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    • 专利摘要:
    The present invention relates to the formulation of adenoviral vectors in an aqueous mixture or a lyophilized composition in the presence of an amorphous sugar and a low concentration of salt, its formulation as well as a process for obtaining the dry composition.
    公开号:BE1023537B1
    申请号:E2016/5605
    申请日:2016-07-20
    公开日:2017-04-26
    发明作者:Erwan Bourles；Frédéric Mathot
    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 an aqueous mixture or a freeze-dried composition, its formulation and a process for obtaining the dry composition.
    CONTEXT
    The adenoviral vectors represent a therapeutic protein delivery platform allowing the incorporation of the nucleic acid sequence coding for the therapeutic protein into the adenoviral genome, said protein being caused to express itself when the adenoviral particle is administered to the treated subject . The development of a stabilization formulation for adenoviral vectors, allowing storage at appropriate storage temperatures with an acceptable shelf life, has been a challenge for the art.
    Stabilization 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.
    SUMMARY OF THE INVENTION
    The inventors have surprisingly discovered that the adenoviral vectors can be particularly sensitive to the presence of salt such as sodium chloride. The invention therefore relates to an aqueous mixture and a freeze-dried composition obtained from said mixture by freeze drying (referred to hereinafter as "dry composition") having low salt concentrations, having in particular sodium chloride 50 mM or less, for the formulation of simian adenoviral vectors. The invention also relates to a method of using the lyophilized composition, the composition being reconstituted with a low salt aqueous liquid, for example, water for injection or an aqueous solution of a nonionic isotonizing agent.
    It has further been found that the inclusion of trehalose, an amorphous sugar, as a cryoprotectant, has other favorable effects on the stability of simian adenoviral vector particles. The invention therefore relates to the aqueous mixture and the dry composition comprising trehalose amorphous sugar or a combination of trehalose with another amorphous sugar as a cryoprotectant.
    In a second aspect, the invention relates to a method of freeze-drying adenoviral vector compositions using an annealing step in the freezing phase, thereby substantially increasing the stability of the adenoviral particles during freeze drying.
    DESCRIPTION OF THE FIGURES
    Figure 1 illustrates the cryodessiccation cycle used in Example 1.
    Figure 2 illustrates the cryodessiccation cycle used in Example 2.
    Figure 3 illustrates the dynamic light scattering (DLS) data as obtained in Experiment 4: Panel I: Composition (a); panel II - composition (b); panel III - composition (c).
    Figure 4 illustrates the PicoGreen® data as obtained in Experiment 5: o - water for injection (EPI), x - 30 mM NaCl, □ - 150 mM NaCl, 0 - 9,25% sucrose, Δ - trehalose 9.25%; Panel I - at T1m4; Panel II - at T2m4;
    Figure 5 illustrates the cryodessiccation cycle used in Example 6.
    FIG. 6 illustrates the PicoGreen® data as obtained in experiment 6: Δ - control adenoviral mother solution, V - negative control degraded adenoviral mother solution, x - data obtained with samples obtained by the cryodessiccation cycle I, o - data obtained with samples obtained by the cryodessiccation cycle II.
    FIG. 7 illustrates the infectivity data as obtained in example 6: Δ - control adenoviral mother solution, V - negative control degraded adenoviral mother solution, x - data obtained with samples obtained by the cryodessiccation cycle I, o - data obtained with samples obtained by the cryodessiccation cycle II,
    FIG. 8 illustrates the data of PicoGreen® and FIG. 9 illustrates the infectivity data as obtained in experiment 7: Δ-control adenoviral mother solution, V -deenoviral mother solution degraded negative control, + - data obtained in FIG. using freeze drying cycle with freezing sequence at -52 ° C with annealing at -10 ° C (3 hours) / primary drying -30 ° C / secondary drying +10 ° C (6 h + 6 h), x - data obtained using a cryodessiccation cycle with the freezing sequence at -52 ° C without annealing / primary drying -30 ° C / secondary drying +10 ° C (6 h + 6 h), 0 - data obtained using a cryodessiccation cycle having the slow freezing sequence at 0.5 ° C / min without annealing / primary drying -30 ° C / secondary drying +10 ° C (6 h + 6 h), o - data obtained using a freeze drying cycle having the sequence freezing at -52 ° C with annealing at -10 ° C (2 hours) / primary drying 30 ° C / secondary drying +10 ° C (6 h + 6 h).
    DETAILED DESCRIPTION
    In contrast to the reports in the art relating to the formulation of adenoviral vectors, the inventors have found that the stabilization formulations developed, for example, for human adenoviral vectors can not be successfully applied to all adenoviral vectors, for example. for example, 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 preserved.
    The new formulation allows the storage of the composition, liquid or dry, 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 dry composition can be stored at 4 ° C for 3 years, at 25 ° C for 3 months or at 37 ° C for 1 month. It will be 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 retained, compared to the infectivity of the starting material.
    The mixtures, compositions and methods described herein 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 maintaining 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 things, be determined by measuring the infectivity of the vector, for example, the retention of infectivity during manipulation (for example, freeze drying) or the storage of the viral vector. The term "infectivity" refers to the ability of the vector to enter a susceptible host, in other words, the cells, and to administer its genetic material for it to be expressed by the host. Infectivity can be expressed as the "50% Cell Culture Infectious Dose" (CCID50), which is the amount of adenoviral vector needed to infect 50% of the cells in a given cell culture. Infectivity can be measured by measuring the proportion of cells in which an adenoviral transgene is expressed. For example, the green fluorescent protein can be used as an infectivity marker, allowing the number of cells expressing the green fluorescent protein to be determined after 24 hours of incubation with the vector. Alternatively, infectivity can be measured by determining the number of cells expressing hexon, adenovirus capsid protein, after 24 hours of incubation with the vector. Adenovirus has been widely used in gene transfer applications because of its ability to achieve highly efficient gene transfer in a variety of target tissues and high transgene capacity. The adenoviral vectors for use in the present invention may be derived from a range of mammalian hosts. More than 100 distinct adenovirus serotypes have been isolated that infect different mammalian species. These adenoviral serotypes have been categorized into six subgenera (A - F, B is subdivided into B1 and B2) based on sequence homology and their ability 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 these adenoviruses of human origin are Ad1, Ad2, Ad4, Ad5, Ad6, Ad11, Ad24, Ad34, Ad35, in particular Ad5, Ad11 and Ad35. Although Ad5-based vectors have been widely used in a number of gene therapy assays, there may be limitations to the use of Ad5 and other adenoviral C-group vectors due to immunity. preexisting in the general population related to a natural infection. Ad5 and other members of human group C tend to be among the most seroprevalent serotypes. In addition, immunity against existing vectors may develop as a result of exposure to the vector during treatment. These types of pre-existing or developed immunity against seroprevalent vectors may limit the effectiveness of gene therapy or vaccination efforts. Variants of adenoviral serotypes thus constitute very important targets in the search for a system of administration of genes capable of evading the immune response of the host.
    Therefore, in another embodiment, the adenoviral vector of the present invention is derived from a non-human simian adenovirus, which is also simply referred to as a simian adenovirus. Many adenoviruses have been isolated from non-human monkeys such as chimpanzees, bonobos, rhesus macaques and gorillas, and the vectors derived from these adenoviruses induce strong immune responses against the transgenes encoded by these vectors (Colloca et al., (2012) Sci., Transl Med.4: 1-9, Roy et al (2004) Virol.324: 361372, Roy et al (2010) J. of Gene Med 13: 17-25). Some advantages of vectors based on non-human simian adenoviruses include the relative absence of antibodies that can induce cross-neutralization against these adenoviruses in the target population. For example, the cross-reactivity of some chimpanzee adenoviruses with pre-existing neutralizing antibody responses is only present in 2% of the target population compared to 35% for some candidate human adenovirus vectors.
    In specific embodiments, the adenoviral vector is derived from a non-human adenovirus, for example a simian adenovirus and in particular a chimpanzee adenovirus such as ChAd3, ChAd63, ChAd8 3, ChAd155, Pan5, Pan6, Pan7. (Also referred to as C7) or Pan 9. Examples of such strains are described in WO03 / 000283, WO2010 / 086189 and GB1510357.5 and are also available from American Type Culture Collection, 10801 University Boulevard. , Manassas, Virginia 20110-2209, and other sources. Alternatively, the adenoviral vectors may be derived from non-human simian adenoviruses isolated from bonobos, such as PanAd1, PanAd2 or PanAd3. Examples of these vectors described herein may for example be found in WO2005 / 017093 and WO2010 / 086189. The adenoviral vectors may also be derived from adenoviruses isolated from gorillas as described in 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 a button fiber (IV), together with a number of other minor proteins, VI , VIII, IX, IIIa and IVa2. Hexon represents the majority of the structural components of the capsid, which consists of 240 trimeric hexomer capsomer and 12 penton bases. The hexon has three double rolls preserved, while the top has three turns, each turn containing a loop of each subunit that forms most of the capsid. The hexon base is highly conserved between adenoviral serotypes, while surface loops are variable (Tatsis and Ertl Molecular Therapy (2004) 10: 616-629). Penton is another protein in the adenoviral capsid that forms a pentamer base to which the fiber binds. The trimer fiber protein protrudes from the base of the penton at the 12 peaks of the capsid and is a rod-like structure. The main role of the fiber protein is anchorage of the viral capsid to the cell surface via the interaction of the button region with a cellular receptor, and variations in the flexible stem as well as the button regions of the fiber are a feature of the different serotypes (Nicklin et al., Molecular Therapy 2005 12: 384-393).
    The adenoviral vectors can be used to deliver the desired RNA or protein sequences, e.g., heterologous sequences, for in vivo expression. A vector may comprise any genetic element comprising 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 other regulatory elements necessary to direct the translation, transcription and / or expression of the gene product in a host cell.
    Typically, an adenoviral vector is designed such that the expression cassette is in a nucleic acid molecule that contains other adenoviral sequences in the native region of a selected adenoviral gene. The expression cassette may be inserted into an existing gene region to disrupt the function of that region, if desired. Alternatively, the expression cassette may be inserted into the site of an adenovirus gene partially or completely deleted. For example, the expression cassette may be at the site of a mutation, insertion or deletion that renders at least one gene of a genomic region selected from the group consisting of E1A, E1B non-functional. , E2A, E2B, E3 and E4. The term "renders non-functional" means that a sufficient amount of the gene region is suppressed or otherwise disrupted, so that the gene region is no longer capable of producing functional gene expression products. If desired, the entire gene region may be removed (and appropriately replaced by the expression cassette). Suitably, the adenovirus E1 genes are deleted and replaced by an expression cassette consisting of the selected promoter, a cDNA sequence of the gene of interest and a poly A signal, resulting in a recombinant virus. deficient for replication.
    In one embodiment, the transgene encoded by the adenoviral vector is a coding sequence for a product that is useful in biology and medicine, for example, therapeutic or immunogenic proteins, RNA, enzymes, or catalytic RNAs. Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, RNA aptamers, and antisense RNAs. An example of a useful RNA sequence is a sequence that disables the expression of a target nucleic acid sequence in the treated animal.
    Thus, in one embodiment, the mixture or composition as described herein is for use in the prophylactic (thus immunogenic or preventive) or therapeutic treatment of a subject, such as a mammal or subject human, depending on the transgene encoded by the adenoviral vector.
    The transgene may encode a polypeptide or protein used for the treatment, for example, of genetic deficiencies, as a therapeutic product or cancer vaccine, for the induction of an immune response, and / or for the purpose of prophylactic vaccination. As used herein, the term induction of an immune response refers to the ability of a protein, also known as an "antigen" or "immunogen," to induce an immune response of T-cells. and / or humoral to the protein.
    Immunogens expressed by the adenoviral vectors formulated as described herein and which are useful for immunizing a human or a non-human animal against other pathogens include, for example, bacteria, fungi, parasitic microorganisms or multicellular parasites that infect human and non-human vertebrates, or a cancer cell or tumor cell. For example, immunogens can be selected from a variety of viral families.
    In one embodiment, the immunogen is derived from a filovirus, for example Ebola virus (Zaire, Sudan, Reston, Budibugyo and Ivory Coast) or Marburg. These antigens can be derived from the viral glycoprotein (transmembrane and / or secreted form) and / or the viral nucleoprotein. Examples of vectors may be encountered, inter alia, in WO2011 / 130627.
    In another embodiment, the immunogens may be selected from respiratory viruses such as respiratory syncytial virus (RSV) and other paramyxoviruses such as human metapneumovirus, hMPV and parainfluenza viruses (PIV). Suitable RSV antigens that are useful as immunogens for immunizing a human or a non-human animal can be selected from: fusion protein (F), binding protein (G), matrix protein (M2) and the nucleoprotein (N). These vectors are described in documents WO2012 / 089833 and PCT / EP2016 / 063297. In one embodiment, the ChAd155-RSV construct as described in PCT / EP2016 / 063297 is considered for the described compositions and methods.
    In another embodiment, the immunogen may be from a retrovirus, for example a lentivirus such as human immunodeficiency virus (HIV). In such an embodiment, the immunogens may be derived from HIV-1 or HIV-2 sequences, such as, for example, Gag, Pol, Nef, Env, and others. These vectors are described, inter alia, in GB1510357.5 and WO2008 / 107370.
    Alternatively or additionally, a transgene sequence may include a reporter sequence, which under the effect of the expression produces a detectable signal. These reporter sequences include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase ( CAT), luciferase, membrane-bound proteins including, for example, CD2, CD4, CD8, influenza hemagglutinin protein, and others well known in the art, against which high affinity antibodies exist or can produced by conventional means, and the fusion proteins comprising a membrane-bound protein appropriately fused to an antigen tag domain from, inter alia, haemagglutinin or Myc. These coding sequences, when associated with regulatory elements that direct their expression, provide detectable signals by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectroscopic assays, FACS cell sorting ( fluorescent activating cell sorting) and immunoassays, including an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), and immunohistochemistry.
    In addition to the transgene, the expression cassette may also comprise conventional control elements that are operably linked to the transgene in a manner that allows its transcription, translation, and / or expression in a cell transfected with the adenoviral vector. As used herein, the term "operably linked" sequences includes both expression control sequences that are contiguous with the gene of interest and control sequences of the gene. expression that act in trans or at a distance to regulate the gene of interest.
    Expression control sequences include appropriate transcription initiation, transcription termination, promoter and amplification sequences; effective RNA processing signals such as splicing and polyadenylation (polyA) signals including rabbit beta-globin polyA; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (for example, the Kozak consensus sequence); sequences that enhance the stability of proteins; and if desired, sequences that enhance the secretion of the encoded product. Among other sequences, chimeric introns can be used.
    A "promoter" is a nucleotide sequence that allows the binding of RNA polymerase and directs the transcription of a gene. Typically, a promoter is in the 5 'non-coding region of a gene near the start site of gene transcription. The elements of the sequence within promoters that function in transcription initiation are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters of bacteria, yeast, plants, viruses, and mammals (including humans). A large number of expression control sequences, including promoters that are internal, native, constitutive, inducible and / or tissue specific, are known in the art and can be used.
    Adenoviral vectors are generated by the modification of wild-type adenovirus to express heterologous genes (transgenes) and / or to delete or inactivate undesirable adenoviral sequences. Adenoviral vectors may also have modified replication competence. For example, the vector may be deficient for replication or have limited replication so that it has reduced replication capacity in non-complementing cells, compared to wild-type virus. This can be caused by the mutation of the virus, for example by deletion of a gene involved in replication, for example by deletion of the gene E1a, E1b, E3 or E4. These modifications are well known to those skilled in the art and are 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 in WO03 / 000283.
    These vectors are generated using techniques known to those skilled in the art. These techniques include conventional cDNA cloning techniques such as those described in the texts, the use of overlapping oligonucleotide sequences of adenoviral genomes, the polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence. . Particularly suitable methods include standard 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. of Gene Med. 13: 17-25; and in WO2010 / 085984 or homologous 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 replication. In particular, complementation cell lines that provide the missing factors in the viral vector that result in its deteriorated replication characteristics (such as E1) can be used. Without limitation, such a cell line may be HeLa cells (ATCC accession number CCL 2), A549 (ATCC access number CCL 185), HEK 293, KB (CCL 17), Detroit (for example , Detroit 510, CCL 72) and WI-38 (CCL 75), among others. These cell lines are 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, for example PER.C6 ™ cells, as represented by the cells deposited under ECACC No. 96022940 in the European Collection of Animal Cell Cultures (ECACC). at the Center for Applied
    Microbiology and Research (WARC, UK) or Her 96 cells (Crucell).
    A particularly suitable complementation cell line is the Procell92 cell line. The Procell92 cell line is based on HEK 293 cells that express the adenoviral E1 genes, transfected with the Tet repressor under the control of the phosphoglycerate kinase 1 (PGK) promoter, and the G418 resistance gene.
    (Vitelli et al., PLOS One (2013) 8 (e55435): 1-9). Procell92.S is suitable for growth under suspension conditions and is also useful for the production of adenoviral vectors expressing toxic proteins (www.okairos.com/e/inners.php m=00084, last accessed on April 13, 2015 ).
    ADENOVIRAL DISTIBUTION METHOD AND ASSAY
    A mixture or composition as described herein comprises one or more recombinant vectors capable of inducing an immune response, for example a humoral response (e.g. antibodies) and / or a cell-mediated response (e.g. cytotoxic T lymphocytes), against a transgene product administered by the vector after administration to a mammal, suitably a human. A recombinant adenovirus may comprise (appropriately in any of its gene deletions) a gene encoding a desired immunogen and may therefore be used in a vaccine. The recombinant adenoviruses can be used as prophylactic or therapeutic vaccines against any pathogen for which the antigen (antigens) critical for the induction of an immune response and capable of limiting the spread of the pathogen has (have) been identified and for which the cDNA is available.
    Thus, in one embodiment, the mixture and / or composition described herein is for use in immunizing a subject, such as a human subject. The immunity levels of the selected gene can be monitored to determine whether or not reminders are needed. After evaluating the antibody titre in the serum, optional booster immunizations may be desired.
    Optionally, a mixture or composition of the invention may be formulated to contain other components, including, for example, adjuvants, stabilizing agents, pH adjusting agents, preservatives and the like. Such an adjuvant can be administered with a primo-immunization DNA vaccine encoding an antigen to enhance the antigen-specific immune response compared to the immune response generated during priming with a DNA vaccine encoding the antigen only . Alternatively, such an adjuvant may be administered with a polypeptide antigen that is administered in a delivery regime involving the adenoviral vectors of the invention.
    In some embodiments, the mixture or composition as described herein is administered to a subject by intramuscular injection, intravaginal administration, intravenous injection, intraperitoneal injection, subcutaneous injection, epicutaneous administration, intradermal administration, nasal administration or oral administration.
    If the therapeutic regimen involves the co-administration of one or more adenoviral vectors and / or another component, these may be co-formulated (in other words, in the same mixture or composition) or may be each formulated in different compositions. When formulated separately, they are favorably administered co-locally at the same site or nearby. For example, the components may be administered (e.g., via a route of administration selected from the intramuscular, transdermal, intradermal, subcutaneous route) to the same side or end ("co-lateral" administration) or opposite sides or ends ("contralateral" administration).
    The viral vector assays will depend mainly on factors such as the condition being treated, the age, the weight and the health of the patient, and may thus vary from one patient to another. For example, a therapeutically effective dose for a human or veterinary adult of the viral vector generally contains 1 x 10 5 to 1 x 10 15 virus particles, for example 1 x 10 8 to 1 x 10 12 (e.g. x 108, 1x109, 5x109, 1x1010, 2.5x1010, 5x1010, 1x1011, 5x1011, 1x1012 particles). Alternatively, a viral vector can be administered at a dose that typically ranges from 1 x 105 to 1 x 1010 plaque forming units (PFU), for example 1 x 105 PFU, 5 x 105 PFU, 1 x 106 PFU, 5 x 106 UFP, 1x107 PFU, 5x107 PFU, 1x108 PFU, 5x108 PFU, 1x109 PFU, 5x109 PFU, or 1x1010 PFU. Doses will vary depending on the size of the animal and the route of administration. For example, a suitable human or veterinary dose (for an animal of about 80 kg) for intramuscular injection is in the range of about 1 x 10 9 to about 5 x 10 12 particles per mL, for a single site. Optionally, the administration can be practiced at multiple sites of administration. In another example, a suitable veterinary or human dose may be in the range of about 1 x 10 11 to about 1 x 10 15 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 the CMV promoter region using as standard curve a serial dilution of plasmid DNA. containing the genome of the vector with the expression cassette comprising the HCMV promoter. The number of copies in the test sample is determined by the parallel series analysis method. Alternative methods of quantifying vector particles may be an analytical HPLC method or a spectrophotometric method based on A260 nm.
    An immunologically effective amount of nucleic acid may conveniently be between 1 ng and 100 mg. For example, an appropriate amount may be from 1 μg to 100 mg. An appropriate amount of the particular nucleic acid (e.g., vector) can be readily determined by one skilled in the art. Examples of effective amounts of a nucleic acid component may be between 1 ng and 100 μg, for example between 1 ng and 1 μg (for example, 100 ng-1 μg), or between 1 μg and 100 μg, for example 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 250 ng, 500 ng, 750 ng, or 1 μg. Effective amounts of a nucleic acid may also comprise from 1 μg to 500 μg, for example from 1 μg to 200 μg, for example from 10 to 100 μg, for example 1 μg, 2 μg, 5 μg, 10 μg, 20 μg. pg, 50 μg, 75 μg, 100 μg, 150 μg, or 200 μg. Alternatively, an example of an effective amount of a nucleic acid may be between 100 μg and 1 mg, for example 100 μg to 500 μg, for example, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg or 1 mg. Generally, a human dose will be contained in a volume of between 0.5 mL and 2 mL. Thus, the mixture and / or composition described herein may be formulated such that a volume of, for example, 0.5, 1.0, 1.5 or 2.0 mL of human dose per individual or combined immunogenic component is administered.
    One 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. Levels of transgene expression, or for an adjuvant, the level of circulating antibodies, can be monitored to determine the frequency of dosing.
    If one or more primary-immunization and / or booster steps are used, this step may include a single dose that is administered on an hourly, daily, weekly or monthly, or yearly basis. For example, mammals may receive one or two doses containing from about 10 μg to about 50 μg of plasmid in a vector. The amount or site of administration is desirably selected based on the mammalian identity and condition.
    Therapeutic levels of, or the level of immune response against, the protein encoded by the selected transgene can be monitored to determine the need, if any, for recalls. Following evaluation of the CD8 + T cell response, or optionally antibody titers, in the serum, optionally optional booster immunizations may be desired. Optionally, the adenoviral vectors may be administered in a single administration or in different combination regimens, for example, in combination with a therapeutic regimen or a treatment involving other active ingredients or in a regimen comprising a primary immunization and a booster.
    The inventors have discovered that the adenoviral vectors can be substantially impacted by the presence of salt, such as sodium chloride, in dry form or in liquid form. The present invention thus relates to formulations, in other words liquid mixtures and dry compositions, taking into account the sensitivity of adenoviral vectors to salt, such as sodium chloride. In one embodiment, simian adenoviral vectors are formulated using the liquid mixtures and compositions described herein.
    The term "salt" as used refers to the ionic compounds that result from the acid and base neutralization reaction, composed of a related number of cations and anions so that the product is without any net charge, eg sodium chloride. The component ions may be inorganic or organic, and may be monoatomic or polyatomic.
    Therefore, according to one embodiment, the amount of salt, especially the amount of NaCl, present in the aqueous mixture is defined to be less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, lower at 15 mM, less than 10 mM, or less than 7.5 mM. Preferably, the composition is not completely salt-free or completely free of sodium chloride. Therefore, according to one embodiment of the invention, the salt, in particular sodium chloride, is present in an amount of at least 0.5 mM, at least 1 mM, of at least 2 mM , at least 3 mM, or at least 4 mM. Alternatively, 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 about 5 mM.
    For purposes of defining ranges, the term "between" as used in this document is considered to include the ends of the range. For example, when sodium chloride is said to be present in an amount of 2.5 to 10 mM, formulations in which NaCl is present at a concentration of 2.5 mM or 10 mM are included.
    According to other embodiments, also the salt content, such as sodium chloride, of the aqueous liquid for the reconstitution of the dry composition is defined. 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.
    The aqueous liquid for reconstitution of the lyophilized composition may be substantially free of salt, for example substantially free of sodium chloride. By substantially free, it is meant that the concentration of salt or sodium chloride is 0 mM or very close to 0 mM.
    In another embodiment, the aqueous liquid for reconstitution of the composition is not completely free of salt or sodium chloride. Accordingly, the salt, such as sodium chloride, may be present in the aqueous liquid used for reconstitution of the dry composition in an amount of at least 0.5 mM, at least 1 mM, minus 2 mM, at least 3 mM, or at least 4 mM. Alternatively, the salt, such as sodium chloride, is present in the aqueous liquid used to reconstitute the composition in an amount of 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 salt, such as sodium chloride, is present in the aqueous liquid used to reconstitute the composition in an amount of 5 mM. The invention thus also relates to a method of using the dry composition as described herein, wherein the dry composition is reconstituted with an aqueous liquid to reconstitute the composition as defined herein.
    The term "cryoprotectant" refers to a class of excipients which prevents alterations related to the freezing of the product that is frozen, in this case, the adenoviral vector.
    A cryoprotectant suitable for use in the present invention is an amorphous sugar, for example selected from sucrose, trehalose, mannose, mannitol, raffinose, lactitol, sorbitol and lactobionic acid, glucose, maltulose , iso-maltulose, lactulose, maltose, lactose, isomaltose, maltitol, palatinit, stachyose, melezitose, dextran, or a combination thereof. In one embodiment, the cryoprotectant is an amorphous sugar selected from sucrose, trehalose, lactose, raffinose, dextran, mannitol, and combinations thereof.
    In a specific embodiment, the cryoprotectant or the amorphous sugar is trehalose, sucrose or trehalose in combination with a second amorphous sugar, for example selected from sucrose, lactose, raffinose, dextran and mannitol. Alternatively, the cryoprotectant is trehalose, sucrose or a combination of sucrose and trehalose. In another embodiment, the cryoprotectant is trehalose or trehalose in combination with sucrose. In yet another embodiment, the cryoprotectant is trehalose.
    The cryoprotectant as selected according to the embodiments in the present invention may be present in defined amounts. In one embodiment, the aqueous mixture contains at least 2.5% (w / v), at least 3% (w / v), at least 3.5% (w / v), at least 4% (w / v). v), at least 4.5% (w / v), at least 5% (w / v), or at least 6% (w / v) of the cryoprotectant as selected above. In another embodiment, the cryoprotectant is present in the aqueous mixture in a total amount of less than 17.5% (w / v), for example less than 15% (w / v), less than 12.5% ( p / v), less than 11% (w / v), less than 10% (w / v), or less than 9.5% (w / v). In other words, the cryoprotectant is present in the aqueous mixture in a total amount of at least 4%, at least 4.5% or at least 5% (w / v), but less than 15%, less than at 12.5%, less than 11% or less than 10% (w / v).
    The total concentration of cryoprotectant in the aqueous mixture is suitably in the range of 5 to 10% (w / v). In one embodiment, at least 5%, between 5 and 15% or between 5 and 10% (w / v) of trehalose are used. In one embodiment, 8%, 8.5%, 9% or 9.25% trehalose is used. In specific embodiments, the aqueous mixture comprises at least 5% (w / v) or between 5 and 10% (w / v) of sucrose, trehalose or a combination thereof. In another specific embodiment, the aqueous mixture comprises at least 5% (w / v) trehalose, optionally further comprising sucrose, lactose, raffinose, dextran and mannitol.
    The aqueous mixture or the dry composition may further comprise a surfactant selected from poloxamer surfactants (e.g., poloxamer 188), polysorbate surfactants (e.g., polysorbate 80 and / or polysorbate 20), octoxidic surfactants, polidocanol surfactants, polyoxylated stearate surfactants, polyoxy castor oil surfactants, N-octyl glucoside surfactants, macrogol 15 hydroxystearate, and combinations thereof. In one embodiment, the surfactant is selected from poloxamer surfactants (e.g., poloxamer 188), polysorbate surfactants (e.g. polysorbate 80 and / or polysorbate 20), particularly 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 at most 0, 5% (w / v) as calculated for the aqueous mixture. The surfactant may be present in an amount 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 of between 0.005% and 0.5% (w / v), for example about 0.02% (w / w). v).
    In another embodiment, a buffer is added to the aqueous mixture or dry composition. The pH is typically adjusted according to 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. In other words, the pH of the aqueous mixture can 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 about 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 may be selected from Tris, succinate, borate, tris-maleate, lysine, histidine, glycine, glycylglycine, citrate, carbonate or combinations thereof. In one embodiment, the buffer is Tris, succinate or borate. In another embodiment, the buffer is Tris.
    The buffer may 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. The buffer may otherwise 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 may be present in an amount of 0.5mM to 50mM, 1mM to 50mM, or 2mM to 20mM. 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 of between 2 and 20 mM, for example about 10 mM.
    In one embodiment, the composition also comprises histidine in an amount of at most or about 20 mM, for example at a concentration of about 10 mM.
    According to other embodiments, the composition also comprises divalent metal ions, for example Mg2 +, Ca2 + or Mg2 + in the form of a salt, for example MgCl2, CaCl2 or MgSCg. In one embodiment, the divalent metal ion is Mg 2+. Typical amounts in which the divalent metal ions are present in the aqueous mixture are between 0.5 and 10 mM, for example 1 or 2 mM, or especially 1 mM.
    For purposes of describing the embodiments of the invention, the specified amounts of the excipients considered for inclusion in the composition (in other words, salt, sodium chloride, cryoprotectant, buffer, surfactant and others described herein) are typically (and unless otherwise specified) expressed in% w / v calculated relative to the volume of the aqueous mixture. Alternatively, 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 one embodiment, the aqueous mixture and / or the (freeze-dried) compositions described herein may be administered to a mammal, for example, to a human subject. In particular, mixtures or compositions comprising an adenoviral vector encoding a transgene (in other words, a recombinant adenoviral vector) which is a therapeutic or immunogenic protein are taken into consideration for their formulation in the aqueous mixture or the freeze-dried compositions described. in this document.
    The aqueous mixture or the dry composition may be contained in a glass bottle, siliconized or non-siliconized. In one embodiment, the aqueous mixture or dry composition is provided in a non-siliconized flask. Suitably, the aqueous mixture may be contained in a non-siliconized and freeze-dried vial when contained in this vial. The invention also relates to a process for freeze drying a liquid containing an adenoviral vector, for example the aqueous mixture as defined herein, to obtain a dry composition, comprising an annealing step. The cryodessiccation cycle is generally composed of three process phases. In the first phase of the process, a predominantly aqueous solution or mixture is frozen. Then, water is first removed by sublimation during primary drying. In the third phase, unfrozen water is removed by diffusion and desorption during secondary drying. The inventors have now discovered that the introduction of an annealing step during the freezing phase of the cryodessiccation cycle has an unexpected positive impact on the stability of the adenoviral vector.
    Accordingly, the invention also relates to a method of freeze drying a liquid containing an adenoviral vector, for example the aqueous mixture as described herein, the freezing step of the cryodessiccation cycle thus comprising an annealing step.
    For purposes of defining the described method, the following terms are used as known in the art. The term "glass transition temperature" or "Tg" is the temperature at which an amorphous solid becomes soft under the effect of heating or friable under the effect of cooling. The term "Tg" refers to the glass transition temperature in the frozen state. The term "collapse temperature" or "Tc" refers to the temperature at which an amorphous material softens to the point that it can no longer support its own structure. The terms "freeze-drying" and "freeze-drying" and "freeze-dried" and "freeze-dried" are used interchangeably and refer to the same process of rapid freezing of a wet substance, followed by dehydration under reduced pressure.
    The term "annealing step" as used herein refers to a process step in the freeze-drying cycles of a composition, wherein during the freezing phase, the product is maintained at a temperature below the freezing point for a predetermined time. As known to those skilled in the art, annealing will lead to Oswald's maturation of ice crystals and cryoconcentration of the amorphous matrix. Typically, the annealing temperature is (slightly) higher than the 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). Be that as it may, the annealing temperature should be between Tg 'and the melting temperature (Tm) during annealing. In specific embodiments, the 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. Annealing may be performed during freezing of the product, in other words when the frozen sample is being formed, provided the product is frozen (solid state) and in a vitreous state (below the Tg '). Alternatively, the annealing is performed after freezing the product.
    In a specific embodiment, the annealing temperature is about -10 ° C (for example, -10 ° C ± 1 ° C), more particularly when the aqueous mixture comprises about or at least 9% (w / v). ) of trehalose.
    In one embodiment, the product is frozen (in other words, the product temperature is below Tg ') before the annealing step. In one embodiment, the freezing is performed by exposing the sample or aqueous mixture to a constant shelf temperature at a freezing temperature that is less than Tg '. In an alternative embodiment, the product may be frozen by applying progressive freezing on the shelf, in other words by gradually reducing the temperature of the shelf to a freezing temperature below Tg. . According to embodiments, the freezing temperature is a temperature below Tg 'minus 5 ° C, lower than Tg' minus 7.5 ° C, or lower than Tg 'minus 10 ° C, for example de - 50 ° C or less. According to one embodiment, the temperature of the product (in other words, the temperature of the sample in the freeze dryer) at the beginning of the freeze-drying cycle is between + 2 ° C and + 8 ° C.
    When applying a freeze by gradually reducing the temperature of the shelf, the temperature is reduced to a rate of at least 0.1 ° C / minute, at least 0.2 ° C / minute at least 0.3 ° C / minute or at least 0.5 ° C / minute, and / or at a rate below 10 ° C / min, 7.5 ° C / min, 5 ° C / minute, or less than 3 ° C / minute. Alternatively, the temperature is reduced at a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / minute, 0.2 to 3 ° C / minute, or 0.3 to 1 ° C / minute. According to other embodiments, the temperature of the shelf reached is maintained for about or at least 1 hour (or 60 minutes).
    In another embodiment in the situation where the product is frozen prior to the application of the annealing step, after initial freezing of the sample or product, the temperature of the shelf is raised to a higher temperature. at Tg 'to initiate the annealing step, for example at a temperature above Tg' plus 0.5 ° C, greater than Tg 'plus 1 ° C, greater than Tg' plus 3 ° C, higher at Tg 'plus 5 ° C, greater than Tg' plus 10 ° C, or greater than Tg 'plus 20 ° C. In any case, the temperature is kept lower than the Tm during annealing. In one embodiment, the temperature is increased at a rate of at least 0.1 ° C / minute, at least 0.2 ° C / minute, at least 0.3 ° C / minute or higher. at least 0.5 ° C / minute, and / or at a rate below 10 ° C / min, 7.5 ° C / min, 5 ° C / min or less than 3 ° C / min. Alternatively, the temperature is increased at a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / minute, 0.2 to 3 ° C / minute, or 0.3 to 1 ° C / minute. In other embodiments, the annealing temperature is maintained for at least 2 and / or up to 4 hours.
    In another embodiment, after the annealing step, the shelf temperature is reduced to a temperature below Tg 'before the initiation of drying under reduced pressure, for example at a temperature below Tg' minus 5 ° C, less than Tg 'minus 7.5 ° C, or less than Tg' minus 10 ° C, for example -50 ° C or less. In one embodiment, to achieve this, the temperature is reduced at a rate of at least 0.1 ° C / minute, at least 0.2 ° C / min, at least 0.3 ° C / minute or at least 0.5 ° C / minute, and / or at a rate of less than 10 ° C / minute, less than 7.5 ° C / minute, less than 5 ° C / minute or less than 3 ° C / minute. Alternatively, the temperature is reduced at a rate of 0.1 to 10 ° C / min, 0.1 to 5 ° C / minute, 0.2 to 3 ° C / minute, or 0.3 to 1 ° C / minute. According to other embodiments, the temperature of the shelf reached is maintained for about or at least 1 hour (or 60 minutes).
    Drying under reduced pressure as envisaged in step b.ii. The cryodessiccation process described herein will typically be conducted in two phases, in other words primary drying and secondary drying. In one embodiment, step b.ii. of the process will include: - step b.ii.1. primary drying at a temperature below the Tc of the product, and - step b.ii.2. secondary drying at a temperature above the Tc of the product and lower than the Tg of the product.
    In another embodiment, the primary drying is carried out at a pressure of less than 90 pbar and / or greater than 40 pbar. Primary drying conditions can be applied for up to 24 hours or more.
    Another embodiment relates to the secondary drying temperature which is achieved by increasing the temperature of the shelf at a rate of 0.1 ° C / minute, at least 0.2 ° C / minute, from minus 0.3 ° C / minute or at least 0.5 ° C / minute, and / or at a rate of less than 3 ° C / minute, less than 2 ° C / minute, or less than 1 ° C / minute. Alternatively, the secondary drying temperature is attained by increasing the temperature of the shelf at a rate of 0.1 to 3 ° C / min, 0.2 to 2 ° C / min, or 0.3 to 1 ° C / minute. According to yet another embodiment, the secondary drying temperature is at least -10 ° C and / or less than 25 ° C. The secondary drying conditions may be applied for at least about 3 hours, at least 4 hours, at least 5 hours, or at least about 6 hours.
    The present invention will now be described in more detail by means of the following non-limiting examples.
    EXAMPLES
    Example 1 The objective of the experiment was to evaluate the impact of an annealing step in the cryodessiccation cycle on the ChAd3 titer expressing green fluorescent protein (GFP). ChAd3 particles were formulated in an aqueous mixture further comprising 10 mM Tris excipients (pH 7.4) - 10 mM histidine - 1 mM MgCl2.6H2O - 0.02% Tween 80 (m / V) - 25 mM NaCl - 8% sucrose (m / V). The concentration of virus particles was 2.5 x 1010 pv / mL. After the formulation step, non-siliconized 3 mL type glass vials were filled using 0.5 mL ± 0.05 of the aqueous mixture. The vials were then partially closed with a bromobutyl stopper
    Helvoet FM460 inserted in cryodessiccation position (partially inserted to allow water vapor to escape during the freeze drying cycle). Half of the samples were transferred to the freeze dryer chamber and subjected to the freeze drying cycle including an annealing step (as shown in FIG. 2) and which consisted of the following steps: 1. Freezing: • the temperature of the shelf has been set at -52 ° C. The filled vials were loaded into the freeze dryer when the shelf temperature was -45 ° C or lower. The samples were then cooled to -52 ° C for a minimum of 1 hour. 2. Annealing step: • (1) The shelf temperature was increased to reach the target annealing temperature (-15 ° C) in one hour. (2) The annealing temperature was maintained for 2 hours. • (3) The shelf temperature was again reduced from -15 ° C to -50 ° C in one hour. (4) The product was held at -50 ° C for at least 1 hour. 3. Primary Drying: • The chamber pressure was set at 80 pbar and the shelf temperature was increased from -52 ° C to -30 ° C over 3 hours. The temperature of the shelf and the pressure of the chamber were maintained for 24 hours. 4. Secondary drying: • The shelf temperature was increased from -30 ° C to 17 ° C over 6 hours, while the chamber pressure was reduced to 40 pbar. When the shelf temperature reached 17 ° C, these conditions were maintained for 6 hours. At the end of the freeze-drying cycle, the chamber was filled with anhydrous nitrogen until a chamber pressure of 825 mbar was reached, then the plugs were fully inserted into the vials (closure). After closing, the pressure of the atmospheric pressure chamber was equilibrated to discharge. The temperature of the chamber was maintained at +2 to + 8 ° C until the vials were discharged. The flasks were then discharged and sealed with removable aluminum capsules.
    In order to evaluate the impact of the annealing step, the second half of the filled vials were loaded into the cryodessicator chamber between step 2 (3) and step 2 (4) of the cryodessiccation cycle. .
    The results of this experiment are presented in Table 1 below:
    Quantitative PCR (qPCR) as reported herein is used to determine the virus content. The test targets the hCMV promoter present in the adenovirus. The DNA sample was extracted with Quiagen QIAmp 96 DNA Blood. The PicoGreen® test measures the degradation of virus particles. Quant-iTTM PicoGreen® DNA Reagent is an ultra-sensitive nucleic acid fluorescent dye for quantifying dsDNA in solution. Infectivity is determined on the basis of the amount of the expressed transgene, which in this example is GFP. The assay will measure cells expressing GFP after 24 hours of infection using flow cytometric detection.
    Example 2
    The objective of this experiment was to evaluate the impact of an annealing step in the cryodessiccation cycle on the ChAd3 titre expressing eGFP. The ChAd3 particles were formulated in an aqueous mixture further comprising 10 mM Tris (pH 7.4) - 10 mM histidine - MgCl2.6H2O 1 mM -Tween 80 0.02% (w / v) - 25 mM NaCl excipients. - sucrose 8% (w / v)
    The concentration of virus particles was 2.5 x 1010 pv / mL. After the formulation step, non-siliconized 3 mL type glass vials were filled using 0.5 mL ± 0.05 of the aqueous mixture. The flasks were then partially closed with a Helvoet FM460 bromobutyl stopper inserted in the freeze-drying position (partially inserted to allow water vapor to escape during the freeze drying cycle). Half of the samples were transferred to the freeze dryer chamber and subjected to the freeze drying cycle including an annealing step (as shown in FIG. 2) and which consisted of the following steps: 1. Freezing: • the temperature of the shelf has been set at -52 ° C. The filled vials were loaded into the freeze dryer when the shelf temperature was -45 ° C or lower. The samples were then cooled to -52 ° C for a minimum of 1 hour. 2. Annealing step: • (1) The shelf temperature was increased to reach the target annealing temperature (-15 ° C) in one hour. (2) The annealing temperature was maintained for 2 hours. • (3) The shelf temperature was reduced again from -15 ° C to -50 ° C in one hour. (4) The product was held at -50 ° C for at least 1 hour. 3. Primary Drying: • The pressure chamber was set at 80 pbar and the shelf temperature was increased from -52 ° C to -30 ° C over 3 hours. The temperature of the shelf and the pressure of the chamber were maintained for 24 hours. 4. Secondary Drying: • The shelf temperature was increased from -30 ° C to 17 ° C in 6 hours, while the chamber pressure was reduced to 40 pbar. When the shelf temperature reached 17 ° C, these conditions were maintained for 3 hours. At the end of the freeze-drying cycle, the anhydrous nitrogen chamber was filled until a chamber pressure of 825 mbar was reached. The flasks were closed, and after closure, the pressure of the atmospheric pressure chamber was relieved for unloading. The temperature of the chamber was maintained at +2 to +8 ° C until the vials were discharged. The flasks were then discharged and sealed with removable aluminum capsules.
    In order to compare the annealed samples with the non-annealed samples, with the same cryodessiccation cycle, the second half of the filled vials were loaded inside the cryosifter chamber between step 2. (3) and step 2. (4) cryodessiccation cycle.
    The results of this experiment are shown in Table 1 below.
    The qPCR, infectivity and PicoGreen® measurements were as defined in Example 1.
    Example 3 The objective of the experiment was to evaluate the stability of an adenoviral vector when formulated in the presence of different amounts of NaCl in the range of 0 to 50 mM. Isotonicity was maintained by supplemental addition of sucrose. In the ChAd3Eboz construct, the adenoviral vector used in the present experiment, chimpanzee adenovirus 3, is used as a vector encoding a Zaire strain Ebola glycoprotein (as described in WO2011 / 130627). Thus, the conditions proposed in Table 1 were tested using a dose of ChAd3Eboz of 5.0 x 109 pv / mL.
    The samples were held at 30 ° C for 3 days after which the impact on the stability of the adenoviral particles was assessed using qPCR (by measuring the virus content by targeting the promoter sequence) and infectivity ( by measuring the infectivity of the adenoviral particles by flow cytometric flow detection of stained cells for hexon adenoviral capsid protein after a 24 hour infection). The test results are shown in Table 1.
    Table 1
    The qPCR, infectivity and PicoGreen® measurements were as defined in Example 1.
    Example 4
    In the present example, three compositions (compositions (a), (b) and (c)) were evaluated by dynamic light scattering (DLS), each after three different storage conditions of the freeze-dried product. The storage conditions tested were 1 month at 4 ° C (T1m4), 1 week at 25 ° C (T1w25) and 3 days at 37 ° C (T3d37). The cryodessiccation cycle applied is the same as for example 1 (FIG. 1).
    Composition (a): ChAd3Eboz 1 x 1011 pv / mL, 10 mM Tris pH 7.5, 10 mM histidine, 25 mM NaCl, 8% sucrose, 1 mM MgCl 2, 0.02% polysorbate 80
    Composition (b): ChAd3Eboz 1 x 1011 pv / mL, 10 mM Tris pH 7.5, 10 mM histidine, 6 mM NaCl (residual), 7% trehalose, 2% sucrose (residual), 1 mM MgCl 2, 0.02 polysorbate 80 %
    Composition (c): ChAd3Eboz 1 x 1011 pv / mL, 10 mM Tris pH 7.5, 10 mM histidine, 6 mM NaCl (residual), 7% trehalose, 2% sucrose (residual), 1 mM MgCl 2, 188 0 poloxamer, 15%
    Two samples of ChAd3Eboz starting material were used, before and after a 30 minute treatment at 60 ° C, as positive and negative controls respectively.
    The results of the experiment are shown in Figure 3.
    Example 5
    ChAd3Eboz was formulated using either (A) 8% sucrose, 25mM NaCl, 10mM Tris pH 7.4, 10mM histidine, 1mM MgCl 2 and 0.02% polysorbate 80, or (B) 7% trehalose, 2% sucrose (residual), 6 mM NaCl, 10 mM Tris pH 7.4, 10 mM histidine, 1 mM MgCl 2 and 0.02% polysorbate 80. The cryodessiccation cycle was applied with the annealing step as described for Example 1 to obtain the freeze-dried samples. The following rehydration medium was tested: after storage of the dry composition for 1 month at 4 ° C. (T1m4): 150 mM NaCl, 30 mM NaCl, and water for injection. after storage of the dry composition for 2 months at 4 ° C. (T2m4): 150 mM NaCl, 9.25% sucrose, 9.25% trehalose and water for injection.
    Two bulk ChAd3Eboz samples were used, before and after a 30 minute treatment at 60 ° C, as positive and negative controls respectively.
    The capsid breakage after reconstitution of the dry composition was evaluated using the PicoGreen® assay. Quant-iTTM PicoGreen® is an ultrasensitive fluorescent nucleic acid to quantify double-stranded DNA in solution.
    As shown in the graph of Figure 4.I (T1m4 ° C time point), the free DNA in the outer phase was directly proportional to the NaCl concentration of the rehydration medium (EPI <30 mM NaCl <NaCl 150 mM). In addition, the trehalose formulation (A) provided better capsid stability compared to the sucrose-based formulation (B) when using a low salt rehydration medium (EPI and 30 mM NaCl).
    As shown in the graph of FIG. 4.II (T2m4 ° C temporal point), results comparable to those obtained with EPI were obtained with a rehydration medium without salt (sucrose 9.25% w / w). v and trehalose 9.25% w / v) with two lyo samples stored for 2 months at 4 ° C. In addition, the trehalose formulation (A) again provided better capsid stability compared to the sucrose-based formulation (B) when using a low-salt rehydration medium ( PPE, trehalose 9.25% w / v and sucrose 9.25% w / v).
    Example 6 The objective of this experiment was to evaluate the feasibility of freeze drying a ChAd155 vector under the same conditions as those described in the previous examples for the ChAd3 vector above. The ChAd155 vector used for the experiment encodes a respiratory syncytial virus viral protein and is described in PCT / EP2016 / 063297.
    The conditions evaluated were: the cryodessiccation cycle applied in example 1 (see FIG. 1, to which reference is made hereinafter as in cycle I) is compared to a cryodessiccation cycle comprising the same sequence as in FIG. but with an annealing step at -10 ° C. and a secondary drying just brought to 10 ° C. in 6 hours and then stopped at this time (see FIG. 5, which is referred to hereinafter as in cycle II). - The impact of the trehalose and histidine content was also evaluated by comparing four compositions:
    Composition (a): ChAd155 1 x 1011 μU / mL, 10 mM Tris pH 8.5, 0.02% polysorbate 80, 1 mM MgCl 2, 9% trehalose, 8 mM NaCl, 2.5% sucrose, 10 mM histidine
    Composition (b): ChAd155 1 x 1011 μU / mL, 10 mM Tris pH 8.5, 0.02% polysorbate 80, 1 mM MgCl 2, 9% trehalose, 8 mM NaCl, 2.5% sucrose
    Composition (c): ChAd155 1 x 1011 μU / mL, 10 mM Tris pH 8.5, 0.02% polysorbate 80, 1 mM MgCl 2, 7% trehalose, 6 mM NaCl (residual), 2.5% sucrose, histidine 10 mM
    Composition (d): ChAd155 1 x 1011 μU / mL, 10 mM Tris pH 8.5, 0.02% polysorbate 80, 1 mM MgCl 2, 7% trehalose, 6 mM NaCl (residual), 2.5% sucrose
    The freeze dried products were reconstituted with water for injection.
    Two samples of bulk ChAd155 were used, before and after a 30 minute treatment at 60 ° C, as positive and negative controls respectively.
    Capsid disruption was assessed after reconstitution of the composition using Quant-iTTM PicoGreen® (ultra-sensitive fluorescent nucleic acid to quantify double-stranded DNA in solution) and infectivity (by measuring the infectivity of adenovirus particles by detection by flow cytometry of stained cells for the hexon capsid protein of adenovirus).
    The results of this experiment are presented in the table below (see graph of figures 5 and 6):
    Example 7
    This experiment is intended to confirm the protective impact of the annealing step on the integrity of the product and to evaluate the impact of the desorption kinetics using secondary drying.
    The cryodessiccation cycle selected in Example 6 (see Figure 5) was used to evaluate the 3 hour plateau annealing step and the desorption kinetics up to 12 hours by adding a 6 hour secondary drying plateau. . The evaluation was carried out on the basis of composition (a) of Example 6: ChAd155 1 x 1011 μU / mL, 10 mM Tris pH 8.5, 0.02% polysorbate 80, 1 mM MgCl 2, trehalose 9 %, 8 mM NaCl, 2.5% sucrose, 10 mM histidine.
    The freeze dried products were reconstituted with water for injection.
    Two samples of bulk ChAd155 were used, before and after a 30 minute treatment at 60 ° C, as positive and negative controls respectively.
    Capsid disruption after reconstitution of the dry composition was evaluated using Quant-iTTM PicoGreen® (ultra-sensitive fluorescent nucleic acid to quantify double stranded DNA in solution) and viral infectivity by CCID50 (quantification of the required virus). to destroy 50% of the infected hosts or to produce cytopathic effect (CPE) on 50% of the inoculated cell culture).
    The results of this experiment are shown in the table below and illustrated in Figures 8 and 9.
    Of course, the invention is not limited to the embodiments described above and shown, from which we can provide other modes and other embodiments, without departing from the scope of the invention. .
    权利要求:
    Claims (61)
    [1]
    An aqueous mixture comprising an adenoviral vector, a cryoprotectant which is an amorphous sugar selected from trehalose, sucrose, lactose, raffinose, dextran, mannitol and combinations thereof, and sodium chloride thereof. a quantity of between zero and 50 mM.
    [2]
    The mixture according to claim 1, wherein the sodium chloride is present in an amount between zero and 40 mM, between 0 and 30 mM, between 0 and 20 mM, between 0 and 15 mM, between 0 and 10 mM or between 2 and 10 mM.
    [3]
    3. The blend of any of the preceding claims, wherein the sodium chloride is present in an amount of 5 mM ± 1 mM.
    [4]
    Mixture according to any one of the preceding claims, wherein the amorphous sugar is present in an amount of at least 2.5% (w / v), at least 3% (w / v), at least 3.5% (w / v), at least 4% (w / v), at least 4.5% (w / v), or at least 5% (w / v) .
    [5]
    Mixture according to any one of the preceding claims, wherein the amorphous sugar is present in an amount of less than 17.5% (w / v), less than 15% (w / v), less than 12.5% (w / v), less than 11% (w / v), less than 10% (w / v), or less than 9.5% (w / v).
    [6]
    Mixture according to any one of the preceding claims, wherein the amorphous sugar is present in an amount of 8%, 8.5%, 9% or 9.25%.
    [7]
    A blend according to any one of the preceding claims, wherein the amorphous sugar is trehalose, sucrose, or trehalose in combination with one or more of sucrose, lactose, raffinose, dextran and mannitol.
    [8]
    Mixture according to any one of the preceding claims, wherein the amorphous sugar is trehalose or trehalose in combination with one or more of sucrose, lactose, raffinose, dextran and mannitol.
    [9]
    Mixture according to any one of the preceding claims, wherein the amorphous sugar is trehalose or trehalose in combination with sucrose.
    [10]
    A blend according to any one of the preceding claims comprising at least 5% (w / v) trehalose, for example about 7% (w / v) or about 9% (w / v) trehalose.
    [11]
    A mixture according to any one of the preceding claims, wherein the adenoviral vector is a simian non-human adenoviral vector, for example a chimpanzee adenoviral vector, a bonobo adenoviral vector or an adenoviral gorilla vector.
    [12]
    A mixture according to any one of the preceding claims, wherein the adenoviral vector is an adenoviral chimpanzee vector, for example ChAd3, ChAd63, ChAd83, ChAd155, Pan5, Pan6, Pan7 or Pan9.
    [13]
    A mixture according to any one of the preceding claims, wherein the adenoviral vector is a simian adenoviral vector selected from ChAd3, ChAd63, ChAd83, ChAd155 and PanAd3.
    [14]
    A blend according to any one of the preceding claims, further comprising a surfactant selected from poloxamer surfactants (e.g., poloxamer 188), polysorbate surfactants (e.g., polysorbate 80 and / or polysorbate 20), octoxidic surfactants, polidocanol surfactants, polyoxyl stearate surfactants, polyoxy castor oil surfactants, N-octyl glucoside surfactants, macrogol 15 hydroxystearate, and combinations thereof.
    [15]
    The blend of any preceding claim, further comprising a surfactant which is a poloxamer surfactant or a polysorbate surfactant.
    [16]
    Mixture according to any one of the preceding claims, further comprising a surfactant which is poloxamer 188 or polysorbate 80.
    [17]
    17. A blend according to any one of the preceding claims, further comprising a surfactant which is polysorbate 80.
    [18]
    The blend of any preceding claim, further comprising a surfactant in an amount of at least 0.001 and at most 0.5% (w / v).
    [19]
    The blend of any preceding claim, further comprising a surfactant in an amount of 0.02% (w / v).
    [20]
    20. A blend according to any of the preceding claims, further comprising a buffer selected from Tris, succinate, borate, tris-maleate, lysine, histidine, glycine, glycylglycine, citrate, carbonate or a combination thereof.
    [21]
    21. The blend of any of the preceding claims, further comprising a buffer selected from Tris, succinate, borate or a combination thereof.
    [22]
    The blend of any preceding claim, further comprising Tris.
    [23]
    23. A blend according to any one of the preceding claims, wherein the pH is at least 6, at least 6.5, at least 7 or at least 7.5.
    [24]
    24. Mixture according to any one of the preceding claims, wherein the pH is less than 10, less than 9.5, less than 9 or less than 8.5.
    [25]
    25. Mixture according to any one of the preceding claims, wherein the pH is between 7.5 and 9.5.
    [26]
    26. A blend according to any one of the preceding claims, wherein the pH is 7.5 (± 0.5).
    [27]
    27. A blend according to any one of the preceding claims, further comprising a buffer in an amount of 0.5mM to 50mM, for example 10mM.
    [28]
    28. The blend of any of the preceding claims, further comprising a bivalent metal ion salt selected from MgCl 2, CaCl 2 or MgSCg.
    [29]
    Mixture according to any one of the preceding claims, further comprising a divalent metal ion salt in an amount of between 0.5 and 10 mM, for example 1 or 2 mM, or 1 mM.
    [30]
    The mixture of any preceding claim, wherein the adenoviral vector comprises a transgene which is an immunogenic transgene.
    [31]
    31. Aqueous mixture comprising a simian adenoviral vector, 5 to 10% (w / v) trehalose, and sodium chloride in an amount of from zero to 10 mM.
    [32]
    The aqueous blend of claim 31, further comprising one or more of the elements of claims 3 to 30.
    [33]
    33. Freeze-dried composition based on one of the mixtures according to claims 1 to 32.
    [34]
    34. A composition according to claim 33 for use in a method of administering the composition to a subject, for example a human subject, after reconstituting the composition with an aqueous liquid containing sodium chloride in an amount less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 10 mM, less than 5 mM or substantially free of sodium chloride.
    [35]
    The composition of claim 33 or 34 for use in a method of administering the composition to a subject after reconstituting the composition with an aqueous liquid containing a nonionic isotonicity modifying agent, such as trehalose or sucrose.
    [36]
    The composition of any one of claims 33 to 35, wherein the adenoviral vector comprises a transgene which is an immunogenic transgene.
    [37]
    37. A composition according to any one of claims 33 to 36, or an aqueous mixture according to any one of claims 1 to 32, for use in a method of inducing an immune response against the immunogenic transgene product in a human subject.
    [38]
    38. A method of immunizing a human subject comprising the step of administering to an aqueous mixture according to any one of claims 1 to 32, or a reconstituted liquid obtained by reconstituting a composition. lyophilized according to any one of claims 33 to 36 with an aqueous liquid containing sodium chloride in an amount of less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 10 mM, less than 5 mM or substantially free of sodium chloride.
    [39]
    39. A method of manufacturing a composition comprising an adenoviral vector comprising the steps of: a. providing an aqueous mixture comprising the adenoviral vector and a cryoprotectant, for example the aqueous mixture according to any one of claims 1 to 32, and, b. freeze drying of the aqueous mixture by i. freezing the aqueous mixture comprising an annealing step and ii. drying the frozen aqueous mixture under reduced pressure.
    [40]
    40. A method of manufacturing a composition comprising an adenoviral vector comprising the steps of: a. providing an aqueous mixture comprising the adenoviral vector and a cryoprotectant, for example the aqueous mixture according to any one of claims 1 to 32, and, b. freeze drying of the aqueous mixture by i. freezing the aqueous mixture comprising the steps of: 1. freezing the aqueous mixture by reducing the temperature to a temperature below the Tg 'of the mixture, 2. applying an annealing step to the frozen mixture by increasing the temperature without doing melt the frozen mixture, 3. reduce the temperature again below the Tg '; and, ii. drying the frozen aqueous mixture under reduced pressure.
    [41]
    41. The method of claim 39 or 40, wherein in step a. the aqueous mixture is supplied in a vial, such as a non-siliconized vial.
    [42]
    42. The method of any one of claims 39 to 41, wherein in step a. the aqueous mixture is supplied at a temperature of between +2 and +8 ° C.
    [43]
    The method of any one of claims 39 to 42, wherein the vial comprises a single dose of the adenoviral vector.
    [44]
    44. The method according to any one of claims 39 to 43, wherein in step b.i.1. the temperature of the product is reduced by applying a plateau temperature at least 10 ° C lower than Tg '.
    [45]
    45. The method according to any one of claims 39 to 44, wherein in step b.i.1. the temperature of the product is reduced by applying a plateau temperature of -50 ° C.
    [46]
    46. The method according to any one of claims 39 to 45, wherein in step b.i.1. the temperature is reduced at a rate of 2 to 10 ° C / minute.
    [47]
    47. The method of any one of claims 39 to 46, wherein in step b.i.1. the temperature is kept below the Tg 'for at least one hour.
    [48]
    48. The method of any one of claims 40 to 47, wherein in step b.i.2. the temperature is raised to a temperature above the Tg 'and below the melting temperature of the frozen mixture.
    [49]
    49. The method of any one of claims 40 to 48, wherein in step b.i.2. the temperature is brought to the annealing temperature at -15 ° C. ± 9 ° C., for example -15 ° C. ± 6 ° C.
    [50]
    50. The method of any one of claims 40 to 49, wherein in step b.i.2. the temperature is raised to the annealing temperature at a rate of 0.1 to 10 ° C / minute.
    [51]
    51. The method of any one of claims 40 to 50, wherein in step b.i.2. the frozen mixture is kept at the annealing temperature for 2 to 4 hours.
    [52]
    52. The method of any one of claims 40 to 51, wherein in step b.i.3. the temperature is reduced to a temperature below Tg '.
    [53]
    53. The method according to any one of claims 40 to 52, wherein in step b.i.3. the temperature is reduced at a rate of 0.1 to 10 ° C / minute.
    [54]
    54. The method according to any one of claims 40 to 53, wherein in step b.i.3. the frozen mixture is kept below the Tg 'for at least 1 hour.
    [55]
    The method of any one of claims 39 to 54, wherein step b.ii. comprises: 1. primary drying at a temperature below the breaking temperature; and 2. secondary drying at a temperature above the break temperature.
    [56]
    The method of claim 55 (above), wherein the pressure during step b.ii.1. is between 40 and 90 pbars.
    [57]
    57. The method of claim 55 or 56, wherein step b.ii.1. is applied for at least 24 hours.
    [58]
    58. The method according to any one of claims 55 to 57, wherein in step b.ii.2. the temperature is brought to a temperature between -10 ° C and 25 ° C, for example, at a rate of 0.1 to 3 ° C / minute.
    [59]
    The process of any one of claims 55 to 58, wherein the elevated temperature in step b.ii.2. is maintained for up to 6 hours.
    [60]
    60. A process according to any one of claims 55 to 59, wherein the pressure during secondary drying is between 15 and 60 bp.
    [61]
    The method of any of claims 39 to 60, further comprising one or more of the elements of claims 1 to 38.
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    同族专利:
    公开号 | 公开日
    MX2018001006A|2018-06-06|
    WO2017013169A1|2017-01-26|
    US20180214379A1|2018-08-02|
    CN108025081A|2018-05-11|
    GB201513010D0|2015-09-09|
    JP6797891B2|2020-12-09|
    BE1023537A1|2017-04-26|
    EP3325016A1|2018-05-30|
    JP2018521078A|2018-08-02|
    CA2992922A1|2017-01-26|
    AR105401A1|2017-09-27|
    US10722470B2|2020-07-28|
    CN108025081B|2021-11-02|
    BR112018001260A2|2018-09-11|
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    法律状态:
    2019-04-01| FG| Patent granted|Effective date: 20170426 |
    2019-04-01| MM| Lapsed because of non-payment of the annual fee|Effective date: 20180731 |
    优先权:
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    GB1513010.7|2015-07-23|
    GBGB1513010.7A|GB201513010D0|2015-07-23|2015-07-23|Novel formulation|
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