A recombinant respiratory syncytial virus vaccine lyophilization protectant, uses and products

Abstract

The application discloses a freeze-drying protective agent for a recombinant respiratory syncytial virus vaccine and application and products, and belongs to the technical field of biological medicines. The technical problem to be solved is to provide a freeze-drying protective agent capable of maintaining the pre-fusion conformation and trimer structure stability of RSV PreF protein, and to solve the technical problem that the pre-fusion conformation of the PreF protein is irreversibly changed into a post-fusion conformation during freeze-drying and storage. The technical solution is characterized in that the freeze-drying protective agent for the recombinant respiratory syncytial virus vaccine comprises the following components in percentage by mass: 6%-8% D-mannitol, 2%-4% sucrose, 0.4%-0.8% L-histidine, 0.8%-1.2% histidine hydrochloride, 0.4%-0.6% arginine, 0.02%-0.05% polysorbate 80, and the balance is a buffer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the field of viral vaccine technology, and specifically relates to a recombinant respiratory syncytial virus vaccine freeze-dried protectant and its application and products. Background Technology

[0002] For understanding the technical content of this invention: Respiratory syncytial virus (RSV) is an enveloped, single-stranded negative RNA virus belonging to the genus Orthopneumovirus of the family Pneumoviridae [1]. It is one of the major pathogens causing acute lower respiratory tract infections (ALRTIs) in infants, immunocompromised individuals, and the elderly worldwide [2-4]. It is highly infectious and pathogenic, and in severe cases, it can lead to bronchitis, pneumonia, and asthma [5-6]. Developing an effective RSV vaccine has become one of the important means of preventing RSV infection. RSV PreF is currently the preferred target antigen for vaccine development [7]. However, long-term storage at room temperature or 2-8°C can cause the F protein to irreversibly change from its pre-fusion conformation to its post-fusion conformation [8]. Therefore, it is necessary to conduct research on screening PreF lyophilized formulations. Freezing removes free and bound water from the PreF buffer system, forming a loose powder cake. In its dehydrated state, PreF maintains its pre-fusion conformation, trimer form, and biological activity more effectively. Compared to storing PreF in a liquid buffer system, freeze-drying results in higher stability, a longer shelf life, and eliminates the excessive need for cold chain transport temperatures [9-11]. RSV fusion proteins (F proteins) exist in two forms: pre-fusion F (PreF) and post-fusion F. The surface of the pre-fusion F protein exposes most of the effective neutralizing antibody epitopes, inducing a stronger humoral immune response than the post-fusion F protein. Therefore, PreF is widely recognized as the preferred target antigen for RSV vaccine development. However, PreF protein is essentially a metastable protein. Prolonged storage at room temperature or 2-8°C leads to an irreversible conformational change—spontaneous conversion from the pre-fusion conformation to the post-fusion conformation. This results in trimer dissociation, loss of key neutralizing epitopes, and a significant reduction in vaccine immunogenicity. Currently, the main RSV vaccines available globally include GSK's Arexvy (approved in 2023) and Pfizer's Abrysvo (approved in 2023). Both vaccines use recombinant PreF protein as the core antigen and are formulated using a lyophilized dosage form. Pfizer's Abrysvo contains recombinant RSV preF A and preF B bivalent antigens in its lyophilized antigen component, and its protective agent formulation includes glycerol, sucrose, mannitol, polysorbate 80(II), and sodium chloride. GSK Arexvy uses trehalose as the main lyophilized protective agent, supplemented with a phosphate buffer system and polysorbate 80(II).

[0003] The lyophilization protection requirements for RSV PreF proteins are highly specific, and general-purpose lyophilization protectant formulations cannot be directly applied. During lyophilization and storage, PreF proteins need to maintain the integrity of their trimer structure while preventing irreversible conformational changes before fusion. The precise ratio of each component in the protectant (such as the ratio of sucrose to mannitol, the type of buffer, and the amino acid form) directly affects the protective effect. The lack of refined formulation research for PreF proteins can easily lead to lyophilized products collapsing, difficulty in reconstitution, or loss of protein activity.

[0004] In view of the above problems, it is urgent to conduct systematic screening and optimization research on lyophilization protectant formulations for recombinant respiratory syncytial virus PreF subunit protein, and to independently develop a lyophilization protectant combination with clearly defined components, systematically optimized ratios, and comprehensive long-term and mandatory condition verification of protective effects, so as to provide key technical support for the research and development and industrialization of RSV PreF subunit vaccine lyophilized formulations.

[0005] Relevant patent documents retrieved: The publication country is China, publication number CN121287901A, publication date 2026.01.09, entitled "Application of SR604 Injection in the Preparation of Drugs for the Prevention and / or Treatment of Human Hemorrhagic Diseases". This document discloses that SR604 injection includes a buffer system selected from histidine / histidine hydrochloride or acetate / sodium acetate; the SR604 injection includes the following protective agents: sugar or sugar alcohol, the sugar selected from trehalose and sucrose, the sugar alcohol selected from sorbitol or mannitol; amino acid, the amino acid selected from arginine hydrochloride or aspartic acid; the SR604 injection includes a surfactant, the surfactant selected from polysorbate 80 and / or P188, for use in liquid formulations of antibody drugs.

[0006] The country of origin is China, publication number CN107625958A, publication date 2018.01.26, entitled "Thermostable Vaccine Composition and Preparation Method Thereof", which discloses the freeze-drying protection of aluminum adjuvant vaccines, and discloses that the glass forming agent can be selected from trehalose, sucrose, mannitol, etc., and the buffer system can be selected from arginine, histidine, etc.

[0007] Relevant non-patent literature retrieved: Journal title: *Virology Journal*, Article title: "Lyophilized Preservation of Respiratory Syncytial Virus", Zhao Sulan et al., Publication date: April 2, 1986. This article discloses that 1% gelatin plus 1% sucrose and 50% skim milk can protect respiratory syncytial virus to withstand lyophilized preservation.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) Existing research on freeze-drying protectants mainly targets whole viruses (such as the non-patent literature "Freeze-drying Preservation of Respiratory Syncytial Virus") or other types of antigens (such as the aluminum-containing adjuvant vaccine in patent literature CN107625958A and the SR604 antibody in patent literature CN121287901A), lacking systematic research on the pre-fusion conformation protection of RSV PreF subunit proteins.

[0009] (2) Patent document CN121287901A is an antibody liquid formulation, which does not involve lyophilized formulations, let alone conformational protection of PreF protein.

[0010] (3) Although patent documents CN107625958A and CN121287901A list a variety of optional excipients in a generalized manner, they do not provide specific formulations and ratios for RSV PreF protein, nor do they reveal the synergistic protective mechanism among the excipients.

[0011] Therefore, developing a lyophilization protectant composition with clearly defined components, systematically optimized ratios, and the ability to maintain the conformational and trimer structural stability of RSV PreF protein before fusion has become a pressing technical challenge in this field.

[0012] [1]AMARASINGHE GK,BÀO Y,BASLER CF,et al.Taxonomy of the orderMononegavirales:update 2017[J].Arch Virol,2017,162(8):2493-2504. [2]Rice E, Oakes DB, Holland C, et al. Respiratory syncytial virus in children: epidemiology and clinical impact post-COVID-19[J]. Curr Opin InfectDis.2023,36(6):522-528. [3]Savic M, Penders Y, Shi T, et al. Respiratory syncytial virusdisease burden in adults aged 60 years and older in high-income countries: Asystematic literature review and meta-analysis[J]. Influenza Other RespirViruses.2023,17(1):e13031. [4]Cantú-Flores K, Rivera-Alfaro G, Muñoz-Escalante JC, et al. Globaldistribution of respiratory syncytial virus A and B infections: a systematicreview. Pathog Glob Health[J].2022,116(7):398-409. [5]SHI T,OOI Y,ZAW E M,et al.Association between respiratorysyncytial virus-associated acute lower respiratory infection in early lifeand recurrent wheeze and asthma in later childhood[J].J Infect Dis,2019:jiz311. [6]Rijsbergen LC, Lamers MM, Comvalius AD, et al. Human RespiratorySyncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway,and Organoid-Derived Respiratory Cultures[J].mSphere.2021,12;6(3):e00237-21. [7]McLellan JS, Chen M, Joyce MG, et al. Structure-based design of afusion glycoprotein vaccine for respiratory syncytial virus[J].Science.2013,342(6158):592-598. [8]Crank MC, Ruckwardt TJ, Chen M, et al; VRC 317 Study Team. A proofof concept for structure-based vaccine design targeting RSV in humans[J].Science. 2019,365(6452):505-509. [9]Al-Hussein A, Gieseler H. Investigation of histidine stabilizing effects on LDH during freeze-drying[J].J Pharm Sci.2013,102(3):813-826.

[10] Kasper JC, Winter G, Friess W. Recent advances and further challenges in lyophilization[J]. Eur J Pharm Biopharm.2013,85(2):162-169.

[11] Ghaemmaghamian Z, Zarghami R, Walker G, et al.Stabilizingvaccines via drying: Quality by design considerations[J]. Adv Drug DelivRev.2022,187:114313. Summary of the Invention The purpose of this invention is to provide: A lyophilized protective agent for recombinant respiratory syncytial virus (RSV) vaccines, its application and products, and related technologies, to address the technical problems in existing technologies, such as the lack of lyophilized protective agent formulations for pre-fusion conformation protection of RSV PreF proteins, and the inability of existing formulations to be directly applied to PreF subunit proteins, or combinations thereof.

[0013] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0014] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0015] Definitions of the standard terminology can be found in the references “Molecular Cloning: A Laboratory Manual (Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, 2001),” “Microbiology Tutorial (4th Edition), Higher Education Press, author: Zhou Deqing, 2020,” “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19,” and “Genetic Engineering, Higher Education Press, 2013-08-01.”

[0016] Unless otherwise stated, conventional methods within the scope of the art, such as mixing, reconstitution, freeze drying, etc., shall be used.

[0017] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0018] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0019] The term "respiratory syncytial virus" as used in this article refers to Respiratory Syncytial Virus (RSV), an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Orthopneumovirus in the family Pneumoviridae. It consists of a nucleocapsid within a lipid membrane and three transmembrane surface glycoproteins (attachment protein G, fusion protein F, and small hydrophobic protein SH). RSV is one of the leading pathogens causing acute lower respiratory tract infections in infants, immunocompromised individuals, and the elderly worldwide.

[0020] The term "F protein" used in this article refers to: the F protein, a major protective antigen on the surface of the RSV virus, belonging to class I fusion proteins, which primarily mediates the fusion of the viral envelope and the target cell membrane. During viral infection, the F protein mediates the fusion of the virus with the host cell membrane, promoting viral entry into the cell. The F protein is highly conserved (>90%) among RSV-A / B subtypes and is a key target for RSV vaccine and drug development.

[0021] The term "prefusion F protein (PreF)" as used in this article refers to the pre-fusion conformation of the F protein, which is in a metastable state. The surface of the prefusion F protein exposes the vast majority of effective neutralizing antibody epitopes, inducing a more robust humoral immune response than the fusion-promoted F protein. PreF has a stronger ability to induce RSV neutralizing antibodies in vivo and is currently the preferred target antigen for RSV vaccine development.

[0022] The term "postfusion F protein" used in this article refers to the stable conformation of the F protein after fusion. During infection, the F protein irreversibly transforms from its metastable prefusion conformation (PreF) to its stable postfusion conformation (PostF).

[0023] The term "PreF Trimer" as used in this article refers to the PreF protein existing in a trimer form in its native state. The integrity of the trimer structure is the structural basis for PreF to exert its immunogenicity. During lyophilization and storage, PreF protein must maintain the integrity of its trimer structure while preventing irreversible conformational changes before fusion.

[0024] The term "lyophilization protectant" as used in this article refers to substances added during the lyophilization preservation of biological products to prevent denaturation of active components. Protectants can alter the physicochemical environment of biological samples during lyophilization, mitigating or preventing damage to bioactive substances caused by lyophilization or rehydration, and preserving as much of their original physiological and biochemical properties and biological activity as possible. Lyophilization protectants have immunomodulatory activity but no pharmacological activity.

[0025] The term "polysorbate 80" as used in this article refers to: in this invention, polysorbate 80 specifically refers to polysorbate 80 (II), which is a pharmaceutical excipient specification included in the Chinese Pharmacopoeia, specifically referring to polysorbate 80 of the injection grade, and is distinguished from polysorbate 80 (ordinary grade) used for oral or topical application.

[0026] The term “L-histidine / histidine hydrochloride buffer” as used in this article refers to a buffer system composed of L-histidine (in its free basic form) and histidine hydrochloride (L-histidine hydrochloride) in a certain proportion. It is widely used in the formulation of biological products such as recombinant proteins, antibodies, and vaccines, mainly as a buffer, antioxidant, and stabilizer.

[0027] In a first aspect, the present invention provides: a lyophilized protective agent for a recombinant respiratory syncytial virus vaccine, comprising the following components by weight percentage: 6%-8% D-mannitol, 2%-4% sucrose, 0.4%-0.8% L-histidine, 0.8%-1.2% histidine hydrochloride, 0.4%-0.6% arginine, 0.02%-0.05% polysorbate 80, with the balance being buffer solution.

[0028] For example, in the lyophilized protective agent of a recombinant respiratory syncytial virus vaccine: The mass percentage of D-mannitol can be 6%, 6.5%, 7%, 7.5%, 8%, or any value within the range of any two of the above values. 7% is preferred.

[0029] The mass percentage of sucrose can be 2%, 2.5%, 3%, 3.5%, 4%, or any value within the range of any two of the above values. 3% is preferred.

[0030] The mass percentage of L-histidine can be 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or any value within the range of any two of the above values. Preferably, it is 0.6%.

[0031] The mass percentage of histidine hydrochloride can be 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, or any value within the range of any two of the above values. Preferably, it is 1.2%.

[0032] The mass percentage of arginine can be 0.4%, 0.5%, 0.6%, or any value within the range of any two of the above values. 0.6% is preferred.

[0033] The mass percentage of polysorbate 80 can be 0.02%, 0.03%, 0.04%, 0.05%, or any value within the range of any two of the above values. Preferably, it is 0.04%.

[0034] Preferably, the recombinant respiratory syncytial virus vaccine lyophilized protectant comprises the following components by weight percentage: 7% D-mannitol, 3% sucrose, 0.6% L-histidine, 1.2% histidine hydrochloride, 0.6% arginine, 0.04% polysorbate 80, with the balance being buffer solution.

[0035] According to some embodiments of the present invention, in the lyophilized protective agent of the recombinant respiratory syncytial virus vaccine, the mass ratio of sucrose to D-mannitol is 1:1.5-4. During lyophilization and storage, sucrose promotes the crystallization of D-mannitol primarily in anhydrous form, effectively inhibiting the formation of hemihydrates. This results in the lyophilized product possessing both good structural strength and protein protection. Exemplarily, the ratio can be 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, or any value within the range of any two of the above values. A sucrose to D-mannitol mass ratio in the range of 1:1.5-2.5 can further enhance the synergistic effect of the two, further improving the protective effect. A further preferred ratio is 1:2.33.

[0036] According to some embodiments of the present invention, the pH of the buffer solution is 6.0-7.0. Exemplarily, it can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, or any value within the range of any two of the above values.

[0037] According to some embodiments of the present invention, the buffer solution is an L-histidine / histidine hydrochloride buffer solution.

[0038] Secondly, the present invention provides the application of the aforementioned recombinant respiratory syncytial virus vaccine freeze-drying protectant in the preparation of recombinant respiratory syncytial virus vaccine freeze-dried formulations.

[0039] Specifically, the applications include, but are not limited to, the following: (1) Application in the preparation of RSV PreF protein lyophilized formulation.

[0040] (2) Application in improving the storage stability of RSV PreF protein.

[0041] (3) Application in maintaining the pre-fusion conformation of PreF protein.

[0042] (4) Application in the preparation of RSV subunit vaccines: The lyophilization protective agent composition of the present invention can be used to prepare lyophilized dosage forms of recombinant RSV PreF subunit vaccines. The lyophilized formulation can be used directly as a finished vaccine product, and can be injected after reconstitution with water for injection.

[0043] (5) Application in vaccine compositions: The lyophilized protective agent composition of the present invention can also be used in combination with other vaccine components (such as adjuvants, other antigens, etc.) to prepare a compound vaccine formulation containing RSV PreF protein. The adjuvants include, but are not limited to, aluminum adjuvants (aluminum hydroxide, aluminum phosphate, etc.), oil emulsion adjuvants, TLR agonists, etc.

[0044] (6) Application in drug delivery systems: The RSV PreF lyophilized formulation prepared from the lyophilized protective agent composition of the present invention can be used as a component of a drug delivery system to prepare novel delivery formulations such as sustained-release implants, microneedle patches, and inhalation powders. The lyophilized powder can be further processed into dosage forms suitable for different routes of administration, including but not limited to powder for injection, powder for inhalation, and lyophilized formulations for external use.

[0045] Thirdly, the present invention provides: a method for preparing a freeze-dried formulation of a recombinant respiratory syncytial virus vaccine, characterized by comprising the following steps: Step S1: Prepare the aforementioned recombinant respiratory syncytial virus vaccine freeze-dried protective agent; Step S2: Mix the RSV PreF antigen stock solution with the recombinant respiratory syncytial virus vaccine freeze-drying protectant to obtain a semi-finished product; Step S3: Freeze-dry the semi-finished product to obtain the freeze-dried formulation of the recombinant respiratory syncytial virus vaccine.

[0046] According to some embodiments of the present invention, in step S2, the RSV PreF antigen stock solution and the recombinant respiratory syncytial virus vaccine lyophilized protectant are mixed at a mass ratio of 1:(0.8-1.2). This mass ratio can be exemplarily 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1, 1:1.15, 1:1.2, or any value within the range of any two of the above values, preferably 1:1.

[0047] According to some embodiments of the present invention, the recombinant respiratory syncytial virus (RSV) vaccine stock solution can be prepared directly (the preparation method can be found in the references in the background art [7]), or a commercially available recombinant RSV vaccine (such as a novel coronavirus subunit vaccine or a herpes zoster virus subunit vaccine) can be selected. The method of obtaining the recombinant RSV vaccine stock solution does not limit the preparation method of the recombinant RSV vaccine lyophilized protectant, the recombinant RSV vaccine lyophilized formulation, or the protection scope of the recombinant RSV vaccine lyophilized formulation of the present invention.

[0048] According to some embodiments of the present invention, the freeze-drying temperature in step S3 is -20°C to -15°C. Exemplarily, it can be -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, or any value within the range of any two of the above values, preferably -18°C.

[0049] According to some embodiments of the present invention, during the freeze-drying process described in step S3, the pH change of the semi-finished product is controlled to be less than 0.1.

[0050] Fourthly, the present invention provides: a lyophilized formulation of recombinant respiratory syncytial virus PreF protein, prepared by the aforementioned preparation method.

[0051] According to some embodiments of the present invention, the recombinant respiratory syncytial virus PreF protein lyophilized preparation appears as a white, loose powder cake, and after reconstitution with water for injection, it is a colorless, clear liquid without insoluble particles, and has a moisture content of ≤3.0%.

[0052] According to some embodiments of the present invention, the recombinant respiratory syncytial virus PreF protein lyophilized preparation, after being stored at 5℃±3℃ for 12 months, or at 25℃±2℃ / RH60%±5% for 6 months, or at 37℃±2℃ / RH60%±5% for 28 days, exhibits a PreF trimer protein concentration loss of less than 10%.

[0053] In this invention, Example 1 at least supports the protection scope of "recombinant respiratory syncytial virus vaccine freeze-dried protectant".

[0054] The term "recombinant respiratory syncytial virus vaccine lyophilized protectant" is derived from the foregoing explanation and / or the corresponding protectant components in Example 1. Therefore, those skilled in the art can reasonably infer that "recombinant respiratory syncytial virus vaccine lyophilized protectant," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "recombinant respiratory syncytial virus vaccine lyophilized protectant."

[0055] The present invention has at least the following beneficial effects: 1. Compared with existing technologies, this invention has developed a systematically optimized lyophilization protectant combination formulation targeting the PreF subunit protein of recombinant respiratory syncytial virus (RSV). Unlike existing general-purpose lyophilization protectant formulations that are mainly based on experience in developing attenuated live virus vaccines or other types of biological products, this invention, for the first time, focuses on the high instability of RSV PreF protein (PreF conformation, easy fusion-prone irreversible changes after forward fusion, and easy dissociation of the trimer) as the core protection target. It provides a composite protectant system composed of six excipients: D-mannitol, sucrose, L-histidine, histidine hydrochloride, arginine, and polysorbate 80(II). After long-term (5℃±3℃ / 12 months), accelerated (25℃±2℃ / 6 months), and forced (37℃±2℃ / 28 days) multi-dimensional stability tests, the key quality attributes of the lyophilized product, such as appearance, reconstitution performance, PreF trimer protein concentration, SEC-HPLC purity, and moisture content, remain stable. The loss of PreF protein concentration and trimer concentration is less than 10%, which is significantly better than the protective effect of existing conventional formulations.

[0056] 2. Compared with the prior art, the present invention is not limited to a generalized list of excipients, but determines the optimal ratio and synergistic mechanism of each excipient through systematic experiments, and determines the content of each component of the recombinant respiratory syncytial virus vaccine freeze-dried protectant, which can synergistically maximize the protective effect of the recombinant respiratory syncytial virus vaccine freeze-dried protectant.

[0057] 3. Based on the established components and proportions, this invention further provides an optimal ratio of sucrose to D-mannitol in the lyophilization protectant of recombinant respiratory syncytial virus vaccines, as well as the synergistic ratio of histidine buffer system, arginine, and polysorbate 80(II). Experiments have shown that when the mass ratio of sucrose to D-mannitol is approximately 1:1.5-2.5, hemihydrate formation can be effectively inhibited, thus enabling the lyophilized product to possess both good structural strength and protein protection. Meanwhile, the use of an L-histidine / histidine hydrochloride buffer system (pH approximately 6.0-7.0) minimized pH changes of less than 0.1 during lyophilization, further reducing the conformational changes of PreF caused by pH drift. Arginine (in its free basic form) increased the solubility of PreF after reconstitution and inhibited aggregation through volume repulsion, avoiding the deterioration of the appearance and stability of the lyophilized product caused by phase separation compared to arginine hydrochloride. Polysorbate 80(II) adsorbed onto the hydrophobic region of the PreF surface at a preferred concentration of 0.04%, further inhibiting protein aggregation induced by gas-liquid and solid-liquid interfaces. The synergistic effects and precise proportions of the above excipients have not been reported in the literature.

[0058] 4. This invention establishes a multi-dimensional evaluation system covering molecular conformational stability, colloidal stability, and long-term storage stability. In the formulation screening process, this invention not only employs conventional indicators such as appearance, reconstitution, moisture content, and SEC-HPLC purity, but also innovatively introduces a protein stability analyzer to detect the activity of PreF in the protective agent solution. T m Value and T agg The influence of candidate formulations on the thermal stability and aggregation tendency of the PreF structure was evaluated using this multi-dimensional evaluation system. The recombinant respiratory syncytial virus vaccine lyophilized protectant provided by this invention exhibits good appearance, reconstitution effect, low moisture content, high purity, and high efficiency. T m Value and High T agg Value, of which the preferred formulation (H2) contains PreF T m The value reached 71.47℃. T agg The values ​​reached 70.66℃, all within the ideal range above 70℃, indicating that this formulation has significant advantages in maintaining the conformational stability of PreF and inhibiting heat-induced aggregation. Combined with long-term, accelerated, and forced stability data, as well as a comparative stability experiment of liquid PreF under the same conditions (the trimer concentration of liquid PreF decreased by approximately 85% after being stored at 5℃±3℃ for 6 months), the significant superiority of the lyophilized formulation combined with the protective agent of this invention over the liquid formulation was systematically demonstrated.

[0059] 5. This invention provides a lyophilization protectant formulation that can be mass-produced and whose components fully comply with the requirements for excipients included in the Chinese Pharmacopoeia. The D-mannitol, sucrose, L-histidine, histidine hydrochloride, arginine, and polysorbate 80(II) selected in this invention are all excipients included in the Chinese Pharmacopoeia, possessing a sound safety and compliance foundation. The formulation process is simple and the system is stable, suitable for the industrial-scale production of lyophilized formulations of recombinant RSV PreF subunit vaccines, providing key technical support for domestically produced RSV vaccines to overcome the technical barriers of lyophilization formulations and achieve independent control. Attached Figure Description

[0060] Figure 1 The graph shows a comparison of the purity of PreF before lyophilization and the lyophilized product after reconstitution by SEC-HPLC for formulation H2. The red curve represents the purity of PreF before lyophilization in group H2, and the blue curve represents the purity of PreF after reconstitution of the lyophilized product in group H2.

[0061] Figure 2 The graph shows a comparison of the purity of PreF before lyophilization and the lyophilized product after reconstitution by SEC-HPLC for formulation H3. The red curve represents the purity of PreF before lyophilization in group H3, and the blue curve represents the purity of PreF after reconstitution of the lyophilized product in group H3.

[0062] Figure 3 The graph shows the results of PreF protein trimer detection for freeze-dried products in groups H2 and H3 under forced stability conditions.

[0063] Figure 4 The image shows the SEC-HPLC purity determination of the H2 lyophilized product after reconstitution under forced stabilization conditions. The red curve represents the H2 lyophilized product after 0 days of forced stabilization conditions, and the blue curve represents the H2 lyophilized product after 28 days of forced stabilization conditions.

[0064] Figure 5 The image shows the SEC-HPLC purity detection of H3 lyophilized product after reconstitution under forced stabilization conditions. The red curve represents H3 lyophilized product stored under forced stabilization conditions for 0 days, and the blue curve represents H3 lyophilized product stored under forced stabilization conditions for 28 days.

[0065] Figure 6 This is a graph showing the denaturation curve of the PreF semi-finished product.

[0066] Figure 7 This is a graph showing the aggregation curve of the PreF semi-finished product.

[0067] Figure 8 The image shows the DLS light intensity distribution of the PreF semi-finished product at 25℃ and 85℃.

[0068] Figure 9 The graph shows the results of the detection of PreF trimer protein concentration at different time points under the condition of 5℃±3℃ for the semi-finished product PreF.

[0069] Figure 10 The graph shows the results of PreF protein concentration detection at different time points under conditions of 5℃±3℃ for the semi-finished product PreF.

[0070] Figure 11 This is a SEC-HPLC purity test result of the semi-finished product PreF after being stored at 5℃±3℃ for 6M.

[0071] Figure 12 The graph shows the results of PreF trimer protein concentration detection at different time points under three conditions for PreF lyophilized products.

[0072] Figure 13 The graph shows the results of PreF protein concentration detection at different time points under three conditions for PreF lyophilized products.

[0073] Figure 14 The image shows the SEC-HPLC purity test results of the PreF lyophilized product at 5℃±3℃. The blue curve represents the PreF lyophilized product after 0 days at 5℃±3℃, and the red curve represents the PreF lyophilized product after 12M at 5℃±3℃.

[0074] Figure 15 The image shows the SEC-HPLC purity detection curve of PreF lyophilized product at 25℃±2℃ and 60%±5%. The red curve represents the 0M value of PreF lyophilized product at 25℃±2℃ and 60%±5%, and the blue curve represents the 6M value of PreF lyophilized product at 25℃±2℃ and 60%±5%.

[0075] Figure 16 The image shows the SEC-HPLC purity detection curve of PreF lyophilized product at 37℃±2℃ and 60%±5%. The red curve represents PreF lyophilized product stored at 37℃±2℃ and 60%±5% for 0 days, and the blue curve represents PreF lyophilized product stored at 37℃±2℃ and 60%±5% for 28 days. Detailed Implementation

[0076] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0077] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0078] All data processing in this invention was performed using OriginPro 2024 software. The analysis between different groups was performed using independent samples t-test or one-way ANOVA test, and P < 0.05 was used to indicate that the difference was statistically significant.

[0079] The main reagents and instruments used in the following examples are: Citric acid, sodium citrate, and polysorbate 80 (II) were purchased from Hunan Ercon Pharmaceutical Co., Ltd.; histidine hydrochloride, arginine, arginine hydrochloride, and trehalose were purchased from Aivit (Shanghai) Pharmaceutical Technology Co., Ltd.; L-histidine, sucrose, and D-mannitol were purchased from Pfanstiehl.; the protein purification system was purchased from GE.; the electronic balance was purchased from Mettler Toledo.; D25 and AM14 antibodies were purchased from Creative Biolabs.; HRP-labeled goat anti-human IgG was purchased from SIGMA.; 96-well ELISA plates were purchased from Corning.; the Spectra Max M3 microplate reader was purchased from Molecular Devices.; vials were purchased from Ningbo Zhengli Pharmaceutical Packaging Co., Ltd.; rubber stoppers were purchased from Jiangsu Hualan Pharmaceutical New Materials Co., Ltd.; aluminum caps were purchased from Hebei Jinhuan Packaging Co., Ltd.; the Tofflon lyo-0.5 freeze dryer was manufactured by Shanghai Dongfulong Testing Technology Co., Ltd.; and the protein stability analyzer was from Unchained. Labs; GatorPrime label-free biomolecule analyzer purchased from Little Crocodile Biotechnology; stability test chamber purchased from MEMMERT; high performance liquid chromatograph purchased from Agilent Technologies; BioCore™ SEC-300 column purchased from Nanospectrum Analytical Systems.

[0080] In this embodiment of the invention, the recombinant respiratory syncytial virus vaccine stock solution (PreF stock solution) was prepared by the applicant (the preparation method is referred to in reference [7] McLellan JS, Chen M, Joyce MG, et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus[J].Science.2013,342(6158):592-598.).

[0081] Example 1: Lyophilized Protectant for Recombinant Respiratory Syncytial Virus Vaccine Using at least one of L-histidine, histidine hydrochloride, citric acid, sodium citrate, arginine, arginine hydrochloride, sucrose, trehalose, D-mannitol, and polysorbate 80(II) as excipients, a total of 33 protective agent formulations were prepared. The composition of the protective agents in the formulations is shown in Table 1. The remainder is a buffer solution containing 200 mM sodium chloride and 20 mM tromethamine, with a pH of 7.2-7.8. Among them, formulations F2, F3, G1, G3, H1, H2, and H3, which contain 6%-8% D-mannitol, 2%-4% sucrose, 0.4%-0.8% L-histidine, 0.8%-1.2% histidine hydrochloride, 0.4%-0.6% arginine, and 0.02%-0.05% polysorbate 80, with the remainder being a buffer solution, are the freeze-drying protective agents for the recombinant respiratory syncytial virus vaccine of this application. The other formulations are comparative examples.

[0082] Table 1

[0083] Note: / indicates that no additives were added.

[0084] Example 2: Lyophilized Formulation of Recombinant Respiratory Syncytial Virus Vaccine (PreF Lyophilized Product) and its Preparation The lyophilization protectant from Example 1 was mixed with an equal volume of PreF stock solution to obtain a semi-finished product. The semi-finished product was dispensed into vials, 0.5 mL / vial, for lyophilization. Lyophilization: After dispensing, the sample was placed in a lyophilizer and pre-frozen to -50°C. The drying process was then started. After lyophilization was completed, the sample was removed to obtain the lyophilized recombinant respiratory syncytial virus vaccine formulation, which was then sealed.

[0085] Example 1: Detection and stability study of PreF lyophilized product (1) Appearance and reconstitution: Five lyophilized samples were taken from each group of recombinant respiratory syncytial virus vaccine lyophilized formulations. The solidification condition was observed under a 40W fluorescent lamp. Then, 0.5mL of sterile water for injection was added, and the dissolution condition and the presence of insoluble matter were observed under a 40W fluorescent lamp.

[0086] (2) PreF protein trimer content determination: The PreF trimer protein content was rapidly detected using the Gator™ system as follows: The reference standard, PreF, and antibody AM14 were diluted with PBS and added to the sample wells respectively. After equilibration, the antibody was immobilized, a baseline was established, and sample binding was performed. A standard curve was constructed using the relationship between the initial slope of the real-time curve of the reference standard and the concentration (R² > 0.99). The PreF trimer concentration was calculated using the standard curve formula. For PreF content detection, antibody D25 was diluted with PBS, and the remaining procedures were the same as before.

[0087] (3) Purity testing: Size exclusion high performance liquid chromatography (SEC-HPLC) was performed as follows: The BioCore™ SEC-300 column was equilibrated with PBS at pH 7.2-7.4. The injection volume was 100 μL. Detection... A 280 The nm value was measured, and the chromatogram was integrated and analyzed after the detection was completed.

[0088] (4) Moisture test: According to the requirements of the Chinese Pharmacopoeia, Volume IV (2020 edition), the moisture content of PreF lyophilized products should not exceed 3.0%.

[0089] (5) Stability study: Long-term stability test of lyophilized products: PreF lyophilized products were placed at 5℃±3℃, and samples were taken for testing at 0M (month), 3M, 6M, 9M, and 12M. The test items included the aforementioned appearance, reconstitution, PreF protein concentration, PreF trimer protein concentration, SEC-HPLC purity, and moisture content.

[0090] Accelerated stability test of lyophilized products: PreF lyophilized products were placed at 25℃±2℃ and 60%±5% humidity. Samples were taken at 0M, 1M, 2M, 3M, and 6M for testing. The test items included the aforementioned appearance, reconstitution, PreF protein concentration, PreF trimer protein concentration, SEC-HPLC purity, and moisture content.

[0091] Forced stability test of lyophilized products: PreF lyophilized products were placed at 37℃±2℃ and 60%±5% humidity, and samples were taken at 0, 7, 14, 21, and 28 days for various tests. The test items included the aforementioned appearance, reconstitution, PreF protein concentration, PreF trimer protein concentration, SEC-HPLC purity, and moisture content.

[0092] (6) Freeze-dried formulation H2 semi-finished product T m , T agg Detection: Detected using a protein stability analyzer T m , T agg , T m and T aggThe stability of lyophilized formulations is evaluated by rapidly exposing the PreF structure through heating stimulation. The procedure is as follows: 9 μL of the semi-finished product is added to the sample well. The heating curve is set at 20-85℃, 0.3℃ / min. During the heating process, the intrinsic fluorescence (IF), static light scattering (SLS266) at 266 nm, static light scattering (SLS473) at 473 nm, and dynamic light scattering (DLS) of PreF are measured. After the measurements, the denaturation curve of PreF is obtained by analyzing the IF signal of the fluorescence spectrum using the barycentric mean (BCM) method. The melting temperature of PreF is then determined based on the denaturation curve. T m The PreF aggregation curve is obtained based on the changes in the SLS266 or SLS473 signal, and the determination is made accordingly. T agg The value is obtained by calculating the PreF average hydration kinetic diameter based on the changes in the DLS signal.

[0093] result: (1) Test results of lyophilized PreF products with different formulations: Please refer to Table 2. The results of experiments in Group A (A1-2) show that, compared to sucrose, trehalose has no positive effect on the appearance of freeze-dried products and its protective effect is slightly worse than that of sucrose. The results of experiments in Group B (B1-B5) show that 6% D-mannitol has a significant positive effect on the appearance of freeze-dried products, while 1% sucrose has a poor effect. The results of experiments in Group C (C1-6) show that the single use of L-histidine, histidine hydrochloride, arginine, and arginine hydrochloride is not conducive to the appearance and moisture sublimation of freeze-dried products. The results of experiments in Group D (D1-D6) show that the combined application of D-mannitol, sucrose, and polysorbate 80(II) has a positive effect on the appearance of freeze-dried products, but the moisture content of the freeze-dried products is all higher than 3%, indicating that L-histidine... The citric acid double buffer system may be detrimental to water sublimation during freeze-drying. SEC-HPLC purity testing showed degradation and aggregation in all reconstituted freeze-dried products, indicating that sucrose and polysorbate 80(II) did not fully exert their protective and protein aggregation-inhibiting effects. Comparative analysis of freeze-dried products in groups E3 and E4 showed that the addition of polysorbate 80(II) had a positive effect on appearance. Compared with freeze-dried products in groups E1, E2, and E4, the lower sucrose concentration resulted in insufficient protection of protein stability in the formulation system. The addition of polysorbate 80(II) had a positive effect on the freeze-drying stability of PreF protein. The experiments in group E determined that the L-histidine buffer-arginine combination was the preferred formulation buffer system. Freeze-dried products in groups F1 (high concentration of polysorbate 80(II)) and F3 (no polysorbate 80(II)) showed aggregation changes in SEC-HPLC purity testing. Low concentration of polysorbate 80(II) could inhibit protein aggregation, but insufficient arginine content may also lead to aggregation. The freeze-drying system contained 4% sucrose and 8% polysorbate 80(II). D-mannitol combination has a significant positive effect on the appearance of PreF freeze-dried products; the freeze-dried products of groups G1 and G3 have good appearance. Compared with the freeze-dried product of group G2, the high concentration of sucrose in the freeze-drying system has no positive effect on the appearance of the freeze-dried products. Compared with group G3, the freeze-dried product of group G1 showed no degradation, aggregation or other changes after reconstitution, and the moisture content was 1.5%. This indicates that increasing the concentration of L-histidine, histidine hydrochloride, arginine and polysorbate 80(II) has a protective effect on protein trimers and pre-fusion conformation, but its inhibitory effect on protein aggregation is not obvious. The experimental results of group H show that compared with group H1, 0.04% polysorbate 80(II) in group H2 can inhibit the aggregation of PreF during the freeze-drying process. At the same time, increasing the content of L-histidine and histidine hydrochloride is more conducive to the protection of protein and the sublimation of moisture during the freeze-drying process. Compared with group H3, the high concentration of histidine hydrochloride in group H2 is more conducive to the protection of protein and the sublimation of moisture during the freeze-drying process.

[0094] The above results indicate that the recombinant respiratory syncytial virus vaccine freeze-drying protectants (such as F2, F3, G1, G3, H1, H2, and H3) provided by this invention contain: 6%-8% D-mannitol, 2%-4% sucrose, 0.4%-0.8% L-histidine, 0.8%-1.2% histidine hydrochloride, 0.4%-0.6% arginine, 0.02%-0.05% polysorbate 80(II), with the balance being buffer solution. They exhibit good appearance, reconstitution effect, no PreF degradation, low moisture content, and high purity.

[0095] Table 2

[0096] Note: / indicates that this test was not performed.

[0097] (2) Forced stability test of groups H2 and H3 The recombinant respiratory syncytial virus vaccine lyophilized protectants provided by this invention, specifically groups H2 and H3, exhibit a white, loose, powdery appearance, rapid reconstitution, clear solutions with no undissolved substances, and a trimer protein content loss of less than 10%. Their moisture contents are 1.5% and 1.2%, respectively, demonstrating superior performance. Therefore, H2 and H3 were used for further stability testing.

[0098] The results show that, as shown in Table 3, the freeze-dried products of groups H2 and H3 were placed in a stability test chamber at 37℃±2℃ and 60%±5% humidity for forced stability testing. After 28 days, they all appeared as white, loose powder cakes. They were rapidly reconstituted upon reconstitution, and the solution was clear with no undissolved substances.

[0099] Please see Figures 1 to 2 The purity of group H2 before and after freeze-drying was 98.77% and 97.36%, respectively; the purity of group H3 before and after freeze-drying was 98.35% and 97.33%, respectively. The purity of groups H2 and H3 decreased by only about 1.41 percentage points and 1.02 percentage points, respectively, after freeze-drying, with the decrease controlled within 1.5 percentage points. Moreover, the purity of both groups after freeze-drying was higher than 97%, indicating that the purity of the samples remained at a high level after freeze-drying.

[0100] like Figure 3 As shown, under forced stability conditions, the PreF trimer protein concentration loss in both H2 and H3 lyophilized products was less than 10% compared to day 0. Figure 4 As shown, SEC-HPLC purity testing results indicated that no aggregation, degradation, or other changes occurred in the H2 group of lyophilized products after reconstitution. Figure 5As shown, a small amount of protein aggregation occurred after reconstitution of the H3 group of lyophilized products. This small aggregation may be due to reversible entanglement caused by excessively high local concentrations. It can be dispersed by gentle shaking or standing. This phenomenon is due to the inherent physical heterogeneity of lyophilized products. This indicates that the H2 group of components has the best lyophilization protection effect on PreF and further research is needed.

[0101] (3) Structural and colloidal stability analysis of PreF in the semi-finished product of group H2 To evaluate the structural and colloidal stability of PreF in semi-finished products (i.e., un-lyophilized) prepared using the H2 group lyophilization formulation, this study used IF assay to detect the properties of PreF. T m Value, based on SLS detection of PreF T agg The values ​​are shown in Table 4.

[0102] Table 4

[0103] like Figure 6 As shown, PreF in H2 formulation buffer T m 1. Temperatures above 70℃ are considered ideal. T m The value indicates that PreF exhibits good structural thermal stability in the H2 formulation. T agg The results show that as the temperature increases, T agg 266 and T agg The intensity of scattered light at 473 nm increased significantly, indicating that PreF formed aggregates after heating. PreF's... T agg value ratio T m The value is slightly lower, and the upward trend of the SLS curve and the BCM curve is basically consistent, indicating that the aggregation and denaturation of PreF occur together.

[0104] like Figure 7 As shown, PreF's T agg Value and T m The close proximity of the value 1 indicates that PreF rapidly aggregates as the first structural domain undergoes transformation.

[0105] like Figure 8As shown, this study also used the DLS principle to characterize the hydration kinetic diameter of PreF in the H2 group of semi-finished products. With the increase in PreF aggregation degree, the average hydration kinetic diameter of PreF also increased. At 25℃, the PreF light intensity distribution showed two particle size peaks, with average hydration kinetic diameters of approximately 11 nm (approximately 99.8%) and 118 nm (approximately 0.2%), indicating the existence of two structural domains in PreF. (Tables 4 and 5 are provided.) Figure 6 The results also show that PreF has two instances. T m The values ​​further verified that PreF itself has two structural domains. At 25℃, the second particle size peak of the PreF light intensity distribution is a non-aggregated peak. When heated to 85℃, the average hydration kinetic diameter of PreF exceeds 1000 nm, which is outside the particle size detection range of DLS (0.3-1000 nm), further verifying that PreF exhibits severe aggregation after heating. T m , T agg The DLS test results show that the composition and ratio of the freeze-drying protectant of the recombinant respiratory syncytial virus vaccine of the present invention are beneficial to enhancing the diffusion effect of PreF, reducing the risk of aggregation, and maintaining the stability of PreF during the freeze-drying process and long-term storage.

[0106] Table 5

[0107] As shown in Table 6, Figure 9 , Figure 10 As shown, when the PreF semi-finished product was placed at 5℃±3℃ for 6 months, there was no significant change in appearance. ELISA analysis showed that the PreF trimer protein concentration decreased by about 85% compared to day 0, and the PreF protein concentration decreased by about 78% compared to day 0.

[0108] As shown in Table 6 and Figure 11 As shown, the SEC-HPLC purity test results showed that the purity did not change significantly, but the position of the main peak shifted forward, indicating that the PreF conformation changed.

[0109] The results in summary indicate that the trimer form and pre-fusion conformation of liquid PreF undergo significant changes during long-term storage, and the long-term stability of liquid PreF is poor at 5℃±3℃.

[0110] Table 6

[0111] (4) Stability evaluation of PreF lyophilized products in group H2 The results showed that under the three stability test conditions (long-term stability test, accelerated stability test, and forced stability test), the appearance of the PreF lyophilized product in group H2 was a white, loose powder cake. After reconstitution, the appearance, the proportion of the main peak, and the position of the main peak did not change, and the moisture content did not exceed 2%. The results are shown in Table 7. Figure 14 , Figure 15 and Figure 16 The loss of PreF trimer protein concentration on day 0 was less than 10% compared to the PreF pre-fusion protein concentration, as shown in the results. Figure 12 , Figure 13 In summary, the components and proportions in the H2 group formulation of this invention provide better protection for PreF during freeze-drying and storage, and are therefore the preferred freeze-drying protectant formulation for freeze-dried PreF.

[0112] Table 7

[0113] Note: / indicates that this test was not performed.

[0114] In summary, the RSV PreF lyophilized product prepared from the H2 group of lyophilization protectant compositions appears as a white, loose, powdery cake. The cake is full, structurally intact, and shows no signs of collapse or shrinkage. It dissolves rapidly when reconstituted with water for injection, resulting in a colorless, clear liquid without insoluble particles or flocculent matter. This excellent appearance and reconstitution properties indicate that D-mannitol in the formulation forms a fully crystalline framework during lyophilization, while sucrose is uniformly dispersed in an amorphous state. The synergistic effect of these two components endows the lyophilized product with good mechanical strength and reconstitution properties.

[0115] The PreF protein trimer structure and activity were significantly preserved. The PreF trimer protein content was detected using the Gator label-free biomolecular analysis system, and the results showed that the trimer protein concentration loss after reconstitution of the H2 group lyophilized product was <10%. SEC-HPLC purity analysis showed that the main peak position and shape of the H2 group lyophilized product after reconstitution were consistent with those before lyophilization, with no abnormal changes, confirming that this protective agent combination can effectively maintain the conformation and trimer structure integrity of PreF before fusion.

[0116] The lyophilized protectant for recombinant respiratory syncytial virus (RSV) vaccine enhances the thermostability of PreF. Protein stability analysis results show that PreF in the H2 group protectant solution... T m The value is 71.47℃. T m The value is 80.80℃. T agg The value of 266 is 70.66℃. T aggThe value of 473 is 71.27℃. T m Value and T agg The values ​​were all within the ideal range above 70℃, indicating that the protective agent combination significantly improved the structural thermal stability of PreF. DLS analysis showed that the average hydration kinetic diameter of PreF was approximately 18.14 nm at 25℃, exhibiting a typical uniform dispersion state. This confirms that the combination of the L-histidine buffer system and arginine, sucrose, and polysorbate 80(II) in the formulation effectively enhanced the intermolecular repulsion of protein molecules, reduced the risk of aggregation, and provided strong protection for stability during the freeze-drying process and long-term storage.

[0117] The residual moisture content of the lyophilized products was well controlled. According to the methods described in the Chinese Pharmacopoeia, Part IV (2020 edition), the moisture content of the H2 group of lyophilized products was approximately 1.2%, far below the quality control upper limit of 3.0%. At the endpoints of long-term (5℃±3℃ / 12 months), accelerated (25℃±2℃ / 6 months), and forced (37℃±2℃ / 28 days) stability tests, the moisture content was 1.3%, 1.6%, and 1.3%, respectively, all below 2%. This indicates that the formulation facilitates sufficient sublimation of moisture during lyophilization, and the lyophilized products exhibit low hygroscopicity during storage, effectively inhibiting water-mediated protein degradation.

[0118] Multi-dimensional stability evaluation verified the long-term protective effect. Long-term stability (5℃±3℃): After 12 months of storage, the H2 group lyophilized product maintained a white, loose, cake-like appearance; the solution was clear and free of particles after reconstitution; the loss of PreF protein concentration and trimer protein concentration was <10%; the SEC-HPLC purity remained at 99.2%-100.0%, with no change in the position and shape of the main peak, and no aggregation or degradation peaks appeared. Accelerated stability (25℃±2℃ / RH60%±5%): After 6 months of storage, all quality indicators were basically consistent with those at 0 months; the loss of PreF protein concentration and trimer concentration was <10%; the SEC-HPLC purity remained above 98.5%; and there were no changes in protein conformation or aggregation. Forced stability (37℃±2℃ / RH60%±5%): After 28 days of storage, the loss of PreF protein concentration and trimer concentration was <10%; the SEC-HPLC purity remained above 97.9%; and there was no aggregation, degradation, or other changes. In contrast, the H3 group (with lower histidine hydrochloride content) showed protein aggregation on day 28 under forced conditions, further verifying the superiority of the H2 group formulation.

[0119] The lyophilized dosage form exhibits significant advantages over liquid formulations. Comparative experiments show that the liquid PreF semi-finished product, stored at 5℃±3℃ for only 6 months, experienced a loss of approximately 85% in trimer protein concentration and approximately 78% in protein concentration, with the SEC-HPLC peak shifting forward, indicating irreversible conformational changes before fusion. In contrast, the H2 group of lyophilized products maintained stable protein activity and structure after 12 months of storage at the same temperature. This comparative result fully demonstrates that the lyophilization protectant combined with the lyophilization process of this invention can fundamentally solve the technical bottleneck of poor stability in RSV PreF liquid formulations, significantly extend vaccine shelf life, and greatly reduce reliance on the cold chain.

[0120] The comprehensive technical effect of synergistic action of each component. In the H2 formulation of this invention, each excipient exhibits a significant synergistic protective effect: the mass ratio of sucrose to D-mannitol is approximately 1.2; sucrose promotes the crystallization of D-mannitol primarily in anhydrous form, inhibiting hemihydrate formation and balancing the structural strength of the powder cake with protein protection; the L-histidine / histidine hydrochloride buffer system (pH approximately 6.0-7.0) exhibits a pH change of less than 0.1 during freeze-drying, effectively preventing conformational changes in PreF caused by pH drift; arginine (in free base form) increases the solubility of PreF after reconstitution through volume repulsion, inhibiting aggregation while simultaneously promoting anhydrous mannitol crystallization; polysorbate 80(II) at an optimized concentration of 0.04% adsorbs onto the hydrophobic region of the PreF surface, significantly inhibiting interface-induced aggregation. All the above excipients are listed in the Chinese Pharmacopoeia, possessing a good safety and compliance foundation, and the formulation process is simple, suitable for industrial-scale production.

[0121] Discussion of Results: This invention provides a lyophilized protective agent for the recombinant respiratory syncytial virus vaccine RSV PreF, which has important guiding significance for the application research of PreF vaccine.

[0122] The protective mechanisms of sucrose and trehalose for PreF in the freeze-drying formulation are mainly based on the water molecule substitution theory and the glass transition theory. The difference in their freeze-drying protection effects on the target protein may be due to the differences in their physicochemical properties. Trehalose has a higher Tg than sucrose, which is beneficial for shortening the freeze-drying cycle. However, sucrose is more difficult to crystallize than trehalose and is less prone to phase separation from PreF during freeze-drying, thus providing better protection for PreF. In the H2 group, the thermal stability of freeze-dried PreF showed no significant difference between day 28 and day 0 (P<0.0001), indicating that sucrose, in its amorphous state, fully played its role as a stabilizer during freeze-drying and storage. Furthermore, the fully crystallized D-mannitol gave the freeze-dried PreF good structural strength. Sucrose may have significantly promoted the crystallization of D-mannitol, primarily in anhydrous form, and inhibited the formation of D-mannitol hemihydrate during freeze-drying and storage, thereby contributing to the high stability of freeze-dried PreF during freeze-drying and storage.

[0123] The buffer pH value used in this invention is beneficial for maintaining the high stability of PreF trimer during freeze-drying. The pH change during freeze-drying is less than 0.1, which helps to further reduce the irreversible conformational changes of PreF before fusion caused by pH changes due to liquid phase separation during freeze-drying, thereby further improving protein activity and stability.

[0124] Meanwhile, this invention uses L-histidine as the main component and adds arginine. Its mechanism of action may be that arginine binds to the side chains of PreF amino acids, increasing the volume repulsion effect between proteins, thereby increasing the solubility of PreF after freeze-drying and reconstitution, and inhibiting PreF aggregation. At the same time, arginine remains in an amorphous state during the pre-freezing process, effectively protecting the PreF structure and promoting the crystallization of D-mannitol mainly in anhydrous form, inhibiting the formation of D-mannitol hemihydrate, which is beneficial to the stability of PreF trimer and the physical appearance of freeze-dried products. Arginine hydrochloride will cause phase separation during freeze-drying, which is not conducive to the stability of PreF and the appearance of freeze-dried powder.

[0125] The polysorbate 80 (II) of the present invention may mainly reduce the adsorption of PreF to gas-liquid, solid-liquid, and solid-vapor interfaces by adsorbing hydrophobic regions onto the surface of PreF, thereby inhibiting the aggregation of PreF during freeze-drying and reconstitution

[14] .

[0126] Studies have shown that adding 6%-8% D-mannitol, 2%-4% sucrose, 0.4%-0.8% L-histidine, 0.8%-1.2% histidine hydrochloride, 0.4%-0.6% arginine, and 0.02%-0.05% polysorbate 80 to the RSV PreF lyophilization protectant achieves good protective effects. The physicochemical properties of the PreF lyophilized product did not significantly decrease after 12M under long-term stability conditions, 6M under accelerated stability conditions, and 28 days under forced stability conditions. This invention provides a simple and stable lyophilization protectant for recombinant respiratory syncytial virus vaccines, suitable for large-scale production, storage, and transportation, providing further data support for the research and application of lyophilized PreF vaccines.

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A lyophilized protective agent for a recombinant respiratory syncytial virus vaccine, characterized in that, It contains the following components by weight percentage: 6%-8% D-mannitol, 2%-4% sucrose, 0.4%-0.8% L-histidine, 0.8%-1.2% histidine hydrochloride, 0.4%-0.6% arginine, 0.02%-0.05% polysorbate 80, and the balance is buffer solution.

2. The lyophilized protective agent for the recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that, In the lyophilized protective agent of the recombinant respiratory syncytial virus vaccine, the mass ratio of sucrose to D-mannitol is 1:1.5-2.

5.

3. The lyophilized protective agent for the recombinant respiratory syncytial virus vaccine according to claim 2, characterized in that, The recombinant respiratory syncytial virus vaccine lyophilized protectant contains the following components by weight percentage: 7% D-mannitol, 3% sucrose, 0.6% L-histidine, 1.2% histidine hydrochloride, 0.6% arginine, 0.04% polysorbate 80, with the balance being buffer solution.

4. The lyophilized protective agent for the recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that, The pH of the buffer solution is 6.0-7.

0.

5. The lyophilized protective agent for the recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that, The buffer solution is an L-histidine / histidine hydrochloride buffer solution.

6. The use of the recombinant respiratory syncytial virus vaccine lyophilization protectant according to any one of claims 1-5 in the preparation of the lyophilized formulation of the recombinant respiratory syncytial virus vaccine.

7. A method for preparing a freeze-dried formulation of a recombinant respiratory syncytial virus vaccine, characterized in that, Includes the following steps: Step S1: Prepare the freeze-drying protectant for the recombinant respiratory syncytial virus vaccine according to any one of claims 1-5; Step S2: Mix the RSV PreF antigen stock solution with the recombinant respiratory syncytial virus vaccine freeze-drying protectant to obtain a semi-finished product; Step S3: Freeze-dry the semi-finished product to obtain the freeze-dried formulation of the recombinant respiratory syncytial virus vaccine.

8. The preparation method according to claim 7, characterized in that, In step S2, the RSV PreF antigen stock solution and the recombinant respiratory syncytial virus vaccine freeze-dried protectant are mixed at a mass ratio of 1:(0.8-1.2).

9. The preparation method according to claim 7, characterized in that, The freeze-drying temperature in step S3 is -20°C to -15°C; and / or During the freeze-drying process described in step S3, the pH change of the semi-finished product is less than 0.

1.

10. A lyophilized formulation of recombinant respiratory syncytial virus PreF protein, characterized in that, It is prepared by the preparation method according to any one of claims 7-9.

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