CD19 antibody pharmaceutical preparation

Abstract

The invention relates to a CD19 antibody pharmaceutical preparation. Specifically, the invention relates to the pharmaceutical preparation of the irnerizumab, and the pharmaceutical preparation comprises the irnerizumab, a buffer solution, a stabilizer and a surfactant. The pharmaceutical preparation can prevent aggregation, degradation, oxidation or denaturation and the like of the irnerizumab, so that the biological activity of the effective components of the irnerizumab is maintained, and the pharmaceutical preparation is suitable for clinical use.

Description

Technical Field

[0001] The present application belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising a monoclonal antibody, and its use as a drug in cancer, transplantation disease and autoimmune disease. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

[0003] The intravenous infusion of inelizumab requires patients to use an intravenous pump, gradually increasing the infusion rate over approximately 90 minutes. Patients must be monitored for infusion reactions during the infusion and continue to be observed for at least one hour after the infusion. In contrast, the subcutaneous injection formulation takes only 5-10 minutes to complete treatment, which is undoubtedly a more convenient treatment experience for patients receiving anti-CD19 therapy.

[0004] Therefore, in order to solve problems such as long dosing time and low patient compliance, as well as to facilitate clinical use and reduce risks, an inerizumab formulation with excellent stability is urgently needed. Summary of the Invention

[0005] The problem to be solved by the present application is to provide a stable pharmaceutical preparation containing high-concentration inerizumab that can be injected subcutaneously.

[0006] The inelizumab and hyaluronidase of the present application can form a stable formulation, and the stable formulation can be injected subcutaneously at a higher volume. First, the preparation of high-concentration antibody preparations is challenging. Higher concentrations of proteins will have great viscosity, increasing the risk of aggregation; secondly, it is very difficult to add hyaluronidase and maintain a high concentration of antibodies and achieve a certain stability. In addition, although antibodies or their antigen-binding fragments have very similar overall structures, antibodies or their antigen-binding fragments differ in amino acid composition and glycosylation, and each antibody or its antigen-binding fragment has different aggregation behavior. Therefore, there is no regularity in obtaining a stable formulation for a certain antibody or its antigen-binding fragment, especially in obtaining a formulation containing hyaluronidase at the same time. The present application provides a stable pharmaceutical formulation comprising high-concentration inelizumab, which can be used for subcutaneous injection.

[0007] The first aspect of the present application provides a CD19 antibody pharmaceutical preparation comprising:

[0008] i) an anti-CD19 antibody or an antigen-binding fragment thereof, comprising a heavy chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a light chain 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, the hyaluronidase content is 1000 to 20000 U / mL; preferably, the hyaluronidase content is 1000 to 10000 U / mL; more preferably, the hyaluronidase content is 1000 to 5000 U / mL; further preferably, the hyaluronidase content is about 1000 U / mL, about 2000 U / mL, about 3000 U / mL, or about 4000 U / mL.

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

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

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

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

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

[0015] In some embodiments, the anti-CD19 antibody or antigen-binding fragment thereof has a concentration of 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 50 mg / mL to 200 mg / mL. In a preferred embodiment, the concentration of the anti-CD19 antibody or its antigen-binding fragment is 100 mg / mL to 200 mg / mL. In a preferred embodiment, the concentration of the anti-CD19 antibody or its antigen-binding fragment is 120 mg / mL to 180 mg / mL.

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

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

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

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

[0021] In some embodiments, the buffer is acetic acid-sodium acetate buffer, and the concentration of the buffer is 5 mM to 50 mM.

[0022] In a preferred embodiment, the buffer is an acetic acid-sodium acetate buffer, and the concentration of the buffer is 10 mM to 40 mM. In a preferred embodiment, the buffer is an acetic acid-sodium acetate buffer, and the concentration of the buffer is about 10 mM, about 20 mM, about 30 mM, or about 40 mM.

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

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

[0025] In some embodiments, the buffer is acetic acid-sodium acetate buffer, the concentration of the buffer is 5 mM to 50 mM, and the pH of the CD19 antibody pharmaceutical preparation is 5.0 to 7.0.

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

[0027] In some embodiments, the CD19 antibody pharmaceutical preparation further comprises a stabilizer, and the stabilizer is 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, and sorbitol. In a preferred embodiment, the stabilizer is selected from sorbitol.

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

[0030] In a preferred embodiment, the stabilizer concentration is 30 mg / mL to 100 mg / mL. In a preferred embodiment, the stabilizer concentration is 35 mg / mL to 55 mg / mL or 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, about 80 mg / mL.

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

[0032] In a preferred embodiment, the stabilizer is selected from sorbitol, and the stabilizer concentration is 30 mg / mL to 100 mg / mL. In a preferred embodiment, the stabilizer is selected from sorbitol, and the stabilizer concentration is 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 pharmaceutical formulation further comprises an antioxidant, wherein the antioxidant is 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 1 mM to 50 mM.

[0035] In a preferred embodiment, the concentration of the antioxidant is 5 mM to 50 mM. In a preferred embodiment, the concentration of the antioxidant is 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, 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 1 mM to 50 mM.

[0037] In a preferred embodiment, the antioxidant is selected from methionine, and the concentration of the antioxidant is 5 mM to 50 mM. In a preferred embodiment, the antioxidant is selected from methionine, and the concentration of the antioxidant is 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 pharmaceutical preparation further comprises a surfactant, wherein the surfactant is 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 0.01 mg / mL to 1.0 mg / mL.

[0041] In a preferred embodiment, the concentration of the surfactant is 0.1 mg / mL to 0.8 mg / mL. In a preferred embodiment, the concentration of the surfactant is 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 concentration of the surfactant is 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 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 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 pharmaceutical formulation comprises the following components: (a) 50 mg / mL to 200 mg / mL of inerizumab, (b) 1 mM to 100 mM buffer, (c) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (d) 20 mg / mL to 150 mg / mL of a stabilizer, (e) 1000 to 20000 U / mL of hyaluronidase, and (f) 1 mM to 50 mM of an antioxidant, and the pH of the CD19 antibody pharmaceutical formulation is 5.0 to 7.0.

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

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

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

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

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

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

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

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

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

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

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

[0056] In a preferred embodiment, the CD19 antibody pharmaceutical formulation comprises the following components: (a) 120 mg / mL inelizumab, (b) 20 mM acetic acid-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. The pH of the CD19 antibody pharmaceutical formulation is 5.5.

[0057] In a preferred embodiment, the CD19 antibody pharmaceutical formulation comprises the following components: (a) 150 mg / mL inelizumab, (b) 20 mM acetic acid-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. The pH of the CD19 antibody pharmaceutical formulation is 5.5.

[0058] In a second aspect, the present application provides a CD19 antibody pharmaceutical preparation comprising: i) inelizumab, whose heavy chain amino acid sequence is shown in SEQ ID NO: 9, and whose light chain amino acid sequence is shown in SEQ ID NO: 10, wherein the concentration of inelizumab is 50 mg / mL to 200 mg / mL; ii) hyaluronidase, wherein the hyaluronidase content is 1000 to 5000 U / mL; iii) 10 mM to 40 mM acetic acid-sodium acetate buffer; iv) 35 mg / mL to 55 mg / mL sorbitol, 5 mM to 15 mM methionine; v) 0.2 mg / mL to 0.6 mg / mL polysorbate 80; the pH of the CD19 antibody pharmaceutical preparation is 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 inelizumab is 100 mg / mL to 200 mg / mL.

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

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

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

[0064] In some embodiments, the concentration of sorbitol is 35 mg / mL, 40 mg / mL, or 55 mg / mL; and the concentration of methionine 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 polysorbate 80 is 0.2 mg / mL, 0.4 mg / mL, or 0.6 mg / mL.

[0067] In a preferred embodiment, the CD19 antibody pharmaceutical preparation comprises the following components:

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

[0069] In a preferred embodiment, the CD19 antibody pharmaceutical preparation comprises the following components:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0094] The third aspect of the present application provides a container comprising the above-mentioned CD19 antibody pharmaceutical preparation; preferably, the container is a small tube or a vial.

[0095] The fourth aspect of the present application provides a medicine box comprising the above-mentioned container or the above-mentioned subcutaneous delivery device.

[0096] In a fifth aspect, the present application provides a method for reducing drug therapy infusion time, comprising subcutaneously administering the above-mentioned CD19 antibody pharmaceutical preparation or the above-mentioned drug kit.

[0097] In a sixth aspect, the present application provides the use of the above-mentioned CD19 antibody pharmaceutical preparation or the above-mentioned drug kit in the preparation of a drug for treating B cell diseases or disorders.

[0098] In preferred embodiments, the B cell disease or disorder is selected from the group consisting of a B cell malignancy, an autoimmune disease, humoral rejection in a human transplant patient, graft-versus-host disease (GVHD), and a post-transplant lymphoproliferative disorder in a human transplant recipient.

[0099] In a preferred embodiment, the autoimmune disease is selected from the group consisting of 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 may be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments obtained by combination are further described in detail below.

[0101] the term

[0102] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0103] The “mM” mentioned in the present disclosure is specifically “mmol / L”.

[0104] As used herein, the terms "about" and "approximately" refer to values ​​within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in each practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0105] When point values ​​are mentioned in this disclosure, it should be understood that the point values ​​include a margin of error. This margin of error is due to factors such as laboratory environment, human operation, instrumentation, methodology, and measurement error.

[0106] Hyaluronidase is a kind of enzyme that degrades hyaluronic acid and reduces the viscosity of hyaluronic acid in extracellular matrix thereby increases tissue permeability.Alternatively, described hyaluronidase is for self-recombinant DNA technology manufacture, people's sample derivative or animal tissue extraction.In the disclosure, hyaluronidase has the common implication of this area, comprises the natural hyaluronidase, recombinant hyaluronidase or their variant that are derived from animal, for example, is derived from the hyaluronidase of rat, mouse, rabbit, cat, dog, monkey, gorilla, people or other primates.Many suitable hyaluronidases of the application are known, and preferred enzyme is human hyaluronidase, such as soluble human PH20 hyaluronidase, is preferably called the recombinant human hyaluronidase of rHuPH20.Exemplary human hyaluronidase is referring to NCBI Reference Sequence:NP_001167515.1, and exemplary recombinant human hyaluronidase is truncated variant or the mutant variant of human hyaluronidase.

[0107] The enzymatic activity of hyaluronidase (including rHuPH20) can be defined by unit / mL (U / mL) or by the total enzyme activity (U) in a particular formulation. The standard definition of the enzymatic activity (U) of a unit is the amount of enzyme that catalyzes the substrate reaction of a limited amount per unit time, for example, one mole or one nanomole substrate / minute. The technology used to measure the activity of hyaluronidase preparations is known in the art, and the activity of hyaluronidase is usually represented by USP units or units, and hereinafter all represents the activity of hyaluronidase by "U / mL".

[0108] The unifying feature of all hyaluronidases is the ability of its depolymerized hyaluronan, regardless of the difference in chemical structure, species origin, tissue origin or the batch of pharmaceutical products derived from same species and tissue. Their uniqueness is that, although having different structures, their activity is identical (except effectiveness). According to the hyaluronidase of the preparation of the present disclosure, it is characterised in that the molecular integrity of the antibody or its Fab in the stable pharmaceutical composition described in the application is not adversely affected. In addition, hyaluronidase only changes the delivery of antibody or its Fab to systemic circulation, but does not have any characteristic of the therapeutic effect of the antibody or its Fab that can provide or contribute to systemic absorption.

[0109] The three-letter and one-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, polyspecific antibodies, dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site with the desired specificity, so long as they exhibit the desired biological activity. Typically, a natural intact antibody consists of a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0111] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended existing technology, mammalian expression systems will lead to glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product should be immediately frozen, such as at -70°C, or freeze-dried.

[0112] "Buffer" refers to a buffering agent that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0113] A "histidine buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate. Histidine acetate buffer is preferred. Histidine acetate buffer is a mixture of histidine and acetic acid, while histidine hydrochloride buffer is a mixture of histidine and hydrochloric acid.

[0114] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0115] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

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

[0117] "Acetate buffer" is a buffer comprising acetate ions. Examples of acetate buffers include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like. A preferred acetate buffer is acetate-sodium acetate.

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

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

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

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

[0122] The pharmaceutical compositions disclosed herein can achieve a stable effect: the antibodies or antigen-binding fragments thereof substantially retain their physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability as well as their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Various analytical techniques are currently available for measuring protein stability after storage at a selected temperature for a selected period of time.

[0123] A stable formulation is one in which no significant change is 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. In addition, a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after being stored at a temperature of 25°C for a period of 1 month, 3 months, or 6 months. A typical example of stability is that no more than about 10%, preferably no more than about 5%, of the antibody monomers aggregate or degrade as measured by SEC-HPLC. By visual analysis, the formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. A decrease of no more than about 10%, preferably no more than about 5%, is typically observed. Aggregations of no more than about 10%, preferably no more than about 5%, are typically formed.

[0124] The term "stable" means that all proteins therein substantially retain their physical, chemical, and biological activities after storage at the intended storage temperature, e.g., 0-40°C. A formulation may be considered stable even if the antibodies therein do not retain 100% of their physical, chemical, or biological activities after storage for a specified period of time. A formulation may be considered "stable" if it retains approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater of the antibody structure and function after storage for a specified period of time.

[0125] An antibody or antigen-binding fragment thereof "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0126] An antibody or antigen-binding fragment thereof "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

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

[0128] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

[0129] The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0130] The term "carrier" as used in the context of the present invention refers to a system that can alter the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, preferably micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules while also having good membrane permeability, making them excellent drug carriers.

[0131] The terms "administer" and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ or a biological fluid refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. "Administer" and "treat" can refer to, for example, treatment, pharmacokinetics, diagnosis, research and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer" and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition or by another cell. "Treatment" as applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0132] The term "treating" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient experiencing one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test commonly used by physicians or other health care professionals to assess the severity or progression of such symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's 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" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may 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 dosage regimen that avoids significant side effects or toxic effects. DETAILED DESCRIPTION

[0134] The following examples are provided so as to provide those skilled in the art with a complete disclosure and description of how to make and use the pharmaceutical formulations of the present invention, but are not to be construed as limiting the scope of the invention.

[0135] This application discloses a stable pharmaceutical composition comprising an anti-CD19 antibody or antigen-binding fragment thereof. The anti-CD19 antibody described herein is inelizumab, a humanized, affinity-optimized, afucosylated IgG1κ monoclonal antibody that binds to the B cell surface antigen CD19. Inelizumab and its preparation method are described in International PCT Patent Application No. PCT / US2007 / 077916, published as WO2008 / 031056, which is hereby incorporated by reference herein. PCT / US2007 / 077916 refers to the anti-CD19 antibody "16C4." The experimental results presented herein were generated using the anti-CD19 antibody inelizumab. Inelizumab described herein 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 complex N-glycoside-linked carbohydrate chains, wherein fucose is not bound to N-acetylglucosamine at the reducing end of the carbohydrate chains (see U.S. patent application Ser. No. 11 / 852,106, filed September 7, 2007, the disclosure of which is incorporated herein in its entirety for all purposes). Inelizumab described herein 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. Purified anti-CD19 antibodies were produced using standard industrial-scale protocols. The relevant sequences are shown in Tables A and B.

[0136] Table A. Inelizumab CDR and variable region sequence information

[0137]

[0138] Note: CDR sequences were defined according to the Kabat definition scheme.

[0139] Table B. Inerizumab full-length sequence information

[0140]

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

[0142] Description of analytical tests used in this application

[0143] 1. Appearance and visible foreign matter

[0144] Visually inspect the sample's appearance, including color, clarity, and visible foreign matter. The procedure is as follows: Wipe the sample bottle clean and observe the appearance using water as a negative control: 1) In a darkroom, place the sample under a clarity meter with the illumination adjusted to 2000-3750 lux. Place the sample at the edge of the light shield while holding the neck of the sample bottle against both black and white backgrounds to observe the sample's color, clarity, and visible foreign matter. 2) Visually inspect the sample's color under fluorescent light, using a white backing plate as the background.

[0145] 2. Protein Concentration

[0146] Using a NanoPhotometer NP80 protein concentration analyzer, add 2.5 μL of sample and measure the absorbance at 280 nm. Measure each sample twice and take the average value.

[0147] 3. pH

[0148] A Mettler S470-K multifunctional parameter analyzer was used for calibration with three standard solutions (pH values ​​of 4.01, 7.00, and 9.21, respectively). The electrode slope was kept within the range of 95%-105%, and 100 μL of sample was taken to determine the pH value.

[0149] 4. Hyaluronidase Activity Assay

[0150] Hyaluronidase activity in the formulation was measured using a SpectraMax M5 multi-function microplate reader. Acidic bovine serum albumin (BSA) precipitates hyaluronic acid. After the reaction between hyaluronic acid and the hyaluronidase in the antibody formulation, acidic BSA was added to precipitate undigested hyaluronic acid. Enzyme activity was assessed by measuring turbidity at 640 nm following the reaction.

[0151] 5. Microfluidics Particle Imaging (MFI)

[0152] The number of particles and particle morphology of the sample were determined using a microfluidics particle imaging system (MFI). Automatic sampling.

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

[0154] 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 detected using a hydrophilic silica size exclusion chromatography column, and the sample purity was calculated using the area normalization method.

[0155] 7. Charge Variant Detection (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 analytical column, and the purity of the acid, base and main component of the samples was calculated using the area normalization method.

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

[0158] The following injection formulations were prepared. Formulation 1 was based on the original low-concentration IV formulation of anti-CD19 antibody, with hyaluronidase added and the antibody concentration increased. The effects of different buffer systems on antibody stability were investigated together with formulations 2-7.

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

[0160] Prescription 2: 120 mg / mL inelizumab, 20 mM acetic acid-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 acetic acid-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 citric acid-sodium citrate, 75 mM sodium chloride, 40 mg / mL trehalose, 0.2 mg / mL polysorbate 80, 2000 U / mL hyaluronidase, pH 6.0;

[0163] Prescription 5: 120 mg / mL inelizumab, 20 mM citric acid-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 formulation was placed in a constant temperature incubator and samples were taken at 0, 1, 2, and 4 weeks to assess stability. Antibody stability and enzyme stability at high temperatures were compared to determine the appropriate buffer system. Protein stability was assessed at 40°C, but since 40°C can damage hyaluronidase activity, enzyme stability was assessed at 30°C.

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

[0168]

[0169]

[0170] The buffer system screening data in Table 1 shows that after one week at 40°C, all formulations showed no visible particles. After two weeks, visible particles were observed in formulations 1 and 7, while the remaining formulations maintained a good appearance. SEC purity data showed that the main peak variation was: formulation 3 < formulation 2 < formulation 5 < formulation 4 < formulation 7 ≈ formulation 1 ≈ formulation 6. CEX purity data showed that the main peak variation was: formulation 3 < formulation 5 < formulation 6 ≈ formulation 4 < formulation 2 < formulation 1 < formulation 7. The decrease in hyaluronidase activity among the groups was within the acceptable range and showed no significant differences.

[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 acetic acid-sodium acetate buffer system exhibited excellent stability. However, studies also found that high pH values ​​were detrimental to its stability. Therefore, the preferred buffer system was acetic acid-sodium acetate at pH 5.5.

[0172] Example 2. Screening of Stabilizers and Antioxidants for Inelizumab Formulations

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

[0174] Prescription 3: 120 mg / mL inelizumab, 20 mM acetic acid-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 inelizumab, 20 mM acetic acid-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 inelizumab, 20 mM acetic acid-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 inelizumab, 20 mM acetic acid-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 inelizumab, 20 mM acetic acid-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 acetic acid-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 formulation was placed in a constant temperature chamber and samples were taken at 0, 1, 2, and 4 weeks to investigate its stability. The antibody stability and enzyme activity stability at high temperature were compared to determine the appropriate stabilizer.

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

[0182]

[0183]

[0184] Table 3. Summary of protein particle number MFI in stabilizer screening

[0185]

[0186] The stabilizer screening data in Table 2 shows that after 4 weeks at 40°C, formulations 8 and 10 showed no visible particles, indicating a good appearance. Formulations 11 and 12 exhibited visible particles. SEC purity data showed that the main peak variation was: Formulation 10 < Formulation 9 < Formulation 3 < Formulation 8 ≈ Formulation 12 ≈ Formulation 11. CEX purity data showed that the main peak variation was: Formulation 10 < Formulation 9 < Formulation 3 < Formulation 12 < Formulation 8 < Formulation 11. The decrease in hyaluronidase activity among the groups was within the acceptable range and showed no significant differences.

[0187] The protein particle count (MFI) data in Table 3 show that the combination of the stabilizer sorbitol and the antioxidant methionine stabilizes the antibody formulation, with sorbitol outperforming trehalose and sucrose, while EDTA is detrimental to its stability. Therefore, the preferred stabilizer is 40 mg / mL sorbitol and the antioxidant is 10 mM methionine.

[0188] Example 3. Screening experiment of stabilizer and antioxidant content of inelizumab formulation

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

[0190] Prescription 10: 120 mg / mL inelizumab, 20 mM acetic acid-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 acetic acid-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 inerizumab, 20 mM acetic acid-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 an accelerated temperature of 40°C. 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 at 40°C, the appearance of each formulation was good. The stability data showed similar trends, with no significant differences overall. Sorbitol in the 35-55 mg / mL range and methionine in the 5-15 mM range both served as effective stabilizers.

[0197] Example 4. Inerizumab concentration screening

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

[0199] Prescription 10: 120 mg / mL inelizumab, 20 mM acetic acid-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 acetic acid-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 inerizumab, 20 mM acetic acid-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 an accelerated temperature of 40°C. 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] The antibody concentration screening data in Table 5 show that after 4 weeks at 40°C, all formulations exhibited good appearance and generally consistent stability trends. Aggregate content at the 180 mg / mL protein concentration increased slightly after 4 weeks, but remained within acceptable limits. The 150 mg / mL protein concentration showed good stability, and considering downstream process friendliness, a 120.0 mg / mL protein concentration was used.

[0207] Example 5. Confirmation of Inerizumab Subcutaneous Injection Prescription

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

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

[0210] 1) Long-term stability study

[0211] The long-term stability was investigated at 25°C and 5°C for 3 months. The results are shown in Table 6 below.

[0212] The long-term stability test results in Table 6 show that there is no obvious quality decline in various data compared with the previous prescription screening, and the appearance, purity, number of protein particles, enzyme activity and other aspects perform well.

[0213] Table 6. Long-term stability study results

[0214]

[0215]

[0216] 2) Vibration factor

[0217] In this study, the shaking stability at 25°C and 200 rpm was investigated. The results are shown in Table 7.

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

[0219] Table 7. 25℃ 200rpm shaking stability test

[0220]

[0221] 3. Freeze-thaw factors

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

[0223] The freeze-thaw stability data in Table 8 show that the protein's basic physicochemical properties and purity remained unchanged after 1, 3, or 5 cycles between -40°C and room temperature. Enzyme activity also remained stable with increasing freeze-thaw cycles. Proteins and hyaluronidase exhibited good freeze-thaw stability when subjected to no more than five freeze-thaw cycles.

[0224] Table 8. Study on the effect of freeze-thaw on stability

[0225]

[0226]

[0227] 4. Light factors

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

[0229] The data from the stability study on the effect of light in Table 9 show that there were no significant differences in the basic physicochemical properties and appearance of the protein when placed under 25°C, 900lx light conditions and the control at 25°C in the dark for 2 weeks. The purity results showed no significant differences between SEC and CEX. There was no obvious decrease in enzyme activity after 2 weeks of light exposure, indicating good light stability.

[0230] Table 9. Study on the effect of light on stability

[0231]

[0232] In summary, after 3 months at 25°C, 2 weeks of shaking at 25°C, 2 weeks of exposure to light at 25°C, and 5 freeze-thaw cycles, inelizumab and hyaluronidase activity remained stable. This formulation demonstrated good protection for inelizumab. Storage at 5°C for 3 months also demonstrated good stability of inelizumab and hyaluronidase activity. The optimized formulation, under multiple factors, ensured good product quality and drug stability during production, storage, and transportation.

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

Claims

1. A CD19 antibody pharmaceutical preparation comprising: i) inelizumab, whose heavy chain amino acid sequence is set forth in SEQ ID NO: 9, and whose light chain amino acid sequence is set forth in SEQ ID NO: 10, wherein the concentration of inelizumab is 50 mg / mL to 200 mg / mL; ii) hyaluronidase, wherein the hyaluronidase content is 1000 to 5000 U / mL; iii) 10 mM to 40 mM acetic acid-sodium acetate buffer; iv) 35 mg / mL to 55 mg / mL sorbitol, 5 mM to 15 mM methionine; v) 0.2 mg / mL to 0.6 mg / mL polysorbate 80; The pH of the CD19 antibody pharmaceutical preparation is 5.0 to 6.

0.

2. The CD19 antibody pharmaceutical preparation according to claim 1, wherein the hyaluronidase content is 1000 U / mL, 2000 U / mL, 3000 U / mL or 4000 U / mL.

3. The CD19 antibody pharmaceutical preparation according to claim 1, wherein the concentration of inelizumab is 100 mg / mL to 200 mg / mL. The CD19 antibody pharmaceutical preparation according to claim 3 , wherein the concentration of inerizumab is 120 mg / mL to 180 mg / mL. 5 . The CD19 antibody pharmaceutical preparation according to claim 1 , wherein the concentration of the acetic acid-sodium acetate buffer is 10 mM, 20 mM, 30 mM or 40 mM. The CD19 antibody pharmaceutical preparation according to claim 1 , wherein the pH of the CD19 antibody pharmaceutical preparation is 5.

5.

7. The CD19 antibody pharmaceutical preparation according to claim 1, wherein the concentration of sorbitol is 35 mg / mL, 40 mg / mL or 55 mg / mL; and the concentration of methionine is 5 mM, 10 mM or 15 mM.

8. The CD19 antibody pharmaceutical preparation according to claim 7, wherein the sorbitol concentration is 40 mg / mL and the methionine concentration is 10 mM; or, sorbitol concentration was 35 mg / mL and methionine concentration was 15 mM; Alternatively, the sorbitol concentration was 55 mg / mL and the methionine concentration was 5 mM.

9. The CD19 antibody pharmaceutical preparation according to claim 1, wherein the concentration of polysorbate 80 is 0.2 mg / mL, 0.4 mg / mL or 0.6 mg / mL.

10. The CD19 antibody pharmaceutical preparation according to any one of claims 1 to 9, comprising the following components: (a) 100 mg / mL to 200 mg / mL of inelizumab, (b) 10 mM, 20 mM or 30 mM acetic acid-sodium acetate buffer, (c) 0.2 mg / mL, 0.4 mg / mL or 0.6 mg / mL of polysorbate 80, (d) 35 mg / mL, 40 mg / mL or 55 mg / mL of sorbitol, (e) 2000 U / mL, 3000 U / mL or 4000 U / mL of hyaluronidase, and (f) 5 mM, 10 mM or 15 mM methionine, wherein the pH of the CD19 antibody pharmaceutical formulation is 5.0 to 6.

0.

11. The CD19 antibody pharmaceutical preparation according to claim 10, comprising the following components: (a) 120 mg / mL to 180 mg / mL of inelizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL of polysorbate, (d) 40 mg / mL of sorbitol, (e) 2000 U / mL of hyaluronidase, and (f) 10 mM methionine, wherein the pH of the CD19 antibody pharmaceutical formulation is 5.5; or (a) 120 mg / mL to 180 mg / mL of inelizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL of polysorbate, (d) 35 mg / mL of sorbitol, (e) 2000 U / mL of hyaluronidase, and (f) 15 mM methionine, wherein the pH of the CD19 antibody pharmaceutical formulation is 5.5; or (a) 120 mg / mL to 180 mg / mL of inerizumab, (b) 20 mM acetate buffer, (c) 0.2 mg / mL of polysorbate, (d) 55 mg / mL of sorbitol, (e) 2000 U / mL of hyaluronidase, and (f) 5 mM methionine. The pH of the CD19 antibody pharmaceutical formulation is 5.5.

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