Stable preparation of human papillomavirus virus-like particle vaccines

Abstract

A stable formulation of human papillomavirus virus-like particle vaccine is provided. The stable formulation is composed of human papillomavirus virus-like particles, a buffer, an osmotic pressure regulator, a surfactant, and an aluminum adjuvant, and the vaccine components include HPV virus-like particles constructed by L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and one or more HPV virus-like particles constructed by L1 proteins of other pathogenic HPV types. The formulation can increase the stability of the vaccine and extend the effective period of the vaccine in the aqueous formulation. It will be published together with Figure 8.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Chinese Patent Application No. 202110049777.7, filed on January 14, 2021, the contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of biopharmaceutical formulations, and in particular to stable human papillomavirus virus-like particle vaccine formulations. [Background technology]

[0003] Cervical cancer is one of the most common malignancies in women, with approximately 500,000 new cases occurring annually worldwide, making it the second most common female cancer. More than 95% of cervical cancers are associated with human papillomavirus (HPV) infection. In addition to directly causing cervical cancer, HPV is also strongly associated with bronchogenic carcinoma, rectal cancer, oral cancer, and skin cancer. Furthermore, HPV is also the primary causative agent of cutaneous and mucosal warts.

[0004] Currently, over 100 HPV types have been discovered, and different HPV types can cause different diseases. According to their association with cervical cancer, HPV can be classified into high-risk, suspected oncogenic, and low-risk types. High-risk and suspected oncogenic types have the potential to cause cancers such as cervical cancer. High-risk types include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. Suspected oncogenic types include types 26, 53, 66, 68, 73, and 82. Low-risk types, including types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and 89, are primarily associated with genital warts, genital condylomata acuminata, and other diseases. HPV types 6, 11, and 16 were the most frequently detected subtypes in patients with genital lesions.

[0005] HPV vaccines are an effective method for preventing papillomavirus infection. Virus-like particle (VLP) vaccines are the most effective vaccine form among many vaccine formats. However, VLP-based HPV vaccines are type-specific; that is, VLP vaccines only provide strong protection against certain HPV types. To provide broad protection, the development of multivalent HPV vaccines is necessary.

[0006] However, prior to administration, human papillomavirus vaccine formulations undergo storage and transportation processes during which the antigens undergo physical and chemical degradation, and these instabilities may reduce the immunogenicity and / or safety of the antigens. Therefore, stable formulations are needed to ensure that the antigens remain immunogenic and safe to meet prophylactic purposes immediately prior to administration. Summary of the Invention [Means for solving the problem]

[0007] In one aspect, the present invention provides a stable formulation of a multivalent human papillomavirus virus-like particle vaccine for preventing HPV-associated disease or infection, comprising a plurality of papillomavirus virus-like particles adsorbed to an adjuvant, a physiologically acceptable concentration of buffer, a osmotic agent, and optionally a surfactant.

[0008] Here, the human papillomavirus virus-like particles include HPV virus-like particles constructed by L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, respectively; The virus is selected from one or more HPV virus-like particles constructed from L1 proteins of other pathogenic HPV types.

[0009] In one embodiment, the buffer is selected from one or more of a citrate buffer, an acetate buffer, or a histidine buffer; the tonicity adjusting agent is selected from one or more of sodium chloride, sodium phosphate or sodium sulfate; the surfactant is a polyethoxy ether, preferably polysorbate 80; The adjuvant is selected from one or more of aluminum hydroxyphosphate (AlPO), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH)), preferably aluminum hydroxyphosphate (AlPO).

[0010] In one embodiment, (a) the total concentration of all types of papillomavirus virus-like particles is 40 μg / mL to 740 μg / mL; (b) the concentration of the buffer is 10 mM to 26 mM, preferably 10 mM, 18 mM, or 26 mM; (c) the concentration of the osmotic pressure regulator is 150 mM to 320 mM, preferably 150 mM to 320 mM; (d) the surfactant concentration is 0 to 0.02 wt %; (e) the concentration of the adjuvant is about 1.0 mg / mL; (f) The pH of the formulation is 5.9 to 6.5, preferably 5.9, 6.2, or 6.5.

[0011] In one embodiment, the concentration of any single type of papillomavirus virus-like particle contained in the multivalent papillomavirus virus-like particle is between 40 μg / mL and 120 μg / mL.

[0012] In one embodiment, the formulation comprises a total of 0.74 mg / mL of all types of papillomavirus virus-like particles, 1.0 mg / mL of aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, a formulation solution pH of 6.2, and optionally, polysorbate 80 at a concentration of 0.3 mg / mL or less.

[0013] In one embodiment, the one or more other pathogenic HPV types are selected from HPV types 35, 39, 51, 56 and 59.

[0014] In one embodiment, wherein at least one of the HPV virus-like particles is a chimeric HPV virus-like particle comprising a chimeric HPV L1 protein, the chimeric HPV L1 protein comprising from its N-terminus to its C-terminus: a. an N-terminal fragment derived from a first type of papillomavirus L1 protein that maintains the immunogenicity of the L1 protein of that type, wherein the first type of papillomavirus is selected from HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and one or more other pathogenic HPV types; b. a C-terminal fragment derived from a second type of papillomavirus L1 protein that has better expression and solubility characteristics than the other type of L1 protein; The chimeric HPV L1 protein has the immunogenicity of the first type of papillomavirus L1 protein.

[0015] In one embodiment, the N-terminal fragment is a fragment obtained by truncating the C-terminus of the native sequence of the L1 protein of a first papillomavirus type at any amino acid position within its α5 region, and a fragment having at least 98% identity thereto, and the C-terminal fragment is a fragment obtained by truncating the N-terminus of the native sequence of the L1 protein of a second papillomavirus type at any amino acid position within its α5 region, as well as functional variants resulting from further mutations, deletions and / or additions to the fragment.

[0016] In one embodiment, the C-terminal fragment comprises one or more nuclear localization sequences.

[0017] In one embodiment, wherein the first type of papilloma L1 protein is selected from HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 or 58, and preferably, its native sequence is an amino acid sequence encoded by a coding gene set forth in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 41, respectively; the second type of papilloma L1 protein is selected from HPV types 16, 28, 33, 59, or 68 L1 proteins; More preferably, the second type of papilloma L1 protein is selected from HPV type 33 or HPV type 59 L1 proteins.

[0018] In one embodiment, the C-terminal fragment is SEQ ID NO: 1 or a fragment thereof having a length of m1 amino acids, preferably a fragment encompassing amino acids 1 to m1 of SEQ ID NO: 1, where m1 is an integer between 8 and 26; or the C-terminal fragment is SEQ ID NO: 2 or a fragment thereof having a length of m2 amino acids, preferably a fragment encompassing amino acids 1 to m2 of SEQ ID NO: 2, where m2 is an integer between 13 and 31.

[0019] In one embodiment, the C-terminal fragment is SEQ ID NO: 3, or a fragment thereof having a length of n amino acids, preferably a fragment encompassing amino acids 1 to n of SEQ ID NO: 3, where n is an integer between 16 and 38.

[0020] In one embodiment, the N-terminal fragment of HPV type 6 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity to a fragment obtained by truncating the C-terminus of the sequence set forth in SEQ ID NO: 4 at any amino acid site within the α5 region thereof; the N-terminal fragment of HPV type 11 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 5 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 16 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 6 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 18 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 7 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 31 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 8 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 35 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 9 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 39 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 10 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 45 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 11 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 51 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 12 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 52 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 13 at any amino acid site within the α5 region; the N-terminal fragment of HPV type 56 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity with a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 14 at any amino acid site within the α5 region; The N-terminal fragment of HPV type 58 L1 protein has 98%, 98.5%, 99%, 99.5%, 99% or 100% identity to a fragment obtained by cleaving the C-terminus of the sequence shown in SEQ ID NO: 15 at any amino acid site within the α5 region.

[0021] In one embodiment, the C-terminus of the N-terminal fragment is connected to the N-terminus of the C-terminal fragment directly or by a linker.

[0022] In one embodiment, when the C-terminus of the N-terminal fragment is joined to the N-terminus of the C-terminal fragment, the contiguous amino acid sequence RKFL is within plus or minus 4 amino acid positions of the splice site, Preferably, the contiguous amino acid sequence LGRKFL is located within plus or minus 6 amino acid positions of the splice site.

[0023] In one embodiment, the chimeric HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58 chimeric HPV L1 proteins have 98%, 98.5%, 99%, 99.5% or 100% identity to SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and the HPV type 33 L1 protein and HPV type 59 L1 protein have 98%, 98.5%, 99%, 99.5% or 100% identity to SEQ ID NO:28 and SEQ ID NO:29, respectively.

[0024] In one embodiment, the formulation comprises a chimeric HPV 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58 HPV L1 protein having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; and and HPV type 33 L1 protein and HPV type 59 L1 protein having the amino acid sequences shown in SEQ ID NO: 28 and SEQ ID NO: 29, respectively.

[0025] In one aspect, the present invention provides a method for preventing HPV-associated disease or infection, comprising administering to a subject a stable formulation of a multivalent human papillomavirus virus-like particle vaccine formulation. Prevention may also be considered treatment, and the two terms are used interchangeably. In one embodiment, the subject is a human.

[0026] In one embodiment, the formulation is stable at 2-8°C for at least 24 months and at 25°C for at least 16 weeks.

[0027] In one aspect, the invention provides the use of a human papillomavirus virus-like particle vaccine formulation in the preparation of a vaccine for preventing HPV-associated disease or infection. [Brief explanation of the drawings]

[0028] [Figure 1] 1 shows the results of the adsorption test for each formulation sample of Example 1. [Figure 2] The analysis results of the antigen content of each formulation sample in Example 1 are shown below. TO: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4. [Figure 3] 1 shows the results of the adsorption test for each formulation sample of Example 2. [Figure 4]The analysis results of the antigen content of each formulation sample in Example 2 are shown below. TO: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4. [Figure 5] 1 shows the results of the adsorption test for each formulation sample of Example 3. [Figure 6] The analysis results of the antigen content of each formulation sample in Example 3 are shown below. TO: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4. [Figure 7] 1 shows the results of the adsorption test for each formulation sample of Example 4. [Figure 8] The analysis results of the antigen content of each formulation sample in Example 4 are shown below. TO: 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides a stable formulation of human papillomavirus vaccine, solving the problem of antibody stability during storage and transportation, and ensuring that the antigen remains immunogenic and safe to meet prophylactic purposes.

[0030] The term "preparation" refers to a composition that maintains the biological activity of the active ingredient in an effective manner and does not contain other ingredients that are unacceptably toxic to the subject. Such a preparation is sterile. The term "sterile" refers to the absence of living bacteria or the absence or substantial absence of all living microorganisms and their spores.

[0031] As used herein, a "stable" formulation refers to a formulation in which the active ingredient substantially retains its physical and / or chemical stability and / or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability and its biological activity after storage.

[0032] The terms "patient" or "subject" are used interchangeably and refer to any mammal, preferably a human, suffering from a condition or disease according to the present invention.

[0033] As used herein, "physiologically acceptable" means that the concentration or ionic strength of a buffer, excipient, or salt makes the formulation biologically compatible with the immunized target host, e.g., a human.

[0034] The stable formulation of the present invention comprises human papillomavirus virus-like particles, a buffer, an osmolality adjusting agent, and an aluminum adjuvant.

[0035] The terms "comprising" and "containing" mean that additional ingredients may be included in addition to the named ingredients.

[0036] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the context clearly dictates otherwise.

[0037] As used herein, "buffer" refers to a buffer solution that resists pH changes due to the action of its conjugate acid-base pair. In one embodiment of the present invention, a histidine buffer is used, and the pH of the formulation solution is preferably about 5.9 to 6.5, more preferably 6.2.

[0038] As used herein, "surfactant" refers to a surface active agent, and in one embodiment, the surfactant herein is polysorbate 80.

[0039] The term "osmolality modifier" refers to a pharmaceutically acceptable osmolality modifier. Suitable osmolality modifiers include, but are not limited to, salts, and in one embodiment of the present invention, a suitable osmolality modifier is sodium chloride (NaCl) having a concentration of about 150 mM to 320 mM.

[0040] The term "adjuvant" refers to a compound or mixture that enhances the immune response. In particular, vaccines may contain an adjuvant. The adjuvant used in the present invention is selected from one or more of aluminum hydroxyphosphate (AlPO), amorphous aluminum hydroxyphosphate sulfate (AAHS), or aluminum hydroxide (Al(OH)), preferably aluminum hydroxyphosphate (AlPO).

[0041] The "stability" of a protein after storage at a selected temperature for a selected period of time can be qualitatively and / or quantitatively assessed in several different ways. In an embodiment of the present invention, an enzyme-linked immunosorbent assay (ELISA) was used to measure the content of active antigen that binds to recombinant human papillomavirus neutralizing antibodies, and the ratio of the active antigen content at each time point to TO was used to compare the stability of the corresponding formulations. An enzyme-linked immunosorbent assay (ELISA) was used to measure the content of antigen that was not adsorbed to the aluminum phosphate adjuvant by centrifugation, and then the absorbance was calculated. EC of human papillomavirus vaccine formulations and positive controls against recombinant human papillomavirus neutralizing antibodies 50 The EC values ​​for the vaccine preparation and the positive control were determined. 50 Calculating the ratio and thereby determining the relative in vitro efficacy of the vaccine.

[0042] The term "immunogenicity" refers to the ability of a substance such as a protein or polypeptide to stimulate an immune response, i.e., a response that results in the production of antibodies, particularly a humoral response, or a response mediated by stimulated cells.

[0043] The term "HPV" or "HPV virus" refers to papillomaviruses of the family Papillomaviridae, which are non-enveloped DNA viruses with a double-stranded, closed-loop DNA genome approximately 8 kb in size and can be generally divided into three regions: (1) the early region (E), which contains six open reading frames encoding nonstructural proteins associated with viral replication, transcription, and transformation: E1, E2, E4-E7, and the E3 and E8 open reading frames; (2) the late region (L), which contains reading frames encoding the major capsid protein L1 and the minor capsid protein L2; and (3) the long regulatory region (LCR), which does not encode any proteins but contains an origin of replication and multiple transcription factor binding sites.

[0044] The terms "HPV L1 protein" and "HPV L2 protein" refer to proteins encoded by the late region (L) of the HPV gene and synthesized during the middle and late phases of the HPV infection cycle. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is a minor capsid protein. 72 L1 pentamers form the shell of the icosahedral HPV virus particle, which encases a closed-loop double-stranded DNA microchromosome. The L2 protein is located in the inner lining of the L1 protein.

[0045] The term "virus-like particle" refers to a hollow particle that contains one or more structural proteins of a virus but does not contain viral nucleic acid.

[0046] The term "concentration of any single type of papillomavirus virus-like particle" refers to the content of any single type of papillomavirus virus-like particle in the formulation, and the term "total concentration of all types of papillomavirus virus-like particles" refers to the sum of the concentrations of each single type of papillomavirus virus-like particle contained in the formulation.

[0047] In one embodiment of the present invention, a human papillomavirus multivalent immunogenic composition is employed, as described in patent application PCT / CN2020 / 102601, filed on July 17, 2020, which is incorporated herein by reference.

[0048] In a particularly preferred embodiment of the present invention, the formulation contains 0.74 mg / mL papillomavirus virus-like particles, 1.0 mg / mL aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, and a pH of 6.2. The vaccine contains polysorbate 80 at a concentration of 0.3 mg / mL or less due to process residues during preparation. The formulation has good stability and can be stored at 2-8°C for at least 24 months and at 25°C for at least 16 weeks.

[0049] The formulations of the present invention may be provided in liquid form or in lyophilized form, which may be reconstituted prior to administration.

[0050] Example The present invention will be more fully understood by reference to the following examples, which should not be construed as limiting the scope of the invention. All documents, patents and patent applications are incorporated herein by reference.

[0051] In the examples below, the preparation, characterization and performance characteristics of various types of papillomavirus virus-like particles used are described in patent application PCT / CN2020 / 102601, filed July 17, 2020.

[0052] In the examples below, the detection methods used were as follows:

[0053] 1) Analysis of antigen content (enzyme-linked immunosorbent assay (ELISA) The positive control (human papillomavirus virus-like particle standard, provided by Sino Cell Tech Ltd.; chimeric HPV types 6, 16, 18, 31, 35, 30, 45, 51, 52, and 56 L1 proteins and HPV types 33 and 59 L1 proteins corresponding to the amino acid sequences of SEQ ID NOs: 16 to 29, respectively; the same applies below) and the analyte were completely dissolved using desorption buffer to serve as the positive control and the analyte to be detected.

[0054] A recombinant human papillomavirus neutralizing antibody (provided by Sino Biological, Inc., hereinafter the same) was combined with a solid-phase carrier to form a solid-phase antibody. The positive control and analyte samples were diluted with sample diluent and then combined with the solid-phase antibody to form a solid-phase antigen-antibody complex. An enzyme-labeled antibody was then added, and a substrate was added for development. The colored product was read at a wavelength of 450 nm. Linear regression was performed for a series of positive control concentrations and their corresponding absorbance values. The absorbance values ​​of the analyte were then substituted into the linear regression equation to determine the antigen content of the sample (M. Shank-Retzlaff, F. Wang, T. Morley et al. Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples. Human Vaccines, 1:5, 191-197).

[0055] 2) Sorption analysis (enzyme-linked immunosorbent assay (ELISA)) A recombinant human papillomavirus neutralizing antibody was combined with a solid-phase carrier to form a solid-phase antibody. The sample to be detected was centrifuged, and the supernatant was used as the analyte. The positive control and analyte were diluted accordingly with sample diluent and then combined with the solid-phase antibody to form a solid-phase antigen-antibody complex. An enzyme-labeled antibody was then added, and a substrate was added for development, and the colored product was read at a wavelength of 450 nm. A linear regression was performed for a series of positive control concentrations and their corresponding absorbances. The absorbance values ​​measured by the analyte were substituted into the linear regression equation to determine the antigen concentration in the supernatant, and the adsorption degree of the sample to be detected was calculated using the following formula (Michael J. Caulfield, Li Shi, Su Wang et al. Effect of Alternative Aluminum Adjuvants on the Absorption and Immunogenicity of HPV16 L1 VLPs in Mice. Human Vaccines 3:4, 139-146).

[0056] Adsorption rate (%) = (1 - antigen concentration in supernatant / antigen concentration in sample to be detected) %.

[0057] 3) Determination of in vitro relative potency, IVRP (IVRP) The desorption buffer was used to completely dissolve the positive control (a 14-valent human papillomavirus vaccine control product (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) obtained from Sino Cell Tech Ltd., which has the same protein sequence as the test sample) and the analyte to be detected. Recombinant human papillomavirus neutralizing antibodies were diluted to a final concentration of 2 μg / mL and added to a 96-well plate at 100 μL / well. The plate was gently tapped to mix the sample, and the sample was coated overnight at 4°C. The plate was washed once with 200 μL / well of washing solution, and the ELISA plate was allowed to dry. The ELISA plate was then blocked with 300 μL / well of blocking solution at room temperature for 1 hour. The samples were washed twice with 300 μL / well of washing solution, and 100 μL of treated blank control (buffer corresponding to the analyte), positive control, and analyte to be detected were added to each well and incubated at room temperature for 1 hour. The plate was washed three times with 200 μL / well of washing solution, followed by the addition of 100 μL / well of diluted enzyme-labeled recombinant human papillomavirus neutralizing antibody. After 1 hour of incubation at room temperature, the plate was washed three times with 200 μL / well of washing solution, and 200 μL / well of developing solution was added and left at room temperature for 20 ± 5 minutes. The reaction was stopped by adding 50 μL / well of stop solution. The absorbance at 450 nm was detected using a microplate reader. The data were processed using the computer program Origin or a four-parameter fitting method, with the concentration of the positive control or analyte on the horizontal axis and the average absorbance on the vertical axis, to determine the EC values ​​of the analyte and positive control. 50 The EC of the analyte is calculated. 50 EC as a positive control 50Divide by this to determine the in vitro relative potency of the analyte (M. Shank-Retzlaff, F. Wang, T. Morley et al. Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples. Human Vaccines, 1:5, 191-197).

[0058] Example 1: Surfactant concentration screening test The composition of the papillomavirus virus-like particle vaccine formulation of this example is shown in the table below:

[0059] [Table 1]

[0060] Method for preparing papillomavirus virus-like particle vaccine formulations: A certain amount of HPV18 virus-like particles suitable for the formulation was taken so that the pH and corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride, and polysorbate 80 in the papillomavirus virus-like particle vaccine formulation met the requirements in Table 1. The particles were then mixed with aluminum phosphate adjuvant and allowed to adsorb overnight at 4°C. Aliquots were then dispensed, marked with the corresponding numbers, and placed in a 37°C incubator. Samples were removed at week 0 for adsorption and antigen content analysis, and at weeks 1, 2, and 4 for antigen content analysis.

[0061] Analytical Test Methods: Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation, analyze its content, and calculate the adsorption degree.

[0062] Antigen content analysis: The detection principle was to measure the content of active antigen capable of binding to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each formulation by comparing the ratio of the active antigen content at each time point to the active antigen content at TO. The higher the ratio, the higher the active antigen content in the formulation and the better the activity was maintained.

[0063] The test results are shown in Tables 2 and 3 and Figs.

[0064] [Table 2]

[0065] [Table 3]

[0066] The test results showed that the adsorption degree of both papillomavirus virus-like particle vaccine formulations was over 99%, and there was no significant difference in the change in antigen content between F1 and F2, i.e., the stability of F1 and F2 was equivalent.

[0067] Example 2: pH screening test The composition of the papillomavirus virus-like particle vaccine formulation of this example is shown in the table below:

[0068] [Table 4]

[0069] Method for preparing papillomavirus virus-like particle vaccine formulations: A certain amount of HPV18 virus-like particles suitable for the formulation was taken so that the pH and corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride, and polysorbate 80 in the papillomavirus virus-like particle vaccine formulation met the requirements in Table 4. The particles were then mixed with aluminum phosphate adjuvant and allowed to adsorb overnight at 4°C. Aliquots were then dispensed, marked with the corresponding numbers, and placed in a 37°C incubator. Samples were removed at week 0 for adsorption and antigen content analysis, and at weeks 1, 2, and 4 for antigen content analysis.

[0070] Analytical Test Methods: Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation, analyze its content, and calculate the adsorption degree.

[0071] Antigen content analysis: The detection principle was to measure the content of active antigen capable of binding to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each formulation by comparing the ratio of the active antigen content at each time point to the active antigen content at TO. The higher the ratio, the higher the active antigen content in the formulation and the better the activity was maintained.

[0072] The test results are shown in Tables 5 and 6 and FIGS.

[0073] [Table 5]

[0074] [Table 6]

[0075] The test results showed that the adsorption rates of the three papillomavirus virus-like particle vaccine preparations were over 99%, and the antigen content change trends of the papillomavirus virus-like particle vaccines of F1 and F2 were better than those of the F3 preparation, i.e., the stability of F1 and F2 was better than that of F3.

[0076] Example 3: Screening test of osmotic agent concentrations The composition of the papillomavirus virus-like particle vaccine formulation of this example is shown in the table below:

[0077] [Table 7]

[0078] Method for preparing papillomavirus virus-like particle vaccine formulations: A certain amount of HPV18 virus-like particles suitable for the formulation was taken so that the pH and corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride, and polysorbate 80 in the papillomavirus virus-like particle vaccine formulation met the requirements in Table 7, and then mixed with aluminum phosphate adjuvant and allowed to adsorb overnight at 4°C. Aliquots were dispensed, marked with the corresponding numbers, and placed in a 37°C incubator. Samples were removed at week 0 for adsorption and antigen content analysis, and at weeks 1, 2, and 4 for antigen content analysis.

[0079] Analytical Test Methods: Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation, analyze its content, and calculate the adsorption degree.

[0080] Antigen content analysis: The detection principle was to measure the content of active antigen capable of binding to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each formulation by comparing the ratio of the active antigen content at each time point to the active antigen content at TO. The higher the ratio, the higher the active antigen content in the formulation and the better the activity was maintained.

[0081] The test results are shown in Tables 8 and 9 and FIGS.

[0082] [Table 8]

[0083] [Table 9]

[0084] The test results showed that the adsorption rate of the two papillomavirus virus-like particle vaccine formulations was over 99%. There was no significant difference in the change in active antigen content between the F1 and F2 formulations after 4 weeks at 37°C, indicating that the stability of F1 and F2 was equivalent.

[0085] Example 4: Screening test of buffer concentration The composition of the papillomavirus virus-like particle vaccine formulation of this example is shown in the table below:

[0086] [Table 10]

[0087] Methods for preparing papillomavirus virus-like particle vaccine formulations; A certain amount of HPV18 virus-like particles suitable for the formulation was taken so that the pH and corresponding concentrations of papillomavirus virus-like particles, aluminum phosphate adjuvant, histidine, sodium chloride, and polysorbate 80 in the papillomavirus virus-like particle vaccine formulation met the requirements in Table 10, and then mixed with aluminum phosphate adjuvant and allowed to adsorb overnight at 4°C. Aliquots were dispensed, marked with the corresponding numbers, and the samples were placed in a 37°C incubator. They were removed for analysis of adsorption and antigen content at week 0 and for antigen content analysis at weeks 1, 2, and 4.

[0088] Analytical Test Methods: Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation, analyze its content, and calculate the adsorption degree.

[0089] Antigen content analysis: The detection principle was to measure the content of active antigen capable of binding to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each formulation by comparing the ratio of the active antigen content at each time point to the active antigen content at TO. The higher the ratio, the higher the active antigen content in the formulation and the better the activity was maintained.

[0090] The test results are shown in Tables 11 and 12 and FIGS.

[0091] [Table 11]

[0092] [Table 12]

[0093] The test results showed that the adsorption rates of the three papillomavirus virus-like particle vaccine preparations were over 99%, and the antigen content change trends of the papillomavirus virus-like particle vaccines of F1 and F2 were better than those of the F3 preparation, i.e., the stability of F1 and F2 was better than that of F3.

[0094] Example 5: Composition confirmation test of each single type (type 6, type 11, type 16, type 18, type 31, type 33, type 35, type 39, type 45, type 51, type 52, type 56, type 58, type 59) papillomavirus virus-like particle vaccine preparation

[0095] Stability monitoring of each single type (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) of papillomavirus virus-like particle vaccine formulations (formulation: 0.74 mg / mL papillomavirus virus-like particles + 1.0 mg / mL aluminum phosphate adjuvant + 18 mM histidine buffer + 320 mM sodium chloride pH 6.2) was performed at 2-8 °C. The vaccines contained polysorbate 80 at concentrations of 0.3 mg / mL or less due to process residues during preparation. The monitoring time points were 3 months, 6 months, 9 months, and 12 months, and adsorption and in vitro relative potency were monitored.

[0096] Analytical Test Methods: Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation, analyze its content, and calculate the adsorption degree.

[0097] In vitro relative potency: The detection principle of this method is to detect the EC50 of the analyte and the positive control using a recombinant human papillomavirus neutralizing antibody, and then calculate the EC 50 The higher the value, the higher the in vitro relative potency and the better the quality of the test sample.

[0098] The experimental results are shown in Table 13.

[0099] The experimental results show that each single type of papillomavirus virus-like particle vaccine formulation disclosed by the present invention has good stability and can be stably stored at 2-8°C for at least 12 months.

[0100] [Table 13]

[0101] Example 6 Preparation and composition confirmation of 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) Preparation method for 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59):

[0102] Each single type (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) of papillomavirus virus-like particle vaccine formulation (composition: 0.74 mg / mL papillomavirus virus-like particles + 1.0 mg / mL aluminum phosphate adjuvant + 18 mM histidine buffer + 320 mM sodium chloride, pH 6.2) was The virus-like particles were collected and mixed at a fixed volume ratio (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 = 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1) to give the following volumes: 0.06 mg / mL, 0.08 mg / mL, 0.12 mg / mL, 0.08 mg / mL, 0.04 mg / mL, 0.04 mg / mL, and 0.04 mg / mL, respectively. A 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) semi-finished product corresponding to concentrations of 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, 0.04 mg / mL, and 0.04 mg / mL was obtained, and then penicillin The vaccines were filled into vials, which were then corked, capped, and labeled to prepare the recombinant 14-valent human papillomavirus vaccines (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), which contained polysorbate 80 at concentrations of 0.3 mg / mL or less due to process residues during the preparation process. Stability monitoring was then performed at 2-8°C (time points were 3, 6, 9, 12, 18, and 24 months) and 25±2°C (time points were 2, 4, 8, and 16 weeks) to monitor in vitro relative potency and adsorption.

[0103] Analytical Test Methods: Adsorption analysis: The detection principle is to obtain the antigen that is not adsorbed to the aluminum phosphate adjuvant by centrifugation, analyze its content, and calculate the adsorption degree.

[0104] Antigen content analysis: The detection principle was to measure the content of active antigen capable of binding to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each formulation by comparing the ratio of the active antigen content at each time point to the active antigen content at TO. The higher the ratio, the higher the active antigen content in the formulation and the better the activity was maintained.

[0105] The experimental results are shown in Tables 14 to 17.

[0106] [Table 14]

[0107] [Table 15]

[0108] [Table 16]

[0109] [Table 17]

[0110] The experimental results show that the 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) preparations disclosed by the present invention have good stability and can be stably stored at temperatures between 2 and 8°C for at least 24 months and at 25°C for at least 16 weeks.

[0111] [Table 18A] [Table 18B] [Table 18C] Table 18D Table 18E Table 18F Table 18G Table 18H

Table 18I

Table 18J

Table 18O

Table 18P

Table 18R

Table 18S

Claims

1. (a) a plurality of human papillomavirus (HPV) virus-like particles, the total concentration of all types of papillomavirus virus-like particles being 740 μg / mL; (b) an adjuvant, which is aluminum adjuvant and is at a concentration of 1.0 mg / mL; (c) a physiologically acceptable concentration of a buffer selected from one or more of a citrate buffer, an acetate buffer, or a histidine buffer, the concentration of which is between 10 mM and 26 mM; (d) a physiologically acceptable concentration of an osmolality adjusting agent selected from one or more of sodium chloride, sodium phosphate, or sodium sulfate, at a concentration of 150 mM or 320 mM; and optionally (e) a physiologically acceptable concentration of a surfactant, the surfactant being a polyethoxy ether and having a concentration of 0 to 0.02% by weight; (f) the pH of the formulation is 5.9 to 6.5; Human papillomavirus virus-like particles are adsorbed to an adjuvant, The HPV virus-like particle is A chimeric HPV virus-like particle formed by an HPV6 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 16; A chimeric HPV virus-like particle formed by an HPV type 11 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 17; A chimeric HPV virus-like particle formed by an HPV16 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 18; A chimeric HPV virus-like particle formed by an HPV18 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 19; A chimeric HPV virus-like particle formed by an HPV type 31 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 20; A chimeric HPV virus-like particle formed by an HPV35 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 21; A chimeric HPV virus-like particle formed by an HPV39 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 22; A chimeric HPV virus-like particle formed by an HPV45 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 23; A chimeric HPV virus-like particle formed by an HPV51 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 24; A chimeric HPV virus-like particle formed by an HPV52 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 25; A chimeric HPV virus-like particle formed by an HPV56 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 26; A chimeric HPV virus-like particle formed by an HPV58 chimeric L1 protein having the amino acid sequence shown in SEQ ID NO: 27; A chimeric HPV virus-like particle formed by an HPV33 chimeric L1 protein having the amino acid sequence set forth in SEQ ID NO: 28, and Contains only chimeric HPV virus-like particles formed by the HPV59 chimeric L1 protein having the amino acid sequence set forth in SEQ ID NO: 29, A stable formulation of a multivalent human papillomavirus virus-like particle vaccine for preventing HPV-associated disease or infection, except where the formulation contains mannitol or sucrose.

2. The adjuvant is aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS) and aluminum hydroxide (Al(OH) 3 2. The formulation of claim 1, wherein the formulation is selected from one or more of:

3. 10. The formulation of claim 1, comprising 0.74 mg / mL of all types of total papillomavirus virus-like particles, 1.0 mg / mL of aluminum phosphate adjuvant, 18 mM histidine buffer, and 320 mM sodium chloride, at a pH of 6.

2.

4. 4. The formulation of any one of claims 1 to 3, comprising polysorbate 80 at a concentration of 0.3 mg / mL or less.

5. A formulation of a papillomavirus vaccine according to any one of claims 1 to 4, characterized in that it can be stably stored at 2-8°C for at least 24 months and at 25°C for at least 16 weeks.

6. 10. Use of a formulation according to any one of claims 1 to 5 in the preparation of a vaccine for preventing HPV-related diseases or infections.

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