Anti-respiratory syncytial virus monoclonal antibody preparation

Abstract

The invention provides an anti-respiratory syncytial virus (RSV) monoclonal antibody preparation as well as a preparation method and application of the preparation. The preparation disclosed by the invention has osmotic pressure similar to physiological conditions, not only contains high-concentration antibodies and can provide a drug level with the highest therapeutic benefit, but also has good storage stability, and better antibody preparations and therapies are provided for prevention and treatment of RSV (Respiratory Syndrome Virus) infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a monoclonal antibody preparation against respiratory syncytial virus. Background Technology

[0002] Respiratory syncytial virus (RSV) is a highly contagious RNA virus that spreads globally and can cause respiratory infections in people of all ages. It is particularly dangerous for children and adults with immunodeficiency or underlying diseases, potentially leading to severe infections and serious respiratory sequelae. The virus is primarily transmitted through direct contact and droplets, with an extremely high infection rate, especially among children, posing a serious threat to their health.

[0003] Respiratory syncytial virus (RSV) is an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Pneumovirus in the family Paramyxoviridae. RSV viral particles consist of a phospholipid bilayer viral envelope and a ribonucleoprotein complex. The viral envelope contains embedded glycoproteins such as a fusion protein (F), an attachment glycoprotein (G), and a small hydrophobic protein (SH). The G protein is the main attachment protein of the virus, interacting with one or more molecules on the surface of the host cell to facilitate the binding of the viral particle to the target cell receptor. Based on the different G proteins, RSV can be divided into two subtypes, A and B. The main function of the F protein is to mediate the fusion of the viral envelope and the cell membrane. The F protein usually exists in trimer form. During the fusion of the virus with the host cell membrane, the F protein undergoes a transition from a pre-fusion (pre-F) conformation to a stable post-fusion (post-F) conformation, allowing the viral genome to enter the host cell and begin replication.

[0004] The RSV F protein plays a crucial role in RSV infection and its sequence is highly conserved. Numerous studies have confirmed that the recognition site of neutralizing antibodies against the F protein is mainly on the pre-fusion F protein. Recombinant proteins designed based on pre-fusion F protein have significantly better immunogenicity and protective efficacy than vaccines designed based on post-fusion F protein. The RSV pre-F protein is the main target for the development of neutralizing antibodies and vaccines.

[0005] Therefore, there is an urgent need in the field for alternative antibody formulations and therapies that provide additional coverage for RSV. Summary of the Invention

[0006] The purpose of this invention is to provide a stable liquid formulation containing a monoclonal antibody against respiratory syncytial virus (RSV).

[0007] In a first aspect of the invention, a monoclonal antibody formulation against respiratory syncytial virus (RSV) is provided, the formulation having an osmotic pressure similar to physiological conditions, in the range of 260~330 mOsmol / kg; the formulation comprises: 75-200 mg / ml anti-RSV monoclonal antibody; pH 5.5 to 6.0, 25-50 mmol / L histidine-histidine hydrochloride buffer; Arginine hydrochloride at a concentration of 50-100 mmol / L; Sodium chloride and / or sucrose, wherein the content of sodium chloride is 0-0.5% and the content of sucrose is 0-5%; and 0.005%-0.02% polysorbate 80(II); The content of the above components is calculated based on the total volume of the preparation.

[0008] In another preferred embodiment, the preparation is an intramuscular injection preparation or an intravenous injection preparation, preferably an intramuscular injection preparation.

[0009] In another preferred embodiment, the viscosity of the formulation is less than 20 cP, making it easy to inject.

[0010] In another preferred embodiment, the osmotic pressure of the formulation is in the range of 280~320 mOsmol / kg.

[0011] In another preferred embodiment, the formulation comprises 80-150 mg / ml of anti-RSV monoclonal antibody; preferably, it comprises 80-100 mg / ml of anti-RSV monoclonal antibody; more preferably, it comprises 100 mg / ml of anti-RSV monoclonal antibody.

[0012] In another preferred embodiment, the formulation comprises a histidine-histidine hydrochloride buffer at pH 5.5 and a concentration of 25-35 mmol / L; preferably, it comprises a histidine-histidine hydrochloride buffer at pH 5.5 and a concentration of 30 mmol / L.

[0013] In another preferred embodiment, the formulation comprises 50-80 mmol / L of arginine hydrochloride; preferably, it comprises 80 mmol / L of arginine hydrochloride.

[0014] In another preferred embodiment, the formulation contains 0.3%-0.5% sodium chloride, preferably 0.4% sodium chloride.

[0015] In another preferred embodiment, the formulation contains 3%-4% sucrose, preferably 4% sucrose.

[0016] In another preferred embodiment, the formulation comprises 0.3% sodium chloride and 3% sucrose.

[0017] In another preferred embodiment, the formulation comprises 80 mmol / L of arginine hydrochloride and 0.4% sodium chloride.

[0018] In another preferred embodiment, the formulation comprises 80 mmol / L of arginine hydrochloride and 4% sucrose.

[0019] In another preferred embodiment, the formulation comprises 50 mmol / L of arginine hydrochloride, 0.3% sodium chloride, and 3% sucrose.

[0020] In another preferred embodiment, the formulation comprises 0.01%-0.02% of polysorbate 80(II); preferably, it comprises 0.01% of polysorbate 80(II).

[0021] In another preferred embodiment, the formulation comprises: 100 mg / ml of anti-RSV monoclonal antibody; 30 mmol / L histidine-histidine hydrochloride buffer at pH 5.5; 80 mmol / L of arginine hydrochloride; 0.4% sodium chloride; and 0.01% polysorbate 80(II); the amounts of the above components are calculated based on the total volume of the formulation.

[0022] In another preferred embodiment, the formulation comprises: 100 mg / ml of anti-RSV monoclonal antibody; 30 mmol / L histidine-histidine hydrochloride buffer at pH 5.5; 80 mmol / L of arginine hydrochloride; 4% sucrose; and 0.01% polysorbate 80(II); the contents of the above components are calculated based on the total volume of the formulation.

[0023] In another preferred embodiment, the formulation comprises: 100 mg / ml of anti-RSV monoclonal antibody; 30 mmol / L histidine-histidine hydrochloride buffer at pH 5.5; 50 mmol / L arginine hydrochloride; 0.3% sodium chloride and 3% sucrose; and 0.01% polysorbate 80(II); the contents of the above components are calculated based on the total volume of the formulation.

[0024] In another preferred embodiment, the total volume of the preparation is 1-2 ml.

[0025] In another preferred embodiment, the viscosity of the formulation was <3cp after being placed at 37°C for 3 weeks, repeatedly frozen and thawed at -70°C / room temperature 5 times, and shaken at 250 rpm for 48 hours. The particle size of DLS was <300 nm, the Tm was higher than 60°C, and the Tagg was higher than 70°C. The content of SEC monomer and polymer, protein content, and non-reducing capillary gel electrophoresis purity did not change significantly under all conditions.

[0026] In a second aspect of the present invention, a method for preparing an anti-RSV monoclonal antibody formulation as described in the first aspect of the present invention is provided, the method comprising the following steps: (1) Replace the anti-RSV monoclonal antibody stock solution with the histidine-histidine hydrochloride buffer solution; (2) Add arginine hydrochloride, sodium chloride and / or sucrose, and polysorbate 80 (II) to the buffer solution and mix well to obtain the preparation.

[0027] In another preferred embodiment, the method further includes the step of dispensing the preparation into containers in volumes of 1-2 ml for storage.

[0028] In a third aspect of the invention, the use of the anti-RSV monoclonal antibody preparation as described in the first aspect of the invention in the preparation of a medicament for the prevention and / or treatment of RSV infection is provided.

[0029] In another preferred embodiment, the drug is an injectable form.

[0030] In another preferred embodiment, the injectable includes an intramuscular injection or an intravenous injection.

[0031] In another preferred embodiment, the drug is an intramuscular injection.

[0032] In a fourth aspect of the invention, a method for preventing and / or treating RSV infection is provided, the method comprising administering to a subject in need an anti-RSV monoclonal antibody preparation as described in the first aspect of the invention.

[0033] In another preferred embodiment, the route of administration of the formulation includes intramuscular injection or intravenous injection, preferably intramuscular injection.

[0034] In another preferred embodiment, the subject in need is at risk of RSV infection or has already been infected with RSV.

[0035] In another preferred embodiment, the desired subject includes humans or non-human mammals (e.g., mice, rats, rabbits, monkeys, etc.).

[0036] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0037] Figure 1 The appearance of the buffer system under PEG pressure is shown.

[0038] Figure 2 The appearance of excipient screening-PEG under pressure is shown.

[0039] Figure 3 The appearance confirms long-term stability of the 5°C formulation.

[0040] Figure 4 The appearance shows the confirmed accelerated stability of the formulation at 25°C.

[0041] Figure 5 The product exhibits a confirmed high-temperature stability at 37°C.

[0042] Figure 6 The appearance after repeated freeze-thaw cycles at -70°C / room temperature is shown.

[0043] Figure 7 The appearance of oscillation at 250 rpm at room temperature (25°C) is shown.

[0044] Figure 8 The trend graph of accelerated stability of the prescription at 25℃ is shown.

[0045] Figure 9 The chart shows the trend of high-temperature stability of the 37℃ prescription.

[0046] Figure 10 The trend graph of the formulation's stability under repeated freeze-thaw cycles at -70℃ / room temperature is shown.

[0047] Figure 11 The trend graph of room temperature oscillation stability for prescription confirmation is shown.

[0048] Figure 12 The purity of monomers by size exclusion chromatography is shown under long-term (5°C) and accelerated (25°C) stability conditions.

[0049] Figure 13 The purity of polymers obtained by size exclusion chromatography under long-term (5°C) and accelerated (25°C) stability conditions is shown.

[0050] Figure 14 The purity of monomers obtained by non-reducing CE-SDS method is shown under long-term (5°C) and accelerated (25°C) stability conditions.

[0051] Figure 15 The purity of low molecular weight impurities obtained by non-reducing CE-SDS method is shown under long-term (5°C) and accelerated (25°C) stability conditions.

[0052] Figure 16 The purity of acid peaks obtained by ion exchange chromatography under long-term (5°C) and accelerated (25°C) stability conditions is shown.

[0053] Figure 17 The purity of the main peak in ion exchange chromatography is shown under long-term (5°C) and accelerated (25°C) stability conditions.

[0054] Figure 18 The purity of the alkali peaks in ion exchange chromatography is shown under long-term (5°C) and accelerated (25°C) stability conditions.

[0055] Figure 19 The relative binding activity (%) was shown under long-term (5°C) and accelerated (25°C) stability conditions. Detailed Implementation

[0056] Through extensive and in-depth research, and through numerous screening and verification processes, the inventors have, for the first time, provided an anti-RSV monoclonal antibody formulation with physiologically similar osmotic pressure, particularly suitable for intramuscular injection. This invention utilizes a PEG-induced precipitation method to screen the buffer system and excipient types in the formulation. Finally, the types and concentrations of excipients in the RSV monoclonal antibody formulation were screened and evaluated through viscosity, DSC, long-term (5°C), accelerated (25°C), and high-temperature (37°C) stability experiments, as well as experiments investigating the effects of repeated freeze-thaw cycles and shaking. The final formulation of the anti-RSV monoclonal antibody was determined. Under this formulation, viscosity <3cp, DLS particle size <300nm, Tm >60°C, Tagg >70°C, and no significant changes were observed under all conditions after 3 weeks at 37°C, 5 freeze-thaw cycles at -70°C / room temperature, and 48 hours of shaking at 250 rpm at room temperature. Therefore, it is particularly suitable for the development of novel RSV drugs. Based on this, this invention was completed.

[0057] the term To better understand this invention, the following terms are defined.

[0058] Unless otherwise stated, all singular terms also include the plural, active, and past tense forms of the term. Thus, for example, referring to "a protein" includes more than one protein, and referring to "a compound" means more than one compound.

[0059] Unless the context clearly indicates otherwise, the term “about” includes values ​​within the standard deviation range of the stated values.

[0060] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value and every tenth of every intermediate integer of the value (unless the context clearly indicates otherwise), between the upper and lower limits of the range, and any other specified or intermediate value within the specified range are included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered in this invention, but are subject to any expressly excluded limitations within the specified range. When a specified range includes one or both of these limits, ranges that do not include (i) any one or (ii) both of the included limits are also included in this invention. For example, “1 to 50” includes “2 to 25”, “5 to 20”, “25 to 50”, “1 to 10”, etc.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0062] Anti-respiratory syncytial virus (RSV) monoclonal antibody The formulation of this invention is not limited to any applicable anti-RSV monoclonal antibody. Generally, any anti-RSV monoclonal antibody known in the art is suitable for formulation into corresponding antibody preparations using the formulation of this invention.

[0063] In a specific embodiment of the present invention, the anti-RSV monoclonal antibody used is derived from the 4F1 modified antibody in Chinese patent application (application number 202111189228.6, publication number CN 115960214 A): heavy chain variable region sequence SEQ ID NO:2 and light chain variable region sequence SEQ ID NO:4.

[0064] Anti-respiratory syncytial virus (RSV) monoclonal antibody preparation This invention provides a monoclonal antibody formulation against respiratory syncytial virus (RSV). As used herein, the terms "formulation of this invention," "RSV monoclonal antibody formulation," "anti-RSV monoclonal antibody formulation," "anti-RSV antibody formulation," and "anti-RSV monoclonal antibody formulation" are used interchangeably and all refer to the formulation described in the first aspect of this invention.

[0065] The formulations of this invention are injectable preparations suitable for both intramuscular and intravenous injection. These formulations are intended for patients requiring intramuscular administration. Given the wide expected dose range of antibody products and the generally limited intramuscular injection volume of 1-2 ml, formulations for intramuscular injection are desirable to contain high antibody concentrations (75-200 mg / ml) to provide maximum therapeutic benefit to patients. However, increased concentration leads to increased intermolecular interactions between protein molecules, potentially causing protein aggregation, precipitation, opalescence, phase separation, and particle formation. Furthermore, increased concentration results in a rapid increase in viscosity, making it difficult to extract the product from its container using a syringe and hindering the injection of the desired dose into the patient (injectability). Excipients commonly used in high-concentration formulations often affect the osmotic pressure of the formulation, and intramuscular injection formulations also require osmotic pressure control, thus complicating the formulation selection of excipients such as pH adjusters, excipients, stabilizers, and viscosity reducers. Therefore, preferred antibody products for intramuscular injection need to balance the effects of concentration and maintain drug levels that provide the highest therapeutic benefit. Ideal products include high protein concentration, low viscosity, osmotic pressure similar to physiological conditions, and low aggregation levels under typical storage conditions. Advantageously, embodiments of the present invention provide an anti-RSV monoclonal antibody formulation comprising a high concentration of antibody and having an acceptable viscosity level upon intramuscular injection. Furthermore, the formulation of the present invention has an osmotic pressure similar to physiological conditions and does not lead to high levels of protein aggregation.

[0066] Specifically, the formulation of the present invention has an osmotic pressure range of 260~330 mOsmol / kg, a viscosity of less than 20 cP, and is easy to inject. It contains 75-200 mg / ml of anti-RSV monoclonal antibody, uses a histidine-histidine hydrochloride buffer system with pH 5.5 to 6.0 and a concentration of 25-50 mmol / L as a buffer system, and contains 50-100 mmol / L of arginine hydrochloride, sodium chloride (0-0.5%) and / or sucrose (0-5%), and 0.005%-0.02% of polysorbate 80 (II); the contents of the above components are calculated according to the total volume of the formulation.

[0067] The content of excipients (arginine hydrochloride, sodium chloride and / or sucrose) and surfactant polysorbate 80(II) in the formulation of the present invention can be appropriately adjusted according to actual application, provided that the osmotic pressure of the formulation is within the range of 260~330mOsmol / kg.

[0068] In one specific embodiment of the present invention, the formulation of the present invention comprises 100 mg / ml of anti-RSV monoclonal antibody, using a pH 5.5, 30 mmol / L histidine-histidine hydrochloride buffer as the buffer system, and comprising 80 mmol / L arginine hydrochloride, 0.4% sodium chloride, and 0.01% polysorbate 80(II).

[0069] In another specific embodiment of the present invention, the formulation of the present invention comprises 100 mg / ml of anti-RSV monoclonal antibody, using a pH 5.5, 30 mmol / L histidine-histidine hydrochloride buffer as the buffer system, and comprising 80 mmol / L arginine hydrochloride, 4% sucrose, and 0.01% polysorbate 80(II).

[0070] In another specific embodiment of the present invention, the formulation of the present invention comprises 100 mg / ml of anti-RSV monoclonal antibody, using a pH 5.5, 30 mmol / L histidine-histidine hydrochloride buffer as a buffer system, and comprising 50 mmol / L arginine hydrochloride, 0.3% sodium chloride, 3% sucrose, and 0.01% polysorbate 80(II).

[0071] Preparation method of the present invention The present invention also provides a method for preparing the anti-RSV monoclonal antibody formulation of the present invention, comprising the following steps: using an ultrafiltration membrane package of RC material with a 30 kDa pore size, the anti-RSV monoclonal antibody protein is replaced into the final buffer system by a large-volume percolation method; arginine hydrochloride, sodium chloride and / or sucrose, and stabilizers, osmotic pressure regulators or viscosity reducers such as polysorbate 80 are added to the buffer containing the anti-RSV monoclonal antibody protein; and the antibody protein is diluted to the final protein concentration using an appropriate buffer to obtain a drug solution; the prepared drug solution is aseptically filtered and dispensed into the final container (vial or pre-filled syringe).

[0072] The main advantages of this invention include: This invention provides, for the first time, a formulation containing a high concentration (up to 100 mg / ml or higher, such as 150 mg / ml or 200 mg / ml) of anti-RSV monoclonal antibody. This formulation, while containing a high concentration of antibody to meet the dosage requirements for small-volume injections, minimizes the formation of antibody aggregates at high concentrations, thus increasing stability. Furthermore, this formulation has an osmotic pressure similar to physiological conditions and a viscosity below 20 cP, making it easy to inject, especially intramuscularly. Moreover, the RSV monoclonal antibody formulated with this method exhibits good storage stability and can effectively maintain the drug's biological activity.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0074] Example 1 Screening of formulation 1.1 Buffer System Screening Experiment (1) Buffer system screening scheme Based on the application of buffer systems in antibody product formulations both domestically and internationally, eight buffer systems were designed for evaluation, with specific formulations shown in Table 1. These eight buffer systems were screened and evaluated using PEG-induced precipitation. PEG-induced precipitation is a commonly used high-throughput method for screening protein solubility. Its principle is based on the size exclusion effect of PEG molecules, which causes protein to precipitate after water loss. Higher protein solubility and stability require a higher concentration of PEG to precipitate the protein.

[0075] Table 1 Buffer System Screening Information RSV monoclonal antibody (batch number: RV231101) was ultrafiltered using an ultrafiltration tube to replace the protein in different buffer systems. The protein concentration was adjusted to 25 mg / ml, and 80 μl samples were aliquoted into 96-well plates, ensuring that each well contained 10 μg of RSV monoclonal antibody protein. Columns 1-8 of the 96-well plate represent buffer systems F1-F8, respectively. Different volumes of PEG stock solution were added to ensure that each well had a final volume of 200 μl. Different volumes of PEG stock solution were added in a gradient to create a PEG concentration gradient of 4%, 6%, 8%, 10%, 12%, 14%, 16%, and 18% in each column. After each formulation sample was shaken to mix, it was allowed to stand for filtration, and the OD500 absorbance was read to measure the turbidity of the solution.

[0076] (2) Buffer system screening results The appearance and OD500 absorbance of the buffer system screening are as follows: Figure 1 As shown in Table 2.

[0077] Table 2 Summary of OD500 absorbance data for screening buffer systems (3) Conclusion of buffer system screening In summary, based on the solubility results, RSV monoclonal antibody molecules showed the best solubility in buffer systems (F1, F2, F4) at pH 5.5. Solubility decreased significantly at pH values ​​below 5.5 and above 6.0. Considering the robustness of the buffer system under high-concentration formulations and the protective effect on proteins, F4 (histidine-histidine hydrochloride, pH 5.5) is recommended as the optimal pH buffer system.

[0078] 1.2 Excipient Screening Experiment (1) Excipient selection scheme Based on the results of the buffer system screening experiment, combined with the application of RSV monoclonal antibody drug excipients at home and abroad, and considering the osmotic pressure control requirements of intramuscular injection formulations (osmotic pressure should be controlled at around 300 mOsmol / kg), four formulations were designed to screen the selected excipients according to the contribution of different excipients to osmotic pressure. The formulations are shown in Table 3.

[0079] Table 3 Excipient Screening Formula RSV monoclonal antibody (batch number: RV231101) was ultrafiltered using an ultrafiltration tube to replace the protein in different excipient systems. The protein concentration was adjusted to 25 mg / ml, and 80 μl samples were dispensed into 96-well plates, ensuring that each well contained 10 μg of RSV monoclonal antibody protein. Columns 1-4 of the 96-well plate represent the F1-F4 buffer systems, respectively. Different volumes of PEG stock solution were added to ensure that each well had a final volume of 200 μl. Different volumes of PEG stock solution were added in a gradient to create a final PEG concentration gradient of 4%, 6%, 8%, 10%, 12%, 14%, 16%, and 18% in each column. After each formulation sample was shaken to mix, it was allowed to pass through the buffer, and the OD500 absorbance was read to measure the turbidity of the solution.

[0080] (2) Results of excipient screening The appearance and OD500 absorbance of the excipients are shown in Table 4 and Figure 2 As shown.

[0081] Table 4 Summary of OD500 Absorbance Values ​​for Excipient Screening (3) Conclusion of excipient screening Except for formulation F1, which had a higher absorbance at a 16% PEG concentration, the other three formulations showed no significant differences at each concentration gradient. Formulations F2 to F4, which contained excipients, were selected for the next step of formulation confirmation experiments.

[0082] 1.3 Formulation Validation Experiment The RSV monoclonal antibody (batch number: RV231101) was added to the prepared buffer and excipient system, and polysorbate 80(II) was added as a surfactant to reduce protein aggregation and particle formation. Specific formulation information is shown in Table 5. After adjusting the protein concentration to 90 mg / ml, 1 mL was dispensed into 2 mL vials, capped, and sealed. The stability of each formulation liquid sample was investigated under the following conditions: 2–8℃ -3W, 25 ± 2℃ -3W, 37 ± 2℃ -3W, -70℃ / room temperature freeze-thaw, and shaking at 200 rpm and 25 ± 2℃.

[0083] Table 5. Formulation Confirmation Information (1) Formulation Validation Experimental Protocol The stability of the selected formulation was investigated through long-term (5℃), accelerated (25℃), and high-temperature (37℃) stability tests, as well as experiments on the effects of repeated freeze-thaw cycles and shaking, to confirm the final formulation. In this experiment, RSV antibody (batch number: RV231101) was concentrated to 40 mg / mL, and after changing the buffer at least 7 times its volume with pH 5.5 histidine buffer, it was further concentrated to 100 mg / mL. After adding appropriate excipients, the protein concentration remained between 80 and 90 mg / mL. The solution was dispensed at 1 mL / bottle into 2 mL vials, capped, and placed under different testing conditions. Based on the stability results, the suitability of the final formulation was confirmed. Specific procedures are shown in Table 6.

[0084] Table 6. Experimental Protocol for Formulation Validation Note: X = appearance, protein concentration, size exclusion chromatography purity, non-reduced sodium dodecyl sulfate capillary gel electrophoresis, charge heterogeneity; Z = viscosity, DLS, Tm / Tagg (2) Results of formulation confirmation experiments The results of stability tests under long-term (5℃), accelerated (25℃), and high-temperature (37℃) conditions are summarized in the table below. Figures 3-5 , Figure 8 , Figure 9 Tables 7-9 show the experimental results regarding the influencing factors of repeated freeze-thaw cycles at -70℃ and room temperature. Figure 6 , Figure 10 The experimental results on the influencing factors of room temperature oscillation are shown in [the table below]. Figure 7 , Figure 11 The results of DLS, Tm / Tagg and viscosity data are shown in Tables 10-12.

[0085] Table 7 Summary of Long-Term Stability Data for 5℃ Prescriptions Table 8 Summary of accelerated stability data for formulation confirmation at 25℃ Table 9 Summary of High-Temperature Stability Data for Formula Confirmation at 37℃ Table 10 Summary of DLS test results for prescription confirmation Table 11 Summary of Tm / Tagg test data for prescription confirmation Table 12 Summary of viscosity test data for prescription confirmation Appearance results showed that, regardless of whether the samples were placed at 5℃ / 25℃ / 375℃ for 3 weeks, subjected to repeated freeze-thaw cycles at -70℃ for 1 / 3 / 5 times, or shaken at 250rpm for 48 hours, all three formulations were colorless, clear and transparent to light, and exhibited a noticeable opalescence against a black background, with no visible particles, compared to T0. There were no significant differences among the three formulations. Charge isomer data showed that, compared to T0, after 3 weeks at 37℃, the F1 acid peak increased by 13.5%, and the main peak decreased by 13.5%; the F2 acid peak increased by 14.0%, and the main peak decreased by 13.8%; the F3 acid peak increased by 14.4%, and the main peak decreased by 14.2%. Under repeated freeze-thaw cycles and shaking conditions, there was no significant trend in the charge isomer content of the three formulations. SEC data showed that after 3 weeks at 37℃, the monomer content was between 99.3% and 99.9% compared to T0, with no significant differences among the three formulations. Under repeated freeze-thaw cycles and shaking conditions, there was no significant trend in the monomer and polymer content of the SEC formulations. Other indicators, such as protein content and non-reducing capillary gel electrophoresis purity, showed no significant changes under all conditions.

[0086] The viscosity of samples placed at 37℃ for 3 weeks, repeatedly frozen and thawed at -70℃ / room temperature 5 times, and placed at 5℃ for 3 weeks was all <3cp, with the viscosity of the F2 formulation being slightly lower; the particle size of DLS was all <300nm, with the F2 formulation having the smallest average particle size; the Tm was all above 60℃, and the Tagg was all above 70℃, with no significant differences among the formulations.

[0087] 1.4 Conclusions of Formulation Screening Study The buffer system and excipients of the formulation were screened using the PEG-induced precipitation method. Finally, the types and concentrations of excipients for the RSV monoclonal antibody formulation were screened and evaluated through viscosity, DSC, accelerated precipitation, high-temperature and freeze-thaw stability tests, and room-temperature shaking stability tests. The optimal RSV monoclonal antibody formulation was ultimately determined to be 100 mg / mL antibody, 30 mmol / L histidine buffer, 0.4% sodium chloride, 80 mmol / L arginine hydrochloride, 0.01% polysorbate 80(II), pH 5.5.

[0088] Example 2: Validation of Formulation Stability The selected formulation F2 was validated through long-term and accelerated stability studies. Results are shown below. Figures 12-19 .

[0089] The results of the long-term stability study at 5℃ showed that after 6 months of storage at 5℃, the F2 formulation RSV monoclonal antibody showed no significant changes in any of the test items (size exclusion chromatography purity (SEC-HPLC), ion exchange chromatography purity (IEX-HPLC), non-reducing CE-SDS purity, etc.), and the relative binding activity was within the acceptable range.

[0090] The results of the accelerated stability study at 25℃ showed that the F2 formulation RSV monoclonal antibody preparation underwent significant changes in physicochemical purity after being stored at 25℃ for 6 months. The content of the main peak in IEX-HPLC decreased significantly, the content of the acidic peak increased significantly, and the content of the basic peak decreased significantly. The content of monomers in SEC-HPLC decreased. The content of light and heavy chains of reduced CE-SDS immunoglobulins decreased. The content of non-reduced CE-SDS immunoglobulins decreased, and the content of low molecular weight impurities increased. The relative binding activity was within an acceptable range.

[0091] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A monoclonal antibody preparation against respiratory syncytial virus (RSV), characterized in that, The formulation has an osmotic pressure similar to physiological conditions, ranging from 260 to 330 mOsmol / kg; the formulation comprises: 75-200 mg / ml anti-RSV monoclonal antibody; pH 5.5 to 6.0, 25-50 mmol / L histidine-histidine hydrochloride buffer; Arginine hydrochloride at a concentration of 50-100 mmol / L; Sodium chloride and / or sucrose, wherein the content of sodium chloride is 0-0.5% and the content of sucrose is 0-5%; and 0.005%-0.02% polysorbate 80(II); The content of the above components is calculated based on the total volume of the preparation.

2. The formulation according to claim 1, characterized in that, The viscosity of the formulation is less than 20 cP, making it easy to inject.

3. The formulation according to claim 1, characterized in that, The preparation is an intramuscular injection preparation or an intravenous injection preparation, preferably an intramuscular injection preparation.

4. The formulation according to claim 1, characterized in that, The formulation comprises a histidine-histidine hydrochloride buffer at pH 5.5 and a concentration of 25-35 mmol / L; preferably, it comprises a histidine-histidine hydrochloride buffer at pH 5.5 and a concentration of 30 mmol / L.

5. The formulation as described in claim 1, characterized in that, The formulation contains 0.3%-0.5% sodium chloride, preferably 0.4% sodium chloride.

6. The formulation according to claim 1, characterized in that, The formulation contains 80 mmol / L of arginine hydrochloride and 0.4% sodium chloride.

7. The formulation according to claim 1, characterized in that, The formulation comprises: 100 mg / ml of anti-RSV monoclonal antibody; pH ​​5.5, 30 mmol / L histidine-histidine hydrochloride buffer; 80 mmol / L arginine hydrochloride; 0.4% sodium chloride; and 0.01% polysorbate 80(II); the contents of the above components are calculated based on the total volume of the formulation.

8. The formulation according to claim 1, characterized in that, The total volume of the preparation is 1-2 ml.

9. A method for preparing the anti-RSV monoclonal antibody formulation as described in claim 1, characterized in that, The method includes the following steps: (1) Replace the anti-RSV monoclonal antibody stock solution with the histidine-histidine hydrochloride buffer solution; (2) Add arginine hydrochloride, sodium chloride and / or sucrose, and polysorbate 80 (II) to the buffer solution and mix well to obtain the preparation.

10. Use of the anti-RSV monoclonal antibody formulation of claim 1 in the preparation of a medicament for the prevention and / or treatment of RSV infection.

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