A CD19 antibody drug formulation

Abstract

This application relates to a CD19 antibody pharmaceutical formulation. Specifically, this application relates to a pharmaceutical formulation of inelolizumab, the formulation comprising inelolizumab, a buffer solution, a stabilizer, and a surfactant. The pharmaceutical formulation of this application can prevent inelolizumab from aggregation, degradation, oxidation, or denaturation, thereby maintaining the biological activity of its active components and making it suitable for clinical use.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical formulations, and specifically relates to a pharmaceutical composition comprising a monoclonal antibody, and its use as a medicine in cancer, transplantation diseases and autoimmune diseases. Background Technology

[0002] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0003] Intravenous infusion of inelolizumab requires patients to use an intravenous infusion pump, with the infusion rate gradually increased, completing the infusion in approximately 90 minutes. Patients' infusion response must be monitored during the infusion, and observation should continue for at least one hour after completion. In contrast, subcutaneous injection can be completed in just 5-10 minutes, offering a significantly more convenient treatment experience for patients receiving anti-CD19 therapy.

[0004] Therefore, in order to solve the problems of long dosing time and low patient compliance, as well as to facilitate clinical use and reduce risks, there is an urgent need for an inelizumab formulation with excellent stability. Summary of the Invention

[0005] The problem this application aims to solve is to provide a stable, subcutaneously injectable pharmaceutical formulation containing a high concentration of inelolizumab.

[0006] The inelolizumab and hyaluronidase of this application can form a stable formulation that allows for subcutaneous injection at higher volumes. First, the preparation of high-concentration antibody formulations is challenging; high protein concentrations result in significant viscosity, increasing the risk of aggregation. Second, maintaining a high antibody concentration and achieving a certain level of stability while incorporating hyaluronidase is very difficult. Furthermore, although antibodies or their antigen-binding fragments have very similar overall structures, they differ in amino acid composition and glycosylation, and each antibody or its antigen-binding fragment exhibits different aggregation behaviors. Therefore, obtaining a stable formulation targeting a specific antibody or its antigen-binding fragment, especially one containing hyaluronidase, is unpredictable. This application provides a stable pharmaceutical formulation containing a high concentration of inelolizumab, which can be used for subcutaneous injection.

[0007] The first aspect of this application provides a CD19 antibody drug formulation comprising:

[0008] i) An anti-CD19 antibody or its antigen-binding fragment, said anti-CD19 antibody or its antigen-binding fragment comprising heavy chains CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and light chains CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and

[0009] ii) Hyaluronidase, wherein the hyaluronidase content is 1,000 to 20,000 U / mL; preferably, the hyaluronidase content is 1,000 to 10,000 U / mL; more preferably, the hyaluronidase content is 1,000 to 5,000 U / mL; and even more preferably, the hyaluronidase content is about 1,000 U / mL, about 2,000 U / mL, about 3,000 U / mL, or about 4,000 U / mL.

[0010] In some embodiments, the anti-CD19 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 7, and the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 8.

[0011] In a preferred embodiment, the anti-CD19 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the sequence of which is shown in SEQ ID NO: 7 and the sequence of which is shown in SEQ ID NO: 8.

[0012] In some embodiments, the anti-CD19 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 9, and the light chain comprising an amino acid sequence as shown in SEQ ID NO: 10.

[0013] In a preferred embodiment, the anti-CD19 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain sequence being as shown in SEQ ID NO: 9, and the light chain sequence being as shown in SEQ ID NO: 10.

[0014] In a preferred embodiment, the anti-CD19 antibody or its antigen-binding fragment is inelizumab.

[0015] In some embodiments, the concentration of the anti-CD19 antibody or its antigen-binding fragment is from 1 mg / mL to 250 mg / mL.

[0016] In a preferred embodiment, the concentration of the anti-CD19 antibody or its antigen-binding fragment is from 50 mg / mL to 200 mg / mL. In a preferred embodiment, the concentration of the anti-CD19 antibody or its antigen-binding fragment is from 100 mg / mL to 200 mg / mL. In a preferred embodiment, the concentration of the anti-CD19 antibody or its antigen-binding fragment is from 120 mg / mL to 180 mg / mL.

[0017] In some embodiments, the CD19 antibody drug formulation further comprises a buffer solution, wherein the buffer solution is one or more of histidine buffer, acetate buffer, phosphate buffer, citrate buffer, and succinate buffer.

[0018] In a preferred embodiment, the buffer solution is one or more of histidine-histidine hydrochloride, acetate-sodium acetate, sodium dihydrogen phosphate-disodium hydrogen phosphate, and citric acid-sodium citrate. In a preferred embodiment, the buffer solution is an acetate-sodium acetate buffer solution.

[0019] In some embodiments, the concentration of the buffer solution is from 1 mM to 100 mM.

[0020] In a preferred embodiment, the concentration of the buffer solution is from 5 mM to 50 mM. In a preferred embodiment, the concentration of the buffer solution is from 10 mM to 40 mM. In a preferred embodiment, the concentration of the buffer solution is about 10 mM, about 20 mM, about 30 mM, or about 40 mM.

[0021] In some embodiments, the buffer solution is an acetate-sodium acetate buffer solution with a concentration of 5 mM to 50 mM.

[0022] In a preferred embodiment, the buffer solution is an acetate-sodium acetate buffer solution with a concentration of 10 mM to 40 mM. In another preferred embodiment, the buffer solution is an acetate-sodium acetate buffer solution with a concentration of approximately 10 mM, approximately 20 mM, approximately 30 mM, or approximately 40 mM.

[0023] In some embodiments, the pH of the CD19 antibody drug formulation is 5.0 to 7.0.

[0024] In a preferred embodiment, the pH of the CD19 antibody drug formulation is 5.0 to 6.5. In a preferred embodiment, the pH of the CD19 antibody drug formulation is 5.0 to 6.0. In a preferred embodiment, the pH of the CD19 antibody drug formulation is 5.5 to 6.0. In a preferred embodiment, the pH of the CD19 antibody drug formulation is approximately 5.5.

[0025] In some embodiments, the buffer solution is an acetate-sodium acetate buffer solution with a concentration of 5 mM to 50 mM, and the pH of the CD19 antibody drug formulation is 5.0 to 7.0.

[0026] In a preferred embodiment, the buffer solution is an acetate-sodium acetate buffer solution with a concentration of 10 mM to 40 mM, and the pH of the CD19 antibody drug preparation is 5.0 to 6.0. In a preferred embodiment, the buffer solution is an acetate-sodium acetate buffer solution with a concentration of 10 mM to 40 mM, and the pH of the CD19 antibody drug preparation is 5.5 to 6.0. In a preferred embodiment, the buffer solution is an acetate-sodium acetate buffer solution with a concentration of about 10 mM, about 20 mM, about 30 mM, or about 40 mM, and the pH of the CD19 antibody drug preparation is about 5.5.

[0027] In some embodiments, the CD19 antibody drug formulation further comprises a stabilizer selected from sugars or polyols.

[0028] In a preferred embodiment, the stabilizer is selected from one or more of trehalose, sucrose, lactose, glucose, mannitol, and sorbitol. In a preferred embodiment, the stabilizer is selected from one or more of trehalose, sucrose, or sorbitol. In a preferred embodiment, the stabilizer is selected from sorbitol.

[0029] In some embodiments, the stabilizer concentration is from 20 mg / mL to 150 mg / mL.

[0030] In a preferred embodiment, the stabilizer concentration is from 30 mg / mL to 100 mg / mL. In a preferred embodiment, the stabilizer concentration is from 35 mg / mL to 55 mg / mL or from 50 mg / mL to 10 mg / mL. In a preferred embodiment, the stabilizer concentration is about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, or about 80 mg / mL.

[0031] In some embodiments, the stabilizer is selected from one or more of sucrose or sorbitol, and the stabilizer concentration is from 30 mg / mL to 100 mg / mL.

[0032] In a preferred embodiment, the stabilizer is selected from sorbitol, and the stabilizer concentration is from 30 mg / mL to 100 mg / mL. In a preferred embodiment, the stabilizer is selected from sorbitol, and the stabilizer concentration is from 35 mg / mL to 55 mg / mL. In a preferred embodiment, the stabilizer is selected from sorbitol, and the stabilizer concentration is about 35 mg / mL, about 40 mg / mL, or about 55 mg / mL.

[0033] In some embodiments, the CD19 antibody drug formulation further comprises an antioxidant selected from at least one of methionine, ascorbic acid, citric acid, and tartaric acid. In a preferred embodiment, the antioxidant is selected from methionine.

[0034] In some embodiments, the concentration of the antioxidant is from 1 mM to 50 mM.

[0035] In a preferred embodiment, the concentration of the antioxidant is from 5 mM to 50 mM. In a preferred embodiment, the concentration of the antioxidant is from 5 mM to 20 mM. In a preferred embodiment, the concentration of the antioxidant is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM.

[0036] In some embodiments, the antioxidant is selected from at least one of methionine, ascorbic acid, citric acid, and tartaric acid, and the concentration of the antioxidant is from 1 mM to 50 mM.

[0037] In a preferred embodiment, the antioxidant is selected from methionine, and the concentration of the antioxidant is from 5 mM to 50 mM. In a preferred embodiment, the antioxidant is selected from methionine, and the concentration of the antioxidant is from 5 mM to 20 mM. In a preferred embodiment, the antioxidant is selected from methionine, and the concentration of the antioxidant is about 5 mM, about 10 mM, or about 15 mM.

[0038] In some embodiments, the CD19 antibody drug formulation further comprises a surfactant, said surfactant being polysorbate, poloxamer, or a combination thereof;

[0039] In a preferred embodiment, the surfactant is selected from one or more of polysorbate 80, polysorbate 20, polysorbate 40, or polysorbate 60. In a preferred embodiment, the surfactant is selected from polysorbate 80.

[0040] In some embodiments, the surfactant concentration is from 0.01 mg / mL to 1.0 mg / mL.

[0041] In a preferred embodiment, the concentration of the surfactant is from 0.1 mg / mL to 0.8 mg / mL. In a preferred embodiment, the concentration of the surfactant is from 0.2 mg / mL to 0.6 mg / mL. In a preferred embodiment, the concentration of the surfactant is about 0.2 mg / mL, about 0.4 mg / mL, or about 0.6 mg / mL.

[0042] In some embodiments, the surfactant is selected from one or more of polysorbate 80, polysorbate 20, polysorbate 40, or polysorbate 60, and the surfactant concentration is from 0.01 mg / mL to 1.0 mg / mL.

[0043] In a preferred embodiment, the surfactant is selected from one or more of polysorbate 80, polysorbate 20, polysorbate 40, or polysorbate 60, and the concentration of the surfactant is from 0.1 mg / mL to 0.8 mg / mL. In a preferred embodiment, the surfactant is selected from polysorbate 80, and the concentration of the surfactant is from 0.2 mg / mL to 0.6 mg / mL. In a preferred embodiment, the surfactant is selected from polysorbate 80, and the concentration of the surfactant is about 0.2 mg / mL, about 0.4 mg / mL, or about 0.6 mg / mL.

[0044] In some embodiments, the CD19 antibody drug formulation comprises the following components: (a) 50 mg / mL to 200 mg / mL inoleizumab, (b) 1 mM to 100 mM buffer, (c) 0.01 mg / mL to 1.0 mg / mL surfactant, (d) 20 mg / mL to 150 mg / mL stabilizer, (e) 1000 to 20000 U / mL hyaluronidase, and (f) 1 mM to 50 mM antioxidant, wherein the pH of the CD19 antibody drug formulation is 5.0 to 7.0.

[0045] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 100 mg / mL to 200 mg / mL inoleizumab, (b) 5 mM to 50 mM buffer, (c) 0.1 mg / mL to 0.8 mg / mL surfactant, (d) 30 mg / mL to 100 mg / mL stabilizer, (e) 1000 to 10000 U / mL hyaluronidase, and (f) 5 mM to 50 mM antioxidant, wherein the pH of the CD19 antibody drug formulation is 5.0 to 6.5.

[0046] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM to 40 mM buffer, (c) 0.2 mg / mL to 0.6 mg / mL surfactant, (d) 35 mg / mL to 55 mg / mL stabilizer, (e) 1000 to 5000 U / mL hyaluronidase, and (f) 5 mM to 20 mM antioxidant, wherein the pH of the CD19 antibody drug formulation is 5.0 to 6.0.

[0047] In some embodiments, the CD19 antibody drug formulation comprises the following components: (a) 100 mg / mL to 200 mg / mL inoleizumab, (b) 5 mM to 50 mM acetate buffer, (c) 0.1 mg / mL to 0.8 mg / mL polysorbate 80, (d) 30 mg / mL to 100 mg / mL sucrose or sorbitol, (e) 1000 to 10000 U / mL hyaluronidase, and (f) 5 mM to 50 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.0 to 7.0.

[0048] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM to 40 mM acetate buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate, (d) 35 mg / mL to 55 mg / mL sucrose or sorbitol, (e) 1000 to 5000 U / mL hyaluronidase, and (f) 5 mM to 20 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.0 to 6.5.

[0049] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM to 40 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate 80, (d) 35 mg / mL to 55 mg / mL sorbitol, (e) 1000 to 5000 U / mL hyaluronidase, and (f) 5 mM to 20 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.0 to 6.5.

[0050] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) about 10 mM, about 20 mM or about 30 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate 80, (d) 35 mg / mL to 55 mg / mL sorbitol, (e) about 2000 U / mL, about 3000 U / mL or about 4000 U / mL hyaluronidase, and (f) 5 mM to 15 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.0 to 6.5.

[0051] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) about 20 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate 80, (d) 35 mg / mL to 55 mg / mL sorbitol, (e) about 2000 U / mL hyaluronidase, and (f) 5 mM to 15 mM methionine, wherein the pH of the CD19 antibody drug formulation is about 5.5.

[0052] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate 80, (d) 35 mg / mL to 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, and (f) 5 mM to 15 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.5.

[0053] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate 80, (d) 35 mg / mL to 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, and (f) 5 mM to 15 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.5.

[0054] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL polysorbate 80, (d) 35 mg / mL to 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, and (f) 5 mM to 15 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.5.

[0055] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL polysorbate 80, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, and (f) 10 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.5.

[0056] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 120 mg / mL inelolizumab, (b) 20 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL polysorbate 80, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, and (f) 10 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.5.

[0057] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components: (a) 150 mg / mL inelolizumab, (b) 20 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL polysorbate 80, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, and (f) 10 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.5.

[0058] A second aspect of this application provides a CD19 antibody drug formulation comprising: i) inelizumab, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 9 and the light chain amino acid sequence of which is shown in SEQ ID NO: 10, wherein the concentration of inelizumab is from 50 mg / mL to 200 mg / mL; ii) hyaluronidase, wherein the hyaluronidase content is from 1000 to 5000 U / mL; iii) 10 mM to 40 mM acetate-sodium acetate buffer; iv) 35 mg / mL to 55 mg / mL sorbitol and 5 mM to 15 mM methionine; v) 0.2 mg / mL to 0.6 mg / mL polysorbate 80; wherein the pH of the CD19 antibody drug formulation is from 5.0 to 6.0.

[0059] In some embodiments, the hyaluronidase content is 1000 U / mL, 2000 U / mL, 3000 U / mL, or 4000 U / mL.

[0060] In some embodiments, the concentration of the inelizumab is from 100 mg / mL to 200 mg / mL.

[0061] In a preferred embodiment, the concentration of inelizumab is from 120 mg / mL to 180 mg / mL.

[0062] In a preferred embodiment, the concentration of the acetate-sodium acetate buffer solution is 10 mM, 20 mM, 30 mM or 40 mM.

[0063] In a preferred embodiment, the pH of the CD19 antibody drug formulation is 5.5.

[0064] In some embodiments, the sorbitol concentration is 35 mg / mL, 40 mg / mL, or 55 mg / mL; and the methionine concentration is 5 mM, 10 mM, or 15 mM.

[0065] In a preferred embodiment, the sorbitol concentration is 40 mg / mL and the methionine concentration is 10 mM; the sorbitol concentration is 35 mg / mL and the methionine concentration is 15 mM; the sorbitol concentration is 55 mg / mL and the methionine concentration is 5 mM.

[0066] In some embodiments, the concentration of the polysorbate 80 is 0.2 mg / mL, 0.4 mg / mL, or 0.6 mg / mL.

[0067] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components:

[0068] (a) 100 mg / mL to 200 mg / mL inoleizumab, (b) 10 mM, 20 mM or 30 mM acetate-sodium acetate buffer, (c) 0.2 mg / mL, 0.4 mg / mL or 0.6 mg / mL polysorbate 80, (d) 35 mg / mL, 40 mg / mL or 55 mg / mL sorbitol, (e) 2000 U / mL, 3000 U / mL or 4000 U / mL hyaluronidase, (f) 5 mM, 10 mM or 15 mM methionine, wherein the pH of the CD19 antibody formulation is 5.0 to 6.0.

[0069] In a preferred embodiment, the CD19 antibody drug formulation comprises the following components:

[0070] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0071] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0072] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0073] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0074] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0075] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0076] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 30 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0077] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 30 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0078] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 30 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0079] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.4 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0080] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.4 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0081] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.4 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or

[0082] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0083] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0084] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0085] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0086] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0087] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 10 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0088] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 30 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0089] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 30 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0090] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 30 mM acetate buffer, (c) 0.2 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0091] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.4 mg / mL polysorbate, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0092] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.4 mg / mL polysorbate, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 6.0; or

[0093] (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.4 mg / mL polysorbate, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody drug formulation is 6.0.

[0094] A third aspect of this application provides a container containing the aforementioned CD19 antibody drug formulation; preferably, the container is a tube or vial.

[0095] A fourth aspect of this application provides a medicine box comprising the aforementioned container or the aforementioned subcutaneous delivery device.

[0096] The fifth aspect of this application provides a method for reducing drug infusion time, the method comprising subcutaneous administration of the aforementioned CD19 antibody drug preparation or the aforementioned drug cartridge.

[0097] The sixth aspect of this application provides the use of the aforementioned CD19 antibody drug formulation or the aforementioned kit in the preparation of a drug for treating B-cell diseases or conditions.

[0098] In a preferred embodiment, the B-cell disease or condition is selected from B-cell malignancies, autoimmune diseases, humoral rejection in human transplant patients, graft-versus-host disease (GVHD), and post-transplant lymphoproliferative disorders in human graft recipients.

[0099] In a preferred embodiment, the autoimmune disease is selected from neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis (MG), and immunoglobulin G4-related disease (IgG4-RD).

[0100] As is well known to those skilled in the art, one, some, or all of the features of the various embodiments described in this disclosure can be further combined to form other embodiments of this disclosure. The above embodiments of this disclosure and other embodiments obtained by combination are further described in detail below.

[0101] the term

[0102] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0103] The “mM” mentioned in this disclosure specifically refers to “mmol / L”.

[0104] As used herein, the terms “about” and “approximately” mean a numerical value within the acceptable margin of error of a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or above 1 standard deviation. Alternatively, “about” or “substantially includes” may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially includes” should be assumed to be within the acceptable margin of error of that specific value.

[0105] When point values ​​are mentioned in this disclosure, it should be understood that the point value includes an error range. This error range is due to factors such as laboratory environment, personnel operation, instruments, methodology, and measurement errors.

[0106] Hyaluronidase is an enzyme that degrades hyaluronic acid and reduces its viscosity in the extracellular matrix, thereby increasing tissue permeability. Optionally, the hyaluronidase is manufactured using recombinant DNA technology, derived from human samples, or extracted from animal tissue. In this disclosure, hyaluronidase has the common meaning in the art, including natural hyaluronidases, recombinant hyaluronidases, or variants thereof derived from animals, such as hyaluronidases derived from rats, mice, rabbits, cats, dogs, monkeys, gorillas, humans, or other primates. Many suitable hyaluronidases are known, with preferred enzymes being human hyaluronidases, such as soluble human PH20 hyaluronidase, preferably recombinant human hyaluronidase referred to as rHuPH20. Exemplary human hyaluronidases are described in NCBI Reference Sequence: NP_001167515.1, and exemplary recombinant human hyaluronidases are truncated or mutant variants of human hyaluronidase.

[0107] The enzyme activity of hyaluronidase (including rHuPH20) can be defined in units / mL (U / mL) or in total enzyme activity (U) in a particular formulation. The standard definition of one unit of enzyme activity (U) is the amount of enzyme that catalyzes a predetermined amount of substrate reaction per unit time, such as one mole or one nanomole of substrate per minute. Techniques for determining the activity of hyaluronidase formulations are known in the art, and hyaluronidase activity is generally expressed in USP units or units; hereinafter, "U / mL" will be used to represent hyaluronidase activity.

[0108] A common characteristic of all hyaluronidases is their ability to depolymerize acetylated hyaluronic acid, regardless of chemical structure, species origin, tissue origin, or batch differences in pharmaceutical products derived from the same species and tissue. What distinguishes them is that, despite their different structures, their activities are identical (except for potency). The hyaluronidases in the formulations according to this disclosure are characterized by having no adverse effect on the molecular integrity of the antibody or its antigen-binding fragment in the stable pharmaceutical composition described in this application. Furthermore, the hyaluronidases only alter the delivery of the antibody or its antigen-binding fragment into the systemic circulation, but do not possess any properties that provide or facilitate the therapeutic effect of the antibody or its antigen-binding fragment for systemic absorption.

[0109] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0110] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, antigen-binding fragments, bispecific or heterospecific antibodies, dimerizing, tetrameric or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen-binding site with desired specificity, provided they exhibit the desired biological activity. Typically, a natural, intact antibody is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0111] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0112] "Buffer solution" refers to a buffer that is resistant to pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0113] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., with histidine-acetate buffer being preferred. Histidine-acetate buffer is prepared by reacting histidine with acetic acid, and histidine-hydrochloride buffer is prepared by reacting histidine with hydrochloric acid.

[0114] "Citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc. A preferred citrate buffer is sodium citrate.

[0115] "Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include sodium succinate, potassium succinate, and calcium succinate. A preferred succinate buffer is sodium succinate. Exemplarily, the sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0116] Phosphate buffer is a buffer that contains phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0117] "Acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include sodium acetate, histidine acetate, potassium acetate, calcium acetate, magnesium acetate, etc. Sodium acetate is a preferred acetate buffer.

[0118] Poloxamer is a block copolymer of ethylene oxide and propylene oxide. It is water-soluble and used as a surfactant in pharmaceutical formulations. Examples of poloxamer include poloxamer 188 (P188).

[0119] The term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof or physiologically / pharmacologically acceptable salts or prodrugs described herein, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the antibody's active ingredient, facilitate administration to the organism, and enhance the absorption of the active ingredient to exert its biological activity.

[0120] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0121] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0122] The pharmaceutical compositions described in this disclosure achieve a stable effect: the antibody or its antigen-binding fragment therein substantially retains its physical and / or chemical stability and / or biological activity after storage; preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0123] A stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, and 6 months. Typical examples of stability include: aggregation or degradation of antibody monomers typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and osmotic pressure of the formulation exhibit variations not exceeding ±10%. A reduction of not more than about 10%, preferably not more than about 5%, is typically observed. Aggregation typically forms at a rate not exceeding about 10%, preferably not more than about 5%.

[0124] The term "stable" means that all proteins in a formulation retain essentially their physical, chemical, and biological activity after storage at the intended storage temperature, such as 0-40°C. A formulation may be considered stable even if the antibodies in it do not retain 100% of their physical, chemical, and biological activity after a certain storage period. A formulation is considered "stable" if it retains approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% of its antibody structure and function after a certain storage period.

[0125] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody or its antigen-binding fragment does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody or its antigen-binding fragment "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0126] If an antibody or its antigen-binding fragment does not exhibit significant chemical changes, then the antibody or its antigen-binding fragment "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0127] If the biological activity of an antibody or its antigen-binding fragment at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody or its antigen-binding fragment "retains its biological activity" in the pharmaceutical formulation.

[0128] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the possibility that the event or circumstances may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but do not have to, be present.

[0129] For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0130] The term "carrier" is used in the context of the drugs disclosed herein, referring to a system that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferably such as micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

[0131] The terms “administer” and “treatment” when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. “Administer” and “treatment” can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. “Administer” and “treatment” also mean, for example, the treatment of cells in vitro and in vitro with a reagent, diagnostic agent, conjugate composition, or another cell. When applied to humans, veterinary, or research subjects, “treatment” refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0132] The term "treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical testing method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0133] The term "effective amount" refers to an amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity. Detailed Implementation

[0134] The following examples are provided to offer a complete disclosure and illustration of how to prepare and use the pharmaceutical formulations of the present invention for those skilled in the art, but do not constitute a limitation on the scope of the invention.

[0135] This application discloses a stable pharmaceutical composition comprising an anti-CD19 antibody or an antigen-binding fragment thereof. The anti-CD19 antibody described in this application is inelolizumab, a humanized, affinity-optimized, fucosylated IgG1κ monoclonal antibody that binds to the B cell surface antigen CD19. Inelolizumab and its preparation method are described in international PCT patent application PCT / US2007 / 077916, published as WO2008 / 031056, which is hereby incorporated by reference. PCT / US2007 / 077916 refers to the anti-CD19 antibody as "16C4". This application uses the anti-CD19 antibody inelizumab to generate the experimental results provided in this application. The inelizumab described in this application comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 07, a light chain variable region having the amino acid sequence of SEQ ID NO: 08, and an Fc region having an N-glycoside-linked complex glycan, wherein fucose is not bound to N-acetylglucosamine at the reducing end of the glycan (see, U.S. Patent Application 11 / 852,106, filed September 7, 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes). The inelizumab described in this application comprises a heavy chain having the amino acid sequence of SEQ ID NO: 09 and a light chain having the amino acid sequence of SEQ ID NO: 10. A standard industrial-scale protocol is used to produce purified anti-CD19 antibody. Specific sequences are shown in Tables A and B.

[0136] Table A. Inelolizumab CDR and Variable Region Sequence Information

[0137]

[0138] Note: CDR sequences are defined using the Kabat definition scheme.

[0139] Table B. Full-length sequence information of inelizumab

[0140]

[0141] The hyaluronidase used in the specific embodiments of this application is recombinant human hyaluronidase rHuPH20, CAS757971-58-7, catalog number GMP-2501-100KU, purchased from Suzhou Nearshore Protein Technology Co., Ltd.

[0142] Description of the analytical tests used in this application

[0143] 1. Appearance and visible foreign objects

[0144] The appearance of the sample is observed visually, including color, clarity, and visible foreign matter. The specific procedure is as follows: Wipe the sample bottle clean, and use water as a negative control to observe the appearance: 1) In a dark room, place the sample under a clarity meter, adjust the illuminance to 2000-3750 Lx, place the sample at the edge of the light shield, hold the neck of the sample vial, and observe the color, clarity, and visible foreign matter of the sample against black and white backgrounds respectively; 2) Under fluorescent light, with a white background, visually inspect the color of the sample.

[0145] 2. Protein concentration

[0146] Using a NanoPhotometer NP80 protein analyzer, 2.5 μL of sample was added, and the absorbance value of the sample at a wavelength of 280 nm was measured. Each sample was measured twice, and the average value was taken.

[0147] 3. pH value

[0148] The electrode slope was measured using a Mettler S470-K multi-parameter instrument with three standard solutions (pH values ​​of 4.01, 7.00, and 9.21) to ensure the electrode slope was within the range of 95%-105%. A 100 μL sample was then taken to measure the pH value.

[0149] 4. Hyaluronidase activity assay

[0150] Hyaluronidase activity in the formulation was determined using a SpectraMax M5 multifunctional microplate reader. Acidic bovine serum albumin precipitates hyaluronic acid. After the reaction of hyaluronidase in the antibody formulation with hyaluronic acid, acidic bovine serum albumin was added to precipitate undigested hyaluronic acid. Turbidity measured at 640 nm after the reaction was used to evaluate enzyme activity.

[0151] 5. Microfluidic particle imaging analysis (MFI)

[0152] The number and morphology of particles in the sample were determined using a microfluidic particle imaging (MFI) system. Automated sample introduction was employed.

[0153] 6. Size Exclusion Chromatography (SEC-HPLC)

[0154] The purity of the sample was determined according to Appendix IIIB of the Pharmacopoeia of the People's Republic of China (2010 edition, Part III), using a hydrophilic silica size exclusion column, and the purity was calculated by the area normalization method.

[0155] 7. Detection of Charge Variants (CEX-HPLC)

[0156] The determination was carried out in accordance with Appendix IIIB of the Pharmacopoeia of the People's Republic of China (2010 edition, Part III). The samples were tested using a weak cation analysis column, and the acidity, baseness and main component purity of the samples were calculated by the area normalization method.

[0157] Example 1. Effect of buffer system on the stability of inelolizumab formulation

[0158] The following prescription injection solutions were prepared. Prescription 1 is based on the original low-concentration IV anti-CD19 antibody prescription, with the addition of hyaluronidase to increase the antibody concentration. Together with prescriptions 2-7, the effects of different buffer systems on antibody stability were investigated.

[0159] Prescription 1: 120 mg / mL inelolizumab, 10 mM histidine-histidine hydrochloride, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 6.0;

[0160] Prescription 2: 120 mg / mL inelolizumab, 20 mM sodium acetate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 6.0;

[0161] Prescription 3: 120 mg / mL inelizumab, 20 mM acetate-sodium acetate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0162] Prescription 4: 120 mg / mL inelizumab, 20 mM sodium citrate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 6.0;

[0163] Prescription 5: 120 mg / mL inelizumab, 20 mM sodium citrate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0164] Prescription 6: 120 mg / mL inelizumab, 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 6.0;

[0165] Prescription 7: 120 mg / mL inelizumab, 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 6.5.

[0166] The above-mentioned formulation was placed in an incubator, and samples were taken at 0, 1, 2, and 4 weeks to examine its stability. Antibody stability and enzyme activity stability under high-temperature conditions were compared to determine a suitable buffer system. Protein stability was accelerated by testing at 40°C; however, since 40°C damages hyaluronidase activity, enzyme activity stability was assessed at 30°C.

[0167] Table 1. Summary of key data for screening buffer systems

[0168]

[0169]

[0170] The buffer system screening data in Table 1 show that after one week of storage at 40℃, no visible particles were observed in any of the formulations. After two weeks, visible particles were observed in formulations 1 and 7, while the remaining formulations remained in good condition. SEC purity data shows that the change in the main peak was: Formulation 3 < Formulation 2 < Formulation 5 < Formulation 4 < Formulation 7 ≈ Formulation 1 ≈ Formulation 6. CEX purity data shows that the change in the main peak was: Formulation 3 < Formulation 5 < Formulation 6 ≈ Formulation 4 < Formulation 2 < Formulation 1 < Formulation 7. The decrease in hyaluronidase activity across all groups was within an acceptable range and showed no significant difference.

[0171] Stability results showed that the original low-concentration (IV) formulation of inelizumab was no longer suitable for the high-concentration (SC) formulation. Among the various buffer systems, the acetate-sodium acetate buffer system exhibited excellent stability, while studies revealed that high pH values ​​were detrimental to its stability. Therefore, the preferred buffer system was the acetate-sodium acetate system at pH 5.5.

[0172] Example 2. Screening experiment for stabilizers and antioxidants in inelolizumab formulation.

[0173] Samples containing different stabilizers were prepared, and the effects of different stabilizers on antibody stability were investigated under the same conditions (buffer solution was 20 mM acetate-sodium acetate, pH 5.5).

[0174] Prescription 3: 120 mg / mL inelizumab, 20 mM acetate-sodium acetate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0175] Prescription 8: 120 mg / mL inelolizumab, 20 mM sodium acetate, 10 mM methionine, 80 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0176] Prescription 9: 120 mg / mL inelolizumab, 20 mM sodium acetate, 10 mM methionine, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0177] Prescription 10: 120 mg / mL inelolizumab, 20 mM sodium acetate, 10 mM methionine, 40 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0178] Prescription 11: 120 mg / mL inelolizumab, 20 mM sodium acetate, 0.005% EDTA, 80 mg / mL trehalose, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0179] Prescription 12: 120 mg / mL inelizumab, 20 mM acetate-sodium acetate, 0.005% EDTA, 40 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5.

[0180] The above-mentioned formulation was placed in an incubator, and samples were taken at 0, 1, 2, and 4 weeks to examine its stability. The antibody stability and enzyme activity stability under high-temperature conditions were compared to determine a suitable stabilizer.

[0181] Table 2. Summary of key data for stabilizer screening

[0182]

[0183]

[0184] Table 3. Summary of Protein Particle Count (MFI) for Stabilizer Screening

[0185]

[0186] Table 2 shows the stabilizer screening data. After 4 weeks at 40℃, formulations 8-10 showed no visible particles and exhibited good appearance. Formulations 11 and 12 showed visible particles. SEC purity data showed the following variation in peak value: Formulation 10 < Formulation 9 < Formulation 3 < Formulation 8 ≈ Formulation 12 ≈ Formulation 11. CEX purity data showed the same variation in peak value: Formulation 10 < Formulation 9 < Formulation 3 < Formulation 12 < Formulation 8 < Formulation 11. The decrease in hyaluronidase activity across all groups was within acceptable limits and showed no significant difference.

[0187] Table 3 shows that the MFI (Mean Function Index) data for protein particles indicates that the antibody formulation is more stable when the stabilizer sorbitol and the antioxidant methionine are present in combination, with sorbitol showing better stability than trehalose and sucrose, while EDTA is detrimental to its stability. Therefore, the optimal combination is 40 mg / mL sorbitol as the stabilizer and 10 mM methionine as the antioxidant.

[0188] Example 3. Screening experiment for stabilizer and antioxidant content in inelolizumab formulation.

[0189] Based on the preliminary screening in Examples 1 and 2, the preferred formulation 10 consisted of 20 mM sodium acetate, 40 mg / mL sorbitol, 10 mM methionine, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, and pH 5.5. Further screening was conducted to investigate the effect of methionine concentration on antibody stability. The screening protocol is as follows:

[0190] Prescription 10: 120 mg / mL inelolizumab, 20 mM sodium acetate, 10 mM methionine, 40 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0191] Prescription 13: 120 mg / mL inelizumab, 20 mM sodium acetate, 5 mM methionine, 55 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0192] Prescription 14: 120 mg / mL inelizumab, 20 mM sodium acetate, 15 mM methionine, 35 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5.

[0193] The stability test was conducted at a high temperature of 40°C using accelerated testing. The protein concentration and final formulation were determined based on the stability results. See the table below for detailed stability data.

[0194] Table 4: Summary of Key Data for Antioxidant Content Screening

[0195]

[0196] The antioxidant content screening data in Table 4 show that after 4 weeks of storage at 40℃, the appearance of each group of formulations was good. The trends of stability data were basically consistent, with no significant overall difference. Sorbitol at 35-55 mg / mL and methionine at 5-15 mM can both serve as effective stabilizers.

[0197] Example 4. Screening of Inelizumab Concentration

[0198] Based on the preliminary screening in Examples 1 and 2, the preferred formulation composition is 20 mM acetate-sodium acetate, 40 mg / mL sorbitol, 10 mM methionine, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5. Further screening studies expanded the protein concentration to 150 mg / mL and 180 mg / mL. The screening protocol is as follows:

[0199] Prescription 10: 120 mg / mL inelolizumab, 20 mM sodium acetate, 10 mM methionine, 40 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0200] Prescription 15: 150 mg / mL inelizumab, 20 mM sodium acetate, 10 mM methionine, 40 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5;

[0201] Prescription 16: 180 mg / mL inelizumab, 20 mM sodium acetate, 10 mM methionine, 40 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, pH 5.5.

[0202] The stability test was conducted at a high temperature of 40°C using accelerated testing. The protein concentration and final formulation were determined based on the stability results. See the table below for detailed stability data.

[0203] Table 5: Summary of Key Data for Antibody Concentration Screening

[0204]

[0205]

[0206] Table 5 shows the antibody concentration screening data. After 4 weeks at 40℃, the appearance of each formulation was good, and the stability data showed a generally consistent trend. The polymer content at a protein concentration of 180 mg / mL increased slightly after 4 weeks, but remained within the acceptable range. The protein concentration of 150 mg / mL showed good stability; considering the friendliness to downstream processes, a protein concentration of 120.0 mg / mL was adopted.

[0207] Example 5. Confirmation of the prescription for subcutaneous injection of inelolizumab

[0208] Based on the prescription screening results, the stability of the preferred prescription of 120 mg / mL inelolizumab, 20 mM sodium acetate, 10 mM methionine, 40 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, hyaluronidase 2000 U / mL, and pH 5.5 under different conditions was verified, and the stability of hyaluronidase was verified simultaneously.

[0209] Inelizumab was ultrafiltered into a 20mM acetate-sodium acetate pH 5.5 buffer system, and excipients were added to prepare the final formulation. In a biosafety cabinet, using a 0.22 µm disposable sterile filter, the sample was aseptically aliquoted into 1.0 mL vials into 3.0 mL vials and capped with a rolled aluminum-plastic composite cap.

[0210] 1) Long-term stability assessment

[0211] The long-term stability was investigated by placing the samples at 25℃ and 5℃ for 3 months. The results are shown in Table 6 below.

[0212] The long-term stability study results in Table 6 show that, compared with the previous prescription screening, there was no significant decline in quality, and the data performed well in terms of appearance, purity, protein particle count, and enzyme activity.

[0213] Table 6. Results of Long-Term Stability Study

[0214]

[0215]

[0216] 2) Vibration factors

[0217] This study investigated the stability of the vibration at 25℃ and 200rpm. The results are detailed in Table 7.

[0218] The shaking stability test data in Table 7 show that, compared with the static test at 25℃, after shaking for 2 weeks, there is no significant difference in basic physicochemical properties, appearance, purity and control, indicating good shaking stability and low risk of being affected by shaking during long-distance transportation.

[0219] Table 7. Stability test of shaking at 25℃ and 200rpm

[0220]

[0221] 3. Freeze-thaw factors

[0222] The research results are detailed in Table 8.

[0223] Table 8 shows the freeze-thaw stability study data, indicating that under conditions ranging from -40℃ to room temperature, after 1, 3, and 5 cycles, the basic physicochemical properties and purity of the protein did not change significantly. With increasing freeze-thaw cycles, enzyme activity showed no significant change. Both the protein and hyaluronidase exhibited good freeze-thaw stability when subjected to up to 5 freeze-thaw cycles.

[0224] Table 8. Stability Study of Freeze-Thaw Effects

[0225]

[0226]

[0227] 4. Lighting factors

[0228] The research results are detailed in Table 9.

[0229] Table 9 shows the stability study data under the influence of light. Under light conditions of 25℃ and 900 lx, and under the control at 25℃ in the dark for 2 weeks, there were no significant differences in the basic physicochemical properties and appearance of the protein. The purity results showed no significant difference in SEC and CEX. There was no significant decrease in enzyme activity after 2 weeks of light exposure, indicating good light stability.

[0230] Table 9. Stability Study of Illumination Effects

[0231]

[0232] In summary, after being stored at 25°C for 3 months, shaken at 25°C for 2 weeks, exposed to light at 25°C for 2 weeks, and subjected to 5 freeze-thaw cycles, inoleizumab and hyaluronidase activity showed good stability. The formulation provides good protection for inoleizumab. Storage at 5°C for 3 months also demonstrated good stability of inoleizumab and hyaluronidase activity. The optimized formulation, under multiple conditions, can ensure good product quality and guarantee the stability of the drug during production, storage, and transportation.

[0233] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A CD19 antibody drug formulation comprising the following components: (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate 80, (d) 40 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 10 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate 80, (d) 35 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 15 mM methionine, wherein the pH of the CD19 antibody formulation is 5.5; or (a) 120 mg / mL to 180 mg / mL inoleizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL polysorbate 80, (d) 55 mg / mL sorbitol, (e) 2000 U / mL hyaluronidase, (f) 5 mM methionine, wherein the pH of the CD19 antibody drug formulation is 5.5; in, The heavy chain amino acid sequence of the inelizumab is shown in SEQ ID NO: 9, and the light chain amino acid sequence is shown in SEQ ID NO: 10.

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