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    • 专利摘要:
    The present invention relates to the formulation of immunogenic or vaccine compositions comprising adjuvants based on liposomes of neutral lipids, the composition being suitable for lyophilization. In particular, the invention relates to the lyophilized forms of these immunogenic or vaccine compositions, the immunogen or vaccine antigen and the adjuvant being both present in one and the same vial, as well as the formulation and preparation of forms. freeze-dried of such an immunogenic or vaccine composition.
    公开号:BE1024188B1
    申请号:E2016/5921
    申请日:2016-12-13
    公开日:2017-12-14
    发明作者:Dominique Ingrid Lemoine；Nicolas Moniotte
    申请人:Glaxosmithkline Biologicals Sa；
    IPC主号:
    专利说明:

    DRY COMPOSITION
    The present invention relates to the formulation of immunogenic or vaccine compositions comprising adjuvants based on liposomes of neutral lipids, the composition being suitable for lyophilization. In particular, the invention relates to lyophilized forms of these immunogenic or vaccine compositions, the immunogen or vaccine antigen and the adjuvant being both present in one and the same vial, as well as the formulation and preparation of the forms. freeze-dried of such an immunogenic or vaccine composition.
    Context of the invention
    Christensen et al. (2007) [Biochim. Biophys. Acta
    1768 (9): 2120-2129 - Trehalose preserves DDA / TDB liposomes and their adjuvant effect during freezedrying] investigated the ability of trehalose and sucrose disaccharides to stabilize a liposomal phospholipid-free adjuvant composed of cationic dimethyldioctadecylammonium (DDA) and 6, Trehalose 6'-dibehenate (TDB) during lyophilization. Trehalose at a concentration greater than or equal to 211 mM was found to protect and preserve DDA / TDB liposomes during lyophilization, while sucrose had to be used at concentrations above 396 mM. The protective effect was not observed in liposomes without TDB.
    Ingvarsson et al. (2013) [J. Controlled Release 167: 256-264, Designing CAF-Adjuvanted Dry Powder Vaccines: Spray drying preserves the adjuvant activity] investigated the spray drying of cationic liposomal adjuvant DDA / TDB using mannitol, lactose or trehalose.
    Mohammed et al. (2006) [Methods 40 (1): 30-8,
    Lyophilization and Sterilization of Liposomal Vaccines to Produce Stable and Sterile Products] is also concerned with lyophilization of cationic liposomal adjuvanted vaccines. It is demonstrated that to protect the liposomes effectively against fusion, the cryoprotectant must be present both inside the liposome and in the outer phase, and that the intra- and extra-liposomal media must have the same osmolarity . For this purpose, the disclosed protocol allows the cryoprotectant to be included in the liposomes during liposome formation.
    Orr et al. (2014) [J. Control Release, 177: 20-6 (published in 2013 electronically) relates to the co-lyophilization of an emulsion-type adjuvant and an antigen.
    WO 99/65465 relates to a method for trapping an agent in liposomes in the presence of a sugar. Summary of the invention
    The inventors have unexpectedly discovered that neutral lipid liposome adjuvants can be lyophilized successfully, thereby conferring thermostabilization to these components and allowing packaging in the same vial of adjuvant and antigen under dry form. The invention thus provides compositions dried under reduced pressure from a liquid mixture comprising an adjuvant which comprises a saponin in a liposomal formulation, wherein the liposomes contain a neutral lipid and a sterol, and a cryoprotectant which is a sugar amorphous.
    In addition, the invention provides methods for preparing such compositions. It has been unexpectedly discovered that to obtain such compositions, it is not necessary that liposome formation be performed in the presence of the cryoprotectant.
    figures
    Figure 1-A illustrates with the continuous line the lyophilization cycle used for the samples of Example 1; the dashed lines delimit the acceptable range of the process for lyophilization of a vaccine composition comprising the RTS, S antigen.
    Figure 1-B illustrates an alternative lyophilization cycle used for lyophilization of vaccine compositions comprising AS01 and an antigen, as described in Example 2; the dashed lines delimit the acceptable range of the process for lyophilization of a vaccine composition.
    Figure 2 illustrates the preclinical immunogenicity data obtained in Example 1: A. Cellular anti-CSP immune response; B. Anti-CSP Antibody; I. Mosquirix ™; II. RTS, S / AS01 co-lyophilized reconstituted with 150 mM NaCl.
    Figure 3 illustrates the nephelometry data obtained in Example 1.
    Figure 4 illustrates the amino acid sequence of the VZV gE as used in Example 2.
    Figure 5 illustrates the SDS-page analysis of the integrity of the VZV gE before and after lyophilization under different circumstances in Example 2; NR means non-reducing conditions, R means reducing conditions, the legend for lanes 1 to 12 is provided in Example 2.
    Figure 6 shows the results of the analysis of the impact of lyophilization on the hemolytic activity of QS21 in Example 2; FB refers to the composition before lyophilization, FC refers to the composition after lyophilization.
    detailed description
    Although lyophilization of vaccines containing proteins, live attenuated or inactivated viruses or bacteria has been reported, to date, successful lyophilization (or drying under reduced pressure in general) and characterization of the thermostability of adjuvants to based on neutral liposomes have not been reported. Apart from the effect on thermostability, lyophilization of such an adjuvant may allow the packaging in the same bottle of the adjuvant and an antigen. Developing vaccines that can reduce the need for maintaining the cold chain would reduce the costs and technological barriers to implementing new vaccines. Packaging in the same bottle of adjuvant and antigen could further reduce costs, logistics and technological barriers to vaccine distribution worldwide.
    The present invention describes the vacuum drying, such as lyophilization, of a composition comprising neutral liposome adjuvants, the formulation of such a composition suitable for lyophilization, as well as lyophilization processes. The inventors have discovered that an adjuvant comprising a saponin, liposomes and optionally a lipopolysaccharide, in which the liposomes are based on neutral lipids, can be lyophilized from a mixture further comprising a cryoprotectant chosen from amorphous sugars, such as sucrose and trehalose. The composition may further comprise an immunogen or an antigen. In particular, the inventors have found that for the claimed liposomal adjuvant composition, it is not necessary that the liposomes be formed in the presence of a cryoprotectant so that the adjuvant retains its structural integrity and its adjuvant or potentiating properties. immunity during drying or lyophilization. An additional advantage of the invention is that the drying gives thermostability to the composition.
    After lyophilization as described herein, the composition can be stored for 12, 24 or 36 months at 30 ° C; for 6 months, or for 12 months or 1 year at 37 ° C; or for three months 45 ° C. Storage ability may be based on the retention of immunogenicity and / or the retention of structural integrity of the components at an acceptable level. The structural integrity of liposomes can be assessed using methods, such as dynamic light scattering (DLS), measuring the size and polydispersity of liposomes, or electron microscopy to analyze the structure of liposomes. In one embodiment, the average particle size (by photon correlation spectroscopy) is between 95 and 120 nm, and / or the polydispersity index (by photon correlation spectroscopy) is not greater than 0.2 . From a functional point of view, the antigenicity of the antigen can be measured by ELISA. Preclinical assays are available to evaluate the overall immunogenicity of the compositions described herein. Immunoassays can quantify a range of responses, such as CD4 T cells and / or CD8 T cells. Immunogenicity refers to the effect of the composition on the immune response when administering the composition to a subject. The adjuvant according to the invention comprises a saponin in a liposomal formulation and optionally a TLR-4 agonist. Definitions "Liposomal Formulation" means that saponin (and optionally a TLR-4 agonist) is formulated with liposomes or, in other words, presented in a liposome-based composition. The liposomes for the present invention contain a neutral lipid or consist essentially of a neutral lipid, i.e., "neutral liposomes". "Neutral lipid" means that the overall net charge of the lipid is (approximately) zero. The lipid may therefore be generally nonionic or may be zwitterionic. In one embodiment, the liposomes comprise a zwitterionic lipid. Examples of suitable lipids are phospholipids, such as phosphatidylcholines. In one embodiment, the liposomes contain a phosphatidylcholine, as the liposome-forming lipid, which is suitably non-crystalline at room temperature. Examples of such non-crystalline phosphatidylcholine lipids include egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine (DLPC). In a particular embodiment, the liposomes of the present invention contain DOPC or consist essentially of DOPC. The liposomes may also contain a limited amount of a charged lipid that increases the stability of the liposome-saponin structure for liposomes composed of saturated lipids. In such cases, the amount of lipid loaded is suitably from 1 to 20% w / w, preferably from 5 to 10% w / w of the liposome composition. Suitable examples of such charged lipids include phosphatidylglycerol and phosphatidylserine. Suitably, the neutral liposomes will contain less than 5% w / w of a loaded lipid, such as less than 3% w / w or less than 1% w / w.
    The liposomes for the present invention further comprise a sterol. Suitable sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In a particular embodiment, the liposomal formulation comprises cholesterol as sterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 11th Edition, page 341, as a naturally occurring sterol found in animal fat. The sterol ratio on phospholipid is 1 to 50% (mole / mole), suitably 20 to 25%.
    When the active saponin fraction is QS21, the QS21 / sterol ratio will usually be in the range of 1/100 to 1/1 (w / w), suitably 1/10 to 1/1 (w / w). weight), and preferably from 1/5 to 1/1 (w / w). Suitably, the sterol is present in excess, the QS21 / sterol ratio being at least 1/2 (w / w). In one embodiment, the QS21 / sterol ratio is 1/5 (w / w). In one embodiment, the sterol is cholesterol.
    The term "liposome" is well known in the art and defines a general class of vesicles that include one or more lipid bilayers surrounding an aqueous space. Liposomes therefore consist of one or more lipid bilayers and / or phospholipids and may contain other molecules, such as proteins or carbohydrates, in their structure. Since both a lipid phase and an aqueous phase are present, the liposomes can encapsulate or entrap a water-soluble substance, a fat-soluble substance and / or amphiphilic compounds.
    As used herein, a "neutral liposome adjuvant" means that the adjuvant comprises neutral liposomes for the presentation of the included immunization potentiating agents.
    As used herein, "essentially consisting of" means that additional components may be present, provided that they do not alter the overall properties or function.
    As used herein, a "vial" refers to a container suitable for use in the packaging, dispensing and use of vaccines or immunogenic compositions. One vial may be a "single dose" vial (i.e., a vial containing an amount of immunogenic or vaccine composition equal to a single dose, such as a single human dose, as will be understood by the human the specific dosage will vary depending on certain factors, such as the specific composition and intended recipient). Alternatively, the vial may contain more than one dose ("multi-dose" vial).
    As used herein, "packaged in one vial" means placing at least two different components, ingredients, or compositions in a single vial. The vial may be a single dose vial (containing a single dose of each component, ingredient or composition), a multi-dose vial.
    As used herein, the terms "mixture" and "mixture" are used interchangeably.
    saponins
    A suitable saponin for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quillaja Saponaria Molina and its adjuvant activity has been described for the first time by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv für die gesamte Virusforschung, Vol 44, Springer Verlag, Berlin, p 243-254). HPLC purified fragments of Quil A that retain adjuvant activity without the Quil A-associated toxicity (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and QA21), have been isolated by HPLC. . QS-21 is a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8 + cytotoxic T lymphocytes (CTL), Th1 lymphocytes and a predominant IgG2a antibody response, and is a preferred saponin in the context of the present invention. In an appropriate form of the present invention, the saponin adjuvant within the immunogenic composition is a quil A derivative of saponaria molina, preferably an immunologically active moiety of Quil A, such as QS-7, QS-17. , QS-18 or QS-21, suitably QS-21.
    Saponin is provided in its least reactogenic composition, where it is quenched with an exogenous sterol, such as cholesterol, and as provided in the liposomal formulation as defined hereinbefore. There are several particular forms of less reactogenic compositions in which QS21 is deactivated with exogenous cholesterol. The saponin / sterol is presented in a liposomal formulation structure. Processes for obtaining saponin / sterol in a liposomal formulation are described in WO 96/33739, particularly in Example 1.
    TLR4 agonists
    In one embodiment of the present invention, the adjuvant comprises a TLR-4 agonist. A suitable example of TLR-4 agonists is a lipopolysaccharide, suitably a nontoxic derivative of lipid A, particularly monophosphorylated lipid A, or more particularly monophosphorylated 3-deacylated A-lipid (3D-MPL).
    3D-MPL is sold as MPL by GlaxoSmithKline Biologicals N.A. and is referred to throughout the document as MPL or 3D-MPL. See, for example, US Patents 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL primarily promotes CD4 + T cell responses with an IFN-g (Th1) phenotype. 3D-MPL can be produced according to the methods described in GB 2 220 211 A. Chemically, it is a mixture of 3-deacylated A-monophosphorylated lipid with 4, 5 or 6 acylated chains. In the compositions of the present invention, small 3D-MPL particles may be used to prepare the aqueous adjuvant composition. 3D-MPL in the form of small particles has a particle size such that it can be sterilized by filtration through a 0.22 μm filter. Such preparations are described in WO 94/21292. Preferably, powdery 3D-MPL is used to prepare the aqueous builder compositions of the present invention. Other ligands of TLR-4 that can be used are alkyl glucosaminide phosphates (AGP), such as those described in WO 98/50399 or US Pat. No. 6,303,347 (AGP preparation methods are also described), appropriately RC527 or RC529 or pharmaceutically acceptable salts of AGP as described in US Pat. No. 6,764,840. Some AGPs are TLR-4 agonists, and some are TLR-4 antagonists. Both are considered useful as adjuvants. Other suitable ligands for TLR-4 are described in WO 2003/011 223 and WO 2003/099 195, such as compound I, compound II and compound III described on pages 4 and 5 of WO 2003/011. 223 or on pages 3 and 4 of the document WO 2003/099 195 and in particular the compounds described in the document WO 2003/011 223, such as ER803022, ER803058, ER803732, ER804053, ER804057m ER804058, ER804059, ER804442, ER804680 and ER804764. For example, a suitable ligand for TLR-4 is ER804057. Other TLR-4 ligands that can be used in the present invention include a glucopyranosyl lipid (GLA) adjuvant as described in WO 2008/153 541 or WO 2009/143 457 or in the articles of literature Coler RN et al. (2011) Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as an Adjuvant Vaccine. PLoS ONE 6 (1): e16333. doi: 10.1371 / journal.pone.0016333 and Arias MA et al. (2012) Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promoted Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140. PLoS ONE 7 (7): e41144. doi: 10.1371 / journal.pone.0041144. WO 2008/153 541 or WO 2009/143457 are hereby incorporated by reference for the purpose of defining TLR-4 ligands for use in the present invention.
    In a specific embodiment, the adjuvant comprises both a saponin and a TLR-4 agonist. In a specific example, the aqueous adjunctive composition comprises QS21 and 3D-MPL. In an alternative embodiment, the aqueous adjuvant composition comprises QS21 and GLA.
    A TLR-4 ligand, such as a lipopolysaccharide, such as 3D-MPL, can be used in an amount between 1 and 100 μg per human dose of the adjuvant composition. 3D-MPL may be used at a level of approximately 50 μg, such as at least 40 μg, at least 45 μg or at least 49 μg, or, less than 100 μg, less than 80 μg, less than 60 μg less than 55 μg or less than 51 μg. Examples of suitable ranges are between 40 and 60 μg, suitably between 45 and 55 μg or between 49 and 51 μg or 50 μg. In another embodiment, the human dose of the adjuvant composition comprises 3D-MPL at a content of about 25 μg, such that at least 20 μg, at least 21 μg, at least 22 μg or at least 24 μg or, less than 30 μg, less than 29 μg, less than 28 μg, less than 27 μg or less than 26 μg. Examples of lower ranges are between 20 and 30 μg, suitably between 21 and 29 μg or between 22 and 28 μg or between 28 and 27 μg or between 24 and 26 μg, or 25 μg.
    A saponin, such as QS21, can be used in an amount between 1 and 100 μg per human dose of the adjuvant composition. QS21 may be used at a level of approximately 50 μg, such as at least 40 μg, at least 45 μg or at least 49 μg, or, less than 100 μg, less than 80 μg, less than 60 μg, lower at 55 μg or below 51 μg. Examples of suitable ranges are between 40 and 60 μg, suitably between 45 and 55 μg or between 49 and 51 μg or 50 μg. In another embodiment, the human dose of the adjuvant composition comprises QS21 at a level of about 25 μg, such as at least 20 μg, at least 21 μg, at least 22 μg or at least 24 μg, or , less than 30 μg, less than 29 μg, less than 28 μg, less than 27 μg or less than 26 μg. Examples of lower ranges are between 20 and 30 μg, suitably between 21 and 29 μg or between 22 and 28 μg or between 28 and 27 μg or between 24 and 26 μg, or 25 μg.
    As for both a TLR-4 agonist and a saponin are present in the adjuvant, then the agonist weight ratio of TLR-4 to saponin is conveniently between 1/5 and 5/1, suitably 1 / 1. For example, when 3D-MPL is present in an amount of 50 μg or 25 μg, then suitably QS21 may also be present in an amount of 50 μg or 25 μg per human dose of the adjuvant.
    liposomes
    The liposomal formulations for the present invention are defined hereinabove. WO 2013/041 572 (also published as US 2014 0 234 403, incorporated herein by reference in its entirety), particularly Examples 3 and 4, discloses methods for preparing a preparation of DOPC-based liposomes additionally containing cholesterol and optionally 3D-MPL, further admixed with QS21, to thereby obtain an adjuvant according to the present invention.
    Antigen
    The composition of the present invention may further comprise an immunogen or an antigen. The antigen may be selected from bacterial, viral or cancer antigens. In one embodiment, the antigen is a recombinant protein, such as a recombinant prokaryotic protein. In one embodiment, the antigen and derivatives of Plasmodium falciparum, Mycobacterium tuberculosis (TB), human immunodeficiency virus (HIV), Moraxella, nontypeable Haemophilus influenzae (ntHi) or varicella zoster virus (VZV). The antigen may comprise or consist of preparations derived from malaria causing parasites, such as Plasmodium falciparum or Plasmodium vivax.
    Suitable antigens derived from Plasmodium falciparum include circumsporozoite protein (CS protein), RTS, PfEMP-I, Pfs 16 antigen, MSP-I, MSP-3, LSA-I, LSA-3, AMA-I and TRAP. Other P. falciparum antigens include EBA, GLURP, RAPI, RAP2, sequestrin, Pf332, STARP, SALSA, PfEXPI, Pfs25, Pfs28, PFS27 / 25, Pfs48 / 45, Pfs230 and their analogs in other Plasmodium spp. . The antigen may be an entire protein or an immunogenic fragment thereof. Alternatively, the antigen may be in the form of a fusion protein.
    An antigen derived from the Plasmodium falciparum CS protein may be in the form of a hybrid fusion protein. The fusion protein may contain a protein derived from the P. falciparum CS protein fused to another protein or fragment thereof. The fusion protein may contain an N-terminal or C-terminal fragment derived from the P. falciparum CS protein. Alternatively, or in addition, the fusion protein may comprise one or more repeating units (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more than 9 repeating units) from the central region of the P. falciparum CS protein. In one embodiment, the fusion protein is a hybrid fusion protein comprising an antigen derived from the CS protein together with a hepatitis B surface antigen (HBsAg) or an immunogenic fragment thereof. Usually, the surface antigen from hepatitis B comprises the major surface protein known as the S antigen, for example the S antigen derived from an adw serotype.
    In particular, the fusion protein can comprise essentially all of the C-terminal portion of the P. falciparum CS protein, four or more tandem repeats of the immunodominant region of the CS protein, and the surface antigen from hepatitis B (HBsAg). In one aspect, the fusion protein comprises a sequence of at least 160 contiguous amino acids having sequence similarity of at least 99%, 98%, 95%, 90% with the C-terminal portion of the CS protein ( Caspers et al (1989) Mol Biochem Parasitol 35, 185190, Gordon et al J Infect Dis (1995), 171 (6): 1576-85). In one aspect, the fusion protein comprises a sequence containing "substantially all" of the C-terminal portion of the CS protein. As used herein, "substantially all" of the C-terminal portion of the CS protein includes the C-terminal sequence lacking the hydrophobic anchoring sequence. In one aspect, the fusion protein comprising a sequence of the CS protein lacking the last 12 to 14 (such as 12) amino acids from the C-terminus is contemplated.
    In one embodiment, the fusion protein useful in the invention is a protein that comprises a contiguous amino acid sequence having sequence similarity of at least 99%, 98%, 95%, 90% with the acids. amines 207 to 395 of P. falciparum clone 3D7, derived from strain NF54 (Caspers et al., supra) fused in frame via a linear linker at the N-terminus of HBsAg. The linker may comprise part or all of the preS2 region of HBsAg.
    A particular fusion protein useful in the invention is the fusion protein known as RTS, as described in WO 93/10152 and WO 98/05355, incorporated herein by reference in their entirety. . The RTS may be in the form of mixed particles RTS, S (where "S" represents an unmerged monomer) or RTS. The RTS, S particles comprise two RTS and S polypeptides that can be simultaneously synthesized and which spontaneously form composite particle structures (RTS, S), for example during purification. These particles can also be called virus-like particles (VLPs).
    Such particles can be prepared in several ways, for example by expressing the fusion protein in a suitable host, such as yeast or bacteria.
    It is believed that the presence of hepatitis B surface antigen and the formation of RTS, S particles enhance the immunogenicity of the CS protein portion of the hybrid protein, facilitate stability and / or allow reproducible protein.
    The CS antigens may be used together with another antigen selected from any antigen that is expressed on the sporozoite or during the pre-erythrocytic stage of the life cycle of the parasite, such as the hepatic stage, for example antigen 1 hepatic stage (LSA-1), hepatic antigen 3 (LSA-3), thrombospondin-related anonymous protein (TRAP), merozoite surface protein 1 (MSP1), merozoite major surface protein , and the merozoite apical antigen 1 (AMA-1). Other suitable antigens for use with CS antigens include PfEMP-I, Pfs 16, MSP-3, LSA-3, AMA-I, TRAP, GLURP, RAPI, RAP2, sequestrin, Pf332, STARP, SALSA , PfEXPI, Pfs25, Pfs28, PFS27 / 25, Pfs48 / 45, Pfs230.
    Suitable antigens from P. vivax include circumsporozoite protein-based antigens (CS protein) and Duffy antigen-binding protein and fragments thereof, such as PvRII (see, for example, WO 02/12 292).
    Suitable antigens based on the CS protein include a fusion protein comprising sequences derived from a P. vivax CS protein. In one embodiment, the fusion protein is a hybrid fusion protein. The hybrid protein may contain a protein derived from P. vivax type I and type II. In particular, the fusion fusion protein may contain a protein derived from P. vivax type I and type II fused to another protein or fragment thereof.
    In one aspect, the hybrid fusion protein comprises a hybrid protein derived from P. vivax CS protein (CSV) and a surface antigen from hepatitis B, such as the major surface protein known as antigen. S, such as S antigen derived from an adw serotype.
    Preferably, the fusion protein is an immunogenic fusion fusion protein comprising: a. at least one repeating unit derived from the central repeat section of a P. vivax type I circumsporozoite protein, b. at least one repeating unit derived from the central repeat section of a P. vivax type II circumsporozoite protein, and c. surface antigen S derived from the hepatitis B virus.
    The CSV derived antigenic component of the invention may be fused to the amino terminus of the S protein. More specifically, the C-terminus of the CSV fragment is fused to the N-terminus of said S antigen.
    For example, a suitable fusion protein is CSV-S, as described in WO 2008/009652.
    In host cells, once expressed, the hybrid fusion protein (including S antigen) is capable of spontaneously assembling to yield a lipoprotein structure / particle composed of many monomers of said proteins (or VLPs). Such particles may be prepared by expression of the fusion protein in a suitable host, such as yeast or bacteria.
    When the selected recipient host cell strain also carries in its genome one or more integrated copies of a hepatitis B S expression cassette, the resulting strain synthesizes a fusion protein in the form of fusion proteins, and also S antigen not fused. They can spontaneously assemble to give lipoprotein particles comprising hybrid fusion protein monomers and S antigen monomers. A suitable host cell for the expression of the fusion protein is, for example, a yeast .
    There is also provided a VLP comprising CSV-S and / or RTS patterns. The particle may consist essentially of CSV-S and RTS motifs. Alternatively, the produced particles comprise or consist essentially of CSV-S, RTS and S motifs. Such mixed particles are described, for example, in WO 2008/009 650.
    In some embodiments, the composition of the invention comprises an antigen derived from Mycobacterium spp., Such as Mycobacterium bovis or
    Mycobacterium tuberculosis, in particular Mycobacterium tuberculosis.
    Antigens of interest in the case of tuberculosis include Rv1196 and Rv0125. Rv1196 (described, for example, by the name Mtb39a in Dillon et al Infection and Immunity 1999 67 (6): 2941-2950) is highly conserved, with a sequence identity of 100% among strains H37Rv, C, Haarlem, CDC1551. , 94-M4241A, 98-R604INH-RIF-EM, KZN605, KZN1435, KZN4207, KZNR506, the F11 strain having a Q30K point mutation (most other clinical isolates have an identity of greater than 90% with H37Rv). Rv0125 (described, for example, by the name Mtb32a in Skeiky et al Infection and Immunity 1999 67 (8): 3998-4007) is also highly conserved, with 100% sequence identity among many strains. Full length Rv0125 includes an N-terminal signal sequence that is cleaved to yield the mature protein.
    In one embodiment, the antigen is derived from Rv1196, e.g. comprises, for example, consists of a sequence having an identity of at least 70% with SEQ ID No: 1, such as at least 80%, at least 90%, more particularly at least 95%, for example at least 98%, such as at least 99%. Typical Rv1196-related antigens will include (eg, will be) a derivative of SEQ ID NO: 1 having a small number of deletions, insertions and / or substitutions. Examples are those with deletions of up to 5 residues at 0 to 5 locations, insertions of up to 5 residues at 0 to 5 locations, and substitutions up to 20 residues. Other derivatives of Rv1196 are those comprising (for example, consisting of) a fragment of SEQ ID No: 1 having a length of at least 200 amino acids, such as at least 250 amino acids, in particular at least 300 amino acids. amino acids, more particularly at least 350 amino acids.
    In one embodiment, the antigen is derived from RvO125, e.g. comprises, e.g., consists of, a sequence having an identity of at least 70% with SEQ ID No: 2, such as at least 80%, at least 90%, more particularly at least 95%, for example at least 98%, such as at least 99%. Typical Rv0125-related antigens will include (for example, will be) a derivative of SEQ ID NO: 2 having a small number of deletions, insertions and / or substitutions. Examples are those with deletions of up to 5 residues at 0 to 5 locations, insertions of up to 5 residues at 0 to 5 locations, and substitutions up to 20 residues. Other derivatives of RvO125 are those comprising (for example, consisting of) a fragment of SEQ ID No: 2 having a length of at least 150 amino acids, such as at least 200 amino acids, in particular at least 250 amino acids, more particularly at least 300 amino acids. Particular derivatives of RvO125 are those comprising (for example, consisting of) the fragment of SEQ ID No: 2 corresponding to residues 1 to 195 of SEQ ID No: 2. Other immunogenic derivatives of RvO125 are those comprising (e.g. consisting of) the fragment of SEQ ID NO: 2 corresponding to residues 192 to 323 of SEQ ID NO: 2. Particularly preferred RvO125 related antigens are derivatives of SEQ ID NO: 2 in which at least one (for example, a two or even three) of the catalytic triads have been replaced or removed so that the protease activity is reduced and the protein is more readily produced - the catalytic serine residue can be deleted or replaced (for example, replaced by a alanine) and / or the catalytic histidine residue can be deleted or replaced and / or replaced, the catalytic aspartic acid residue can be deleted or replaced. Particularly interesting derivatives of SEQ ID No. 2 are derivatives in which the catalytic serine residue has been replaced (for example, replaced by an alanine). Antigens also of interest are Rv0125-related antigens which comprise, for example, consist of a sequence having an identity of at least 70% with SEQ ID No: 2, such as at least 80%, in particular at least 90%. , more particularly at least 95%, for example at least 98%, such as at least 99%, and in which at least one of the catalytic triads has been replaced or deleted or those comprising, for example consisting of, a fragment of SEQ ID No: 2 having a length of at least 150 amino acids, such as at least 200 amino acids, in particular at least 250 amino acids, more particularly at least 300 amino acids, and in which at least one of the catalytic triads has been replaced or deleted. Other immunogenic derivatives Rv0125 are those comprising (for example, consisting of) the fragment of SEQ ID NO: 2 corresponding to residues 192 to 323 of SEQ ID No: 2 in which at least one (for example, one, two or three) catalytic triads have been replaced or deleted. Particular immunogenic derivatives of Rv0125 are those comprising (e.g., consisting of) the fragment of SEQ ID NO: 2 corresponding to residues 1 to 195 of SEQ ID NO: 2 in which the catalytic serine residue (position 176 of SEQ ID No: 2) has been replaced (for example, replaced by alanine).
    Suitably, the antigen will comprise, for example, a sequence having an identity of at least 70% with SEQ ID NO: 3, such as at least 80%, in particular at least 90%, more particularly minus 95%, such as at least 98%, for example at least 99%. Typical M72-related antigens will include, for example, a derivative of SEQ ID NO: 3 having a small number of deletions, insertions and / or substitutions. Examples are those with deletions of up to 5 residues at 0 to 5 locations, insertions of up to 5 residues at 0 to 5 locations, and substitutions up to 20 residues. Other M72 derivatives are those comprising, for example, a fragment of SEQ ID No: 3 at least 450 amino acids in length, such as at least 500 amino acids, such as at least 550 amino acids, such as at least 600 amino acids, such as at least 650 amino acids or at least 700 amino acids. Since M72 is a fusion protein derived from the two individual Rv0125 and Rv1196 antigens, any fragment of at least 450 residues will comprise a plurality of epitopes from the full length sequence (Skeiky et al., J. Immunol. : 7618-7628; Skeiky Infect Immun., 1999 67 (8): 3998-4007; Dillon Infect Immun., 1999 67 (6): 2941-2950).
    An M72-related antigen will include, for example, a sequence having an identity of at least 70% with SEQ ID NO: 3, such as at least 80%, in particular at least 90%, more preferably at least 95%. %, such as at least 98%, for example at least 99%.
    Typical M72-related antigens will include, for example, a derivative of SEQ ID NO: 3 having a small number of deletions, insertions and / or substitutions. Examples are those with deletions of up to 5 residues at 0 to 5 locations, insertions of up to 5 residues at 0 to 5 locations, and substitutions up to 20 residues.
    In particular embodiments, the M72-related antigen will include residues 2 to 723 of SEQ ID NO: 3, for example, will comprise (or consist of) SEQ ID NO: 3 or will comprise (or consist of) SEQ ID NO: 4.
    Another antigen that can be used according to the present invention is the tuberculosis antigen Rv1753 and its variants, as described in WO 2010 010 180, for example a
    Rv1753 selected from Seq ID No: 1 and 2 to 7 of WO 2010 010 180, in particular Seq ID No: 1. Another antigen of interest in the case of tuberculosis is Rv2386 and its variants, as described in FIG. WO 2010 010 179, for example a sequence of Rv2386 selected from Seq ID No: 1 and 2 to 7 of WO 2010 010 179, in particular Seq ID No: 1. Other antigens of interest in the case of tuberculosis are Rv3616 and its variants, as described in WO 2011 092 253, for example a natural sequence of Rv3616 selected from Seq ID No: 1 and 2 to 7 of WO 2011/092253 or a modified sequence of Rv3616, as those selected from Seq ID No: 161 to 169, 179 and 180 of WO 2011/092253, in particular Seq ID No: 167. An additional antigen of interest is HBHA, as described in WO 97 044 463, WO 03 044 048 and WO 2010 149 657. Other antigens of interest are those comprising (or consist of ant en): Rv1174, also called DPV, as represented by SEQ ID No. 8 in WO 2010 010 177; Rv1793, also called MTI or Mtb9.9, as represented by SEQ ID No. 10 in WO 2010 010 177; Rv2087, also called MSL or Mtb9.8, as represented by SEQ ID No. 9 in WO 2010 010 177; Rv3616, also referred to as HTCC1 or Mtb40, as represented by SEQ ID Nos. 1 and 2 to 7 in WO 2010 010 177 or SEQ ID Nos. 161 to 169, 179 or 180 in WO 2011/092253; and / or Rv3874, also known as CFP10 or Tb38.1, as represented by SEQ ID No. 9 in WO 2010 010 177; or an immunogenic portion (such as at least 20, 50, 75 or 100 residues thereof) or a variant thereof (eg having an identity of at least 70%, 80%, 90% or 95% with these). (WO 2010/010177 and WO 2011/092253 are hereby incorporated by reference in their entirety).
    The tuberculosis antigens are suitably used in the form of a polypeptide, but may alternatively be provided in the form of a polynucleotide encoding said polypeptide.
    An additional antigen that can be used according to the present invention is derived from varicella-zoster virus (VZV). The VZV antigen useful in the invention may be any suitable VZV antigen or an immunogenic derivative thereof, suitably a purified VZV antigen.
    In one embodiment, the VZV antigen is the glycoprotein gE of VZV (also called gp1) or an immunogenic derivative thereof. The wild type or full length gE protein consists of 623 amino acids comprising a signal peptide, the main portion of the protein, a hydrophobic anchoring region (residues 546 to 558) and a C-terminal tail. In one aspect, a C-terminal truncated gE protein (also referred to as truncated gE) is used, whereby truncation removes 4 to 20 percent of the total amino acid residues at the carboxy terminus. In another aspect, the truncated gE protein lacks the carboxy-terminal anchoring region (suitably approximately amino acids 547 to 623 of the wild-type sequence). In a further aspect, the gE protein is a truncated gE protein having the sequence of SEQ ID NO. 1. The antigen gE, its non-anchored derivatives (which are also immunogenic derivatives) and their production are described in EP 0 405 867 and the references cited therein [see also Vafai A., Antibody binding sites on truncated forms of varicalla-zoster virus gpl (gE) glycoprotein, Vaccine 1994 12: 1265-9]. EP 192902 also describes gE and its production. A truncated gE protein is also described by Haumont et al. Virus Research (1996) Vol 40, p 199-204, incorporated herein by reference in its entirety. An adjuvanted composition of VZV gE suitable for use according to the present invention is described in WO 2006/094756, i.e. a carboxy terminally truncated VZV gE protein in combination with a adjuvant comprising QS21, 3D-MPL and liposomes additionally containing cholesterol. Leroux-Roels I. et al. (J. Infect, Dis 2012, 206: 1280-1290) reported a Phase I / II clinical trial evaluating the VZV truncated gE adjuvanted subunit vaccine.
    In another embodiment, the compositions of the present invention may comprise an immunogen or antigen that is a derivative of any of the antigens described herein. As used herein, the term "derivative" refers to an antigen that is modified from its naturally occurring form. The derivatives of the present invention are sufficiently similar to native antigens to retain antigenic properties and remain capable of eliciting an immune response directed against the native antigen. Whether or not a given derivative triggers such an immune response can be measured by an appropriate immunoassay, such as ELISA or flow cytometry.
    cryoprotecteur
    A cryoprotectant suitable for use in the present invention is an amorphous sugar, such as a sugar selected from sucrose, trehalose, lactose, raffinose, and combinations thereof. In one embodiment, the cryoprotectant and sucrose or trehalose or a combination thereof. The cryoprotectant may further comprise sugars enhancing the structure of the freeze-drying cake, such as dextran.
    In one embodiment, the liquid mixture for drying, for example by lyophilization, contains at least 3% (w / v), at least 4% (w / v), at least 5% (w / v). ), at least 6% (w / v) of the cryoprotectant. In another embodiment, the cryoprotectant is present in the liquid mixture in a total amount of less than 10%, less than 8%, less than 7%, less than 6% or less than 5.5% (wt%). volume). Expressed otherwise, the cryoprotectant is present in the liquid mixture in a total amount of at least 4%, at least 4.5% or at least 5% (% w / v), but less than 10%, less than 8%, less than 7% or less than 6% (% by weight / volume).
    The total cryoprotectant concentration in the liquid mixture is suitably from 5 to 10% (w / v), whereby at least 5% sucrose, trehalose or a combination thereof is present. In one embodiment, 5% sucrose is used. In one embodiment, 5% trehalose is used. In specific embodiments, the liquid mixture comprises at least 5% (w / v%) or between 5 and 10% (w / v%) of sucrose, trehalose or a combination thereof.
    In another embodiment, the reconstituted vaccine contains at least 0.6% (w / v), at least 0.8% (w / v), at least 1% (w / v) or at least 1.2% (w / v) of the cryoprotectant. In another embodiment, the cryoprotectant is present in the reconstituted vaccine in a total amount of less than 2%, less than 1.6%, less than 1.4%, less than 1.2% or less than 1.1% ( % by weight / volume). Expressed in another manner, the cryoprotectant is present in the reconstituted vaccine in a total amount of at least 0.8%, at least 0.9% or at least 1% (% w / v) but less than 2%, less than 1.6%, less than 1.4% or less than 1.2% (% by weight / volume).
    The total cryoprotectant concentration in the reconstituted vaccine is suitably 1 to 2% (w / v), whereby at least 1% sucrose, trehalose or a combination thereof is present. In one embodiment, 1% sucrose is used. In one embodiment, 1% trehalose is used. In specific embodiments, the reconstituted vaccine comprises at least 1% (w / v%) or 1% to 2.5% (w / v%) of sucrose, trehalose or a combination thereof. this.
    In another embodiment, the ratio of cryoprotectant concentration to that of liposome lipid is from 10 to 20. In specific embodiments, the ratio is 10, 15 or 20.
    In an alternative embodiment, the amorphous sugar also contributes to tonicity or provides tonicity in the reconstituted vaccine, which requires a higher concentration of amorphous sugar, such as 8 to 10% (e.g., 9.25%). sucrose (w / v) in the reconstituted vaccine, or 8-10% trehalose (w / v) (e.g., 9.25%), or a combination of sucrose and trehalose in an amount total of 8 to 10% (w / v).
    Additional excipients
    In one embodiment, the liquid mixture is a substantially aqueous mixture optionally including other solvents, such as ethanol or isopropanol.
    In another embodiment, a buffer is added to the composition. The pH of the liquid mixture is adjusted taking into account the therapeutic components of the composition. Suitably, the pH of the liquid mixture is at least 4, at least 5, at least 5.5, at least 5.8, at least 6. Expressed differently the pH of the liquid mixture may be less than 9, less than 8, less than 7.5 or less than 7. In other embodiments, the pH of the liquid mixture is between 4 and 9, between 5 and 8 between 5.5 and 7.5 or between 5.8 and 6.4.
    A suitable buffer may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. In one embodiment, the buffer is a phosphate buffer, such as Na / Na2PO4, Na / K2PO4 or K / K2PO4.
    The buffer may be present in the liquid mixture in an amount of at least 6 mM, at least 10 mM or at least 40 mM. Or, the buffer may be present in the liquid mixture in an amount less than 100 mM, less than 60 mM or less than 40 mM.
    In specific embodiments, the buffer is a phosphate buffer present in the liquid mixture in an amount of between 6 and 40 mM, such as about 10 mM. Suitably, the buffer is selected from Na / K2PO4, K / K2PO4 or succinate. In particular, K / K2PO4 is used as a buffer.
    The formulation of a protein antigen for lyophilization purposes according to the present invention may comprise a surfactant. Surfactants particularly suitable for use in the present invention include polysorbates, particularly polysorbate 80 (PS80), and poloxamer 188.
    In another embodiment, the liquid mixture or the dried composition contains a limited amount of NaCl, for example less than 60 mM, less than 50 mM, less than 40 mM, less than 30 mM, less than 25 mM, or less than 20 mM NaCl in the liquid mixture. In specific embodiments, the liquid mixture contains less than 10% cryoprotectant, for example sucrose, and less than 50 mM NaCl. Alternatively, the liquid mixture contains less than 5% cryoprotectant, for example sucrose, and less than 30 mM NaCl.
    In another embodiment, the liquid mixture or the dried composition contains a limited amount of salts, for example less than 60 mM, less than 50 mM, less than 40 mM, less than 30 mM, less than 25 mM, or less than 20 mM NaCl in the liquid mixture.
    The tonicity of the composition after reconstitution can be adjusted using methods known to those skilled in the art, for example by providing sufficient isotonicity agents in the dried composition, for example by reconstituting the dried composition with at least one solvent isotonic. In particular embodiments, the tonicity of the reconstituted composition may be adjusted by the addition of appropriate amounts of NaCl upon reconstitution, for example by reconstituting the dried composition with saline, or by increasing the initial amount of cryoprotectant to levels giving isotonicity when reconstituted with water for injection. Alternatively, the dried composition is reconstituted with an isotonic aqueous solution of a nonionic isotonic agent, for example sorbitol. In a preferred embodiment, the lyophilized composition is reconstituted with saline.
    It is well known that for parenteral administration, the solutions must have a pharmaceutically acceptable osmolality to prevent cell deformation or lysis. A pharmaceutically acceptable osmolality will generally mean that the solutions have an osmolality that is approximately isotonic or slightly hypertonic. Suitably, the compositions of the present invention, when reconstituted, will exhibit an osmolality in the range of 250 to 750 mOsm / kg, for example the osmolality may be in the range of 250 to 550 mOsm / kg, as in the range of 280 to 500 mOsm / kg. The osmolality can be measured according to techniques known in the art, for example using a commercially available osmometer, for example the Advanced Model 2020 available from Advanced Instruments Inc. (USA).
    The present invention further provides a composition as described herein for use in the treatment or prevention of disease, the composition being an immunogenic composition or a vaccine composition. In a specific example of this embodiment, the invention provides an immunogenic composition, such as a vaccine composition, for use in the treatment or prevention of a disease associated with one or more antigens described above. In one embodiment, the invention provides an immunogenic composition as described herein for use in the treatment or prevention of a disease selected from malaria, tuberculosis, COPD, HIV and herpes .
    The present invention further provides methods of treating or prophylaxis of malaria, tuberculosis, COPD, HIV or herpes in an individual in need thereof, including the step of providing said individual with an amount of effective immunogenic or vaccine composition as described herein. The invention also provides a method for producing a dried composition as described herein, comprising the following steps: i. mixing a plurality of components to obtain a liquid mixture, said components comprising: a. a liposomal liquid preparation comprising liposomes, said liposomes comprising a neutral lipid and a sterol; b. a saponin; and c. a cryoprotectant; and ii. drying the mixture under reduced pressure.
    In one embodiment, the liquid liposomal preparation of step i (a) further comprises a lipopolysaccharide. Expressed in another manner, the liquid liposomal preparation optionally comprises a lipopolysaccharide. The lipopolysaccharide is as defined hereinabove.
    In another embodiment, the mixed liquid composition of step (i) further comprises (or optionally) one or more components selected from antigens, immunogens, buffers and surfactants. Therefore, the method of producing a dried composition as described herein may comprise the following steps: i. mixing a plurality of components to obtain a liquid mixture, said components comprising: a. a liposomal liquid preparation comprising liposomes, said liposomes comprising a neutral lipid and a sterol; b. a saponin; vs. a cryoprotectant; and D. one or more ingredients selected from an antigen, a buffer and a surfactant; and ii. drying the mixture under reduced pressure.
    As used herein, a mixed liquid composition is a composition comprising multiple components, wherein an isolated component is not necessarily liquid, but the resulting blended composition (mixture) is in liquid form, that is, that is, the mixed composition is amorphous and flows freely, and its volume is constant under a given pressure.
    In one embodiment, the mixed liquid composition of step (i) comprises any two elements or the three elements among: an antigen, a buffer and a surfactant. Expressed in another manner, the mixed liquid composition optionally comprises any two elements or the three elements among: an antigen, a buffer and a surfactant.
    In one embodiment of the present invention, the liposomes in the liquid liposome preparation do not contain any cryoprotectants, for example they have not been formed in the presence of a cryoprotectant. In one embodiment of the present invention, the liquid liposomal formulation contains no cryoprotectant, for example it does not contain amorphous sugar, such as an amorphous sugar selected from sucrose, trehalose, lactose, raffinose and their combinations.
    In an alternative embodiment, the components of the liquid composition are mixed in a specific order. First, a solution of the cryoprotectant in water is provided, to which (if present) the buffer solution is added. Second, the liquid liposomal preparation is added. Third, the saponin component is added. Fourth, (if present) the surfactant is added and, fifth, the antigen (if present) is added. Between certain process steps, the mixture may be stirred for a period of time, for example 10 minutes or more, 15 minutes or more, 30 minutes or more, 45 minutes or more or 15 to 45 minutes. In one embodiment, the mixture is stirred after adding the saponin. In another embodiment, the mixture is stirred for at least 15 minutes after adding the surfactant. In another embodiment, the mixture is stirred for at least 15 minutes after the addition of the antigen. In a further embodiment, the mixture is stirred for at least 15 minutes after each of the saponin, surfactant and / or antigen addition steps.
    In one embodiment, some or all of the activities of step i. is carried out at room temperature.
    The drying under reduced pressure of a liquid mixture as obtained in step ii. can be achieved using different methodologies known in the art. In one embodiment, drying in step ii. is carried out by lyophilization. The terms "freeze drying" or "lyophilization" 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 freeze-drying or freeze-drying cycle usually consists of three process steps. In the first phase of the process, a mainly aqueous solution or mixture is frozen, i.e. "freezing the mixed liquid composition of step i. ". Then, the water is removed, i.e. "drying the frozen composition", first by sublimation during primary drying. In the third phase, unfrozen water is removed by diffusion and desorption during secondary drying.
    In order to define the described method, the following terms are used because they are known in the art. The term "glass transition temperature" or "Tg" is the temperature at which an amorphous solid softens after heating or becomes brittle after cooling. The term "Tg" refers to the glass transition temperature in the frozen state. The term "collapse temperature" or "Tc" refers to the temperature applied during primary drying and at which an amorphous material softens to a point where it can no longer support its own structure.
    In lyophilization of step ii., The mixed liquid composition of step i. is frozen before drying by bringing the temperature of the product below the Tg 'of the composition. In one embodiment, freezing is accomplished by exposing the sample or aqueous mixture to a constant storage temperature at a freezing temperature that is less than Tg '. In an alternative embodiment, the product may be frozen by applying a ramp time freeze, i.e. gradually reducing the storage temperature to a freezing temperature below Tg '. According to embodiments, the freezing temperature is a temperature lower than 5 ° C with respect to Tg ', lower than 7.5 ° C with respect to Tg', or lower than 10 ° C with respect to Tg ', such that than or below -50 ° C.
    Drying the frozen composition under reduced pressure as envisaged in the lyophilization of step ii. described herein will generally be performed in two phases, i.e., primary drying and secondary drying. In one embodiment, the drying will include: - primary drying at a temperature below the Tc of the product, and - secondary drying at a temperature above the Tc of the product and less than the Tg of the product.
    In one embodiment, step ii. The drying process described herein is completed in less than 48 hours, in less than 36 hours, in less than 30 hours, in less than 28 hours. In a specific embodiment, step ii. is over in less than 28 hours.
    In one embodiment, the antigen is RTS, S and the primary drying is performed at a pressure of less than 90 μbar and / or greater than 45 μbar. In the same embodiment, the conditions of the primary drying can be applied for 19 hours and should be applied for at least 15 hours.
    In an alternative embodiment, for example when the antigen is a VZV gE derivative, a more conservative lyophilization cycle is used, as illustrated in Figure 1-B.
    The dried composition obtained by the method described is capable of eliciting an immune response in a subject. Said immune response is in correspondence with the adjuvant, and with any antigen present in the composition.
    In one embodiment, the cryoprotectant (which may be in liquid form or in another form) is mixed with the liposomal preparation prior to mixing with the saponin. In a further embodiment, the surfactant is mixed prior to the antigen. According to another embodiment, the order of the mixture is first of all the mixture of the cryoprotectant and the buffer, then the addition of the liquid liposome preparation, the saponin, the surfactant and the antigen in the order respective.
    In the process description, each of the terms has the same meaning as for the compositions described herein.
    Processes for obtaining or preparing the liposomal preparation are described in WO 2013/041 572, which is incorporated herein by reference in its entirety. A suitable method described in this document comprises: (a) producing a lipid film by (i) dissolving a mixture of lipids in isopropanol to form a homogeneous mixture, and (ii) removing the solvent from the homogeneous mixture to form a lipid film, wherein the lipid mixture comprises lipid and sterol; (b) hydrating the lipid film with a hydration solution to form a suspension of large liposomes; (c) reducing the size of the large liposome suspension produced in step (b) with a high shear and high pressure homogenizer to form liposomes; and optionally (d) sterilizing the liposomes. Suitably, step (c) comprises the steps of: (c ') pre-homogenizing the large liposome suspension solution with a high shear mixer, and (c' ') homogenizing the produced solution in step (c ') with a high pressure homogenizer.
    Unexpectedly, in order for the dried composition to retain its ability to potentiate immunity or its immunogenicity, it was not necessary for the liposomes of the adjuvant to be formed and / or formulated, for example during the preparation of the liposomal preparation, in the presence of a cryoprotectant.
    The following examples illustrate the invention.
    Examples 1. Example 1 - Co-lyophilization of a vaccine based on RTS, S / AS01 (quadridosis)
    Vaccine based on RTS, S / AS01
    The RTS, S antigen consists of two polypeptide chains, RTS and S. The RTS polypeptide contains a portion (aa 207 to 395) of the P. falciparum CS protein fused to the surface antigen (S) of the hepatitis B. The RTS fusion protein and the S polypeptide are coexpressed in Saccharomyces cerevisiae and assemble spontaneously to give pseudoviral particles called RTS, S. These purified particles constitute the RTS, S antigen used in the formulation of the vaccine. Further details for obtaining the RTS, S antigen are available in WO 93/10152, incorporated herein by reference in its entirety. AS01 refers to a vaccine adjuvant comprising QS21, 3D-MPL in a liposomal formulation containing cholesterol.
    Concentrated mass of liposomes
    The concentrated mass of liposomes was prepared as described in Example 3 of WO 2013/041 572 (hereby incorporated by reference in its entirety). In brief, the concentrated mass of liposomes was prepared in 2 steps. The first step was the preparation of a lipid film. DOPC (dioleoyl phosphatidylcholine), 3D-MPL and cholesterol were sequentially dissolved in isopropanol. Then, the isopropanol was removed with stirring and with a reduced pressure gradient in a heating bath at 55 ° C to obtain a film residue. The pressure was then gradually reduced and final drying was applied to obtain a lipid film. The second step was the preparation of the concentrated mass of liposomes. For this purpose, the lipid film was rehydrated in PBS to form a coarse suspension of liposomes. The liposome suspension was then homogenized with a high shear mixer in-line with a high pressure homogenizer to produce the desired nanoscale liposomes. The resulting concentrated liposome mass is filtered through a 0.22 μm PES membrane. The concentrated mass of liposomes usable in the example contained 40 mg / ml of DOPC, 10 mg / ml of cholesterol, 2 mg / ml of MPL in a 10 mM phosphate buffer (pH 6.1) and 150 mM NaCl.
    Vaccine formulation The antigen, ie, RTS, S, and adjuvant, ie AS01, were co-formulated for freeze-drying in water for injection. adding 1) 30% sucrose (add 5%) 2) 100 mM buffer, PO4 (K / K2) or succinate, pH 6.1 (add 10 mM), 3) 40 mg / ml mass of liposomes ( add 5 mg / ml), 4) 5 mg / ml of QS21 (add 0.25 mg / ml), then the adjuvant preparation thus obtained was stirred for 15-45 minutes at room temperature. Then, 3% (w / v) polysorbate 80 (add 0.0312%) was added and the mixture was stirred for 15-45 minutes at room temperature. RTS, S antigen was added at 0.25 mg / ml and the resulting solution was stirred for 15-45 minutes at room temperature. The pH was measured and adjusted to 6.1 if necessary.
    Control formulations containing either adjuvant or antigen were also prepared. The tested samples are as follows: 1. RTS, S (lot A) PO4 (Na / Na2) pH 6.8 2. RTS, S (lot B) PO4 (Na / Na2) pH 6.8 3. AS01E3 PO4 ( K / K2) pH 6.1 4. colyo RTS, S (lot A) / AS succinate 10 mM pH 6.1 5. colyo RTS, S (lot A) / AS PO4 (K / K2) 10 mM pH 6, 1 6. colyo RTS, S (lot B) / AS succinate 10 mM pH 6.1 7. colyo RTS, S (lot B) / AS PO4 (K / K2) 10 mM pH 6.1 8. RTS, S ( batch A) succinate pH 6.1 9. RTS, S (lot A) PO4 (K / K2) pH 6.1 10. RTS, S (lot B) succinate pH 6.1 11. RTS, S (lot B) PO4 (K / K2) pH 6.1 12. AS01 succinate pH 6.1 13. AS01 PO4 (K / K2) pH 6.1 14. Sucrose 5%
    Freeze-drying
    The resulting formulations were filled into glass vials (0.5 ml fill volume) and lyophilized by applying a 28-hour lyophilization cycle as shown in Figure 1-A.
    Evaluation
    Several aspects were judged to evaluate the thermal stability of samples for 1 year at 4 and 30 ° C, 6 months at 37 ° C and 3 months at 45 ° C. 1. Visual appearance of cakes
    The cakes had an elegant pharmaceutical appearance (similar to that of the bidose formulation of Mosquirix) for all formulation groups. Curiously, the formulations containing the RTS, S but not the adjuvant showed a slight retraction. The appearance of the cakes was found to be stable for 12 months at 30 ° C and 6 months at 37 ° C. A slight contraction was observed after 6 months at 45 ° C, which is probably due to a decrease in Tg due to an increase in the moisture content of the cake. 2. Morphology of liposomes by electron microscopy
    The liposome structure was also analyzed by transmission electron microscopy using a Zeiss Libra120. Negative staining analysis was performed according to the conventional two-step negative staining method using sodium phosphotungstate as a contrast agent (Hayat M.A. & Miller S.E., 1990, Negative Staining, Mc
    Graw - Hill ed.), Using carbon-Formvar carbon dioxide coated 200-mesh gratings and performing the 100 kV analysis. The samples were also analyzed by cryo-microscopy at 80 kV, without any contrast agent, after vitrification at 107 ° K in the holes of a carbon-coated plastic mesh (Dubochet et al., 1987, in Cryotechniques in Biological EM, RA Steinbrecht and K. Zierold, eds Springer Verlag). The analysis showed that in the phosphate buffered solutions, liposome morphology was retained after co-lyophilization of RTS, S / AS01 and stable for 6 months at 45 ° C. 3. Antigen-adjuvant interactions The interaction between the RTS antigen, S and the AS01, DOPC and cholesterol components is investigated by ultracentrifugation in a sucrose gradient followed by quantification of RTS, S, DOPC and cholesterol in the fractions collected. The tested samples reconstituted in 150 mM NaCl are compared to Mosquirix ™ (RTS, S lyophilized and reconstituted with AS01 liquid). As for the
    Mosquirix ™, no interaction was observed between RTS, S and adjuvant components. 4. Particle size of liposomes
    Colloidal stability was assessed by nephelometry, which showed a slightly higher stability of the phosphate buffered formulation compared to the succinate buffered formulations. The size of the AS01 liposomes in the co-lyophilized samples was measured by DLS, which showed a hydrodynamic radius of about 95 nm (the hydrodynamic radius in the control liquid formulation is 110 nm). This is most likely due to the presence of PS80 in the formulation (and is not due to the lyophilization step, nor to the presence of RTS, S). The size of the AS01 liposomes remained stable over time at elevated temperature. The results of nephelometry after incubation at different temperatures are shown in Figure 3.
    5. Particle size of RTS, S
    The particle size of RTS, S was measured by SEC-HPLC on a TSKgel G5000PWXL with fluorescence detection (ÀEx: 280 nm / AtEm: 320 nm) to avoid
    Interference with adjuvant components when UV detection is used. The retention time of the RTS, S particles in the samples where the adjuvant and the antigen were co-lyophilized was identical to the RTS, S control purified in bulk and remained stable for 1 year at 4 and 30 ° C. month at 37 ° C and 3 months at 45 ° C.
    6. RTS and S Proteins The integrity of RTS and S proteins has been demonstrated by SDS-PAGE and ELISA. SDS-PAGE profiles did not change for 1 year at 4 and 30 ° C, 6 months at 37 ° C. At 45 ° C, slight streaks were visible after 3 months of storage. However, antigenicity by ELISA remained stable for 1 year at 4 and 30 ° C, 6 months at 37 ° C and 3 months at 45 ° C. The antigenicity of RTS, S was measured by a CS-S sandwich capture ELISA assay (coating with a monoclonal anti-CSP and revealing with a polyclonal anti-S). 7. Chemical integrity of the adjuvant components The chemical integrity of the AS01 components (QS21 and MPL) was evaluated as it is known that both components are sensitive to hydrolysis. The hydrolysed congeners of QS21 (QS21H) and MPL are quantified by HPLC methods. The concentration and hydrolysis of QS21 (QS21H) was determined by reverse phase HPLC on a Symetry RP18 column, with UV detection at 214 nm. MPL congeners were determined after derivatization with DNBA and RP-HPLC on a C18 Waters Symmetry column and fluorescence detection (excitation at 345 nm and 515 nm emission).
    QS21H remained below 3% in all freeze-dried samples. In contrast, the content of QS21H in the reference liquid adjuvant formulation (1 mg / ml of DOPC, 0.25 mg / ml of cholesterol, 50 μg / ml of QS21, 50 μg / ml of MPL in 10 mM of buffer). phosphate (pH 6.1), 150 mM NaCl) rapidly increased at elevated temperature (greater than 3% after 1 month at 37 ° C and after 3 months at 30 ° C).
    The MPL congeners also remained stable for 12 months at 30 ° C and for 6 months at 45 ° C in all lyophilized formulations. In contrast, the reference liquid adjuvant formulation is not stable at elevated temperature, as indicated by deacylation of MPL (decrease in the proportion of penta and hexa congeners, together with an increase in the proportion of tetra congeners). The proportion is greater than 35% after 1 month at 45 ° C, after 3 months at 37 ° C or after 6 months at 30 ° C. 8. Preclinical Immunogenicity The immunogenicity of the co-lyophilized samples was compared to the immunogenicity of Mosquirix ™ in a mouse model. Antibody-like responses and CD8 T cell responses directed against both S antigen and CS antigen were evaluated, and CD4 responses directed against S antigen were evaluated in CB6F1 mice.
    Fresh groups of leukocytes collected at different time points were stimulated for 6 hours with 15-mer peptide pools covering the CSP or HBs sequence. Specific cellular responses of CSP and HBs were assessed by ICS which measures the amount of CD4 + and CD8 + T lymphocytes expressing IFN-γ and / or IL-2 and / or TNFα. All ICS analyzes were performed using the FlowJo software.
    The results of the study show that colyophilization of RTS, S and AS01 has no impact on immunogenicity (identical cellular and humoral responses for both RTS and S to T0, compared with Mosquirix ™ current.
    In addition, co-lyophilization RTS, S / AS01 was stable for 1 year at 30 ° C (and for 1 year at 30 ° C plus 1 month at 45 ° C), 6 months at 37 ° C and 3 ° C. month at 45 ° C (except for a slight increase in Hbs-specific CD8 + T cell responses observed after 3 months at 37 ° C, but not at 45 ° C). After reconstitution of RTS, lyophilized in the reference liquid adjuvant formulation previously incubated for 3 months at 37 ° C, a slight increase in CSP specific CD4 + T cell responses was observed). The reference liquid adjuvant formulation incubated for 3 months at 45 ° C could not be injected because it was found to be hemolytic. The immune response is illustrated in FIG. 2. EXAMPLE 2 Co-lyophilization of a VZV / AS01 gE-based vaccine (unidose) The VZV gE antigen (also referred to herein as gE) is a truncated form of the varicella zoster virus glycoprotein E, has the sequence disclosed in FIG. 4 and is obtained as disclosed in example 2 of WO 2006/094 756. AS01 refers to the adjuvant of the vaccine, comprising QS21, 3D-MPL in a liposomal formulation containing cholesterol.
    The concentrated mass of liposomes used is the same as that described in Example 1. The antigen, that is to say, gE of VZV, and an adjuvant, that is to say AS01, have been co- formulated for lyophilization in water for injection by mixing: 1) 30% sucrose (add 5%) 2) 100 mM buffer, PO4 (K / K2), pH 6.1 (add 10 mM) 3) 40 mg / ml mass of liposomes (add 2.5 mg / ml), and 4) 5 mg / ml of QS21 (add 0.125 mg / ml), then the adjuvant preparation thus obtained was stirred for 15 to 45 minutes at room temperature. Then, 3% (w / v) polysorbate 80 (add 0.02%) was added and the mixture was stirred for 15-45 minutes at room temperature. The VZV gE antigen was added at 0.125 mg / ml and the resulting solution was stirred for 15-45 minutes at room temperature. The pH was measured and adjusted to 6.1 if necessary.
    Control formulations containing either adjuvant or antigen were also prepared. The tested samples were as follows: 1) gE of VZV / AS01
    2) gE of the VZV 3) AS01.
    Freeze-drying
    The resulting formulations were filled into glass vials (0.5 ml fill volume) and lyophilized by applying a 40 hour lyophilization cycle as shown in Figure 1-B.
    Evaluation
    The purpose of this experiment was to evaluate the feasibility of co-lyophilization of another antigen (VZV gE) with AS01 adjuvant. The integrity of both antigen and adjuvant was evaluated directly after co-lyophilization.
    The lyophilized material was analyzed after reconstitution with 150 mM NaCl and compared to a control vaccine based on VZV gE (freeze-dried VZV gE and reconstituted in liquid AS01 or in buffered saline (10 mM phosphate, 150). mM NaCl, pH 6.1) 1. Injection capacity
    The pH values in the different groups are slightly lower (e.g., about 0.4 units) than that of the control Shingrix vaccine, although the pH was set at 6.1 in the corresponding final masses (before lyophilization). The osmolality determined in the 3 groups is similar to that of the control.
    2. Morphology of liposomes by electron microscopy
    The structure of the liposomes was also analyzed by transmission electron microscopy with negative staining, using the same protocol as that of Example 1. The adjuvant exhibited the morphology characteristic of the structural advantage of AS01 at the EM level. i.e. liposomes of various sizes and shapes, with clearly visible perforations in the membranes. Putative gE antigens have been observed as a very small amorphous material dispersed between liposomes.
    The same profile was observed in sample gE / AS01 before and after lyophilization, and in the control gE cake reconstituted in AS01 adjuvant, indicating that liposome morphology is retained after co-lyophilization of gE / AS01. 3. Particle size of liposomes
    The size of the AS01 liposomes in the samples was measured by DLS (in the AS01 containing groups), showing a hydrodynamic radius of about 90 nm similar to the hydrodynamic radius in the control liquid formulation. The value obtained was close to the expected value for the AS01 liposome
    4. Size of gE in solution
    The size of the antigen gE was measured by SEC-HPLC on a TSKgel G4000PWXL with fluorescence detection (ÀEx: 280 nm / AtEm: 320 nm) in order to avoid interference with adjuvant components when UV detection is used. The retention time of the VZV gE in the samples where the adjuvant and the antigen were co-lyophilized was identical to the bulk purified control gE, indicating the co-lyophilization process has no impact on the size of the gE in solution.
    5. Integrity of the gE Protein The integrity of the VZV gE protein was demonstrated by SDS-PAGE analysis of the samples before and after lyophilization. The SDS-PAGE profiles (see Figure 5) were similar for the co-lyophilized samples and the control gE of the VZV (purified mass and drug product), both in reducing condition (R) and in non-reducing condition (NR ). A small band of higher molecular weight was observed in the co-freeze-dried sample, which probably corresponds to aggregation, but does not represent a significant amount of protein. Legend for Figure 5
    6. In Vitro Therapeutic Activity (Antigen Activity by ELISA) In vitro therapeutic activity was measured by ELISA in samples containing VZV gE. The test is an ELISA by inhibition based on human polyclonal antibodies against varicella and shingles antigens (VARITECT®).
    Briefly, serial dilutions of the samples containing the VZV gE antigen are incubated with a fixed amount of VARITECT®. After incubation, human anti-gE antibodies, which do not react with the VZV gE antigen samples, are detected by incubation on a microplate coated with the antigen gE. The antigen / antibody complex is revealed by the addition of a peroxidase labeled anti-human IgG rabbit antibody followed by the addition of tetramethylbenzidine. The antigenic activity of the VZV gE is obtained by dividing the gE content of the VZV by the protein content (measured by Lowry). The therapeutic activity, which can only be applied to the final containers, is obtained by dividing the gE content of the VZV by the titer of the standard that was used for the validation of the process.
    The ratio between the in vivo therapeutic activity and the gE content of the VZV was close to 1 in all the groups tested, as in the control. These results further confirm the integrity of the VZV gE antigen after lyophilization in the presence of AS01.
    7. Chemical integrity of the adjuvant components
    As in Example 1, the chemical integrity of AS01 components (QS21 and MPL) was evaluated in samples containing AS01. The hydrolysed congeners of QS21 (QS21H) and MPL are quantified by HPLC methods. QS21H remained below 3% in all freeze-dried samples. There was no impact of the freeze-drying process on the chemical integrity of the MPL, as indicated by similar proportions of tetra, penta and hexa congeners before and after lyophilization in both groups.
    QS21 triggers hemolytic activity when it is not deactivated with cholesterol within the liposomal membrane. Hemolytic activity was therefore evaluated in AS01 containing formulations before and after lyophilization. Regardless of the conditions tested, the haemolytic rate remained below the acceptable baseline of 1% (see Figure 6). None of them were responsible for removing the deactivation of QS21.
    权利要求:
    Claims (44)
    [1]
    A composition dried under reduced pressure from a liquid mixture comprising an adjuvant which comprises a saponin in a liposomal formulation in which the liposomes contain a neutral lipid and a sterol, and a cryoprotectant which is an amorphous sugar.
    [2]
    2. Composition according to claim 1, wherein the drying under reduced pressure is carried out by lyophilization.
    [3]
    3. A composition according to any one of the preceding claims, wherein the liquid mixture is an aqueous mixture.
    [4]
    4. A composition according to any one of the preceding claims, wherein the neutral lipid is a phosphatidylcholine selected from egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine, preferably DOPC.
    [5]
    5. The composition according to any one of the preceding claims, wherein the saponin is QS21.
    [6]
    The composition of any one of the preceding claims, wherein the sterol is cholesterol.
    [7]
    The composition of any of the preceding claims, wherein the adjuvant further comprises a TLR-4 agonist.
    [8]
    The composition of claim 7, wherein the TLR-4 agonist is a lipopolysaccharide.
    [9]
    The composition of claim 7 or 8, wherein the TLR-4 agonist or lipopolysaccharide is 3D-MPL.
    [10]
    10. A composition according to any one of the preceding claims, further comprising an antigen.
    [11]
    The composition of claim 10 wherein the antigen is a protein, such as a recombinant protein.
    [12]
    The composition of claim 10 or 11, wherein the antigen is derived from Plasmodium falciparum, Mycobacterium tuberculosis, HIV, Moraxella, ntHi or varicella zoster virus.
    [13]
    13. A composition according to any one of claims 10 to 12, wherein the antigen is selected from RTS, S, M72, UpsA, PiLa, PE-Pila and VZV gE or a truncated form thereof.
    [14]
    14. Composition according to any one of claims 10 to 13, contained in a single bottle.
    [15]
    15. A composition according to any one of the preceding claims, wherein the cryoprotectant is an amorphous sugar or a mixture of amorphous sugars.
    [16]
    A composition according to any one of the preceding claims, wherein the cryoprotectant is selected from the group of amorphous sugars consisting of sucrose, trehalose, lactose, raffinose and combinations thereof.
    [17]
    A composition according to any one of the preceding claims, wherein the cryoprotectant is present in an amount sufficient to allow storage for 12 to 24 to 36 months at 30 ° C, 6 to 12 months at 37 ° C, or month at 45 ° C.
    [18]
    18. A composition according to any one of the preceding claims, wherein the cryoprotectant is present in an amount of 3 to 10% (w / v) of the liquid mixture.
    [19]
    19. A composition according to any one of the preceding claims, wherein the cryoprotectant comprises sucrose.
    [20]
    20. A composition according to any one of the preceding claims, wherein the cryoprotectant comprises trehalose.
    [21]
    21. A composition according to any one of the preceding claims, wherein the cryoprotectant is a combination of at least two cryoprotectants selected from sucrose, trehalose and dextran.
    [22]
    22. A composition according to any one of the preceding claims, wherein the cryoprotectant is present in the mixture in a total amount of at least 3%, at least 4%, at least 5% or at least 6% (% by weight / volume).
    [23]
    23. A composition according to any one of the preceding claims, wherein the cryoprotectant is present in the mixture in a total amount of less than 10%, less than 8%, less than 7%, less than 6% or less than 5.5%. % (% by weight / volume).
    [24]
    24. A composition according to any one of the preceding claims, wherein the cryoprotectant is present in the mixture in a total amount of at least 4%, at least 4.5% or at least 5%, and less than 10%, less than 8%, less than 7% or less than 6% (% by weight / volume).
    [25]
    25. A composition according to any one of the preceding claims, wherein the liquid mixture comprises at least 5% or between 5 and 10% (w / v%) of sucrose, trehalose or a combination thereof.
    [26]
    26. A composition according to any one of the preceding claims, further comprising a buffer.
    [27]
    27. A composition according to any one of the preceding claims having a pH of at least 4, at least 5, at least 5.5, at least 5.8, at least 6.
    [28]
    28. Composition according to any one of the preceding claims, having a pH of less than 9, less than 8, less than 7.5, or less than 7.
    [29]
    29. A composition according to any one of the preceding claims, having a pH between 4 and 9, between 5 and 8, between 5.5 and 7.5 or between 5.8 and 6.4.
    [30]
    30. Composition according to any one of the preceding claims, comprising a buffer selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS.
    [31]
    31. A composition according to any one of the preceding claims, comprising a buffer which is a phosphate, such as Na / Na2PO4, Na / K2PO4 or K / K2PO4.
    [32]
    32. A composition according to any one of claims 25 to 30, wherein the buffer is present in the liquid mixture in an amount of at least 6mM, at least 10mM or at least 40mM.
    [33]
    33. A composition according to any one of claims 25 to 31, wherein the buffer is present in the liquid mixture in an amount of less than 100 mM, less than 60 mM or less than 40 mM.
    [34]
    34. The composition according to any one of claims 25 to 32, wherein the buffer is a phosphate present in the liquid mixture in an amount of between 6 and 40 mM, such as about 10 mM.
    [35]
    35. Composition according to any one of the preceding claims, further comprising a surfactant.
    [36]
    36. The composition of claim 34, wherein the surfactant is polysorbate 80 or poloxamer 188.
    [37]
    37. Composition according to any one of the preceding claims, for a reconstitution / reconstitution with an isotonic solution, such as a saline solution, before administration to a mammal.
    [38]
    38. Composition according to any one of the preceding claims, for its use in vaccination.
    [39]
    39. A composition according to any one of the preceding claims, wherein the composition comprises multiple doses, such as at least 4 doses, for vaccinating subjects in need thereof.
    [40]
    40. Process for the preparation of a composition according to any one of claims 1 to 39, comprising the following steps: i. the mixture: a. a liquid liposome preparation comprising liposomes containing a neutral lipid and a sterol, and optionally lipopolysaccharide; b. saponin; vs. the cryoprotectant; d. optionally the antigen; e. optionally the buffer; f. optionally a surfactant; and, ii. drying the liquid mixture obtained in step (i) under reduced pressure.
    [41]
    41. The method of claim 40, wherein the liquid liposome preparation does not contain a cryoprotectant.
    [42]
    42. A process according to any one of claims 40 to 41, wherein the drying in step ii is carried out by lyophilization.
    [43]
    43. Method according to the preceding claim, wherein the duration of the lyophilization cycle is less than 48 hours, such as less than 30 hours.
    [44]
    44. Process according to any one of claims 40 to 43, in which the order for the mixture is firstly the mixture of the cryoprotectant (ic) and the buffer (ie), followed by the addition of the liposomal preparation. liquid (ia), saponin (ib), surfactant (if) and antigen (id) respectively.
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    同族专利:
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    US10702594B2|2020-07-07|
    BE1024188A1|2017-12-05|
    GB201522068D0|2016-01-27|
    EP3389698A1|2018-10-24|
    WO2017102737A1|2017-06-22|
    US20200390875A1|2020-12-17|
    引用文献:
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    EP3658118A4|2017-07-24|2021-03-31|Novavax, Inc.|Methods and compositions for treating respiratory disease|
    WO2022031359A1|2020-08-07|2022-02-10|Infectious Disease Research Institute|Purified saponins and chromatographic process for purification of same|
    法律状态:
    2018-02-15| FG| Patent granted|Effective date: 20171214 |
    优先权:
    申请号 | 申请日 | 专利标题
    GBGB1522068.4A|GB201522068D0|2015-12-15|2015-12-15|Dried composition|
    GB1522068.4|2015-12-15|
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