Formulation containing Anti-tetanus toxin antibody

Abstract

Disclosed in the present invention is a formulation containing an anti-tetanus toxin antibody, wherein the formulation comprises the anti-tetanus toxin antibody at a concentration of 1-100 mg / mL, and has a pH of 5.0-7.0; and optionally, the formulation further comprises one or more of a surfactant, a pH regulator, a protein protective agent, and an osmotic pressure regulator. The formulation provided by the present invention can effectively maintain the stability of the antibody, so that the antibody is not liable to aggregate, thereby preventing oxidation, denaturation and degradation. In addition, the formulation also maintains the high titer of the antibody against tetanus toxin, and thus has the high biological activity required for preventing or treating tetanus.

Description

A preparation containing anti-tetanus toxin antibodies

[0001] Cross-reference to related applications

[0002] This patent application claims priority to Chinese invention patent application No. CN202411723505.0, filed on November 28, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention belongs to the field of biopharmaceutical technology, specifically, this invention relates to preparations containing anti-tetanus toxin antibodies. Background Technology

[0004] Tetanus is a specific infection caused by Clostridium tetani, which invades the human body, multiplies in an anaerobic environment, and produces a toxin (tetanus toxin), leading to muscle spasms. Tetanus toxin primarily attacks motor neurons in the nervous system; therefore, tetanus is clinically characterized by trismus, paroxysmal spasms, and tonic spasms. Globally, the mortality rate of tetanus is as high as 30-50%, with a mortality rate as high as 80% for severe cases and newborns. Although the incidence of tetanus is low in developed countries, it remains prevalent in low- and middle-income countries and underdeveloped regions, posing a significant public health problem.

[0005] Proper wound care and the appropriate use of tetanus immunoglobulins after injury are crucial for preventing tetanus infection. Tetanus toxin is highly toxic, acts rapidly, and has a high mortality rate. When a patient is diagnosed with tetanus infection, a large number of bacteria and toxins are usually already present in their body. Therefore, timely injection of anti-tetanus toxin antibodies, which neutralize the tetanus toxin and inactivate it, is one of the effective methods for preventing or treating tetanus.

[0006] Chinese patent publication CN108218984B discloses an anti-tetanus toxin antibody, which is a fully human monoclonal antibody. Experiments have shown that this antibody is a neutralizing antibody against tetanus toxin, capable of resisting lethal doses of tetanus toxin in animals, with protective efficacy essentially consistent with standard antitoxins. Furthermore, due to its very high titer against tetanus toxin, the actual dosage required is far lower than that of standard antitoxins. Developing a pharmaceutical formulation based on this antibody for clinical application has enormous potential for the prevention and treatment of tetanus.

[0007] However, antibody molecules are biological macromolecules with complex structures, making them susceptible to environmental physical and chemical influences during production, storage, and use. These influences can lead to various forms of alteration in their physicochemical properties, such as degradation, aggregation, denaturation, and oxidation, resulting in reduced safety and efficacy, or even inactivation. Furthermore, as proteins, antibody molecules inherently possess a tendency to aggregate, driven by intermolecular charge interactions, which can cause instability. They are also affected by factors such as the ionic strength of the solution system. Therefore, developing stable, safe, and effective antibody formulations has always been a key technical challenge in the field of antibody pharmaceuticals.

[0008] Currently, high-concentration antibody formulations are generally considered more challenging to develop because higher protein concentrations in the formulation lead to stronger protein-protein interactions, making aggregate formation more likely. However, some studies have found that certain antibodies, due to their unique structures, cannot be formulated into high-concentration formulations and can only be formulated and applied at specific low concentrations. Although it is generally believed that antibodies present at low concentrations in formulations will result in fewer aggregate formations, this still requires the selection of appropriate formulation excipients for these antibody molecules to ensure the long-term stability of the antibody formulation and guarantee that it retains the necessary biological activity for prevention or treatment and remains safe when used in practice.

[0009] Therefore, for the tetanus monoclonal antibody with its specific structure, preparing a suitable drug formulation that can be stored for a long time, is stable, safe and effective also presents a challenge. Summary of the Invention

[0010] To address the aforementioned technical problems, the present invention aims to provide a pharmaceutical preparation of an anti-tetanus toxin antibody, wherein the anti-tetanus toxin antibody is present at a low concentration and has few aggregates, thereby maintaining the effective concentration of the antibody protein and the efficacy of the anti-tetanus toxin, and ensuring long-term stability of the preparation.

[0011] The inventors of this invention selected the antibody disclosed in the aforementioned Chinese patent publication CN108218984B as the active ingredient in the pharmaceutical formulation. In the context of this invention, this antibody is referred to simply as "antibody TRN0011". For antibody TRN0011, the inventors screened pH adjusters, excipients (such as protein protectants and osmotic pressure regulators), and surfactants, and conducted stability tests under conditions including high temperature, freeze-thaw cycles, and shaking, selecting formulations with superior stability. Then, by analyzing the appearance, protein concentration, pH value, SE-HPLC, icIEF, and reduced and non-reduced CE-SDS of different formulation systems, the stability was evaluated, leading to the selection of the optimal formulation for the TRN0011 antibody molecule.

[0012] Therefore, the present invention provides the following technical solution.

[0013] On one hand, the present invention provides a formulation comprising an anti-tetanus toxin antibody, the formulation comprising the anti-tetanus toxin antibody at a concentration of 1-100 mg / mL and having a pH value of 5.0-7.0; optionally, the formulation further comprises one or more of a surfactant, a pH adjuster, a protein protectant, and an osmotic pressure adjuster.

[0014] In this regard, the antibody comprises: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, respectively comprising the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and light chain CDR1, light chain CDR2, and light chain CDR3, respectively comprising the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. Preferably, the antibody comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:4; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:9.

[0015] More preferably, the antibody is a monoclonal antibody comprising two heavy chains and two light chains. Preferably, the antibody is an IgG1 type antibody. More preferably, the anti-tetanus toxin antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO:5; and the anti-tetanus toxin antibody comprises a light chain having the amino acid sequence shown in SEQ ID NO:10.

[0016] In this regard, the formulation comprises the antitetracycline antibody at a concentration not exceeding 50 mg / mL. Preferably, the formulation comprises the antitetracycline antibody at a concentration of 3-50 mg / mL, more preferably 5-50 mg / mL, more preferably 10-30 mg / mL, and even more preferably 10-20 mg / mL.

[0017] In specific embodiments, the concentration of the antibody may be 3 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, etc.

[0018] The formulation provided by the present invention has a pH value of 5.0-7.0, within which the antibody maintains high stability.

[0019] In a specific embodiment, the pH value of the preparation can be 5.5-6.5. In a specific embodiment, the pH value of the preparation can be 5.5±0.2, 6.0±0.2, or 6.5±0.2, preferably 6.0±0.2, and more preferably 6.0.

[0020] In this regard, the formulation comprises a nonionic surfactant as the protein protectant. Preferably, the nonionic surfactant is polysorbate or poloxamer, such as polysorbate 80, polysorbate 20, or poloxamer P188, and more preferably polysorbate 80.

[0021] In this regard, the formulation comprises the surfactant at a concentration of 0-0.4%, preferably 0-0.02%, more preferably 0.005-0.02%.

[0022] In specific embodiments, the concentration of the surfactant can be 0%, 0.005%, 0.02%, 0.2%, 0.3%, 0.4%, etc. The percentage is a weight-volume percentage, i.e., w / v content.

[0023] In a specific embodiment, the surfactant is polysorbate 80 with a concentration of 0-0.02%.

[0024] In this respect, the pH adjuster is a buffer solution.

[0025] Preferably, the formulation provided by the present invention comprises histidine / histidine hydrochloride buffer.

[0026] In a specific embodiment, the concentration of the histidine / histidine hydrochloride buffer is 10-100 mM.

[0027] In a specific embodiment, the concentration of the histidine / histidine hydrochloride buffer solution can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, etc., preferably 10-30mM, and more preferably 20mM.

[0028] In this regard, the osmotic pressure regulator is one or more selected from sucrose, trehalose, mannitol, sorbitol, sodium chloride, and arginine hydrochloride.

[0029] In a specific embodiment, the osmotic pressure regulator is a sugar, such as sucrose and / or trehalose listed above; or it may be mannitol and / or sorbitol.

[0030] Preferably, the osmotic pressure regulator can be one or more of sucrose, trehalose, and mannitol. In a specific embodiment, the concentration of the osmotic pressure regulator can be 2-8%, such as 2%, 4%, 6%, 8%, etc., preferably 4-6%, and more preferably 4%.

[0031] Preferably, the osmotic pressure regulator can be sucrose. In a specific embodiment, the osmotic pressure regulator is sucrose with a concentration of 4%.

[0032] In a specific embodiment, the osmotic pressure regulator is sodium chloride and / or arginine hydrochloride, with a concentration of 0-140 mM. In a specific embodiment, the concentration of the sodium chloride or arginine hydrochloride can be 0, 5 mM, 10 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, etc., preferably 70-100 mM, and most preferably 70 mM.

[0033] Alternatively, the formulation of the present invention may simultaneously contain sucrose and sodium chloride. Preferably, the formulation contains 2-8% sucrose and / or 0-140 mM sodium chloride. The sucrose concentration may be 2%, 4%, 6%, 8%, etc., preferably 4-6%, more preferably 4%; the sodium chloride concentration may be 0, 5 mM, 10 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, etc., preferably 70-100 mM, most preferably 70 mM.

[0034] In a specific embodiment, the osmotic pressure regulator is a combination of sodium chloride and sucrose, wherein the concentration of sodium chloride is 70 mM and the concentration of sucrose is 4%.

[0035] The antibody formulations provided by this invention, in addition to the antibodies, pH adjusters, protein protectants, and surfactants, may optionally contain other functional excipients depending on the application.

[0036] Furthermore, the formulation is a liquid preparation administered parenterally, preferably an injectable preparation, and more preferably an intravenous or subcutaneous injection preparation. Preferably, the formulation is prepared using sterile water for injection.

[0037] In a specific embodiment, the formulation comprises:

[0038] (1) The antitetracheotoxin antibody at a concentration of 1-100 mg / mL, preferably not exceeding 50 mg / mL;

[0039] (2) 0-0.02% polysorbate 80;

[0040] (3) 10-30mM histidine / histidine hydrochloride buffer;

[0041] (4) 4-6% of any one of sucrose, trehalose, mannitol, and sorbitol;

[0042] (5) Any one of sodium chloride or arginine hydrochloride at 70-140mM;

[0043] (6) The pH value is 5.5-6.5, for example 6.0±0.2.

[0044] Preferably, in a specific embodiment, the formulation comprises:

[0045] (1) 10-30 mg / mL of the antitetracycline toxin antibody;

[0046] (2) 0-0.02% polysorbate 80;

[0047] (3) 10-30mM histidine / histidine hydrochloride buffer;

[0048] (4) 4-6% of any one of sucrose, trehalose, mannitol, and sorbitol;

[0049] (5) Any one of sodium chloride or arginine hydrochloride at 70-140mM;

[0050] (6) The pH value is 5.5-6.5, for example 6.0±0.2.

[0051] Preferably, in a specific embodiment, the formulation comprises:

[0052] (1) 10-30 mg / mL of the antitetracycline toxin antibody;

[0053] (2) 0-0.02% polysorbate 80;

[0054] (3) 10-30mM histidine / histidine hydrochloride buffer;

[0055] (4) 4-6% sucrose and / or trehalose;

[0056] (5) 70-140mM sodium chloride and / or arginine hydrochloride;

[0057] (6) The pH value is 5.5-6.5, for example 6.0±0.2.

[0058] Preferably, in a specific embodiment, the formulation comprises:

[0059] (1) 10-30 mg / mL of the antitetracycline toxin antibody;

[0060] (2) 0-0.02% polysorbate 80;

[0061] (3) 10-30mM histidine / histidine hydrochloride buffer;

[0062] (4) 4-6% mannitol and / or sorbitol;

[0063] (5) 70-140mM sodium chloride and / or arginine hydrochloride;

[0064] (6) The pH value is 5.5-6.5, for example 6.0±0.2.

[0065] Preferably, in a specific embodiment, the formulation comprises:

[0066] (1) 10-30 mg / mL of the antitetracycline toxin antibody;

[0067] (2) 0-0.02% polysorbate 80;

[0068] (3) 10-30mM histidine / histidine hydrochloride buffer;

[0069] (4) 4-6% sucrose;

[0070] (5) 70-140mM sodium chloride;

[0071] (6) The pH value is 5.5-6.5, for example 6.0±0.2.

[0072] Preferably, in a specific embodiment, the formulation comprises:

[0073] (1) 20 mg / mL of the antitetracycline toxin antibody;

[0074] (2) 0-0.02% polysorbate 80;

[0075] (3) 10-30mM histidine / histidine hydrochloride buffer;

[0076] (4) 4-6% sucrose;

[0077] (5) 70-140mM sodium chloride;

[0078] (6) The pH value is 5.5-6.5, for example 6.0±0.2.

[0079] Preferably, in a specific embodiment, the formulation comprises:

[0080] (1) 20 mg / mL of the antitetracycline toxin antibody;

[0081] (2) 0.02% polysorbate 80;

[0082] (3) 20mM histidine / histidine hydrochloride buffer;

[0083] (4) 4% sucrose;

[0084] (5) 70mM sodium chloride;

[0085] (6) The pH value is 6.0.

[0086] In another aspect, the present invention provides a method for preparing the formulation containing anti-tetanus toxin antibodies, the method comprising:

[0087] The anti-tetanus toxin antibody is concentrated and replaced in the pH adjuster through ultrafiltration. Optionally, the amount of pH adjuster to be added is calculated according to the composition of the formulation. The added pH adjuster, as well as surfactants, protein protectants and osmotic pressure regulators, are added to complete the preparation of the formulation solution.

[0088] Optionally, the prepared formulation may be sterilized, filtered, dispensed, capped, and crimped.

[0089] In a specific embodiment, the preparation method includes:

[0090] The anti-tetanus toxin antibody (TRN0011 protein), after being filtered to remove the virus, was concentrated using ultrafiltration. Based on the formulation, the required pH adjuster, surfactant, and osmotic pressure adjuster were calculated to prepare the TRN0011 formulation solution. The prepared solution was then filtered through a 0.22μm sterile filter and dispensed into 2R (or 2mL) vials. A 13mm butyl chloride rubber stopper was added, and a 13mm aluminum-plastic composite cap was formed. This constitutes the TRN0011 formulation.

[0091] In another aspect, the present invention provides the use of the formulation containing anti-tetanus toxin antibodies in the preparation of medicaments for treating tetanus toxin or Clostridium tetani infection.

[0092] This invention also provides other related products.

[0093] In another aspect, the present invention provides a container containing the formulation containing anti-tetanus toxin antibodies provided by the present invention. For example, the container is a 2R (or 2mL) vial.

[0094] Alternatively, the present invention provides a medicine box containing the formulation containing anti-tetanus toxin antibodies provided by the present invention or the container; further, the medicine box optionally also includes instructions for using the formulation containing anti-tetanus toxin antibodies.

[0095] Alternatively, the present invention provides a method for treating tetanus toxin or Clostridium tetani infection, the method comprising administering to a subject in need a therapeutically effective amount of the preparation provided by the present invention, and optionally other drugs or means. The subject is a mammal; preferably, the subject is a human.

[0096] Compared with existing technologies, this invention has developed a low-concentration formulation suitable for antibody TRN0011 through multiple experiments. The formulation provided by this invention is specifically designed for low concentrations of antibody TRN0011. This formulation effectively maintains the stability of the antibody, making it less prone to aggregation, oxidation, denaturation, and degradation, thus facilitating long-term storage. Furthermore, the formulation provided by this invention maintains the high titer of antibody TRN0011 against tetanus toxin, possessing the high biological activity required for the prevention or treatment of tetanus. Attached Figure Description

[0097] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0098] Figure 1 shows the Fc / 2 molecular weight analysis results of formulation 4 in the PS80 concentration study;

[0099] Figure 2 shows the Fc / 2 molecular weight analysis results of formulation 5 in the PS80 concentration study.

[0100] The best way to implement an invention

[0101] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0102] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0103] (I) Antibodies

[0104] The antibody used in the following examples is a recombinant natural fully human monoclonal antibody against tetanus toxin, disclosed in Chinese patent publication CN108218984B. In the context of this invention, this antibody may be abbreviated as "antibody TRN0011" or "TRN0011" and may be used interchangeably with "protein".

[0105] The amino acid sequence of antibody TRN0011 is shown in Table 1.

[0106] Table 1. Amino acid sequence of anti-tetanus toxin antibody TRN0011

[0107] As an immunoglobulin G1 (IgG1) monoclonal antibody, TRN0011 can bind to the specific surface of tetanus toxin protein with high affinity, thereby preventing the enzymatic cleavage activity of tetanus toxin by neutralizing the toxin and thus protecting the body.

[0108] The stability of various formulations containing TRN0011 was evaluated through physicochemical property analysis, including appearance, protein concentration, pH, osmotic pressure, purity (SE-HPLC, icIEF, reduced and non-reduced CE-SDS), and binding activity. Optimal formulations, particularly those containing TRN0011 at low concentrations, were screened. The detection methods used included:

[0109] 1. Appearance

[0110] In a dark room, visually inspect the appearance of the sample using a clarity meter. The sample bottle must be wiped clean, and the illuminance should be adjusted to 2000–3750 lx. Place the sample at the edge of the light shield (25 cm), hold the neck of the sample (cilene) and visually inspect the color, clarity, and visible foreign matter against black and white backgrounds.

[0111] 2. pH value

[0112] The pH meter was calibrated with a standard solution using the potentiometric method. A 250 μL sample was taken to measure the pH value. The measurement was performed twice, and the average value was taken.

[0113] 3. Protein concentration

[0114] The absorbance of the diluted sample at 280 nm was measured using a Shimadzu UV-1900, and the absorbance was calculated based on the sample's extinction coefficient (1.431 AU * mL * mg). -1 *cm -1 Calculate the protein concentration. Repeat the measurement twice and take the average as the final result.

[0115] 4. Size Exclusion Chromatography (SE-HPLC)

[0116] An Agilent 1260 high-performance liquid chromatograph (HPLC) and a TSKgel G3000SWXL column (5 μm, 7.8 mm × 300 mm) were used. The column temperature was set to 25 °C, the injection plate temperature to 2–8 °C, and the flow rate to 1.0 mL / min. The mobile phase consisted of 50 mM phosphate buffer, 300 mM sodium chloride, and pH 6.8 ± 0.1. 100 μg of protein was injected into the HPLC system, and isocratic elution was performed for 20 min. Detection was performed at 280 nm. Data acquisition and analysis were performed using the instrument's built-in software, and the purity of each component was calculated using the peak area normalization method.

[0117] 5. Capillary gel permeation chromatography (CE-SDS)

[0118] 5.1 Non-reducing capillary gel chromatography (non-reducing CE-SDS)

[0119] The standard and sample were diluted with PB-CA buffer to 4 mg / mL, making a total volume of 25 μL. Then, 75 μL of 1% SDS sample buffer was added, making a total volume of 100 μL. 5 μL of NEM (100 mM) was added to the sample tube, making a total volume of 105 μL. The mixture was centrifuged at 1300 rpm for 1 minute, incubated at 70°C for 10 minutes, removed, cooled to room temperature for at least 3 minutes, centrifuged at 1300 rpm for 1 minute, and 95 μL of the prepared sample was transferred to a sample vial for injection. The sample tray temperature was set to 15°C. The capillary temperature was maintained at 25°C using a condenser. Sample injection was performed using voltage, -5 kV for 30 seconds. Constant voltage separation was used; the voltage applied across the capillary depended on the capillary length. In this method, a positive voltage of -17 kV was applied across the capillary for 40 minutes of separation, using a PDA detector at a detection wavelength of 220 nm.

[0120] 5.2 Reducing Capillary Gel Chromatography (Reduced CE-SDS)

[0121] The standard and sample were diluted with PB-CA buffer to a concentration of 4 mg / mL, bringing the total volume to 25 μL. Then, 75 μL of 1% SDS sample buffer was added, bringing the total volume to 100 μL. 5 μL of BME was added to the sample tube to bring the total volume to 105 μL. The mixture was then centrifuged at 1300 rpm for 1 minute, incubated at 70°C for 10 minutes, removed, cooled to room temperature for at least 3 minutes, centrifuged at 1300 rpm for 1 minute, and 90 μL of the prepared sample was transferred to a sample vial for injection. The sample tray temperature was set to 15°C. The capillary temperature was maintained at 25°C using a condenser. Sample injection was performed using voltage, applying -5 kV for 30 seconds. Constant voltage separation was used; the voltage applied across the capillary depended on the capillary length. In this method, a positive voltage of -17 kV was applied across the capillary for 40 minutes of separation, using a PDA detector at a detection wavelength of 220 nm.

[0122] 6. Differential Scanning Calorimetry (DSC)

[0123] The sample was diluted to 1 mg / mL with buffer. 400 μL of sample was added to each even-numbered well of a 96-well plate, and 400 μL of the corresponding buffer solution was added to each odd-numbered well. Experimental parameters were set as follows: scan temperature from 10–95 °C, scan rate at 90 °C / h. Heatmap analysis was performed using DSC automated data analysis software.

[0124] 7. Isostatic capillary electrophoresis (icIEF)

[0125] Sample premix preparation: 0.5 μL PI label 7.55, 0.5 μL PI label 9.77, 1 μL amphoteric electrolyte 3-10, 3 μL amphoteric electrolyte 8-10.5, 2.5 μL ultrapure water, 35 μL 1% methylcellulose, 37.5 μL 8M urea.

[0126] Sample preparation and analysis:

[0127] Dilute the sample with ultrapure water to 1 mg / mL, then add 20 μL of the sample to 80 μL of premixed solution, mix and centrifuge, then take 90 μL of the sample into the inner liner tube for loading and detection. The sample plate temperature is 15℃, the focusing parameters are 1500V for 1 minute, 3000V for 8 minutes, and the injection time is 100 seconds.

[0128] 8. Subunit-level analysis

[0129] After ultrafiltration twice, 20 μg of sample was taken out and 1.0 μL of Ides enzyme was added. The mixture was reacted at 37℃ for 30 min to cleave the antibody protease into F(ab')2 and 2xFc / 2. Subsequently, 0.5 μL of Rapid PNGsae F enzyme was added and the mixture was heated in a metal bath at 50℃ for 15 min to remove the N-sugar from 2xFc / 2. The sample was then diluted to 0.4 mg / mL. The diluted sample was detected using liquid chromatography-mass spectrometry (Waters, ACQUITY UPLC I-Class / Xevo G2-XS QTof), and the data were analyzed using UNIFI software to determine the subunit levels of the sample.

[0130] 9. Weak cation exchange chromatography (WCX-HPLC)

[0131] Samples were loaded and adsorbed under low salt and pH conditions below the isoelectric point. Elution was performed using a gradually increasing pH gradient and salt concentration gradient, so that different charged heterogeneous components in the product were eluted in order of the strength of ion interactions. Peaks were detected at a wavelength of 280 nm, and the relative content of each component was calculated using the peak area normalization method.

[0132] Example 1: Screening of pH-buffer system

[0133] (I) Buffer Screening Experiment

[0134] The first round of research designed four commonly used buffer systems, resulting in a total of 12 formulation buffers. The antibody concentration was tentatively set at 10 mg / mL. The 12 formulations were screened by studying their thermodynamic stability, examining their appearance, pH value, and protein content at 40°C, and investigating changes in their stability using SE-HPLC, reduced and non-reduced CE-SDS, and icIEF.

[0135] The screening criteria are shown in Table 2.

[0136] Table 2. Screening Scheme for pH-Buffer Systems

[0137] The anti-tetanus toxin antibody stock solution was changed to 20mM glacial acetic acid / sodium acetate buffer (pH 4.5, 5.0, 5.5), 20mM succinic acid / sodium succinate buffer (pH 5.0, 5.5, 6.0), 20mM histidine / histidine hydrochloride buffer (pH 5.5, 6.0, 6.5), and 20mM sodium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 6.5, 7.0, 7.5), respectively. After the buffer changes, the antibody concentration was adjusted to approximately 10 mg / mL. After sample preparation, the samples were stored in a constant temperature and humidity incubator at 40℃ for 4 weeks. Samples were taken at weeks 0, 1, 2, and 4 for analysis. The screening results are shown in Table 3.

[0138] Table 3. Screening Results of pH-Buffer Systems

[0139] As shown in Table 3, in the pH-buffer system screening experiment, the formulations at 40℃:

[0140] Appearance: Visible particles were observed in F04-F06 (succinic acid / sodium succinate buffer system) and F10-F12 (sodium dihydrogen phosphate / disodium hydrogen phosphate buffer system) after 2 and 4 weeks of storage, while no visible particles were observed in F01-F03 (glacial acetic acid / sodium acetate buffer system) and F07-F09 (histidine / histidine hydrochloride buffer system) after 2 weeks of storage. After 4 weeks, a small number of particles appeared in F03, while no visible particles were observed in F07-F09.

[0141] SE-HPLC results: After 4 weeks of storage, all formulations showed varying degrees of decrease in the main peak (1.8%-5.2%) and increase in fragments. Among them, F01 (glacial acetic acid / sodium acetate buffer system, pH 4.5) showed the largest decrease in the main peak at 5.2%, while the decrease in the main peak of other formulations was less than or equal to 4.2%.

[0142] icIEF results: After 4 weeks of storage, the main peak of all preparations decreased (6.5%-26.5%) and the acid peak increased. Among them, the main peak of F07-F09 (histidine / histidine hydrochloride buffer system) decreased the least (6.5%-7.4%).

[0143] Results of reduced and non-reduced CE-SDS: The purity of all formulations decreased to varying degrees after 4 weeks of storage. The purity of non-reduced CE-SDS decreased by 0.1%-5.4%, and the purity of reduced CE-SDS decreased by 1.2%-7.0%. Among them, the purity of non-reduced CE-SDS of F01 (glacial acetic acid / sodium acetate buffer system, pH 4.5) decreased by 5.4%, the purity of reduced CE-SDS of F12 (sodium dihydrogen phosphate / disodium hydrogen phosphate buffer system, pH 7.5) decreased by 7.0%, and the purity of other formulations decreased by less than 5%.

[0144] In summary, based on appearance, no visible particles were observed in formulations F01 (glacial acetic acid / sodium acetate buffer system, pH 4.5), F02 (glacial acetic acid / sodium acetate buffer system, pH 5.0), F07 (histidine / histidine hydrochloride buffer system, pH 5.5), F08 (histidine / histidine hydrochloride buffer system, pH 6.0), and F09 (histidine / histidine hydrochloride buffer system, pH 6.5) after 4 weeks of storage, indicating good stability. SE-HPLC data showed that all formulations exhibited varying degrees of peak reduction (1.8%-5.2%) and fragment increase after 4 weeks of storage. Among these, formulation F01 (glacial acetic acid / sodium acetate buffer system, pH 4.5) showed the most significant increase. 4.5) The main peak decreased by the most, by 5.2%; non-reducing CE-SDS results showed that after 4 weeks of storage, the purity of formulation F01 decreased the most; icIEF data showed that after 4 weeks of storage, the main peak of formulation F07-09 (histidine / histidine hydrochloride buffer system) decreased the least (6.5%-7.4%).

[0145] Therefore, the preferred buffer system is the histidine / histidine hydrochloride buffer system. Considering the operability and robustness of the production process, the intermediate pH value of 6.0 in the histidine / histidine hydrochloride buffer system (pH 5.5 to 6.5) was selected as the preferred pH value for the next round of screening experiments.

[0146] (II) Screening of Buffer System Concentration

[0147] As shown in Table 4, formulations with different concentrations of histidine / histidine hydrochloride buffer were prepared and then placed at 50°C for 4 weeks. Samples were taken at T0 and after 4 weeks, and stability was evaluated using different methods to determine the appropriate concentration of histidine / histidine hydrochloride buffer. The results are shown in Table 5.

[0148] Table 4. Formulations under investigation

[0149] Table 5. Results of formulation stability studies with buffer concentration as the variable. Note: The + or - numbers in parentheses in the table indicate the increase (+) or decrease (-) value compared to the T0 result; the same applies below.

[0150] As shown in Table 5, in the histidine / histidine hydrochloride buffer system, when the buffer concentration (mM) varied from 10mM to 100mM, the content of the main peak, aggregates and fragments did not change significantly. However, when the buffer concentration was 50mM or higher, the appearance of the solution changed from colorless and slightly opalescent to pale yellow and slightly opalescent after storage at 50°C.

[0151] Therefore, the data shows that when the monoclonal antibody concentration remains constant and the buffer concentration varies within a specific range, the aggregate and fragment contents do not increase significantly. Thus, the factor causing the change in solution color cannot be clearly identified (it is speculated that it may be due to the unique spatial structure of the antibody or other components in the system besides the protein).

[0152] In summary, the concentration range of histidine / histidine hydrochloride buffer is 10-100 mM, preferably 10-30 mM. Considering the operability and robustness of the production process, the intermediate value of 20 mM was used for subsequent experiments.

[0153] (III) Investigation of pH value of buffer system

[0154] As shown in Table 6, formulations with different pH values ​​were prepared and then placed at 50°C for 2 weeks. Samples were taken and their stability was evaluated using different methods to determine the suitable pH for the formulation. The results are shown in Table 7.

[0155] Table 6. Formulations under investigation

[0156] Table 7. Formulation stability results with pH as a variable

[0157] As shown in Table 7, the SEC-HPLC results indicate that the polymer content in the formulation reaches 82.8% at pH 4.0, and 11.4% at pH 8.0.

[0158] When the pH value is above 4.0 and 7.5, the charge heteroform (icIEF) of the formulation product is ND (low signal), indicating that the acid-base peak of the antibody may have changed significantly and the response value is too low to be integrated normally.

[0159] Based on the above information and the SEC-HPLC results in Table 7, it can be seen that the polymer content is lowest and the purity is highest when the sample pH is 6.0. Monoclonal antibody polymers can affect drug efficacy, immunogenicity, and PK / PD ratio, making variations in polymer content particularly important. Therefore, based on the results in Tables 6 and 7, the suitable pH range is determined to be 5.0-7.0, with 6.0 being particularly preferred.

[0160] Therefore, a histidine / histidine hydrochloride buffer solution was selected as the buffer system, with a histidine / histidine hydrochloride concentration of 10-30 mM and a pH range of 5.0-7.0. Considering the operability and robustness of the production process, the median histidine / histidine hydrochloride concentration of 20 mM and pH of 6.0 were used for subsequent experiments.

[0161] Example 2: Investigation of antibody protein concentration in the formulation

[0162] As shown in Table 8, different amounts of buffer, osmotic pressure regulator, and surfactant were added to the product after ultrafiltration concentration using TRN0011 to prepare formulations with different antibody protein concentrations. The formulations were then placed at 50°C for 4 weeks, and samples were taken to evaluate stability using the following methods to determine the appropriate protein concentration. The results are shown in Table 9.

[0163] Table 8. Formulations under investigation

[0164] Table 9. Results of formulation stability studies with antibody protein concentration as the variable.

[0165] According to the results in Table 9, visually, when the antibody protein concentration is 30 mg / mL or less, the solution appears colorless or pale yellow with a slight opalescence; however, when the antibody protein concentration is 50 mg / mL or higher, the formulation appears pale yellow with an opalescence. Opalescence, also known as protein opalescence, is caused by the presence of only antibody TRN0011 in the solutions of formulations 1 through 9. A slight opalescence in protein solutions is normal, but the appearance of opalescence suggests a possible conformational change in the protein. Therefore, it is speculated that the opalescence changes in formulations 7 through 9 are due to a conformational change in the TRN0011 protein after examination, and this conformational change may directly affect the efficacy of the antibody. Thus, these visual results suggest that the stability of the formulation is uncertain when the concentration of antibody TRN0011 in the formulation is 50 mg / mL or higher.

[0166] SEC-HPLC results showed that the content of mesomer in formulations 1, 8, and 9 was significantly higher than that in other formulations. WCX-HPLC results showed that the acid-base peak detection signal of formulation 1 was lower, indicating a drastic change in the sample's charge heterogeneity, which likely also affected efficacy. Non-reducing CE-SDS results also showed a significant increase in fragment content in formulations 7, 8, and 9.

[0167] Based on the results and taking all factors into consideration, the concentration range of the antibody protein is below 50 mg / mL, preferably 10-30 mg / mL, and particularly preferably 10-20 mg / mL.

[0168] Example 3: Concentration of polysorbate 80 (PS80) in the formulation

[0169] Extensive preliminary experiments have shown that the molecular weight of the protein polypeptide fragments of antibody TRN0011 increases to varying degrees, presumably due to protein oxidative modification. Therefore, as shown in Table 10, formulations with different PS80 concentrations were prepared and then placed at 50°C for 4 weeks. The changes in the molecular weight of the protein polypeptide fragments were then monitored to investigate whether PS80 can protect against protein oxidative modification.

[0170] Table 10. Formulations under investigation

[0171] The changes in the molecular weight of protein peptides in the formulation under different PS80 concentrations were detected. Samples were digested sequentially with Ides enzyme and Rapid PNGsae F enzyme, and then diluted to 0.4 mg / mL. The diluted samples were detected using liquid chromatography-mass spectrometry (Waters, ACQUITY UPLC I-Class / Xevo G2-XS QTof), and the data were analyzed using UNIFI software.

[0172] The results show that the theoretical molecular weight of Fc / 2 is 23787.6 Da (including the molecular weight lost due to the loss of the C-terminal lysine), as shown in Figures 1 and 2. The measured molecular weights of formulations 4 and 5 are both 23835.5 Da, which is approximately 48 Da higher than the theoretical molecular weight. Therefore, it is speculated that this difference in mass is due to the oxidative modification of antibody Fc / 2.

[0173] Therefore, the data show that polysorbate 80 in the concentration range of 0-0.02% has a certain protective effect against the oxidative modification of antibody TRN0011, and the stability of formulations 1, 2 and 3 is much better than that of formulations 4 and 5.

[0174] In addition, after the formulations were placed at 50°C for 4 weeks, samples were taken to evaluate stability indicators such as SEC-HPLC (%) and non-reducing CE-SDS. The results showed that there was no significant difference in the stability of formulations 1-3, while the polymer content of formulations 4 and 5 increased abnormally, the purity was low, and the stability was poor.

[0175] Therefore, the preferred range for PS80 is 0-0.02%.

[0176] Example 4: Investigation of other excipients in the formulation

[0177] As shown in Table 12, formulations with different sugars and salts were prepared and then placed at 50°C for 4 weeks. Samples were taken and the following methods were used to evaluate stability in order to determine the suitable types and concentrations of sugars and salts. The results are shown in Table 13.

[0178] Table 12. Formulations under investigation

[0179] Table 13. Results of stability studies on different sugars

[0180] The experimental results showed that the color of formulations 1 and 2 turned pale yellow after being stored for 4 weeks.

[0181] As can be seen from the SEC-HPLC (%) results in Table 13, the purity of formulations 2 to 4 is higher than that of formulation 1 (formulation 1 has a higher polymer content), therefore the sugar content range is selected as 2-8%, preferably 4-6%.

[0182] The results from formulations 5 to 7 and formulation 10 show that sugars can be sucrose, mannitol, trehalose, and sorbitol.

[0183] Comparing the results of formulations 9 to 11, formulation 11 showed the highest degree of aggregate aggregation and lower purity. In contrast, the appearance of formulation 9 changed from colorless to pale yellow, which may have been due to a partial reaction that caused the color change. Therefore, the salt concentration range was 70-100 mM, which could be sodium chloride or arginine hydrochloride.

[0184] Example 5: Further investigation and verification of formulation stability

[0185] Different formulations were prepared according to the formulation composition and pH shown in Table 14, and samples were taken after shaking at 25°C for 2 weeks. The stability of the different formulations was confirmed by examining the formulations with different sugar and salt types and concentrations, and the results are shown in Table 15.

[0186] Table 14. Formulations under investigation

[0187] Table 15. Results of formulation stability study under 2-week shaking conditions

[0188] The experimental results above show that formulations 1 to 9 have good stability, especially when the protein concentration is below 50 mg / mL, the formulations have fewer aggregates and better stability, which further verifies that the formulations maintain high stability when the concentration is below 50 mg / mL.

[0189] In addition, based on the above experimental results, formulations 8 to 11 were all colorless and slightly opalescent in appearance, indicating that the formulations had few aggregates and high purity. This further verifies that when the PS80 range is preferably 0.005-0.02%, the sugar content range is preferably 4-6%, and the salt concentration range is preferably 70-100mM, the formulations have better stability.

[0190] Example 6: Formulation Efficacy Data

[0191] Based on the above results, a formulation with the following composition was prepared: 20 mg / mL antibody, 20 mM histidine / histidine hydrochloride concentration, 0.02% PS80, 4% sucrose, 70 mM sodium chloride, pH 6.0. Further investigation was conducted to determine whether this antibody formulation, while enhancing protein stability, did not affect antibody titer.

[0192] The potency of the antibody in the formulation against tetanus antitoxin was determined. In short, based on the principle that antitoxins, i.e. antibodies, can neutralize toxins, a comparative experiment was conducted between the test sample (antibody formulation) and a standard (antibody standard) to calculate the number of international units (IU / ml) of antitoxin contained in each 1 ml of the test sample.

[0193] 1. Reagents: Borate buffer saline solution: Weigh 8.5g sodium chloride, 4.5g boric acid, and 0.5g sodium tetraborate (Na2B4O7·10H2O), dissolve in water and dilute to 1000ml, filter, and sterilize to a pH of 7.0–7.2.

[0194] 2. Preparation of tetanus antitoxin (antibody TRN0011 standard solution and toxin)

[0195] (1) Preparation of diluted antitoxin standard: Tetanus antitoxin standard is diluted with borate buffered saline to contain 0.5 IU per 1 ml, that is, after mixing with an equal amount of toxin, each 0.4 ml injection volume contains 1 / 10 IU.

[0196] (2) Preparation of diluted toxin: Tetanus toxin was diluted with borate buffered saline to contain 5 test doses (1 / 10L+) per 1 ml, that is, after mixing with an equal amount of antitoxin, each 0.4 ml injection volume contained 1 test dose (1 / 10L+).

[0197] 3. Preparation of the test solution

[0198] The test sample was diluted to several dilutions using borate-buffered saline, so that each 1 ml contained approximately 0.5 IU of antitoxin, or approximately 1 / 10 IU of antitoxin per 0.4 ml injection volume after mixing with an equal volume of the toxin. The intervals between dilutions were approximately 5%.

[0199] 4. Determination method

[0200] Quantitatively pipette the diluted antitoxin standard solution and the test solution of different dilutions into small test tubes, add an equal amount of diluted toxin solution to each tube, mix well, stopper, incubate at 37°C for 1 hour, and then inject immediately.

[0201] Administer 0.4 ml subcutaneously to the abdomen or groin of 17–19 g mice. Inject at least three mice for each dilution of the standard and test sample. Administer from high to low dilutions sequentially, washing the mice 2–3 times with the next dilution solution each time a dilution is changed. Observe the mice at least once daily (morning and afternoon) for five consecutive days, and record morbidity and mortality.

[0202] The results showed that all control mice (injected with toxin and standard) died within 72–120 hours; the potency of the test product was the highest dilution of the mice that died simultaneously with the control mice or showed the most severe tetanus neurotoxicity symptoms. The final confirmed potency of antibody TRN0011 in the formulation was 9000 IU / ml.

[0203] This demonstrates that the formulation not only maintains good stability against low concentrations of protein, but also effectively preserves the anti-tetanus toxin titer of antibody TRN0011.

[0204] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. A formulation comprising anti-tetanus toxin antibodies, characterized in that, The formulation comprises the antitetracycline toxin antibody at a concentration of 1-100 mg / mL and has a pH of 5.0-7.0; and optionally, the formulation further comprises one or more of a surfactant, a pH adjuster, a protein protectant, and an osmotic pressure regulator.

2. The formulation according to claim 1, characterized in that, The antitetracycline toxin antibody comprises: heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and light chain CDR1, light chain CDR2 and light chain CDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:

8. Preferably, the antitetracycline toxin antibody comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:4; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:9; More preferably, the anti-tetanus toxin antibody is a monoclonal antibody containing two heavy chains and two light chains; even more preferably, the anti-tetanus toxin antibody is an IgG1 type antibody. More preferably, the heavy chain of the antitetracycline toxin antibody comprises the amino acid sequence shown in SEQ ID NO:5; and the light chain of the antitetracycline toxin antibody comprises the amino acid sequence shown in SEQ ID NO:

10.

3. The formulation according to claim 1 or 2, characterized in that, The formulation contains the antitetracycline toxin antibody at a concentration not exceeding 50 mg / mL; Preferably, the formulation comprises the antitetracycline toxin antibody at a concentration of 3-50 mg / mL, more preferably 5-50 mg / mL, more preferably 10-30 mg / mL, and even more preferably 10-20 mg / mL.

4. The formulation according to any one of claims 1 to 3, characterized in that, The pH value of the preparation is 5.0-7.0, preferably 5.5-6.5; Preferably, the pH value of the preparation is 5.5±0.2, 6.0±0.2 or 6.5±0.

2.

5. The formulation according to any one of claims 1 to 3, characterized in that, The formulation contains a nonionic surfactant as a protein protectant; Preferably, the nonionic surfactant is polysorbate or poloxamer, such as polysorbate 80, polysorbate 20 or poloxamer P188. More preferably, the formulation comprises the surfactant at a concentration of 0-0.4%, preferably 0-0.02%, more preferably 0.005-0.02%.

6. The formulation according to any one of claims 1 to 5, characterized in that, The pH adjuster is a buffer solution; Preferably, the formulation comprises a histidine / histidine hydrochloride buffer; Preferably, the formulation comprises 10-100mM, more preferably 10-30mM, and more preferably 20mM of histidine / histidine hydrochloride buffer.

7. The formulation according to any one of claims 1 to 6, characterized in that, The osmotic pressure regulator is one or more of sucrose, trehalose, mannitol, sorbitol, sodium chloride, and arginine hydrochloride; Preferably, the osmotic pressure regulator is one or more of sucrose, trehalose, sorbitol and mannitol, and its concentration is 2-8%, preferably 4-6%, and more preferably 4%. Alternatively, the osmotic pressure regulator is sodium chloride and / or arginine hydrochloride, with a concentration of 0-140 mM, preferably 70-100 mM, and most preferably 70 mM; Preferably, the osmotic pressure regulator is sucrose and / or sodium chloride, with a concentration of 2-8% sucrose and / or 0-140mM sodium chloride.

8. The formulation according to any one of claims 1 to 7, characterized in that, The preparation is a liquid preparation administered parenterally, preferably an injectable preparation, and more preferably an intravenous or subcutaneous injection preparation; Preferably, the preparation is prepared using sterile water for injection.

9. The formulation according to any one of claims 1 to 8, characterized in that, The formulation comprises: (1) The antitetracheotoxin antibody at a concentration of 1-100 mg / mL, preferably not exceeding 50 mg / mL; (2) 0-0.02% polysorbate 80; (3) 10-30mM histidine / histidine hydrochloride buffer; (4) 4-6% of any one of sucrose, trehalose, mannitol, and sorbitol; (5) Any one of sodium chloride or arginine hydrochloride at 70-140mM; (6) The pH value is 5.5-6.

5.

10. A method for preparing the formulation according to any one of claims 1 to 9, the method comprising: The anti-tetanus toxin antibody is concentrated and replaced in the pH adjuster through ultrafiltration. Optionally, the amount of pH adjuster to be added is calculated based on the composition of the formulation, and the added pH adjuster, as well as surfactants, protein protectants and osmotic pressure regulators, are added.

11. The use of the formulation of any one of claims 1 to 9 in the preparation of a medicament for treating tetanus toxin or Clostridium tetani infection.

12. A container comprising the formulation according to any one of claims 1 to 9.

13. A medicine box comprising the formulation of any one of claims 1 to 9, or the container of claim 12; Furthermore, the kit may optionally include instructions for using the preparation containing anti-tetanus toxin antibodies.

14. A method for treating tetanus toxin or Clostridium tetani infection, the method comprising administering to a subject in need a therapeutically effective amount of the preparation of any one of claims 1 to 9, and optionally other drugs or means.

15. The method according to claim 14, characterized in that, The subject is a mammal; preferably, the subject is a human.

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