Recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation and preparation method thereof

Abstract

The invention provides a recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation and a preparation method thereof. The injection preparation is prepared from 1.0 mg / mL to 10 mg / mL of recombinant anti-BCMA and CD3 full human bispecific antibody, 0.02% to 0.04% (w / w) of surfactant, 40 mmol / L to 240 mmol / L of stabilizer, a pH regulator and a solvent, and the pH of the injection preparation is 4.5 to 8.0. The recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation is a liquid which is free of preservatives, colorless to faint yellow and clarified to microemulsion, the physical and chemical stability of the double antibodies can still be maintained within a long time, and the storage life is effectively prolonged.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody drugs, and specifically relates to bispecific antibodies, and in particular to a recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation and a preparation method thereof. Background Art

[0002] BCMA (B-cell maturation antigen, TNFRSF17, CD269) is a transmembrane protein belonging to the TNF receptor superfamily. BCMA is a non-glycosylated transmembrane protein that is involved in B cell maturation, growth and survival. It is expressed on the surface of mature B cells and plasma cells and is essential for maintaining normal humoral immunity. BCMA is mainly expressed in plasma cells and mature B lymphocytes, and is basically undetectable in other normal human cells. BCMA is highly expressed in blood tumors such as non-Hodgkin's lymphoma, multiple myeloma, and chronic lymphocytic leukemia.

[0003] Multiple myeloma (MM) is a malignant tumor of plasma cells and the second most common tumor in the blood system. Its incidence rate is second only to diffuse large B-cell lymphoma (DLBCL) among blood tumors, accounting for about 10% of blood system malignancies. In recent years, despite great progress in chemotherapy, proteasome inhibitors, immunomodulatory agents thalidomide derivatives and CD38-targeted antibodies, the high relapse rate and low effective drug clearance rate of patients have not been solved. As the disease progresses, the aggressiveness of each relapse increases, and the remission period obtained by new therapies gradually shortens. BCMA is selectively expressed at high levels in plasma cells and is the most selectively expressed receptor on MM cell lines. Its expression level gradually increases with the differentiation of B cells and also gradually increases during the course of multiple myeloma. It is an ideal target for MM treatment.

[0004] So far, the approved BCMA targeted therapies include: BCMA antibody-drug conjugate BLENREP (GSK), BCMA targeted CRA-T therapies including Abecma (BMS) and Carvykti (Legend Biotech and Johnson & Johnson), and Johnson & Johnson's CD3×BCMA bispecific antibody Teclistamab. In addition, Pfizer's CD3×BCMA bispecific antibody Elranatamab, BCMA targeted CRA-T therapy Anti-BMCA CAR-T cell therapy Ikilencel (Nanjing Reindeer and Innovent Biologics) and Zevorcabtageneautoleucel (Coherus Biotech) have applied for listing.

[0005] In recent years, bispecific antibodies have become a research hotspot in immunotherapy. Bispecific antibodies are artificial antibodies containing two specific antigen binding sites, which can build a bridge between target cells (tumor cells) and effector cells (immune cells) to produce targeted tumor-killing effector functions.

[0006] Compared with chemotherapy and antibody-drug conjugates, bispecific antibody drugs avoid the side effects of chemotherapy drugs. Compared with personalized therapies such as CAR-T cells, bispecific antibody drugs are safer on the one hand, by adjusting the dosing cycle and dosage to reduce side effects, avoiding uncontrollable risks after cell infusion into the body; on the other hand, they are ready-to-use, universal drugs that do not require a long production cycle.

[0007] Biopharmaceuticals are subject to various destructive conditions during production, transportation, storage and use, such as high temperature, light and freeze-thaw. In order to ensure the safety and effectiveness of drugs, the integrity of the drug structure, especially the integrity of the higher-order structure, must be maintained. Polymers and insoluble particles of biopharmaceuticals are key factors in causing immune responses to biopharmaceuticals. The appearance of biopharmaceuticals is also a very important indicator. Pharmacopoeias of various countries stipulate that biopharmaceuticals must not contain visible foreign matter, because the presence or absence of visible foreign matter in biopharmaceuticals and the amount of insoluble particles are directly related to the safety of biopharmaceuticals, and they are considered to be one of the most important factors causing immune responses to biopharmaceuticals.

[0008] Therefore, different biopharmaceutical preparations affect the immune response of biological drugs, and it is necessary to explore preparations for different biological drugs. Summary of the invention

[0009] In view of this, the present invention aims to study the stability of samples under different conditions by SEC-HPLC, DSF, cIEF, CEX-HPLC, CE-SDS and MFI, and screen out a recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation and a preparation method thereof, so as to solve the problem that the pharmaceutical composition of the recombinant anti-BCMA and CD3 fully human bispecific antibody is prone to form aggregates and insoluble particles during long-term storage.

[0010] The purpose of the present invention is to provide a recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation.

[0011] The injection preparation provided by the present invention comprises a recombinant anti-BCMA and CD3 fully humanized bispecific antibody, a surfactant, a stabilizer, a pH regulator and a solvent;

[0012] Wherein, the concentration of the recombinant anti-BCMA and CD3 fully human bispecific antibody in the injection preparation is 1.0-10 mg / mL;

[0013] The mass percentage of the surfactant in the injection preparation is 0.02%-0.04% (w / w, g / 100g);

[0014] The stabilizer is selected from a combination of at least one or more of sodium chloride, sucrose, mannitol, glycine, and proline; the concentration of each component in the stabilizer is 40-240 mmol / L;

[0015] The pH range of the injection preparation is 4.5-8.0.

[0016] In the above-mentioned recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation, the concentration of the recombinant anti-BCMA and CD3 fully human bispecific antibody is 5-10 mg / mL. This protein concentration can make the injection preparation of the present invention more stable and significantly prolong the shelf life of the recombinant anti-BCMA and CD3 fully human bispecific antibody.

[0017] In the above-mentioned recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation, the pH value of the injection preparation ranges from 5.0 to 6.0;

[0018] Furthermore, the pH value ranges from 5.2 to 5.8, such as 5.2, 5.5, 5.8, etc.

[0019] Furthermore, the pH regulator does not react with the recombinant anti-BCMA and CD3 fully human bispecific antibody itself, and can provide a stable environment so that the pH value of the preparation is always maintained at 5.2-5.8. The above pH regulator has good physiological compatibility with blood, has low vascular irritation caused by injection, and has good patient compliance with medication.

[0020] In the above-mentioned recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation, the pH adjuster is any one of sodium citrate / citric acid buffer solution, disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution, sodium acetate / acetic acid buffer solution, L-histidine / L-histidine hydrochloride buffer solution, and L-histidine / aspartic acid buffer solution;

[0021] Furthermore, the pH regulator is any one of L-histidine / L-histidine hydrochloride buffer solution, L-histidine / aspartic acid buffer solution, and sodium citrate-citric acid buffer solution;

[0022] Furthermore, the pH regulator is L-histidine / L-histidine hydrochloride buffer solution.

[0023] In an embodiment of the present invention, the concentration of the L-histidine / L-histidine hydrochloride buffer solution may be 20 mmol / L, specifically 20 mM His / His-HCl.

[0024] In the above-mentioned recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation, the stabilizer is selected from at least one or a combination of proline and mannitol, and the concentration range of the stabilizer component is 40mmol / L-240mmol / L;

[0025] Furthermore, the stabilizer is selected from at least one or a combination of proline and mannitol, specifically proline, and the concentration of the proline is 240mmol / L; or the stabilizer is a combination of proline and mannitol, and the concentration is 40mmol / L proline and 200mmol / L mannitol.

[0026] The stabilizer can be combined with the pH regulator to make the injection preparation of the present invention have an osmotic pressure equivalent to that of plasma, reduce injection pain and / or alleviate gastrointestinal adverse reactions caused by medication in some patients; at the same time, maintain the stability of the spatial structure of the recombinant anti-BCMA and CD3 fully human bispecific antibody, significantly extend the shelf life of the recombinant anti-BCMA and CD3 fully human bispecific antibody, and maintain stable drug efficacy.

[0027] In the recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation described above, the surfactant is selected from at least one of polysorbate 20 and polysorbate 80, or a combination of two thereof; the surfactant can increase the solubility of the recombinant anti-BCMA and CD3 fully human bispecific antibody, and improve the stability of the preparation by reducing the surface tension and system free energy between proteins and between proteins and vials.

[0028] Further, the surfactant is polysorbate 20;

[0029] Furthermore, the mass percentage of polysorbate 20 in the injection preparation is 0.02%-0.04%, and the preferred concentration is 0.02%.

[0030] In the above-mentioned recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation, the solvent is selected from at least one of the following: water for injection and purified water. When normal saline is used instead of water for injection or purified water, the effect of normal saline on osmotic pressure should be considered, and the dosage of osmotic pressure regulators such as proline and mannitol should be appropriately reduced.

[0031] In one embodiment of the present invention, the injection preparation comprises 10 mg / mL of recombinant anti-BCMA and CD3 fully human bispecific antibody, 0.04% (w / w) polysorbate 20, 240 mmol / L proline, a pH adjuster (20 mmol / L His / His-HCl) and a solvent; the pH of the injection preparation is 5.2.

[0032] In a second aspect, the present invention provides a method for preparing the recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation described in the first aspect, which is used to prepare each preparation in the examples, and the method comprises the following steps:

[0033] 1) Weigh the pH adjuster, stabilizer, surfactant and solvent in proportion to the prescription;

[0034] 2) Adding a solvent and a pH adjuster to a preparation container to adjust the pH value to a preset value of the preparation, which is solution A;

[0035] 3) Take a portion of solution A and add a stabilizer, stir and dissolve to obtain solution B;

[0036] 4) taking a portion of solution A and adding the surfactant, stirring and dissolving to obtain solution C;

[0037] 5) Using ultrafiltration, the recombinant anti-BCMA and CD3 fully human bispecific antibody sample was ultrafiltered and exchanged into part of solution A to serve as solution D; the remaining solution A was mixed with the solutions B, C, and D, filtered through a 0.22 μm filter membrane, and then bottled.

[0038] In the method described above,

[0039] In the steps 3) and 4), the stirring time is 15-30 min;

[0040] In the aforementioned step 5), the ultrafiltration method is to use a 30KD ultrafiltration tube to ultrafilter the recombinant anti-BCMA and CD3 fully human bispecific antibody sample into partial solution A.

[0041] In a third aspect, the present invention provides the use of the recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation described in the first aspect in the preparation of a drug for treating multiple myeloma.

[0042] SEC-HPLC is an analytical method that uses the relative relationship between the pore size of the gel pores and the size of the sample to separate. Molecules with larger molecular weight in the sample are completely excluded because they cannot enter the gel pores, and can only pass through the chromatographic column along the gaps between the porous gel particles, so they are first eluted from the column by the mobile phase. Molecules with smaller molecular weight can enter the gel cavities, are more strongly retained in the column, and are not easily eluted, thereby achieving the separation of solute molecules of different sizes. For the analysis of biopharmaceuticals, it is mainly used to measure the content of polymers, which is mainly reflected in the reduction of monomer content and the increase of polymers. DSF is a more advanced differential fluorescence scanning technology. This technology studies protein stability by detecting small changes in tryptophan autofluorescence. The fluorescence intensity of tryptophan in proteins is closely related to the environment around them. Therefore, by detecting the fluorescence changes of tryptophan, the chemical and thermal stability of proteins can be evaluated without labeling. cIEF can focus and separate heterogeneous bodies with different charges according to their different isoelectric points, and the focused bands are detected at 280nm to obtain the charge distribution spectrum. The isoelectric point of the sample to be tested is calibrated by using two markers (pI maker) with known isoelectric points added to the sample. CEX-HPLC (cation exchange chromatography) is separated based on the difference in the number of charges carried by the analyte. The sample is positively charged at low salt and neutral pH, adsorbed on the chromatographic column, and eluted using salt concentration or pH gradient. Depending on the strength of the charge, the acidic isomer is eluted first, followed by the monomer and alkaline isomer. Then the peak area normalization method can be used to determine the main peak content, acidic peak content and alkaline peak content of the test sample. CE-SDS uses a high-voltage direct current electric field as the driving force and a capillary as the separation channel. The capillary is first filled with gel, and the gel will form a molecular sieve in the capillary. The sample is separated according to the molecular weight and the movement speed in the capillary is different, which can effectively reduce the diffusion of components, and the resulting peak shape is sharp and the separation efficiency is high. MFI (microfluidic imaging particle analysis system) is when the sample passes through the detection area, MFI captures images from the sample, analyzes each particle in each image, and creates a database of particle number, size, transparency, and morphology (or shape). In order to conduct immediate visual verification, the system displays the image of the monitoring system in real time, and then generates a system analysis report to understand the distribution of particles in the sample and identify and analyze micron-sized particles. Biological activity: The Fab end of the recombinant anti-BCMA and CD3 fully human bispecific antibody has two targets CD3+BCMA. CD3 can specifically recognize PBMC cell receptors, and BCMA specifically recognizes NCI-H929 cell surface receptors. After the dual targets are connected, PBMC cells are activated to kill NCI-H929 cells. After the cells are killed, the intracellular LDH (lactate dehydrogenase) will be released into the culture medium. The activity of the antibody drug is indirectly reflected by detecting the LDH content in the culture medium.

[0043] Compared with the prior art, the recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation of the present invention has the following advantages:

[0044] (1) Compared with the reported anti-BCMA and CD3 fully human bispecific antibody analogs, this antibody has two BCMA binding sites and can better target myeloma cells;

[0045] (2) Compared with reported anti-BCMA and CD3 fully human bispecific antibody analogs, this antibody has optimized anti-CD3 activity and reduced cytokine release;

[0046] (3) Compared with the reported anti-BCMA and CD3 fully humanized bispecific antibody analogs, this antibody has a longer half-life and is more convenient to use than CART drugs;

[0047] (4) The recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation provided by the present invention is a preservative-free, colorless to light yellow, clear to slightly opalescent liquid, which can maintain the physical and chemical stability of the bispecific antibody for a long time, effectively extending the storage period;

[0048] (5) The recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation provided by the present invention has simple auxiliary material components and stable and controllable quality.

[0049] The components of the recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation described in the present invention have a synergistic effect: alcohols (mannitol) and sugars (sucrose) can not only form hydrogen bonds with proteins to increase protein stability, but also can be used as drug osmotic pressure regulators; salts (NaCl) can bind to protein molecules through electrostatic effects to improve protein colloid stability, and NaCl is also a commonly used osmotic pressure regulator; amino acids (glycine and proline) can interact electrostatically with chains with opposite charges, thereby increasing protein solubility and / or conformational stability, while minimizing protein-protein interactions, and amino acids can also be used as drug osmotic pressure regulators; surfactants (Tween 20, Tween 80) can compete with proteins in the adsorption layer to reduce the surface adsorption effect of proteins, while increasing protein solubility and reducing the amount of large protein particles. The combined use of recombinant anti-BCMA and CD3 fully humanized bispecific antibodies can not only maintain the physical and chemical stability of the bispecific antibodies, but also effectively extend the storage period. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a comparison chart of the change rates of cIEF results of the six groups of samples in Example 2 compared with the T0 point. From left to right, the prescriptions are F1, F2, F3, F4, F5, and F6.

[0051] Figure 2 This is a comparison chart of the results of the 37°C stability test monomer (SEC-HPLC) in Example 3, and each prescription group represents T0, 1W, 2W, and 4W from left to right.

[0052] Figure 3 This is a comparison chart of the results of the 25°C stability test monomer (SEC-HPLC) in Example 3, and each prescription group represents T0, 1W, 2W, and 4W from left to right.

[0053] Figure 4 This is a comparison chart of the results of the 2-8°C stability test monomer (SEC-HPLC) in Example 3, and each prescription group represents T0, 1W, and 4W from left to right.

[0054] Figure 5 This is a comparison chart of the results of the 37°C stability experiment (SEC-HPLC) in Example 4, and each prescription group represents T0, 1W, 2W, 3W, and 4W from left to right.

[0055] Figure 6 This is a comparison chart of the results of the 25°C stability experiment (SEC-HPLC) in Example 4, and each prescription group represents T0, 1M, 2M, 3M, and 6M from left to right.

[0056] Figure 7 This is a comparison chart of the monomer (SEC-HPLC) results of the 2-8°C stability experiment in Example 4, and each prescription group represents T0, 1M, 2M, 3M, 6M, 9M, and 12M from left to right.

[0057] Figure 8 This is a comparison chart of the results of the photostability experiment (SEC-HPLC) in Example 4, and each prescription group represents T0, 5D, and 10D from left to right.

[0058] Fig. 9 This is a comparison chart of the main peak (SEC-HPLC) results of the oscillation stability experiment in Example 4, and each prescription group represents T0, 5D, and 10D from left to right.

[0059] Fig.10 This is a comparison chart of the freeze-thaw stability test monomer (SEC-HPLC) results in Example 4, and each prescription group represents T0, 3C, and 6C from left to right.

[0060] Note: In the attached figure, "W" stands for "week"; "M" stands for "month"; "D" stands for "day"; and "C" stands for "time". DETAILED DESCRIPTION

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0062] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0063] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are all conventional methods unless otherwise specified.

[0064] The recombinant anti-BCMA and CD3 fully human bispecific antibody of the present invention adopts the recombinant anti-BCMA and CD3 fully human bispecific antibody (ie, PR003178 bispecific antibody) prepared in Example 5 of Chinese Patent Publication No. CN114502591A.

[0065] The other raw materials mentioned above can be obtained from public commercial channels unless otherwise specified.

[0066] Example 1, pH screening

[0067] The thermal stability of the anti-BCMA and CD3 fully human bispecific antibodies was observed in the pH range of 4.5 to 8.0. Specific information is shown in Table 1 below.

[0068] The recombinant anti-BCMA and CD3 fully human bispecific antibody was ultrafiltered into the buffer system in Table 1 using a 30KD ultrafiltration tube, and the concentration of the bispecific antibody was as shown in the third column of Table 1.

[0069] Table 1 is a summary of pH screening information

[0070]

[0071]

[0072] As can be seen from Table 1, a thermal stability test was performed during pH screening. This detection method screened out that the pH range with better thermal stability for the anti-BCMA and CD3 fully human bispecific antibody was pH 4.5-6.0, but the Tm Onset and Tagg473 temperatures were basically the same at pH 4.5-pH 5.0. In the case of consistent thermal stability, the pH value closer to neutral (blood pH) was selected, so the selected pH range was pH 5.0-6.0.

[0073] Example 2, buffer screening

[0074] The experimental design for buffer type screening is shown in Table 2.

[0075] The recombinant anti-BCMA and CD3 fully human bispecific antibodies were ultrafiltered and exchanged into the buffer system of Table 2 using a 30KD ultrafiltration tube, and the corresponding stabilizer was added to complete the preparation, which was then dispensed into vials; the stability test was performed by placing at 37°C for 4 weeks.

[0076] The dual antibody solution after 4 weeks of storage was detected by SEC-HPLC, cIEF, reduced CE-SDS and non-reduced CE-SDS. The results are shown in Tables 3-5 and Figure 1 shown.

[0077] Table 2 is the buffer screening experiment design table

[0078]

[0079] Table 3 is a summary of SEC-HPLC test results

[0080]

[0081]

[0082] Note: “-” in the table indicates a decrease.

[0083] Table 4 is a summary of cIEF test results

[0084]

[0085] Note: “↑” in the table indicates an increase in the rate of change compared to point T0, and “↓” indicates a decrease in the rate of change compared to point T0.

[0086] Table 5 is a summary of the results of reduced CE-SDS purity and non-reduced CE-SDS purity test

[0087]

[0088]

[0089] From the above, it can be seen that based on the results of SEC-HPLC, cIEF, reduced CE-SDS purity and non-reduced CE-SDS purity tests, samples F3 (His, pH 5.5) and F5 (His + Asp, pH 5.5) have good stability. In subsequent experiments, the effects of buffer and protective agent on the stability of the protein can be observed by appropriately changing the buffer system (pH 5.0 to pH 6.0) and adding protective agent.

[0090] Example 3: Preparation prescription screening experiment

[0091] The experimental prescription design based on the results of Example 2 is shown in Table 6.

[0092] Table 6 is the formulation formula design table for the formulation formula screening experiment

[0093]

[0094] Note: “-” in the table means that the substance is not added; the solvent used in the preparation of the samples in this table is water for injection or purified water.

[0095] The recombinant anti-BCMA and CD3 fully human bispecific antibodies were ultrafiltered and exchanged into the buffer system of Table 6 using a 30KD ultrafiltration tube, and the corresponding stabilizer was added to complete the preparation to obtain the preparation sample, which was filtered using a 0.2 μm filter and then dispensed into a vial.

[0096] The packaged samples were placed in a 37℃ / 25℃ / 2-8℃ constant temperature box for stability testing. The experimental results are shown in Table 7, Table 8, Table 9, Figure 2-Figure 4 shown.

[0097] Table 7 is a summary of the results of the 37°C stability test for the formulation screening experiment.

[0098]

[0099]

[0100] Note: "-" in the above table means that there is no test result for this item. Sample 7 became turbid after being placed at 37℃ for 1 week, and samples 13 and 14 became turbid after being placed at 37℃ for 2 weeks, so the formulations of samples 7, 13 and 14 were discarded.

[0101] Table 8 is a summary of the results of the 25°C stability test for the formulation screening experiment

[0102]

[0103]

[0104]

[0105] Table 9 is a summary of the results of the 2-8℃ stability test for the formulation screening experiment

[0106]

[0107]

[0108] Based on the stability test results at 37°C, 25°C, and 2-8°C, the optimal buffer selected was 20mM His / His-HCl, and the optimal additives selected were proline, mannitol, and polysorbate 20. The sample stability was slightly better when the protein concentration was 1mg / mL, but this concentration was too low to be conducive to subsequent research. There was no significant difference in the stability of the sample with a protein concentration of 5mg / mL and that of the sample with a protein concentration of 10mg / mL.

[0109] Example 4: Preparation prescription optimization experiment

[0110] According to the prescription screening results, the pH selected in this experiment was pH 5.2 and pH 5.8, the additives were proline and mannitol, and the surfactant was polysorbate 20. Combined with the prescription screening results, the protein concentration in this experiment was positioned at 10 mg / mL, and the specific formulation prescription design is shown in Table 10.

[0111] The preparation method is as follows: Use a 30KD ultrafiltration tube to ultrafilter the recombinant anti-BCMA and CD3 fully human bispecific antibody into the buffer system in Table 10, add the corresponding stabilizer and surfactant, and obtain the preparation solution after the preparation is completed. Then, the preparation solution is divided into vials. The divided samples are placed in a 37℃ / 25℃ / 2-8℃ constant temperature box for stability experiments, and experiments on influencing factors such as light (25℃, 5000lx) / oscillation (25℃, 100rpm) / freeze-thaw (-40℃ / room temperature) are carried out. The experimental results are shown in Tables 11-16 and Figure 5-Figure 10 shown.

[0112] Table 10 is the formulation formulation design table for the formulation formulation optimization experiment

[0113]

[0114] Table 11 is a summary of the results of the 37°C stability test for the formulation optimization experiment.

[0115]

[0116] Table 12 is a summary of the results of the 25°C stability test for the formulation optimization experiment.

[0117]

[0118] Table 13 is a summary of the results of the 2-8℃ stability test for the formulation optimization experiment

[0119]

[0120]

[0121] Table 14 is a summary of the results of the light stability test for the formulation optimization experiment

[0122]

[0123]

[0124] Table 15 is a summary of the results of the oscillation stability test of the formulation optimization experiment

[0125]

[0126] Table 16 is a summary of the freeze-thaw stability test results of the formulation optimization experiment

[0127]

[0128]

[0129] Based on the results of stability experiments on the samples under illumination, shaking, freeze-thaw, 37°C, 25°C and 2-8°C, when the buffer was 20mmol / LHis / His-HCl, pH5.2-5.8, the excipients were proline (240mmol / L) or a combination of proline (40mmol / L) and mannitol (200mmol / L), and the surfactant was Tween 20 (0.02%), the samples had good overall stability in terms of monomer purity, charge heterogeneity (main peak content), purity (non-reduced CE-SDS) and biological activity.

Claims

1. A recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation, the injection preparation comprising a recombinant anti-BCMA and CD3 fully human bispecific antibody, a surfactant, a stabilizer, a pH adjuster and a solvent; in, The concentration of the recombinant anti-BCMA and CD3 fully human bispecific antibody in the injection preparation is 1.0-10 mg / mL; The mass percentage of the surfactant in the injection preparation is 0.02%-0.04%; The stabilizer is selected from a combination of at least one or more of sodium chloride, sucrose, mannitol, glycine, and proline; the concentration of each component in the stabilizer is 40-240 mmol / L; The pH range of the injection preparation is 4.5-8.

0.

2. The recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation according to claim 1, characterized in that: In the injection preparation, the concentration of the recombinant anti-BCMA and CD3 fully human bispecific antibody is 5-10 mg / mL.

3. The recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation according to claim 1 or 2, characterized in that: The pH value of the injection preparation ranges from 5.0 to 6.0; Furthermore, the pH value range is 5.2-5.

8.

4. The recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation according to any one of claims 1 to 3, characterized in that: The pH regulator is any one of sodium citrate / citric acid buffer solution, disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution, sodium acetate / acetic acid buffer solution, L-histidine / L-histidine hydrochloride buffer solution, and L-histidine / aspartic acid buffer solution; Furthermore, the pH regulator is any one of L-histidine / L-histidine hydrochloride buffer solution, L-histidine / aspartic acid buffer solution, and sodium citrate-citric acid buffer solution; Furthermore, the pH regulator is specifically L-histidine / L-histidine hydrochloride buffer solution.

5. The recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation according to any one of claims 1 to 4, characterized in that: The stabilizer is selected from at least one or a combination of proline and mannitol, and the concentration range of the stabilizer component is 40mmol / L-240mmol / L; Furthermore, the stabilizer is proline, and the concentration of the proline is 240 mmol / L; or the stabilizer is a combination of proline and mannitol, and the concentrations of the proline and mannitol are 40 mmol / L proline and 200 mmol / L mannitol.

6. The recombinant anti-BCMA and CD3 fully humanized bispecific antibody injection preparation according to any one of claims 1 to 5, characterized in that: The surfactant is selected from at least one of polysorbate 20 and polysorbate 80; Further, the surfactant is polysorbate 20; Furthermore, the mass percentage of polysorbate 20 in the injection preparation is 0.02%-0.04%.

7. The recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation according to any one of claims 1 to 6, characterized in that: The solvent is selected from at least one of the following: water for injection and purified water.

8. A method for preparing the recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation according to any one of claims 1 to 7, comprising the following steps: 1) Weighing the pH adjuster, the stabilizer, the surfactant and the solvent in any one of claims 1 to 7 in proportion; 2) adding the solvent and the pH adjuster into a preparation container to adjust the pH value to a preset value of the preparation, as solution A; 3) taking a portion of solution A and adding the stabilizer, stirring and dissolving to obtain solution B; 4) taking a portion of solution A and adding the surfactant, stirring and dissolving to obtain solution C; 5) Using ultrafiltration, the recombinant anti-BCMA and CD3 fully human bispecific antibody sample was ultrafiltered and exchanged into part of solution A to serve as solution D; the remaining solution A was mixed with the solutions B, C, and D, filtered through a 0.22 μm filter membrane, and then bottled.

9. The preparation method according to claim 8, characterized in that: In the steps 3) and 4), the stirring time is 15-30 min; In the aforementioned step 5), the ultrafiltration method uses a 30KD ultrafiltration tube to ultrafilter the recombinant anti-BCMA and CD3 fully human bispecific antibody sample into solution A.

10. Use of the recombinant anti-BCMA and CD3 fully human bispecific antibody injection preparation according to any one of claims 1 to 7 in the preparation of a drug for treating multiple myeloma.

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