Stable preparation of human papillomavirus virus-like particle vaccine

Abstract

To provide a stable formulation of a multivalent human papillomavirus virus-like particle vaccine for the prevention of HPV-related diseases or infections.SOLUTION: The above problem is solved by a stable formulation of a multivalent human papillomavirus virus-like particle vaccine for the prevention of HPV-related diseases or infections as defined in claim 1.SELECTED DRAWING: 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, in particular to stable human papillomavirus virus-like particle vaccine formulations. [Background technology]

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

[0004] Currently, more than 100 HPV types have been discovered, and different HPV types can cause different diseases. According to their close relationship with cervical cancer, HPV can be classified into high-risk types, suspected carcinogenic types, and low-risk types. High-risk types and suspected carcinogenic types can induce cancers such as cervical cancer. High-risk types included types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. Suspected carcinogenic 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 mainly associated with genital warts, genital warts, and other diseases. HPV types 6, 11, and 16 were the subtypes most frequently detected in patients with genital lesions.

[0005] HPV vaccines are an effective method to prevent papillomavirus infection. Virus-like particle (VLP) vaccines are the most effective vaccine form among many vaccine forms. However, VLP-based HPV vaccines are type-specific, i.e., VLP vaccines only show strong protection against certain HPV types. In order to provide broad protection, the development of multivalent HPV vaccines is necessary. Summary of the Invention [Problem to be solved by the invention]

[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 the prophylactic purpose immediately prior to administration. [Means for solving the problem]

[0007] In one aspect, the 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 a 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, and The HPV virus-like particles are 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 aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH) 3 ), preferably aluminum hydroxyphosphate (AlPO 4 ) are selected from one or more of the following:

[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 concentration of the surfactant is 0 to 0.02% by weight; (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 aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, pH of the formulation solution 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 and maintaining the immunogenicity of the L1 protein of said 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 having 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, the native sequence thereof is the amino acid sequence encoded by the coding gene shown 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 protein.

[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 from 8 to 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 from 13 to 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 from 16 to 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 shown 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 truncating 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 truncating 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 truncating 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 truncating 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 truncating 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 truncating 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 truncating 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 truncating 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 truncating 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 truncating 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 truncating 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 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 sequence 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 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 as 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 description of the drawings]

[0028] [Figure 1] 1 shows the results of the adsorption test for each formulation sample of Example 1. [Diagram 2] The analysis results of the antigen content of each preparation sample of Example 1 are shown below. T0: 37°C, 0th week; 37°C_1W: 37°C, 1st week; 37°C_2W: 37°C, 2nd week; 37°C 4W: 37°C, 4th week. [Diagram 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 preparation sample of Example 2 are shown below. T0: 37°C, 0th week; 37°C_1W: 37°C, 1st week; 37°C_2W: 37°C, 2nd week; 37°C 4W: 37°C, 4th week. [Diagram 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 preparation sample of Example 3 are shown below. T0: 37°C, 0th week; 37°C_1W: 37°C, 1st week; 37°C_2W: 37°C, 2nd week; 37°C 4W: 37°C, 4th week. [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 preparation sample of Example 4 are shown below. T0: 37°C, 0th week; 37°C_1W: 37°C, 1st week; 37°C_2W: 37°C, 2nd week; 37°C 4W: 37°C, 4th week. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 pre-administration antigen remains immunogenic and safe to meet prophylactic purposes.

[0030] The term "formulation" 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 formulation is sterile. The term "sterile" refers to the absence of living bacteria or the absence or substantial absence of any 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 suffering from a condition or disease according to the present invention. Preferably, a human.

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

[0034] The stable formulation of the present invention comprises a human papillomavirus virus-like particle, a buffer, a osmotic 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 pairs. 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 pharma- ceutically 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, the vaccine may contain an adjuvant. The adjuvant used in the present invention is aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH) 3 ), preferably aluminum hydroxyphosphate (AlPO 4 ) are selected from one or more of the following:

[0041] The "stability" of a protein after storage at a selected temperature for a selected period of time can be qualitatively and / or quantitatively evaluated 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 antigens that bind 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 antigens that were 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-coated 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, as well as 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 stages 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 the minor capsid protein. Seventy-two 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" is 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" is 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 by reference into this specification and the claims.

[0048] In a particularly preferred embodiment of the invention, the formulation comprises 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, where 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 stably stored at 2-8°C for at least 24 months and at 25°C for at least 16 weeks.

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

[0050] Working 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 on 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 chimeric HPV 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 as below) and the analyte were completely dissolved using a desorption buffer to prepare the positive control and the analyte to be detected.

[0054] Recombinant human papillomavirus neutralizing antibody (provided by Sino Biological, Inc., the same as below) was combined with a solid-phase carrier to form a solid-phase antibody. The positive control and the analyte to be detected were diluted with a sample diluent and then combined with the solid-phase antibody to form a solid-phase antigen-antibody complex. Then, an enzyme-labeled antibody was added, 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 the corresponding absorbance, and the absorbance value of the analyte was substituted into the linear regression equation to obtain the antigen content of the sample to be detected (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)) The recombinant human papillomavirus neutralizing antibody is combined with a solid-phase carrier to form a solid-phase antibody. The sample to be detected is centrifuged, and the supernatant is the analyte. The positive control and the analyte are diluted accordingly by sample diluent, and then combined with the solid-phase antibody to form a solid-phase antigen-antibody complex. Then, enzyme-labeled antibody is added, and a substrate is added for development, and the colored product is read at a wavelength of 450 nm. A linear regression is performed for a series of positive control concentrations and the corresponding absorbance, and the absorbance value measured by the analyte is substituted into the linear regression equation to obtain the antigen concentration of the supernatant, and the adsorption degree of the sample to be detected is calculated by 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 degree (%) = (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 (14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) control product obtained from Sino Cell Tech Ltd, which has the same sequence as the protein sequence in the test sample) and the analyte, which were used as the positive control and the analyte to be detected. The recombinant human papillomavirus neutralizing antibody was diluted to a final concentration of 2 μg / mL and added to the 96-well plate at 100 μL / well, the plate was tapped to mix the sample, and the sample was coated overnight at 4°C. The plate was washed once with a dose of 200 μL / well of washing solution, and the ELISA plate was dried. 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 h. The plate was washed three times with 200 μL / well of washing solution, and then diluted enzyme-labeled recombinant human papillomavirus neutralizing antibody was added at 100 μL / well. After incubation at room temperature for 1 h, the plate was washed three times with 200 μL / well of washing solution, developer was added at 200 μL / well, and the plate was placed at room temperature for 20 ± 5 min. The reaction was stopped by adding 50 μL / well of stop solution. The absorbance at 450 nm was detected by a microplate reader. The data were processed using the computer program Origin or a four-parameter fitting method, and the EC values ​​of the analyte and positive control were calculated by taking the concentration of the positive control or analyte on the horizontal axis and the average absorbance on the vertical axis. 50 Calculate the EC of the analyte. 50 EC of positive control 50and dividing by this to obtain 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: Screening test of surfactant concentration 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 a papillomavirus virus-like particle vaccine formulation: A certain amount of HPV 18 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 respectively met the requirements in Table 1, then mixed with aluminum phosphate adjuvant, adsorbed overnight at 4°C, and aliquots were dispensed, which were marked by the corresponding numbers, and the samples were placed in a 37°C incubator and taken out for analysis of adsorption degree and antigen content at week 0, and for antigen content analysis at weeks 1, 2 and 4.

[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 is to measure the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to the active antigen content at T0. The higher the ratio, the higher the active antigen content in the preparation, and the better the activity was maintained.

[0063] The test results are shown in Tables 2 and 3 and in Figs. 1 and 2.

[0064] [Table 2]

[0065] [Table 3]

[0066] The test results showed that the adsorption degree of both two papillomavirus virus-like particle vaccine formulations was more than 99%, and there was no significant difference in the change of 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 a papillomavirus virus-like particle vaccine formulation: A certain amount of HPV 18 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 respectively met the requirements in Table 4, then mixed with aluminum phosphate adjuvant, adsorbed overnight at 4°C, and aliquots were dispensed, which were marked by corresponding numbers, and the samples were placed in a 37°C incubator and taken out for analysis of adsorption degree and antigen content at week 0, and for antigen content analysis at weeks 1, 2 and 4.

[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 is to measure the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to the active antigen content at T0. The higher the ratio, the higher the active antigen content in the preparation, and the better the activity was maintained.

[0072] The test results are shown in Tables 5 and 6 and in 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 above 99%, and the antigen content change trends of the papillomavirus virus-like particle vaccine in F1 and F2 were better than that of the F3 preparation, that is, the stability of F1 and F2 was better than that of F3.

[0076] Example 3: Screening test of osmotic agent concentration 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 a papillomavirus virus-like particle vaccine formulation: A certain amount of HPV 18 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 respectively met the requirements in Table 7, then mixed with aluminum phosphate adjuvant, adsorbed overnight at 4°C, and aliquots were dispensed, which were marked by corresponding numbers, and the samples were placed in a 37°C incubator and taken out for analysis of adsorption degree and antigen content at week 0, and for antigen content analysis at weeks 1, 2 and 4.

[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 is to measure the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to the active antigen content at T0. The higher the ratio, the higher the active antigen content in the preparation, and the better the activity was maintained.

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

[0082] [Table 8]

[0083] [Table 9]

[0084] The test results showed that the adsorption degree of the two papillomavirus virus-like particle vaccine formulations was more than 99%. There was no significant difference in the change in active antigen content of the F1 and F2 formulations at 37℃ for 4 weeks, i.e., 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 HPV 18 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 respectively met the requirements of Table 10, then mixed with aluminum phosphate adjuvant, adsorbed overnight at 4°C, and aliquots were dispensed, which were marked by corresponding numbers, and the samples were placed in a 37°C incubator and taken out for analysis of adsorption degree 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 is to measure the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to the active antigen content at T0. The higher the ratio, the higher the active antigen content in the preparation, and the better the activity was maintained.

[0090] The test results are shown in Tables 11 and 12 and in 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 above 99%, and the antigen content change trends of the papillomavirus virus-like particle vaccine in F1 and F2 were better than that of the F3 preparation, that is, 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] 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 and type 59) of papillomavirus virus-like particle vaccine formulation (formulation: 0.74 mg / mL papillomavirus virus-like particle + 1.0 mg / mL aluminum phosphate adjuvant + 18 mM histidine buffer + 320 mM sodium chloride pH 6.2) was subjected to stability monitoring at 2-8 °C. Here, the vaccine contained polysorbate 80 at a concentration of 0.3 mg / mL or less due to process residues during preparation. The monitoring time points were 3 m (3 months), 6 m (6 months), 9 m (9 months) and 12 m (12 months), and the degree of 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, respectively, using recombinant human papillomavirus neutralizing antibodies, and then 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 preparation disclosed by the present invention has good stability and can be stably stored under conditions of 2 to 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] Papillomavirus virus-like particle vaccine formulation (composition: 0.74 mg / mL papillomavirus virus-like particle + 1.0 mg / mL aluminum phosphate adjuvant + 18 mM histidine buffer + 320 mM sodium chloride, pH value: 6.2) was used for each single type (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). The samples were mixed at a fixed volume ratio (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 = 1.5:2:3:2:1:1:1:1:1:1:1:1:1:1:1) and diluted with water to obtain 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 0.06 mg / mL, respectively, for each type of virus-like particle. 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, 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 recombinant 14-valent human papillomavirus vaccines (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59), where the vaccines contained concentrations of polysorbate 80 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 is to measure the active antigen content that can bind to recombinant human papillomavirus neutralizing antibodies by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to the active antigen content at T0. The higher the ratio, the higher the active antigen content in the preparation, 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 a temperature of 2-8°C for at least 24 months and at a temperature of 25°C for at least 16 weeks.

[0111] [Table 18A] [Table 18B] [Table 18C]

Table 18D

Table 18E

Table 18I

Table 18J

Table 18K

Table 18L

Table 18M

Table 18N

Table 18O

Table 18P

Table 18Q

Table 18R

Table 18S

Claims

1. (a) a plurality of human papillomavirus (HPV) virus-like particles, the concentration of any single type of papillomavirus virus-like particle in the multivalent papillomavirus virus-like particles being between 40 μg / mL and 120 μg / mL; (b) preferably an aluminum adjuvant, more preferably aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH) 3 ), most preferably aluminum hydroxyphosphate (AlPO 4 ) at a concentration of about 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 being between 10 mM and 26 mM, preferably 10 mM, 18 mM, or 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 to 320 mM, preferably 150 mM or 320 mM; and, optionally, (e) a physiologically acceptable concentration of a surfactant, which is a polyethoxy ether, preferably polysorbate 80, at a concentration of 0 to 0.02% by weight; (f) the pH of the formulation is 5.9 to 6.5, preferably 5.9, 6.2 or 6.5; Human papillomavirus virus-like particles are adsorbed to an adjuvant, the human papillomavirus virus-like particle is selected from HPV virus-like particles constructed with chimeric L1 proteins of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, or 58, and from one or more HPV virus-like particles constructed with L1 proteins of other pathogenic HPV types, preferably selected from HPV types 33 and 59; The chimeric HPV L1 protein consists, from N-terminus to C-terminus: a. an N-terminal fragment of an L1 protein of a first human papillomavirus type, the first human papillomavirus type being selected from HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56, and 58, and having an amino acid sequence selected from the sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; and b. a C-terminal fragment derived from the L1 protein of a second papillomavirus type, the C-terminal fragment being a fragment consisting of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; Including, The chimeric HPV L1 protein has the immunogenicity of the L1 protein of a first papillomavirus type. A stable formulation of a multivalent human papillomavirus virus-like particle vaccine for the prevention of HPV-associated disease or infection, except where the formulation contains mannitol or sucrose.

2. 2. The formulation of claim 1 comprising 0.74 mg / mL total papillomavirus virus-like particles of all types, 1.0 mg / mL aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, pH 6.2, and optionally polysorbate 80 at a concentration of 0.3 mg / mL or less.

3. The formulation of claim 1 or 2, wherein the HPV virus-like particles also include HPV virus-like particles assembled by L1 proteins of other pathogenic HPV types.

4. The formulation of any one of claims 1 to 3, wherein the C-terminus of the N-terminal fragment is directly connected to the N-terminus of the C-terminal fragment or is connected via a linker.

5. the chimeric HPV L1 proteins of chimeric HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 and 58 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; 5. The formulation of claim 1, wherein the HPV type 33 L1 protein and the 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.

6. HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 and 58 chimeric HPV L1 proteins having the amino acid sequences shown 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; 6. The formulation of claim 5, comprising an HPV type 33 L1 protein and an HPV type 59 L1 protein having the amino acid sequences set forth in SEQ ID NO: 28 and SEQ ID NO: 29, respectively.

7. A formulation of a papillomavirus vaccine according to any one of claims 1 to 6, 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.

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