the frequency of dosing.
If one or more awareness and / or recall steps are used, these steps may include a single dose that is administered hourly, daily, weekly or monthly, or annually. For example, mammals can receive one or two doses containing between about 10 μg and about 50 μg of a plasmid in a support. The amount or site of delivery is desirably chosen based on the identity and condition of the mammal.
The therapeutic levels of, or the level of immune response against, the protein encoded by the selected transgene can be monitored to determine the need for booster injection, if any. After an estimate of a CD8 + T cell response, or optionally, antibody titers, in serum, optional booster immunizations may be desired. Optionally, the adenoviral vector can be delivered in a single administration or in various combined regimens, for example, in combination with a diet or treatment involving other active ingredients in an awareness-boosting regimen.
Unless otherwise indicated, "therapy" or "therapy" may relate to one or both of a preventive and curative therapy.
The aqueous mixture or the desiccated composition can be contained in a glass bottle, either silicone or non-silicone. In one embodiment, the aqueous mixture or the dried composition is supplied in a non-silicone bottle. Suitably, the aqueous mixture can be contained in a non-siliconized and freeze-dried bottle when it is contained in this bottle. The invention also provides a kit comprising two containers, of which a first container comprises the adenoviral composition as defined here and a second container comprises the liquid as defined here for reconstitution of the desiccated composition. The invention also provides a method for freeze-drying or freeze-drying a liquid containing an adenoviral vector, such as the aqueous mixture as defined here, to obtain a lyophilized composition as defined here, the method comprising an annealing step. The freeze-drying or freeze-drying cycle usually consists of three process phases.
In the first phase of the process, a mainly aqueous (aqueous) solution or mixture is frozen. Subsequently, the water is removed first by sublimation during a primary desiccation. In the third phase, unfrozen water is removed by diffusion and desorption during secondary desiccation. The inventors have also found that the introduction of an annealing step during the freezing phase of the lyophilization cycle has an unexpected positive impact on the stability of the adenoviral vector. Consequently, the invention also provides a method of cryodessiccation of a liquid containing an adenoviral vector, such as the aqueous mixture as described here, where the step of freezing the cryodessiccation cycle comprises an annealing step.
The temperature and the freezing and drying time will ultimately determine the moisture content of the lyophilized composition. In one embodiment, the moisture content of the lyophilized composition is 1.4% (w / w) or more, for example between 1.4 and 10% (w / w), between 1.4 and 8 % (w / w), between 1.7 and 8% (w / w), between 1.9 and 8% (w / w), between 1.4 and 5% (w / w), between 1.7 and 5% (w / w), between 1.9 and 5% (w / w), between 1.4 and 3% (w / w), between 1.7 and 3% (w / w), or between 1.9 and 3% (w / w). In a specific embodiment, the moisture content of the lyophilized composition is 1.7% (w / w) or more, 1.8% (w / w) or more, or, 1.9% (w / p) or more.
In order to define the method described, the following terms are used as they are known in the art. The expression "glass transition temperature" or "Tg" is the temperature at which an amorphous solid becomes soft when heated or brittle when cooled. The term "Tg '" refers to the glass transition temperature in the frozen state. The term "slump temperature" or "Tc" refers to the temperature at which an amorphous material softens to the extent that it can no longer support its own structure. The terms "freeze-drying" and "freeze-dried", and, "freeze-dried" and "freeze-dried" are used interchangeably and refer to the same rapid freezing process of a moist substance, followed by dehydration under reduced pressure. The term "annealing step" as used herein, refers to a process step in freeze-drying cycles of a composition, in which during the freezing phase, the product is maintained at a sub-freezing temperature specified for a predetermined period of time. As is known to those skilled in the art, annealing will lead to Ostwald ripening of the ice crystals and cryoconcentration of the amorphous matrix. Typically, the annealing temperature is (slightly) above Tg '. In one embodiment, the annealing is carried out at a temperature between (Tg '+ 0.5 ° C) and (Tg' + 20 ° C), for example at a temperature of -15 ° C +/- 9 ° C or -15 ° C +/- 6 ° C, or between (Tg '+ 0.5 ° C) and (Tg' + 10 ° C). In any case, the annealing temperature should be between Tg 'and the melting temperature (Tm) during annealing. In specific embodiments, annealing is carried out at a temperature between -4 ° C and -24 ° C, alternatively between -4 ° C and -20 ° C, alternatively between -4 ° C and -15 ° C, or alternatively between -8 ° C and -15 ° C, for example at -10 ° C +/- 0.5 ° C.
Annealing may be carried out during the freezing of the product, that is to say while the frozen sample is being formed, provided that the product is frozen (solid state) and in a glassy state (below Tg '). Alternatively, the annealing is carried out post-freezing the product.
In a specific embodiment, the annealing temperature is approximately -10 ° C (for example -10 ° C +/- 1 ° C), more particularly when the aqueous mixture comprises sorbitol and trehalose in a sorbitol ratio on trehalosis between 4/14 and 4 / 16.5.
In one embodiment, the product is frozen (that is to say product temperature below Tg ') before the annealing step. In one embodiment, freezing is accomplished by exposing the sample or aqueous mixture to a constant storage temperature at a freezing temperature which is below Tg '. In a variant embodiment, the product can be frozen by application of a storage ramp freezing, that is to say a progressive reduction of the storage temperature to a freezing temperature below Tg '. According to embodiments, the freezing temperature is a temperature below Tg 'minus 5 ° C, below Tg' minus 7.5 ° C, or below Tg 'minus 10 ° C, such as at below -50 ° C. According to one embodiment, the product temperature (that is to say the temperature of the sample in the freeze-dryer) at the time when the freeze-drying cycle is started is between +2 ° C and +8 ° C.
When applying a shelf ramp freezing, the temperature is reduced to a rate of at least 0.1 ° C / min, at least 0.2 ° C / min, at least 0.3 ° C / min or at least 0.5 ° C / min, and / or a rate of less than 10 ° C / min, 7.5 ° C / min, 5 ° C / min or less than 3 ° C / min. Alternatively, the temperature is reduced to a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / min, 0.2 to 3 ° C / min, or 0.3 to 1 ° C / min. According to additional embodiments, the storage temperature reached is maintained for approximately or at least 1 hour (or 60 minutes).
In an additional embodiment to the situation where the product is frozen before application of the annealing step, at the end of the initial freezing of the sample or of the product, the storage temperature is increased to a temperature above above Tg 'to initiate the annealing step, such that at a temperature above Tg' plus 0.5 ° C, above Tg 'plus 1 ° C, above Tg' plus 3 ° C, above Tg 'plus 5 ° C, above Tg' plus 10 ° C or above Tg 'plus 20 ° C. In any case, the temperature is kept below Tm during the annealing. In one embodiment, the temperature is raised to a rate of at least 0.1 ° C / min, at least 0.2 ° C / min, at least 0.3 ° C / min or at least 0.5 ° C / min, and / or a rate of less than 10 ° C / min, 7.5 ° C / min, 5 ° C / min or less than 3 ° C / min. As a variant, the temperature rose at a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / min, 0.2 to 3 ° C / min, or 0.3 to 1 ° C / min.
According to additional embodiments, the annealing temperature is maintained for at least two and / or up to four hours.
In an additional embodiment, at the end of the annealing step, the storage temperature is reduced to a temperature below Tg 'before initiation of drying under reduced pressure, such as to a temperature below Tg 'minus 5 ° C, below Tg' minus 7.5 ° C, or below Tg 'minus 10 ° C, such as below -50 ° C. In one embodiment, to achieve this, the temperature is reduced to a rate of at least 0.1 ° C / min, at least 0.2 ° C / min, at least 0.3 ° C / min or at minus 0.5 ° C / min, and / or a rate of less than 10 ° C / min, less than 7.5 ° C / min, less than 5 ° C / min or less than 3 ° C / min. Alternatively, the temperature is reduced to a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / min, 0.2 to 3 ° C / min, or 0.3 to 1 ° C / min. According to additional embodiments, the storage temperature reached is maintained for approximately or at least 1 hour (or 60 minutes).
Desiccation under reduced pressure as envisaged in step b.ii. the lyophilization process described here will typically be carried out in two phases, that is to say a primary desiccation and a secondary desiccation. In one embodiment, step b.ii. of the process will include: - a step b.ii.1. primary drying at a temperature below the Tc of the product, and, - a step b.ii.2. secondary drying at a temperature above the Tc of the product and below the Tg of the product.
In specific embodiments, the primary drying of the compositions described here is carried out at -30 ° C +/- 5 ° C, the secondary drying of the compositions described here is carried out at 10 ° C +/- 5 ° C, or, the primary drying of the compositions described here is carried out at -30 ° C +/- 5 ° C and the secondary drying is carried out at 10 ° C +/- 5 ° C.
In specific embodiments, during a freeze-drying of the compositions described here, primary drying conditions are applied for 24 hours or more, between 24 and 40 hours, or, between 30 and 40 hours.
In an additional embodiment, primary drying is carried out at a pressure of less than 90 μbar and / or above 40 μbar. Primary Drying Conditions can be applied for up to 24 hours or more.
Another embodiment concerns the secondary drying temperature reached by increasing the storage temperature at a rate of 0.1 ° C / min, at least 0.2 ° C / min, at least 0.3 ° C / min or at least 0.5 ° C / min, and / or a rate of less than 3 ° C / min, less than 2 ° C / min, or less than 1 ° C / min. Alternatively, the secondary drying temperature is reached by increasing the storage temperature at a rate of 0.1 to 3 ° C / min, 0.2 to 2 ° C / min, or 0.3 to 1 ° C / min . According to yet another embodiment, the secondary drying temperature is at least -10 ° C and / or below 30 ° C. In a specific embodiment, the secondary drying temperature for the compositions containing sorbitol is 25 ° C. +/- 5 ° C. In an alternative embodiment, during the freeze-drying of the compositions described here, the secondary drying temperature is 10 ° C. +/- 5 ° C.
Secondary drying conditions may be applied for at least or for about three hours, at least about four hours, at least about five hours, or, at least, for about six hours.
Particular embodiments of the invention include:
Embodiment 1: a composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v) and (viii) sorbitol 3.5% (w / v).
Embodiment 2: a composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi) 2% sucrose (w / v), (vii) trehalose 23% (w / v).
Embodiment 3: a composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v) and (viii) sorbitol 3.5% (w / v) and (ix) TWEEN 80 0.02% (w / v).
Embodiment 4: a composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi) 2% sucrose (w / v), (vii) trehalose 23% (w / v) and (viii) TWEEN 80 0.02% (w / v).
Embodiment 5: a lyophilized or lyophilized composition comprising (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM, (vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v) and (viii) sorbitol 3.5% (w / v).
Embodiment 6: a lyophilized or lyophilized composition comprising (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM, (vi) 2% sucrose (w / v), and (vii) trehalose 23% (w / v).
Embodiment 7: a lyophilized or lyophilized composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi) 2% sucrose (w / v), (vii) trehalose 23% (w / v), (viii) sorbitol 3.5% (w / v) and (ix) TWEEN 80 0.02 % (w / v).
Embodiment 8: a lyophilized or lyophilized composition comprising (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM, (vi) 2% sucrose (w / v), (vii) trehalose 23% (w / v) and (viii) TWEEN 80 0.02% (w / v).
Embodiment 9: a composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, ( vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v), (viii) sorbitol 3.5% (w / v).
Embodiment 10: a composition consisting essentially of (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM, ( vi) 2% (w / v) sucrose and (vii) trehalose 23% (w / v).
Embodiment 11: a composition essentially consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, ( vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v), (viii) sorbitol 3.5% (w / v) and (ix) TWEEN 80 0.02% (w / v).
Embodiment 12: a composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, ( vi) 2% sucrose (w / v), (vii) trehalose 23% (w / v) and (viii) TWEEN 80 0.02% (w / v).
Embodiment 13: a lyophilized or lyophilized composition consisting essentially of (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) Histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM, (vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v) and (viii) sorbitol 3.5% (w / v).
Embodiment 14: a lyophilized or lyophilized composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) MgCl2 at 1 mM, (vi) 2% sucrose (w / v) and (vii) trehalose 23% (w / v).
Embodiment 15: a lyophilized or lyophilized composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) MgCl2 at 1 mM, (vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v), (viii) sorbitol 3.5% (w / v) and (ix) TWEEN 80 to 0 , 02% (w / v).
Embodiment 16: a lyophilized or lyophilized composition consisting essentially of (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM, (vi) 2% sucrose (w / v), (vii) trehalose 23% (w / v) and (viii) TWEEN 80 0.02% (w / v).
Embodiment 17: a composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi ) 2% sucrose (w / v), (vii) trehalose 18% (w / v), (viii) sorbitol 3.5% (w / v) and (ix) water for injection.
Embodiment 18: a composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi ) 2% (w / v) sucrose, (vii) trehalose 23% (w / v), and (viii) water for injection.
Embodiment 19: a composition consisting of (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM, (vi ) 2% sucrose (w / v), (vii) trehalose 18% (w / v), (viii) sorbitol 3.5% (w / v), (ix) TWEEN 80 0.02% ( w / v) and (ix) water for injection. Embodiment 20: a composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2, (vi ) 2% sucrose (w / v), (vii) trehalose 23% (w / v), (ix) TWEEN 80 at 0.02% (w / v) and (viii) water for injection.
Embodiment 21: a lyophilized or lyophilized composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM histidine, (iv) 5 mM NaCl, (v) 1 mM MgCl2 , (vi) 2% sucrose (w / v), (vii) trehalose 18% (w / v) and (viii) sorbitol 3.5% (w / v).
Embodiment 22: a lyophilized or lyophilized composition consisting of (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM , (vi) 2% sucrose (w / v) and (vii) trehalose 23% (w / v).
Embodiment 23: a lyophilized or lyophilized composition consisting of (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM , (vi) sucrose at 2% (w / v), (vii) trehalose at 18% (w / v), (viii) sorbitol at 3.5% (w / v) and (ix) TWEEN 80 at 0, 02% (w / v).
Embodiment 24: a lyophilized or lyophilized composition consisting of (i) an adenoviral vector, (ii) TRIS at 10 mM, (iii) histidine at 10 mM, (iv) NaCl at 5 mM, (v) MgCl2 at 1 mM , (vi) 2% sucrose (w / v), (vii) trehalose 23% (w / v) and (viii) TWEEN 80 0.02% (w / v).
Embodiment 25: The composition of any of embodiments 1 to 24, wherein the adenoviral vector is a simian adenovector, such as a chimpanzee adenovector. In particular, an adenoviral vector chosen from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 or Pan 9, more particularly still an adenoviral vector is chosen from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, and PanAd3 and even more particularly, the adenoviral vector is ChAd155.
The present invention will now be described further using the following non-limiting examples.
EXAMPLES
EXAMPLE 1 Evaluation of the Effect of Sorbitol and Comparison of Trehalose and Sucrose
The objectives of the experiment were to evaluate a protective effect of sorbitol on the adenovirus during freeze-drying, and to evaluate the impact of replacing the total trehalose load with sucrose. The vector ChAd155 used in the experiment codes for a respiratory syncytial viral protein (ChAd155-RSV) and is described in document PCT / EP2016 / 063297. The particles of ChAd155 were formulated in an aqueous mixture further comprising the excipients Tris at 10 mM - histidine at 10 mM -MgCl2.6H2O at 1 mM - Tween 80 at 0.02% (w / v) - NaCl at 5 mM - Trehalose or sucrose at 23% (w / v) - Sorbitol at 2% (w / v). The sugar concentration was calculated to reach the maximum osmolality allowed for a pediatric injection, c. to d. 900 mOsm / kg. The concentration of the viral particles was 1.1 x 1011 pU / ml. This composition was calculated so that it is reached after reconstitution of the lyophilized material with 0.625 ml of water for injection in glass vials of non-silicone type filled with 0.5 ± 0.02 ml. Next, the vials were partially sealed with a Helomet FM460 bromobutyl stopper inserted in a freeze-dry position (partially inserted to allow water vapor to escape during the freeze-dry cycle).
The freeze-drying cycle used included the following steps (as shown in Figure 1): 1. Freezing: - The storage temperature was set at -52 ° C. The filled vials were loaded into the cryosensor when the storage temperature was at or below -45 ° C. The samples were then cooled to -52 ° C for a minimum of one hour. 2. Annealing step: - The storage temperature has been raised so that it reaches the target annealing temperature (-10 ° C) in one hour. - The annealing temperature was maintained for two hours. - The storage temperature was again reduced from -10 ° C to -50 ° C within one hour. - The product was kept at -50 ° C for at least one hour. 3. Primary drying: - The chamber pressure was fixed at 80 pbar and the storage temperature was raised from -50 ° C to -25 ° C over three hours. The storage temperature and chamber pressure were maintained for 24 hours. 4. Secondary drying: - The storage temperature was increased from -25 ° C to +10 ° C over six hours, while the chamber pressure was reduced to 40 pbar. When the storage temperature reached +10 ° C, these conditions were maintained for six hours. At the end of the freeze-drying cycle, the chamber was filled with dry nitrogen until a chamber pressure of 825 mbar was reached, then the caps were fully inserted into the bottles (stoppering). Once the closure was completed, the chamber pressure was balanced to atmospheric pressure for discharge. The chamber temperature was maintained at +2 to +8 ° C until the vials were unloaded. The vials were then unloaded and over-sealed with removable aluminum caps.
The results of this experiment are presented in the table below:
Two samples of purified crude ChAd155, diluted to reach the concentration of the reconstituted vaccine, were used, before and after treatment for 30 minutes at 60 ° C. as positive control (fresh purified crude control) and negative control (degraded purified crude control) respectively.
The PicoGreen assay measures the degradation of viral particles. The Quant-iTTM PicoGreen dsDNA reagent is an ultra-sensitive fluorescent nucleic acid dye for quantifying double-stranded DNA in solution.
The HEXON infectivity of the adenoviral particles is measured by cytometric detection of stained cell flow for an adenovirus hexes capsid protein. The unit concentration of adenovirus particles is also measured using a high performance anion exchange liquid chromatography system (AEX-HPLC for “Anion Exchange High Performance Liquid Chromatography”) coupled to a fluorescence detector and using a commercial adenovirus standard as a reference. Systems
Chromatographs used were Dionex Ultimate 3000 and Waters Acquity UPLC biocompatible (class H).
Compositions containing trehalose exhibited an increase in the glass transition temperature inducing better stability at high temperature storage of the product. Likewise, infectivity increased (10-20%) in trehalose formulations compared to sucrose formulations.
For formulations containing sorbitol, it has been noted that the presence of sorbitol leads to a decrease in the glass transition temperature (Tg), which could affect the appearance of the resulting cake. As illustrated in Figure 3, the infectivity improved by 10 to 20% for compositions containing sorbitol.
Example 2 - Determination of statistical PE for the formulation containing trehalose, sorbitol and NaCl The objective of the experiment was to evaluate several ranges of concentration in trehalose, sorbitol and NaCl to determine optimal conditions for the freeze-dried adenovirus candidate. The adenovirus used was ChAd155-RSV. The protective effect of trehalose and sorbitol as observed in Example 1 was further evaluated in conjunction with the impact of NaCl.
The particles of ChAd155-RSV were formulated in an aqueous mixture further comprising the excipients Tris at 10 mM - histidine at 10 mM - MgCl2.6H2O at 1 mM - Tween 80 at 0.02% (w / v) - NaCl ( variable: 5, 25 or 45 mM) - trehalose (14, 18.5 or 23% (v / p)) - sorbitol (0, 2 or 4% (v / p)). The concentration of the viral particles evaluated was 1.1 x 1011 pU / ml. It was calculated that the compositions were reached after reconstitution of the lyophilized composition with 0.625 ml of water for injection in glass vials of non-silicone type filled with 0.5 ± 0.02 ml. The bottles were then partially sealed with a Helvoet FM460 bromobutyl stopper inserted in the drying position (partially inserted to allow water vapor to escape during the freeze-drying cycle).
The following compositions were put to the test: - sample 1: trehalose (T) at 18.5% (v / p) -
sorbitol (S) 0% (v / p) - NaCl (N) 25 mM
- sample 2: T 14% - S 0% - N 5 mM
- sample 3: T 18.5% - S 4% - N 25 mM
- sample 4: T 18.5% - S 2% - N 25 mM
- sample 5: T 23% - S 0% - N 5 mM
- sample 6: T 23% - S 4% - N 45 mM
- sample 7: T 18.5% - S 2% - N 25 mM
- sample 8: T 14% - S 4% - N 5 mM
- sample 9: T 23% - S 0% - N 45 mM
- sample 10: T 14% - S 2% - N 25 mM
- sample 11: T 18.5% - S 2% - N 5 mM
- sample 12: T 14% - S 0% - N 45 mM
- sample 13: T 18.5% - S 2% - N 25 mM
- sample 14: T 14% - S 4% - N 45 mM
- sample 15: T 23% - S 2% - N 25 mM
- sample 16: T 18.5% - S 2% - N 45 mM
- sample 17: T 23% - S 4% - N 5 mM - sample 18: fresh purified raw control - sample 19: degraded purified raw control
Two samples of purified crude ChAd155, diluted to reach the concentration of reconstituted vaccine, were used, before and after treatment for 30 minutes at 60 ° C. as positive controls (fresh purified crude control) and negative (degraded purified crude control) respectively.
The freeze-drying cycle used included the following steps (as shown in Figure 4): 1. Freezing: - The storage temperature was set at -52 ° C. The filled vials were loaded into the cryosensor when the storage temperature was -45 ° C or below. The samples were then cooled to -52 ° C for a minimum of one hour 2. Annealing step: - The storage temperature was raised so that it reached the target annealing temperature (-10 ° C) in one hour. - The annealing temperature was maintained for two hours. - The storage temperature was again reduced from -10 ° C to -50 ° C within one hour. - The product was kept at -50 ° C for at least one hour. 3. Primary drying: - The chamber pressure was fixed at 80 μbar and the storage temperature was raised from -50 ° C to -30 ° C over three hours. The storage temperature and chamber pressure were maintained for 24 hours 4. Secondary drying: - The storage temperature was increased from -25 ° C to +10 ° C over six hours, while the pressure was reduced from chamber at 40 μbar. When the storage temperature reached +10 ° C, these conditions were maintained for six hours. At the end of the freeze-drying cycle, the chamber was filled with dry nitrogen until a chamber pressure of 825 mbar was reached, then the caps were fully inserted into the bottles (stoppering). Once the closure was completed, the chamber pressure was balanced to atmospheric pressure for discharge. The chamber temperature was maintained at +2 to +8 ° C until the vials were unloaded. The vials were then unloaded and over-sealed with removable aluminum caps.
All samples were analyzed at T0 and after storage for one week at + 4 ° C, + 25 ° C (T1W25) or + 30 ° C.
The results of this experiment are presented in the table below:
The PicoGreen assay and the HEXON infectivity were as described for Example 1. A quantitative PCR (qPCR) as reported here allows us to determine the virus content. The test targets the hCVM promoter present in the adenovirus. The Quiagen QIAmp 96 DNA Blood DNA sample was extracted. The results are expressed as genomic equivalents per milliliter (gE / mL). The best results were obtained taking into account the global parameters (for example the PicoGreen value is at the lowest, the infectivity and the content by HPLC at the highest, the osmolality lower than 900 mOsm / kg), with compositions comprising 14-18.5% trehalose, 4% sorbitol and <25 mM NaCl.
By following a statistical analysis, the following optimized compositions have been identified according to sets of parameters of different constraints (see FIG. 11 for diagram 1 of the experimental design (PE)): - by not taking into account the constraints relating to Tg, the best candidate was Trehalose = 14% / Sorbitol = 4% / NaCl = 5 mM. - taking into account that the Tg should be> 25 ° C, the best candidates were trehalose = 14% to 16% / sorbitol = 3% / NaCl = 5 mM.
For compositions of which the higher trehalose content is desirable, the
following optimized compositions (see FIG. 12 for PE diagram 2): - trehalose = 22%, sorbitol = 2% and NaCl = 5 mM. The effect of the sorbitol content on the Tg of the lyophilized composition affected the appearance of the resulting cake and the stability of the adenoviral particle stored therein. The decrease in the glass transition temperature of the candidates including sorbitol in their composition led to a bad appearance of the cake (melting / sagging) over a week at +25 ° C and +30 ° C. It has been observed that the reduced glass transition temperature is directly correlated with the moisture content (residual moisture) measured in the cake after the freeze-drying step. Nevertheless, surprisingly, at the same time, it was observed that the samples with higher moisture content (measured by Karl Fisher titration) better maintained the infectivity of the adenoviral particle during storage. Despite the melted appearance of the cakes, the infectivity remained higher, provided that a minimum moisture content was maintained of at least 1.8% w / w.
Example 3 - Statistical PE for the trehalose / sorbitol / NaCl formulation
The results of Example 2 supported optimal ranges for trehalose (14-16%), sorbitol (3-4%) and NaCl (5mM).
Additional compositions were tested to supplement the data of Example 2 using the same ChAd155-RSV. For the compositions evaluated, the molarity of NaCl was fixed at 5 mM. The trehalose and sucrose content was varied as follows:
* Resulting osmolality of 910 mOsm / kg
Two diluted crude purified ChAd155 samples were used to reach the concentration of reconstituted vaccine before and after treatment for 30 minutes at 60 ° C as positive and negative controls respectively.
The freeze-drying cycle was also evaluated in order to improve the stability of the appearance of the cake after storage (in particular at +25 ° C. (room temperature) in order to cover the time of reconstitution and administration to the patient after storage. at cold temperature).
In Example 2, it was determined that the impact of a decreased Tg in the presence of sorbitol was directly correlated to the moisture content (residual moisture) measured in the cake after the freeze-drying step. Although the data has shown that
the moisture content had a protective effect on the infectivity of the adenovirus at stability, a bad appearance of the cakes (melted appearance) is undesirable. To this end, the freeze-drying or freeze-drying cycle was further optimized as follows: - lyo cycle (1): a longer primary freeze-drying phase (+ 10H) was used in combination with a higher secondary desiccation temperature (at +25 ° C) (see Figure 13). - lyo cycle (2): a longer primary drying phase (+ 10H) was used in combination with a secondary drying temperature of +10 ° C (see Figure 14).
The freeze-dried products were evaluated at T0 and after stability for one week at +4 ° C (T1W4), +25 ° C (T1W25) and +30 ° C (T1W30).
The results of this experiment are presented in the tables below:
The PicoGreen assay, the HEXON infectivity and AEX-HPLC were as described for example 1. The CCID50 infectivity is a measure of the titer of adenovirus. The 50 cell culture infectious dose (CCID50) is a limiting dilution assay that quantifies the amount of virus required to produce a cytopathic effect in 50% of cells in a certain volume. In the present example, the titer is expressed in a log 10 scale (Log CCID50 / mL). It is measured by inoculating the serial dilution samples on indicator cells. After the incubation time (7 days at 37 ° C. for the adenovirus), the adenovirus hexon was immunostained and the results were determined under the microscope.
The best results were obtained taking the global parameters into account (for example PicoGreen value at the lowest, infectivity and HPLC content at the highest, osmolality lower than 900 mOsm / kg) using the cryodessiccation cycle 2. The compositions 2 (14% trehalose - 4% sorbitol), 4 (16% trehalose - 3.5% sorbitol) and 5 (16% trehalose - 4% sorbitol) performed best on the overall parameters.
EXAMPLE 4 Stability of Lyophilized Adenovirus
Stability studies have been performed on lyophilized compositions over five months at 25 ° C or seven months at 15 ° C. Extrapolation of real-time data to three years was also performed using statistical models based on the assumption that degradation would follow a similar pattern. Two stability models were used to extrapolate the viral content, measured by HPLC and infectivity, measured by sorting of fluorescently labeled cells (FACS for "Fluorescent Activated Cell Sorting"), expressed in international unit (IU). (1) Linear model (2) first-order decay model:
and (2) first-order decay (with asymptote):
Both measured and extrapolated data are shown in the following table.
Secondary drying at 15 ° C resulted in greater stability, as measured in loss of adenovirus, than secondary drying at 25 ° C. Two profiles were observed, which are shown in Figures 18 and 19. Figure 18 shows the stability of the adenovirus formulated with either 18% trehalose + 3.5% sorbitol or 23% trehalose at 4 ° C over 200 days , at a secondary drying temperature of 15 ° C. Figure 19 shows the stability of the adenovirus formulated with either 18% trehalose + 3.5% sorbitol or 23% trehalose at 4 ° C over 200 days at a secondary drying temperature of 25 ° vs.
As shown in Figures 18 and 19, a concentration of trehalose at 23% resulted in a relatively higher loss (approximately 45%) than a concentration of trehalose at 18% + sorbitol at 3.5% during freeze-drying, with little subsequent loss at 4 ° C over time. In contrast, 18% trehalose + 3.5% sorbitol resulted in an adenoviral vector loss of only approximately 30%.
EXAMPLE 5 Physical Stability of Lyophilized Adenovirus
We examined the effect of mechanical stresses, by replicating the vibration stress emanating from road and / or air transport, on the stability of
lyophilized compositions. More particularly, the physical stability of the compositions in the lyophilized state was tested after exposure to shaking stress.
Two formulations were prepared as previously described and tested: (1) 18% sucrose + 3.5% sorbitol (2) 23% trehalose
For each test condition, ten glass vials, either silicone or non-silicone, containing lyophilized compositions were attached to the tape horizontally inside a light insulated container (Sofribox) at 4 ° C. Three replicas were used for each test. The samples were vigorously shaken for two hours at "level 2" followed by two more hours at "level 1" using a Lansmont Model 1000 vibration test system. The experimental design is intended to replicate the vibration stress experienced for two hours of road transport and two hours of air transport respectively.
The vibrations are supposed to gradually disintegrate the powder cake according to the composition of the cake, and the intensity and duration of the vibrations. The physical integrity of the lyophilized compositions was determined by visual analysis using Axiovision (CQR &amp; photo) at times 0, 2 hours and 4 hours (Figures 20 and 21).
Figure 20 shows the proportion of lyophilized samples which remained intact (O), were cracked (+) or were fragmented (X) after a simulated transport of two hours by road transport and two hours by air transport. The formulation with 18% trehalose + 3.5% sorbitol resulted in lyophilized compositions which were less affected by the vibration stress than the formulations with 23% trehalose. A secondary desiccation temperature of 15 ° C also resulted in lyophilized compositions which were less affected by the vibration stress during transport. The formulation with 18% trehalose + 3.5% sorbitol, with secondary drying temperature of 15 ° C and the use of siliconized bottles resulted in the best conditions for keeping the lyophilized cakes intact.
FIG. 21 shows the consistency of lyophilized samples which remained non-powdery (O), slightly powdery (+) or powdery (X) after a simulated transport of two hours by road transport and two hours by air transport. The formulation with 18% trehalose + 3.5% sorbitol resulted in lyophilized compositions which were less affected by the vibration stress than formulations with 23% trehalose. A secondary desiccation temperature of 15 ° C also resulted in lyophilized compositions which were less affected by the vibration stress during transport. When formulated with 18% trehalose + 3.5% sorbitol, a secondary desiccation temperature of 15 ° C resulted in less powder formation than a secondary desiccation temperature of 25 ° C.

权利要求:
Claims (36)
[1]
1. Composition comprising an adenoviral vector, sorbitol and an amorphous sugar chosen from trehalose, sucrose, mannose, mannitol, raffinose, dextran, and combinations thereof.
[2]
2. Composition according to claim 1, which is a lyophilized composition.
[3]
3. Composition according to claim 1 or 2, in which the sorbitol to amorphous sugar ratio is less than 4/14.
[4]
4. Composition according to any one of the preceding claims, in which the sorbitol to amorphous sugar ratio is between 4/14 and 3/18, or between 4/14 and 3.5 / 16.
[5]
5. Composition according to any one of the preceding claims, further comprising a salt, for example NaCl.
[6]
6. Lyophilized composition from an aqueous mixture comprising an adenoviral vector, sorbitol and an amorphous sugar chosen from trehalose, sucrose, mannose, mannitol, raffinose, dextran, and combinations thereof.
[7]
7. Composition according to claim 6, in which the sorbitol to amorphous sugar ratio is between 4/14 and 3/18, or between 4/14 and 3.5 / 16.
[8]
8. Composition according to claims 6 or 7, in which the aqueous mixture has an NaCl concentration below 25 mM, below 20 mM, below 15 mM, below 10 mM, or between 2 and 10 mM.
[9]
9. Composition according to claims 6 to 8, in which the concentration of amorphous sugar in the aqueous mixture is below 18% (w / v).
[10]
10. Composition according to claims 6 to 9, in which the concentration of amorphous sugar in the aqueous mixture is between 10 and 20% (w / v), or between 14 and 18% (w / v).
[11]
11. Composition according to claims 6 to 10, in which the amorphous sugar is present in an amount corresponding to a concentration in the aqueous mixture of between 10 and 20% (w / v), or between 14 and 18% (w / v ).
[12]
12. Composition according to Claims 6 to 11, in which the concentration of sorbitol in the aqueous mixture is between 3 and 4% (w / v).
[13]
13. Composition according to claims 6 to 12, in which the sorbitol is present in an amount corresponding to a concentration of sorbitol in the aqueous mixture of between 3 and 4% (w / v).
[14]
14. Composition according to any one of claims 6 to 13, in which the NaCl is present in an amount corresponding to a concentration in the aqueous mixture of between 3 and 20 mM, between 3 and 10 mM, or between 4 and 6 mM .
[15]
15. Composition according to any one of the preceding claims, in which the additional amorphous sugar is trehalose or sucrose.
[16]
16. Composition according to any one of the preceding claims, in which the additional amorphous sugar is trehalose.
[17]
17. Composition according to any one of the preceding claims, in which the adenoviral vector is a simian adenovector, such as a chimpanzee adenovector.
[18]
18. Composition according to any one of the preceding claims, in which the adenoviral vector is chosen from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan5, Pan6, Pan 7 or Pan 9.
[19]
19. Composition according to any one of the preceding claims, in which the adenoviral vector is chosen from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, and PanAd 3.
[20]
20. Composition according to any one of the preceding claims, in which the adenoviral vector is ChAd155.
[21]
21. Composition according to any one of the preceding claims, in which the composition further comprises a surfactant, a buffering agent and / or a bivalent metal salt.
[22]
22. Composition according to any one of the preceding claims, in which the composition comprises a surfactant chosen from poloxamer surfactants (for example poloxamer 188), polysorbate surfactants (for example polysorbate 80 and / or polysorbate 20), octoxinal surfactants, polidocanol surfactants, polyoxyl stearate surfactants, polyoxyl castor oil surfactants, N-octyl glucoside surfactants, macrogol hydroxy stearate 15, and combinations thereof.
[23]
23. Composition according to any one of the preceding claims, in which the composition comprises a surfactant which is a poloxamer surfactant or a polysorbate surfactant.
[24]
24. Composition according to any one of the preceding claims, in which the composition comprises a surfactant which is poloxamer 188 or polysorbate 80.
[25]
25. Composition according to any one of the preceding claims, in which the composition comprises a surfactant which is polysorbate 80.
[26]
26. Composition according to any one of the preceding claims, in which the composition comprises a buffer chosen from Tris, succinate, borate, Tris-maleate, lysine, histidine, glycine, glycylglycine, citrate, carbonate, phosphate or one of their combination.
[27]
27. Composition according to any one of the preceding claims, in which the composition comprises a buffer chosen from Tris, succinate, borate or one of their combination.
[28]
28. Composition according to any one of the preceding claims, in which the composition comprises Tris.
[29]
29. Composition according to any one of the preceding claims, in which the composition comprises a divalent metal ion salt chosen from MgCl2, CaCl2 or MgSO4.
[30]
30. Composition according to any one of the preceding claims, in which the composition comprises MgCl2.
[31]
31. Composition according to any one of the preceding claims, which is lyophilized and has a moisture content of 1.40% (w / w) or more, such as between 1.40 and 10% (w / w).
[32]
32. Composition according to any one of the preceding claims, for rehydration / reconstitution with water for injection.
[33]
33. Kit comprising two containers in which a first container contains a lyophilized composition defined by any one of claims 1 to 32 and a second container contains water for injection.
[34]
34. Kit according to the preceding claim, in which the content of the second container is used to rehydrate / reconstitute the content of the first container.
[35]
35. Aqueous mixture as defined in any one of claims 6 to 32 for cryodessiccation.
[36]
36. A method of preparing a composition according to any one of claims 32, comprising the steps of freeze-drying the aqueous mixture have a. freezing the aqueous mixture including an annealing step and b. drying of the frozen aqueous mixture under reduced pressure.

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MX2019008798A|2019-09-11|

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JP2020506691A|2020-03-05|

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BE1025187A1|2018-11-27|

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GB201701239D0|2017-03-08|

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法律状态:
2018-12-17| FG| Patent granted|Effective date: 20181203 |

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2020-10-15| MM| Lapsed because of non-payment of the annual fee|Effective date: 20200131 |

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GB1701239.4|2017-01-25|

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GBGB1701239.4A|GB201701239D0|2017-01-25|2017-01-25|Novel formulation|

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