Stable bispecific antibody composition

Abstract

Provided is a pharmaceutical composition, comprising a CD20 / CD3 bispecific antibody at 5-40 mg / ml, a buffer at 10-50 mM, a protective agent at 100-300 mM, an antioxidant at 2-30 mM, and a surfactant at 0.05-1.00 mg / ml. Also provided is pharmaceutical use of the described pharmaceutical composition.

Description

A stable bispecific antibody composition Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a stable pharmaceutical composition containing a CD20 / CD3 bispecific antibody. Background Technology

[0002] Bispecific antibodies are artificial antibodies containing two specific antigen-binding sites. They can bridge the gap between target cells and functional molecules (cells), stimulating a targeted immune response. They are a type of genetically engineered antibody and have become a hot topic in the field of antibody engineering, with broad application prospects in the immunotherapy of tumors.

[0003] CD20 is a glycosylated protein expressed on the surface of B lymphocytes. Its expression gradually increases during the B cell development cycle, but it is not expressed in differentiated normal tissues. More importantly, CD20 is expressed in the vast majority of B-cell non-Hodgkin lymphomas, a characteristic that makes it an ideal target for the treatment of B-cell-related tumors.

[0004] CD3 is a transmembrane protein present on the surface of T cells. The CD3 molecule is linked to T cell antigen receptors via salt bridges and participates in T cell signal transduction. CD3 plays a role in recruiting T cells at tumor cell sites. However, if the affinity of dual anti-inflammatory molecules for CD3 is too strong, it can lead to overactivation of immune T cells, inducing cytokine release syndrome (CRS).

[0005] The CD20 / CD3 bispecific antibody (hereinafter referred to as "CD20 / CD3 bispecific antibody") of this application has low CD3 affinity and high CD20 affinity, killing tumor cells through ADCC and CDC. In in vitro and in vivo model studies, the CD20 / CD3 bispecific antibody can significantly inhibit the proliferation of rituximab-resistant cancer cells. In addition, the CD20 / CD3 bispecific antibody has a low probability of inducing CRS in animal models. Therefore, the CD20 / CD3 bispecific antibody shows promise as a highly effective and safe anti-tumor drug.

[0006] For bispecific antibodies, finding formulations that are both conducive to the stability of their protein higher-order structure and easy to administer is crucial for biopharmaceutical formulations. Furthermore, due to the unique structure of bispecific antibody molecules, improving their stability presents a significant challenge.

[0007] Summary of the Invention

[0008] The present invention aims to provide a CD20 / CD3 bispecific antibody drug composition (hereinafter referred to as "CD20 / CD3 bispecific antibody drug composition") that has enhanced stability (including molecular size stability, charge stability, etc.) not only under conventional storage conditions, but also under various conditions such as high temperature, low temperature, shaking, transportation, and simulated clinical administration.

[0009] The first aspect of this invention relates to a pharmaceutical composition comprising:

[0010] (a) 5–40 mg / ml CD20 / CD3 bispecific antibody;

[0011] (b) 10–50 mM buffer;

[0012] (c) 100–300 mM protective agent;

[0013] (d) 2-30 mM antioxidants;

[0014] (e) 0.05–1.00 mg / ml surfactant;

[0015] The buffer is selected from histidine / histidine hydrochloride, citric acid / sodium citrate, glutamic acid / monosodium glutamate, or acetic acid / sodium acetate;

[0016] The protective agent is selected from one or more of polyols, sugars, and amino acids;

[0017] The antioxidants mentioned therein are selected from one or more of amino acids and metal ion chelating agents;

[0018] The surfactants mentioned therein are one or more of the following: polysorbates, poloxamers, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerol fatty acid esters, polyethylene glycol fatty acid esters, and polyoxyethylene polyoxypropylene alkyl ethers;

[0019] The CD20 / CD3 bispecific antibody comprises:

[0020] The first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2 and 3, wherein the VH1 CDR1-CDR3 respectively contain amino acid sequences that are at least 80% identical, preferably at least 90% identical, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99% identical, and more preferably completely identical to SEQ ID NO.:1-3.

[0021] The second heavy chain variable region (VH2) contains CDRs 1, 2 and 3, wherein the VH2 CDRs 1-3 each contain an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to that of SEQ ID NO.:4-6.

[0022] The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein CDRs 1-3 of VL1 each contain an amino acid sequence that is at least 80% identical, preferably at least 90% identical, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99% identical, and more preferably completely identical to that in SEQ ID NO.:7-9; and

[0023] The second light chain variable region (VL2) contains CDRs 1, 2 and 3, wherein the VL2 CDRs 1-3 each contain an amino acid sequence that is at least 80% identical, preferably at least 90% identical, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99% identical, and more preferably completely identical to SEQ ID NO.:7-9.

[0024] The specific sequences are shown in the table below:

[0025] In a preferred embodiment, the first heavy chain variable region (VH1) contains an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to, SEQ ID NO.:10; the second heavy chain variable region (VH2) contains an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to, SEQ ID NO.:12; and the first light chain variable region (VL1) and the second light chain variable region (VL2) contain an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to, SEQ ID NO.:12.

[0026] The specific sequences are shown below:

[0027] SEQ ID No.:10(VH1):

[0028] SEQ ID No.:11(VH2):

[0029] SEQ ID No.:12 (VL1 or VL2):

[0030] In a preferred embodiment, in the CD20 / CD3 bispecific antibody: the full length of the CD20 heavy chain is as shown in sequence SEQ ID NO.:13;

[0031] The full length of the CD3 heavy chain is shown in sequence SEQ ID NO.:14;

[0032] The full length of the common light chain shared by both is shown in sequence SEQ ID NO.:15. The specific sequences are shown below:

[0033] SEQ ID No.:13 (Anti-CD20 Heavy Chain):

[0034] SEQ ID No.:14 (Anti-CD3 Heavy Chain):

[0035] SEQ ID No.:15 (Common Light Chain):

[0036] In a preferred embodiment, the buffer is histidine / histidine hydrochloride.

[0037] In a preferred embodiment, the protective agent is selected from one or more of trehalose, sucrose, arginine, glycine, and mannitol.

[0038] In a further preferred embodiment, the protective agent is arginine, preferably arginine hydrochloride.

[0039] In a preferred embodiment, the antioxidant is methionine or EDTA-2Na, with methionine being the preferred antioxidant.

[0040] In a preferred embodiment, the surfactant is polysorbate 20.

[0041] In a preferred embodiment, the pH of the pharmaceutical composition is 5.5-7.0, preferably 6.0-7.0, more preferably 6.0-6.6, and most preferably 6.3.

[0042] In a preferred embodiment, the pharmaceutical composition comprises:

[0043] -5 to 40 mg / ml CD20 / CD3 bispecific antibody; preferably 15 to 25 mg / ml; more preferably 18 to 22 mg / ml;

[0044] -10 to 50 mM histidine / histidine hydrochloride buffer; preferably 10 to 40 mM; more preferably 10 to 30 mM;

[0045] -100-300 mM arginine hydrochloride; preferably 110-200 mM; more preferably 112-168 mM;

[0046] -2 to 30 mM methionine; preferably 3 to 25 mM; more preferably 5 to 20 mM;

[0047] -0.05 to 1.00 mg / ml polysorbate 20; preferably 0.10 to 0.50 mg / ml; more preferably 0.10 to 0.30 mg / ml.

[0048] In a preferred embodiment, the pharmaceutical composition is an aqueous solution with a pH of 6.3 comprising the following components:

[0049] -20 mg / ml CD20 / CD3 bispecific antibody;

[0050] -20mM histidine / histidine hydrochloride buffer;

[0051] -140mM arginine hydrochloride;

[0052] -10mM methionine;

[0053] -0.20 mg / mL polysorbate 20.

[0054] The second aspect of the present invention relates to the use of the pharmaceutical composition according to the first aspect of the present invention in the preparation of a medicament for treating cancer.

[0055] In a preferred embodiment, the cancer is chronic lymphocytic leukemia or non-Hodgkin's lymphoma.

[0056] A third aspect of the invention relates to a method of treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to the first aspect of the invention.

[0057] In a preferred embodiment, the cancer is chronic lymphocytic leukemia or non-Hodgkin's lymphoma.

[0058] In a preferred embodiment, the subject is a mammal, preferably a human.

[0059] Beneficial effects of the present invention

[0060] The CD20 / CD3 bispecific antibody drug composition of the present invention exhibits significantly enhanced stability (including molecular size stability and charge stability) not only under conventional storage conditions, but also under various conditions such as high temperature, low temperature, shaking, transportation, and simulated clinical administration, achieving unexpected technical effects and possessing significant clinical application value.

[0061] To facilitate understanding of this invention, certain terms used herein are defined or described. Unless otherwise indicated or defined, scientific and technical terms used in conjunction with this invention will have the meanings commonly understood by one of ordinary skill in the art.

[0062] DSF Differential Scanning Calorimetry

[0063] SEC-HPLC (Size Exclusion High Performance Liquid Chromatography)

[0064] CEX-HPLC Cation Exchange High Performance Liquid Chromatography

[0065] HMW high molecular weight peak group (polymer)

[0066] LMW low molecular weight peak group (fragment)

[0067] PS20 Polysorbate 20

[0068] NT not detected

[0069] ND not detected

[0070] CDR Complementary Determinant Region

[0071] The term "stability" refers to the ability of a pharmaceutical preparation or composition to maintain its physical, / or chemical, and / or biological stability when stored.

[0072] The term "buffer" refers to a substance that maintains a substantially constant pH value of a solution within a certain range. As used herein, the buffer includes, but is not limited to, one or more of the following: citric acid, sodium citrate, glutamic acid, monosodium glutamate, acetic acid, sodium acetate, histidine, histidine hydrochloride, phosphate, phosphate-buffered saline, tris(hydroxymethyl)aminomethane (Tris) buffer.

[0073] The term "protectant" refers to a substance that enables a component in a pharmaceutical preparation or composition to remain stable within a certain range. As used herein, the protectant includes, but is not limited to, one or more of polyols, sugars, amino acids, etc.

[0074] The term "antioxidant" refers to a substance that can prevent the oxidation of components in a pharmaceutical preparation or composition within a certain range. As used herein, the antioxidant is selected from one or more of amino acids, metal ion chelators, etc.

[0075] The term "surfactant" refers to a substance that causes a significant change in the interfacial state of a solution system. As used herein, the surfactants include, but are not limited to, one or more of the following: polysorbates (polysorbate 80 (Tween 80), polysorbate 60 (Tween 60), polysorbate 40 (Tween 40), polysorbate 20 (Tween 20), etc.), poloxamers (poloxamer 188, etc.), sorbitan fatty acid esters (sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate, etc.), polyoxyethylene sorbitan fatty acid esters (polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate, etc.), glycerol fatty acid esters (glycerol monocaprylate, glycerol monomyristicate, glycerol monostearate, etc.), polyethylene glycol fatty acid esters (polyethylene glycol distearate, etc.), and polyoxyethylene polyoxypropylene alkyl ethers (polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene hexadecyl ether, etc.). Attached Figure Description

[0076] Figure 1 shows the SEC-HPLC results of CD20 / CD3 bispecific antibody drug compositions at different pH values ​​after accelerated degradation at 40°C (left: polymer ratio; right: fragment ratio).

[0077] Figure 2 shows the CEX-HPLC results of CD20 / CD3 bispecific antibody drug compositions at different pH values ​​after accelerated testing at 25°C (left: acid peak ratio; right: polymer ratio).

[0078] Figure 3 shows the CEX-HPLC results of CD20 / CD3 bispecific antibody drug compositions under different buffer systems (left: acid peak ratio, right: main peak ratio).

[0079] Figure 4 shows the results of nitrogen-filled CEX-HPLC comparison of the CD20 / CD3 bispecific antibody drug composition (left: acid peak ratio; right: main peak ratio). Detailed Implementation

[0080] To better understand the present invention, it will be described in detail below with reference to embodiments. However, it should be understood that these embodiments are merely illustrative examples and are not intended to limit the invention. In this invention, unless otherwise stated, all operations are performed at room temperature and normal pressure.

[0081] Example 1: Effect of each component on the stability of the pharmaceutical composition of the present invention

[0082] The inventors conducted extensive experiments to investigate the effects of pH, buffer systems, protective agents (including sugars and amino acids, etc.) and antioxidants on the stability of the exemplary CD20 / CD3 bispecific antibody drug composition of the present invention. Through formulation design of experiment (DoE) and other methods, a CD20 / CD3 bispecific antibody drug composition with excellent stability was obtained, which has the following composition: 20 mg / ml CD20 / CD3 bispecific antibody, 20 mM histidine / histidine hydrochloride buffer, 140 mM arginine hydrochloride, 10 mM methionine, 0.20 mg / mL polysorbate 20, pH 6.3.

[0083] The full length of the CD20 heavy chain (also known as "anti-CD20 heavy chain" or "CD20 heavy chain") of the CD20 / CD3 bispecific antibody (also known as "CD20 / CD3 bispecific antibody") is shown in sequence SEQ ID NO.:13; the full length of the CD3 heavy chain (also known as "anti-CD3 heavy chain" or "CD3 heavy chain") is shown in sequence SEQ ID NO.:14; and the full length of the common light chain shared by both is shown in sequence SEQ ID NO.:15.

[0084] 1. pH

[0085] Based on the initial formulation of 5 mg / ml CD20 / CD3 bispecific antibody, 20 mM histidine / histidine hydrochloride, 50 mg / ml trehalose dihydrate, and 0.01% polysorbate 20, the changes in the physicochemical properties of the above composition in five different pH values ​​(pH 5.5, 6.0, 6.5, 7.0, and 7.5) histidine buffer systems were investigated.

[0086] First, the thermal stability of the above composition under various pH conditions was investigated by DSF testing, and the results are shown in Table 1 below.

[0087] Table 1: DSF Detection Results

[0088] As shown in Table 1, the CD20 / CD3 bispecific antibody drug composition had the lowest DSF values ​​(Th1, Th2) at pH 5.5, and its corresponding thermal stability was lower than that of samples under other pH conditions.

[0089] In addition, the above-mentioned pharmaceutical compositions were subjected to freeze-thaw cycling experiments, accelerated degradation experiments at 40℃±2℃, and accelerated degradation experiments at 25℃±2℃. The freeze-thaw cycling experiment involved placing the samples in a freeze-thaw cycle from -80℃ to -60℃ to 25℃±2℃ for three cycles before sampling. The accelerated degradation experiment involved placing the samples in a constant temperature chamber at 40℃±2℃ and sampling after 1, 2, 3, and 4 weeks. The accelerated degradation experiment involved placing the samples in a constant temperature chamber at 25℃±2℃ and sampling after 3 months. The changes in molecular size and charge heterogeneity of each formulation after degradation by the above influencing factors were detected, and the experimental results are shown in Table 2 below:

[0090] Table 2: Effect of pH on stability

[0091] As shown in Table 2, after three freeze-thaw cycles from -80℃ to -60℃ and then to 25℃±2℃, the purity changes of the composite samples were not significantly different, except for the pH 5.5 sample which had a higher polymer ratio. After four weeks of accelerated high-temperature treatment at 40℃±2℃, the purity changes were similar, with an increase in the CEX-HPLC acid peak for all pH samples. Mass spectrometry analysis confirmed this was related to the formation of a certain proportion of oxidizing substances. The pH 5.5 sample had a significantly higher SEC-HPLC polymer ratio, while the pH 7.0 and 7.5 samples had significantly higher fragment ratios (see Figure 1). After 3 months of accelerated treatment at 25℃±2℃, the proportion of acidic peaks in all pH samples increased significantly with increasing treatment time, with slightly lower increases in pH 5.5 and pH 6.0 samples. The low pH (pH 5.5) samples showed a significantly higher proportion of aggregates, while the pH 6.0 and pH 6.5 samples showed slightly lower proportions. High pH (pH 7.0, 7.5) samples showed higher proportions of both aggregates and degradation fragments, but the proportion of the main peak was significantly lower than other formulations (see Figure 2). The pH 5.5 sample, after 3 months of accelerated treatment at 25℃±2℃, exhibited an abnormal phenomenon of a sharp initial increase followed by a decrease in aggregate size, which was due to reversible antibody aggregation. Overall, the CD20 / CD3 bispecific antibody composition showed the least change in charge purity and molecular size purity within the pH range of 6.0–6.5.

[0092] Therefore, for the stability of protein physicochemical properties, a pH range of 6.0–6.5 is preferred. To avoid the tendency of samples to aggregate below pH 6.0, the center point and acceptable range of pH for CD20 / CD3 bispecific antibody drug compositions are preferably pH 6.3 ± 0.3 (pH 6.0–6.6), which can avoid sample aggregation caused by excessively low pH limits.

[0093] 2. Buffer system

[0094] Based on the preliminary determination of the suitable pH value for the CD20 / CD3 bispecific antibody drug composition, the effect of buffer systems (histidine / histidine hydrochloride system and citric acid / citric acid system) on the stability of the composition was investigated.

[0095] Table 3: Composition of CD20 / CD3 bispecific antibody drug compositions with different buffering systems

[0096] The above compositions were subjected to accelerated degradation experiments at 40°C, i.e., the samples were placed in a 40°C constant temperature chamber, and samples were taken after 2 and 4 weeks. The changes in the charge heterostructure of each sample after degradation by the above influencing factors were detected, and the results are shown in Table 4 below:

[0097] Table 4: Effect of buffer system on stability at 40℃±2℃

[0098] As can be seen from the data (Table 4 and Figure 3), the growth rate of the acidic peak in the CEX-HPLC of the histidine / histidine hydrochloride system was significantly slower than that of the citric acid / citrate system: In the citric acid / citrate system, after 4 weeks at 40℃±2℃, the acidic peak percentage increased from 18.2% at T0 to 44.0%, an increase of 25.8%, while in the histidine / histidine hydrochloride system, the acidic peak percentage increased from 17.9% at T0 to 33.1%, an increase of only 15.2%. Therefore, the histidine / histidine hydrochloride system is preferred as a buffer system for the CD20 / CD3 bispecific antibody drug composition, which can significantly improve the stability of the drug composition of the present invention.

[0099] 3. Protectants and antioxidants

[0100] To confirm the reason why the acidic peaks of each composition sample increased after treatment at 40℃±2℃ and 25℃±2℃ in the pH study, a CD20 / CD3 bispecific antibody composition with the following components was selected: 5 mg / ml CD20 / CD3 bispecific antibody, 20 mM histidine / histidine hydrochloride, 50 mg / ml trehalose dihydrate, 0.01% PS20, pH 6.5. After treatment at 40℃±2℃ for 3 weeks, mass spectrometry peptide mapping analysis of the sample revealed that multiple sites of the CD20 / CD3 bispecific antibody were oxidized, especially W106 of heavy chain CD20 and M254 of heavy chain CD3, with oxidation rates exceeding 50%, which was specifically reflected in the change of the proportion of acidic peaks in CEX-HPLC.

[0101] To further confirm the oxidation phenomenon quickly, a nitrogen-filled comparative study was conducted on the CD20 / CD3 bispecific antibody drug compositions at pH 6.0 and pH 6.5. The CEX-HPLC results after acceleration at 45℃±2℃ for 1 week are shown in Table 5.

[0102] Table 5: Results of Nitrogen Purging Comparison Experiment (CEX-HPLC)

[0103] The results in Table 5 and Figure 4 show that the acid peak growth in nitrogen-filled samples was slower than that in unfilled samples, but oxidation could not be completely inhibited, further indicating that CD20 / CD3 bispecific antibodies are prone to oxidation. Therefore, when screening protective agents, the addition of antioxidants should be considered to inhibit antibody oxidation.

[0104] The protective effects of methionine, EDTA-2Na, arginine (using arginine hydrochloride), glycine, and mannitol on the CD20 / CD3 dual-antibody drug combination were further investigated. The composition of the protective agents and optional antioxidants used is shown in Table 6.

[0105] Table 6: Composition of Protective Agent

[0106] The above formulations were subjected to high-temperature accelerated degradation experiments and freeze-thaw cycle experiments, respectively. In the high-temperature accelerated degradation experiment, samples were placed in a constant temperature chamber at 40℃±2℃, and samples were taken after 2 and 4 weeks, respectively. In the freeze-thaw cycle experiment, samples were subjected to a freeze-thaw cycle from -80℃ to -60℃ to 25℃±2℃ for 6 cycles, and samples were taken afterward. The changes in molecular size and charge heterogeneity of each formulation after being subjected to the above influencing factors were detected, and the experimental results are shown in Table 7 below:

[0107] Table 7: Effects of Protectants and Antioxidants on Stability

[0108] As can be seen from the table above, after the CD20 / CD3 bispecific antidote composition of the present invention was stored at 40℃±2℃ in the dark for 2 weeks and 4 weeks respectively under different protective agent conditions, the CEX-HPLC purity changed significantly under the condition of no antioxidant in the protective agent ("antioxidant-free group"). For example, for the sample in the antioxidant-free group, the "acid peak (%)" increased from 27.0 to 27.5 at T0 to 42.6 to 50.1 after 4 weeks at 40℃±2℃, while the change was smaller under the condition of adding antioxidant ("antioxidant-added group").

[0109] After six freeze-thaw cycles at -80°C to -60°C / 25°C, the SEC-HPLC purity of CD20 / CD3 bispecific antibody compositions under different cryoprotectant conditions showed significant changes with the addition of only glycine or mannitol as cryoprotectant (e.g., for samples with only glycine or mannitol, the %HMW (% high molecular weight substance) increased from 1.2 at T0 to 2.5 (glycine only) and 2.4 (mannitol only) after six freeze-thaw cycles at -80°C to -60°C / 25°C), while there was almost no change with the addition of arginine hydrochloride + methionine or arginine hydrochloride + EDTA-2Na (the %HMW remained at 1.0 after six freeze-thaw cycles at -80°C to -60°C / 25°C). This indicates that cryoprotectant formulations containing arginine can effectively protect CD20 / CD3 bispecific antibodies from damage under freeze-thaw conditions and prevent aggregation.

[0110] Therefore, the combination of arginine and antioxidants (methionine or EDTA-2Na) in the pharmaceutical composition results in significantly better sample stability than samples using protective agents such as glycine and mannitol, and is even more effective than using arginine alone. This significantly improves the stability of the pharmaceutical composition of the present invention and achieves unexpected technical effects.

[0111] 4. DoE Investigation

[0112] Through formulation DoE analysis, the optimal CD20 / CD3 bispecific antibody drug composition was obtained: 20 mg / ml CD20 / CD3 bispecific antibody, 20 mM histidine / histidine hydrochloride buffer, 140 mM arginine hydrochloride, 10 mM methionine, 0.20 mg / mL polysorbate 20, pH 6.3.

[0113] Example 2 - Preparation of the pharmaceutical composition

[0114] The pharmaceutical composition of the present invention was prepared according to the following steps: 20 mg / ml CD20 / CD3 bispecific antibody (the antibody sequence information used is the same as in Example 1), 20 mM histidine / histidine hydrochloride buffer, 140 mM arginine hydrochloride, 10 mM methionine, 0.2 mg / mL polysorbate 20, and pH adjusted to 6.3 with sodium hydroxide or dilute hydrochloric acid if necessary.

[0115] Laboratory preparation: Take a low concentration (generally 1-5 mg / mL) of the CD20 / CD3 bispecific antibody cationic chromatography sample and concentrate it using a 15 / 10K ultrafiltration centrifuge tube until the protein concentration of the concentrate is close to 20 mg / mL. Then, use a 10KD dialysis card to replace the buffer with 20 mM histidine / histidine hydrochloride, pH 6.3 buffer. Continue to concentrate the sample using an ultrafiltration centrifuge tube to 25-30 mg / mL. Add the buffer concentrate and protein concentrate sample according to the above prescription information to ensure that the protein and excipient concentrations reach the prescribed concentrations. After sterilization filtration through a 0.22 μm filter, aliquot the sample into borosilicate glass vials under a local Grade A environment, and then cap and stopper the vials.

[0116] Production under GMP conditions: The virus filtration intermediate is subjected to ultrafiltration washing steps containing 20mM histidine / histidine hydrochloride buffer, 140mM arginine hydrochloride, 10mM methionine, and pH 6.3 buffer. After concentration and displacement, a surfactant is added to the intermediate to achieve a concentration of 0.2mg / mL of polysorbate 20, obtaining the stock solution of the formulation described in this invention, which is then frozen at -80℃ to -60℃. During production, the frozen stock solution is thawed and mixed at room temperature in the workshop, then sterilized by a 0.22μm sterile filter into sterile containers. Under a B+A grade environment, it is filled into borosilicate glass vials according to the set filling volume, and then fully stoppered with a membrane-coated rubber stopper and crimped with an aluminum-plastic composite cap. After crimping, the qualified products are inspected by light, labeled, packaged, and stored.

[0117] Example 3 - Stability under high temperature, acceleration and oscillation conditions

[0118] The stability of the pharmaceutical composition of Example 2 produced under GMP conditions was investigated sequentially under high temperature (40℃±2℃), accelerated (upright and inverted at 25℃±2℃, respectively) and oscillation (oscillation at 200 rpm for 7 days at room temperature). The results are shown in Tables 8, 9-10 and 11 below.

[0119] Table 8: Stability under the influence of high temperature (40℃±2℃) factors

[0120] Table 11: Stability under Oscillation Influencing Factors

[0121]

NT

[0122] As shown in Example 3, when the CD20 / CD3 bispecific antibody drug composition of the present invention was stored at 40℃±2℃ in the dark for 1 month (Table 8), the purity of SEC-HPLC, rCE-SDS, nrCE-SDS, CEX-HPLC, and CDC activity decreased only slightly, but still met the release and shelf-life quality standards for the CD20 / CD3 bispecific antibody drug composition. Other quality indicators showed no significant changes.

[0123] When the CD20 / CD3 bispecific antibody drug composition of the present invention was stored at 25°C ± 2°C in the dark for 1, 2, 3, and 6 months (Tables 9-10), no significant changes in particle or molecular size heterogeneity or charge heterogeneity were observed, regardless of whether it was stored upright or upside down. No changes were observed in the biological activity results. All quality indicators met the release and shelf-life quality standards for the CD20 / CD3 bispecific antibody drug composition.

[0124] Furthermore, under room temperature shaking conditions for 7 days (Table 11), no significant changes were observed in the molecular size heterogeneity, charge heterogeneity, and insoluble microparticles of the CD20 / CD3 bispecific antibody drug composition of the present invention. All quality indicators met the release and shelf-life quality standards for the CD20 / CD3 bispecific antibody drug composition.

[0125] In summary, the CD20 / CD3 bispecific antibody composition exhibits some sensitivity to high temperatures, which is an inherent characteristic of biological products. However, based on current stability study data, it can be determined that the finished CD20 / CD3 bispecific antibody composition maintains long-term stability under conditions of 25℃±2℃ and 2~8℃ in the dark. Under normal use (stored at 2~8℃ in the dark for 24 months) and even under certain degree of overheating (less than 40℃±2℃ in the dark for 1 month and 25℃±2℃ in the dark for 6 months), its quality remains stable, and all quality indicators meet its release standards and shelf-life quality standards. These results demonstrate that the CD20 / CD3 bispecific antibody composition of this invention possesses excellent physicochemical and biological stability.

[0126] Example 4 - Stability under simulated transportation conditions

[0127] The stability of the pharmaceutical composition of the present invention from Example 2, produced under GMP conditions, was investigated under simulated transportation conditions. The laboratory-scale simulated transportation experiment was conducted under cold chain conditions of 2–8°C. Shaking (250 rpm / min) was used to simulate the shear forces experienced by the product during transportation, and upright and inverted placement methods were used to simulate different placement states that the product might be in during actual transportation. Simultaneously, three cold chain removal stages were designed in the experiment, with cold chain removal conditions at a temperature of 40°C ± 2°C and a humidity of 75% RH ± 5%. The experimental cycle consisted of 2 days of simulated cold chain (2–8°C) transportation and 2 days of simulated cold chain removal (temperature 40°C ± 2°C, humidity 75% RH ± 5%); this constituted one cycle, and a total of 3 cycles were performed, for a total duration of 12 days.

[0128] The test results included properties, clarity, protein content, insoluble particles (photoresistance method), visible foreign matter, molecular-size heterogeneity (SEC-HPLC), charge heterogeneity (CEX-HPLC), and biological activity. After undergoing simulated transportation including cold chain removal, the above biochemical and physicochemical indicators of the sample did not change significantly compared with the sample before simulated transportation. The specific results are shown in Table 12 below.

[0129] Table 12: Stability under simulated transportation conditions

[0130] In addition, the stability of the pharmaceutical composition of the present invention produced under GMP conditions in Example 2 under long-term upright and inverted conditions at low temperature (2-8°C) was investigated in turn, and the results are shown in Tables 13 and 14 below.

[0131] Example 5 - Stability under simulated clinical dosing conditions

[0132] The stability of the pharmaceutical composition of the present invention, produced under GMP conditions in Example 2, was investigated under simulated clinical dosing conditions. Simulating clinical dosing procedures, appropriate amounts of the CD20 / CD3 bispecific antibody composition were injected into 250 ml of 0.9% sodium chloride injection to prepare high- and medium-concentration samples of 4.0 mg / ml and 0.5 mg / ml, respectively, according to clinically likely concentration ranges. Appropriate amounts of the CD20 / CD3 bispecific antibody composition were injected into 0.9% sodium chloride injection containing 10 ml of CD20 / CD3 bispecific antibody injection diluent to prepare a low-concentration sample of 0.04 mg / ml. Stability was investigated for 24 hours and 3 hours under two storage conditions: 2–8°C and room temperature / light. Infusion sets were connected at specified time points, and infusions were performed under room temperature / light to examine changes in product quality properties and assess product compatibility with the infusion system. The CD20 / CD3 bispecific antibody injection diluent is identical to the finished CD20 / CD3 bispecific antibody product except that it does not contain the active ingredient. It contains: 20mM histidine / histidine hydrochloride buffer, 140mM arginine hydrochloride, 10mM methionine, and 0.2mg / mL polysorbate 20. If necessary, the pH can be adjusted to 6.3 with sodium hydroxide or dilute hydrochloric acid.

[0133] As shown in the table, the CD20 / CD3 bispecific antibody product, after being diluted with physiological saline to a concentration of 4.0 mg / ml or 0.5 mg / ml, or after being diluted with CD20 / CD3 bispecific antibody injection diluent and physiological saline to a concentration of 0.04 mg / ml, can be stored in infusion bags at 2–8°C for 24 hours or at room temperature with light for 3 hours. Furthermore, it can be infused within 9 hours at room temperature with light using an infusion set with a 0.2 μm online filter, without affecting product quality. Regardless of the CD20 / CD3 bispecific antibody concentration (4.0 mg / ml, 0.5 mg / ml, or 0.04 mg / ml), the infusion system showed no adsorption of the product. Therefore, the composition of this invention exhibits good compatibility with commonly available infusion bags and sets.

[0134] Table 15: Product Compatibility Study with Infusion System

[0135] *Note: Bacterial endotoxin (EU / mL) result for medium and low concentration groups = test result (EU / mg) × protein content (mg / ml) Clinical use conditions A: Infusion bag: 5℃±3℃, 24 hours; Infusion set: room temperature and light for 9 hours.

[0136] Clinical use conditions B: Infusion bag: 3 hours at room temperature and light; Infusion set: 9 hours at room temperature and light.

[0137] As shown in Example 5, after dilution and infusion under simulated clinical use conditions, the protein concentration and SEC-HPLC purity of the CD20 / CD3 bispecific antibody composition of the present invention showed no significant changes, and the level of insoluble particles remained low. No protein instability caused by dilution was observed, demonstrating excellent stability. During clinical use, the product quality was not affected by processes and factors such as dilution, filtration, infusion, room temperature, and light, indicating that the formulation of the CD20 / CD3 bispecific antibody composition of the present invention is robust and reliable.

[0138] In summary, the CD20 / CD3 bispecific antibody drug composition of the present invention exhibits excellent stability under various conditions such as high temperature, acceleration, low temperature, shaking, transportation, freeze-thaw, and simulated clinical use, achieving unexpected technical effects and possessing significant clinical application value.

[0139] While various embodiments of the invention have been described above, it should be understood that they are provided by way of example only and not as limitations. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications will fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition comprising: (a) 5–40 mg / ml CD20 / CD3 bispecific antibody; (b) 10–50 mM buffer; (c) 100–300 mM protective agent; (d) 2-30 mM antioxidants; (e) 0.05–1.00 mg / ml surfactant; The buffer is selected from histidine / histidine hydrochloride, citric acid / sodium citrate, glutamic acid / monosodium glutamate, or acetic acid / sodium acetate; The protective agent is selected from one or more of polyols, sugars, and amino acids; The antioxidants mentioned therein are selected from one or more of amino acids and metal ion chelating agents; The surfactants mentioned therein are one or more of the following: polysorbates, poloxamers, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, glycerol fatty acid esters, polyethylene glycol fatty acid esters, and polyoxyethylene polyoxypropylene alkyl ethers; The CD20 / CD3 bispecific antibody comprises: The first heavy chain variable region (VH1) contains CDRs 1, 2 and 3, wherein the VH1 CDRs 1-3 each contain an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to SEQ ID NO.:1-3. The second heavy chain variable region (VH2) contains CDRs 1, 2 and 3, wherein the VH2 CDRs 1-3 each contain an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to that of SEQ ID NO.:4-6. The first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein CDRs 1-3 of VL1 each contain an amino acid sequence that is at least 80% identical, preferably at least 90% identical, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99% identical, and more preferably completely identical to that in SEQ ID NO.:7-9; and The second light chain variable region (VL2) comprises CDRs 1, 2, and 3, wherein each of the VL2 CDRs 1-3 contains at least 80% identical to, preferably at least 90% identical to, at least 97% identical to, more preferably at least 98% identical to, and more preferably at least 99% identical to, SEQ ID NO.:7-9. The same, or more preferably, identical amino acid sequences.

2. The pharmaceutical composition according to claim 1, wherein in the CD20 / CD3 bispecific antibody: The first heavy chain variable region (VH1) contains an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to, SEQ ID NO.:10; the second heavy chain variable region (VH2) contains an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to, SEQ ID NO.:12; and the first light chain variable region (VL1) and the second light chain variable region (VL2) contain an amino acid sequence that is at least 80% identical to, preferably at least 90% identical to, more preferably at least 97% identical to, more preferably at least 98% identical to, more preferably at least 99% identical to, and more preferably completely identical to, SEQ ID NO.:

12. Preferably, the full length of the CD20 heavy chain is as shown in sequence SEQ ID NO.:13; The full length of the CD3 heavy chain is shown in sequence SEQ ID NO.:14; The full length of the common light chain shared by both is shown in sequence SEQ ID NO.:

15.

3. The pharmaceutical composition according to claim 1, wherein the buffer is histidine / histidine hydrochloride; wherein the pH of the pharmaceutical composition is 5.5-7.0, preferably 6.0-7.0, more preferably 6.0-6.6, and most preferably 6.

3.

4. The pharmaceutical composition according to claim 1, wherein the protective agent is selected from one or more of trehalose, sucrose, arginine, glycine, and mannitol, preferably arginine, more preferably arginine hydrochloride.

5. The pharmaceutical composition according to claim 1, wherein the antioxidant is methionine or EDTA-2Na, preferably methionine.

6. The pharmaceutical composition according to claim 1, wherein the surfactant is polysorbate 20.

7. The pharmaceutical composition according to claim 1, comprising: -15~25mg / ml CD20 / CD3 bispecific antibody; -10~40mM histidine / histidine hydrochloride buffer; -110~200mM arginine hydrochloride; -3~25mM methionine; -0.10~0.50mg / ml polysorbate 20; Preferably, it comprises: -18~22mg / ml CD20 / CD3 bispecific antibody; -10~30mM histidine / histidine hydrochloride buffer; -112~168mM arginine hydrochloride; -5~20mM methionine; -0.10~0.30mg / ml polysorbate 20.

8. The pharmaceutical composition according to any one of claims 1-7, wherein it is an aqueous solution comprising the following components at a pH of 6.3: -20 mg / ml CD20 / CD3 bispecific antibody; -20mM histidine / histidine hydrochloride buffer; -140mM arginine hydrochloride; -10mM methionine; -0.20 mg / mL polysorbate 20.

9. Use of the pharmaceutical composition according to any one of claims 1-8 in the preparation of a medicament for treating cancer.

10. The use according to claim 9, wherein the cancer is chronic lymphocytic leukemia or non-Hodgkin's lymphoma.

11. A method of treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1-8.

12. The method of claim 11, wherein the cancer is chronic lymphocytic leukemia or non-Hodgkin's lymphoma.

13. The method according to claim 11 or 12, wherein the subject is a mammal, preferably a human.

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